Energetic basis for selective recognition of T·G mismatched base pairs in DNA by imidazole-rich polyamides

PubMed Central

Lacy, Eilyn R.; Nguyen, Binh; Le, Minh; Cox, Kari K.; O'Hare, Caroline; Hartley, John A.; Lee, Moses; Wilson, W. David

2004-01-01

To complement available structure and binding results and to develop a detailed understanding of the basis for selective molecular recognition of T·G mismatches in DNA by imidazole containing polyamides, a full thermodynamic profile for formation of the T·G–polyamide complex has been determined. The amide-linked heterocycles f-ImImIm and f-PyImIm (where f is formamido group, Im is imidazole and Py is pyrrole) were studied by using biosensor-surface plasmon resonance (SPR) and isothermal titration calorimetry (ITC) with a T·G mismatch containing DNA hairpin duplex and a similar DNA with only Watson–Crick base pairs. Large negative binding enthalpies for all of the polyamide–DNA complexes indicate that the interactions are enthalpically driven. SPR results show slower complex formation and stronger binding of f-ImImIm to the T·G than to the match site. The thermodynamic analysis indicates that the enhanced binding to the T·G site is the result of better entropic contributions. Negative heat capacity changes for the complex are correlated with calculated solvent accessible surface area changes and indicate hydrophobic contributions to complex formation. DNase I footprinting analysis in a long DNA sequence provided supporting evidence that f-ImImIm binds selectively to T·G mismatch sites. PMID:15064359

Vital Roles of the Second DNA-binding Site of Rad52 Protein in Yeast Homologous Recombination*

PubMed Central

Arai, Naoto; Kagawa, Wataru; Saito, Kengo; Shingu, Yoshinori; Mikawa, Tsutomu; Kurumizaka, Hitoshi; Shibata, Takehiko

2011-01-01

RecA/Rad51 proteins are essential in homologous DNA recombination and catalyze the ATP-dependent formation of D-loops from a single-stranded DNA and an internal homologous sequence in a double-stranded DNA. RecA and Rad51 require a “recombination mediator” to overcome the interference imposed by the prior binding of single-stranded binding protein/replication protein A to the single-stranded DNA. Rad52 is the prototype of recombination mediators, and the human Rad52 protein has two distinct DNA-binding sites: the first site binds to single-stranded DNA, and the second site binds to either double- or single-stranded DNA. We previously showed that yeast Rad52 extensively stimulates Rad51-catalyzed D-loop formation even in the absence of replication protein A, by forming a 2:1 stoichiometric complex with Rad51. However, the precise roles of Rad52 and Rad51 within the complex are unknown. In the present study, we constructed yeast Rad52 mutants in which the amino acid residues corresponding to the second DNA-binding site of the human Rad52 protein were replaced with either alanine or aspartic acid. We found that the second DNA-binding site is important for the yeast Rad52 function in vivo. Rad51-Rad52 complexes consisting of these Rad52 mutants were defective in promoting the formation of D-loops, and the ability of the complex to associate with double-stranded DNA was specifically impaired. Our studies suggest that Rad52 within the complex associates with double-stranded DNA to assist Rad51-mediated homologous pairing. PMID:21454474

H3K9me3 demethylase Kdm4d facilitates the formation of pre-initiative complex and regulates DNA replication

PubMed Central

Wu, Rentian; Wang, Zhiquan; Zhang, Honglian; Gan, Haiyun; Zhang, Zhiguo

2017-01-01

DNA replication is tightly regulated to occur once and only once per cell cycle. How chromatin, the physiological substrate of DNA replication machinery, regulates DNA replication remains largely unknown. Here we show that histone H3 lysine 9 demethylase Kdm4d regulates DNA replication in eukaryotic cells. Depletion of Kdm4d results in defects in DNA replication, which can be rescued by the expression of H3K9M, a histone H3 mutant transgene that reverses the effect of Kdm4d on H3K9 methylation. Kdm4d interacts with replication proteins, and its recruitment to DNA replication origins depends on the two pre-replicative complex components (origin recognition complex [ORC] and minichromosome maintenance [MCM] complex). Depletion of Kdm4d impairs the recruitment of Cdc45, proliferating cell nuclear antigen (PCNA), and polymerase δ, but not ORC and MCM proteins. These results demonstrate a novel mechanism by which Kdm4d regulates DNA replication by reducing the H3K9me3 level to facilitate formation of pre-initiative complex. PMID:27679476

Binding characteristics and protective capacity of cyanidin-3-glucoside and its aglycon to calf thymus DNA.

PubMed

Zhang, Chao; Guo, Xiaofei; Cai, Wenqian; Ma, Yue; Zhao, Xiaoyan

2015-04-01

The binding characteristics and protective capacity of cyanidin (Cy) and cyanidin-3-glucoside (C3G) to calf thymus DNA were explored for the first time. The Cy and C3G gave a bathochromic shift to the ultraviolet-visible spectra of the DNA, indicating the formation of the DNA-Cy and DNA-C3G complexes. The complexes were formed by an intercalative binding mode based on the results of the fluorescence spectra and competitive binding analysis. Meanwhile, the Cy and C3G protected the DNA from the damage induced by the hydroxyl radical. The binding capacity and protective capacity of the C3G were stronger than that of the Cy. Furthermore, the formation of the DNA-anthocyanin complexes was spontaneous when the hydrogen bond and hydrophobic force played a key role. Hence, the Cy and C3G could protect the DNA automatically from the damage induced by the hydroxyl radical. © 2015 Institute of Food Technologists®

Structures of RNA Polymerase Closed and Intermediate Complexes Reveal Mechanisms of DNA Opening and Transcription Initiation.

PubMed

Glyde, Robert; Ye, Fuzhou; Darbari, Vidya Chandran; Zhang, Nan; Buck, Martin; Zhang, Xiaodong

2017-07-06

Gene transcription is carried out by RNA polymerases (RNAPs). For transcription to occur, the closed promoter complex (RPc), where DNA is double stranded, must isomerize into an open promoter complex (RPo), where the DNA is melted out into a transcription bubble and the single-stranded template DNA is delivered to the RNAP active site. Using a bacterial RNAP containing the alternative σ 54 factor and cryoelectron microscopy, we determined structures of RPc and the activator-bound intermediate complex en route to RPo at 3.8 and 5.8 Å. Our structures show how RNAP-σ 54 interacts with promoter DNA to initiate the DNA distortions required for transcription bubble formation, and how the activator interacts with RPc, leading to significant conformational changes in RNAP and σ 54 that promote RPo formation. We propose that DNA melting is an active process initiated in RPc and that the RNAP conformations of intermediates are significantly different from that of RPc and RPo. Copyright © 2017 The Author(s). Published by Elsevier Inc. All rights reserved.

PEG and mPEG-anthracene induce DNA condensation and particle formation.

PubMed

Froehlich, E; Mandeville, J S; Arnold, D; Kreplak, L; Tajmir-Riahi, H A

2011-08-18

In this study, we investigated the binding of DNA with poly(ethylene glycol) (PEG) of different sizes and compositions such as PEG 3350, PEG 6000, and mPEG-anthracene in aqueous solution at physiological conditions. The effects of size and composition on DNA aggregation and condensation as well as conformation were determined using Fourier transform infrared (FTIR), UV-visible, CD, fluorescence spectroscopic methods and atomic force microscopy (AFM). Structural analysis showed moderate complex formation for PEG 3350 and PEG 6000 and weaker interaction for mPE-anthracene-DNA adducts with both hydrophilic and hydrophobic contacts. The order of ± stability of the complexes formed is K(PEG 6000) = 1.5 (±0.4) × 10(4) M(-1) > K(PEG 3350) = 7.9 (±1) × 10(3) M(-1) > K(m(PEG-anthracene))= 3.6 (±0.8) × 10(3) M(-1) with nearly 1 bound PEG molecule per DNA. No B-DNA conformational changes were observed, while DNA condensation and particle formation occurred at high PEG concentration.

Detection of DNA via the fluorescence quenching of Mn-doped ZnSe D-dots/doxorubicin/DNA ternary complexes system.

PubMed

Gao, Xue; Niu, Lu; Su, Xingguang

2012-01-01

This manuscript reports a method for the detection of double-stranded DNA, based on Mn:ZnSe d-dots and intercalating agent doxorubicin (DOX). DOX can quench the photoluminescence (PL) of Mn:ZnSe d-dots through photoinduced electron transfer process, after binding with Mn:ZnSe d-dots. The addition of DNA can result in the formation of the Mn:ZnSe d-dots-DOX-DNA ternary complexes, the fluorescence of the Mn:ZnSe d-dots-DOX complexes would be further quenched by the addition of DNA, thus allowing the detection of DNA. The formation mechanism of the Mn:ZnSe d-dots-DOX-DNA ternary complexes was studied in detail in this paper. Under optimal conditions, the quenched fluorescence intensity of Mn:ZnSe d-dots-DOX system are perfectly described by Stern-Volmer equation with the concentration of hsDNA ranging from 0.006 μg mL(-1) to 6.4 μg mL(-1). The detection limit (S/N = 3) for hsDNA is 0.5 ng mL(-1). The proposed method was successfully applied to the detection of DNA in synthetic samples and the results were satisfactory.

The interaction of DiaA and DnaA regulates the replication cycle in E. coli by directly promoting ATP–DnaA-specific initiation complexes

PubMed Central

Keyamura, Kenji; Fujikawa, Norie; Ishida, Takuma; Ozaki, Shogo; Su’etsugu, Masayuki; Fujimitsu, Kazuyuki; Kagawa, Wataru; Yokoyama, Shigeyuki; Kurumizaka, Hitoshi; Katayama, Tsutomu

2007-01-01

Escherichia coli DiaA is a DnaA-binding protein that is required for the timely initiation of chromosomal replication during the cell cycle. In this study, we determined the crystal structure of DiaA at 1.8 Å resolution. DiaA forms a homotetramer consisting of a symmetrical pair of homodimers. Mutational analysis revealed that the DnaA-binding activity and formation of homotetramers are required for the stimulation of initiation by DiaA. DiaA tetramers can bind multiple DnaA molecules simultaneously. DiaA stimulated the assembly of multiple DnaA molecules on oriC, conformational changes in ATP–DnaA-specific initiation complexes, and unwinding of oriC duplex DNA. The mutant DiaA proteins are defective in these stimulations. DiaA associated also with ADP–DnaA, and stimulated the assembly of inactive ADP–DnaA–oriC complexes. Specific residues in the putative phosphosugar-binding motif of DiaA were required for the stimulation of initiation and formation of ATP–DnaA-specific–oriC complexes. Our data indicate that DiaA regulates initiation by a novel mechanism, in which DiaA tetramers most likely bind to multiple DnaA molecules and stimulate the assembly of specific ATP–DnaA–oriC complexes. These results suggest an essential role for DiaA in the promotion of replication initiation in a cell cycle coordinated manner. PMID:17699754

H3K9me3 demethylase Kdm4d facilitates the formation of pre-initiative complex and regulates DNA replication.

PubMed

Wu, Rentian; Wang, Zhiquan; Zhang, Honglian; Gan, Haiyun; Zhang, Zhiguo

2017-01-09

DNA replication is tightly regulated to occur once and only once per cell cycle. How chromatin, the physiological substrate of DNA replication machinery, regulates DNA replication remains largely unknown. Here we show that histone H3 lysine 9 demethylase Kdm4d regulates DNA replication in eukaryotic cells. Depletion of Kdm4d results in defects in DNA replication, which can be rescued by the expression of H3K9M, a histone H3 mutant transgene that reverses the effect of Kdm4d on H3K9 methylation. Kdm4d interacts with replication proteins, and its recruitment to DNA replication origins depends on the two pre-replicative complex components (origin recognition complex [ORC] and minichromosome maintenance [MCM] complex). Depletion of Kdm4d impairs the recruitment of Cdc45, proliferating cell nuclear antigen (PCNA), and polymerase δ, but not ORC and MCM proteins. These results demonstrate a novel mechanism by which Kdm4d regulates DNA replication by reducing the H3K9me3 level to facilitate formation of pre-initiative complex. © The Author(s) 2016. Published by Oxford University Press on behalf of Nucleic Acids Research.

Structure of a bacterial RNA polymerase holoenzyme open promoter complex

DOE Office of Scientific and Technical Information (OSTI.GOV)

Bae, Brian; Feklistov, Andrey; Lass-Napiorkowska, Agnieszka

2015-09-08

Initiation of transcription is a primary means for controlling gene expression. In bacteria, the RNA polymerase (RNAP) holoenzyme binds and unwinds promoter DNA, forming the transcription bubble of the open promoter complex (RPo). We have determined crystal structures, refined to 4.14 Å-resolution, of RPo containing Thermus aquaticus RNAP holoenzyme and promoter DNA that includes the full transcription bubble. The structures, combined with biochemical analyses, reveal key features supporting the formation and maintenance of the double-strand/single-strand DNA junction at the upstream edge of the -10 element where bubble formation initiates. The results also reveal RNAP interactions with duplex DNA just upstreammore » of the -10 element and potential protein/DNA interactions that direct the DNA template strand into the RNAP active site. Addition of an RNA primer to yield a 4 base-pair post-translocated RNA:DNA hybrid mimics an initially transcribing complex at the point where steric clash initiates abortive initiation and σA dissociation.« less

Structure of a bacterial RNA polymerase holoenzyme open promoter complex

DOE PAGES

Bae, Brian; Feklistov, Andrey; Lass-Napiorkowska, Agnieszka; ...

2015-09-08

Initiation of transcription is a primary means for controlling gene expression. In bacteria, the RNA polymerase (RNAP) holoenzyme binds and unwinds promoter DNA, forming the transcription bubble of the open promoter complex (RPo). We have determined crystal structures, refined to 4.14 Å-resolution, of RPo containing Thermus aquaticus RNAP holoenzyme and promoter DNA that includes the full transcription bubble. The structures, combined with biochemical analyses, reveal key features supporting the formation and maintenance of the double-strand/single-strand DNA junction at the upstream edge of the -10 element where bubble formation initiates. The results also reveal RNAP interactions with duplex DNA just upstreammore » of the -10 element and potential protein/DNA interactions that direct the DNA template strand into the RNAP active site. Additionally a RNA primer to yield a 4 base-pair post-translocated RNA:DNA hybrid mimics an initially transcribing complex at the point where steric clash initiates abortive initiation and σ A dissociation.« less

Mechanism of introduction of exogenous genes into cultured cells using DEAE-dextran-MMA graft copolymer as non-viral gene carrier.

PubMed

Eshita, Yuki; Higashihara, Junko; Onishi, Masayasu; Mizuno, Masaaki; Yoshida, Jun; Takasaki, Tomohiko; Kubota, Naoji; Onishi, Yasuhiko

2009-07-23

Comparative investigations were carried out regarding the efficiency of introduction of exogenous genes into cultured cells using a cationic polysaccharide DEAE-dextran-MMA (methyl methacrylate ester) graft copolymer (2-diethylaminoethyl-dextran-methyl methacrylate graft copolymer; DDMC) as a nonviral carrier for gene introduction. The results confirmed that the gene introduction efficiency was improved with DDMC relative to DEAE-dextran. Comparative investigations were carried out using various concentrations of DDMC and DNA in the introduction of DNA encoding luciferase (pGL3 control vector; Promega) into COS-7 cells derived from African green monkey kidney cells. The complex formation reaction is thought to be directly proportional to the transformation rate, but the complex formation reaction between DDMC and DNA is significantly influenced by hydrophobic bonding strength along with hydrogen bonding strength and Coulomb forces due to the hydrophobicity of the grafted MMA sections. It is thought that the reaction is a Michaelis-Menten type complex formation reaction described by the following equation: Complex amount = K1 (DNA concentration)(DDMC concentration). In support of this equation, it was confirmed that the amount of formed complex was proportional to the RLU value.

NET formation induced by Pseudomonas aeruginosa cystic fibrosis isolates measured as release of myeloperoxidase-DNA and neutrophil elastase-DNA complexes.

PubMed

Yoo, Dae-goon; Floyd, Madison; Winn, Matthew; Moskowitz, Samuel M; Rada, Balázs

2014-08-01

Cystic fibrosis (CF) airway disease is characterized by Pseudomonas aeruginosa infection and recruitment of neutrophil granulocytes. Neutrophil granule components (myeloperoxidase (MPO), human neutrophil elastase (HNE)), extracellular DNA and P. aeruginosa can all be found in the CF respiratory tract and have all been associated with worsening CF lung function. Pseudomonas-induced formation of neutrophil extracellular traps (NETs) offers a likely mechanism for release of MPO, HNE and DNA from neutrophils. NETs are composed of a DNA backbone decorated with granule proteins like MPO and HNE. Here we sought to examine whether CF clinical isolates of Pseudomonas are capable of inducing NET release from human neutrophil granulocytes. We used two methods to quantify NETs. We modified a previously employed ELISA that detects MPO-DNA complexes and established a new HNE-DNA ELISA. We show that these methods reliably quantify MPO-DNA and HNE-DNA complexes, measures of NET formation. We have found that CF isolates of P. aeruginosa stimulate robust respiratory burst and NET release in human neutrophils. By comparing paired "early" and "late" bacterial isolates obtained from the same CF patient we have found that early isolates induced significantly more NET release than late isolates. Our data support that Pseudomonas-induced NET release represents an important mechanism for release of neutrophil-derived CF inflammatory mediators, and confirm that decreased induction of NET formation is required for long-term adaptation of P. aeruginosa to CF airways. Copyright © 2014 Elsevier B.V. All rights reserved.

Kinetics of DSB rejoining and formation of simple chromosome exchange aberrations

NASA Technical Reports Server (NTRS)

Cucinotta, F. A.; Nikjoo, H.; O'Neill, P.; Goodhead, D. T.

2000-01-01

PURPOSE: To investigate the role of kinetics in the processing of DNA double strand breaks (DSB), and the formation of simple chromosome exchange aberrations following X-ray exposures to mammalian cells based on an enzymatic approach. METHODS: Using computer simulations based on a biochemical approach, rate-equations that describe the processing of DSB through the formation of a DNA-enzyme complex were formulated. A second model that allows for competition between two processing pathways was also formulated. The formation of simple exchange aberrations was modelled as misrepair during the recombination of single DSB with undamaged DNA. Non-linear coupled differential equations corresponding to biochemical pathways were solved numerically by fitting to experimental data. RESULTS: When mediated by a DSB repair enzyme complex, the processing of single DSB showed a complex behaviour that gives the appearance of fast and slow components of rejoining. This is due to the time-delay caused by the action time of enzymes in biomolecular reactions. It is shown that the kinetic- and dose-responses of simple chromosome exchange aberrations are well described by a recombination model of DSB interacting with undamaged DNA when aberration formation increases with linear dose-dependence. Competition between two or more recombination processes is shown to lead to the formation of simple exchange aberrations with a dose-dependence similar to that of a linear quadratic model. CONCLUSIONS: Using a minimal number of assumptions, the kinetics and dose response observed experimentally for DSB rejoining and the formation of simple chromosome exchange aberrations are shown to be consistent with kinetic models based on enzymatic reaction approaches. A non-linear dose response for simple exchange aberrations is possible in a model of recombination of DNA containing a DSB with undamaged DNA when two or more pathways compete for DSB repair.

Formation of monofunctional cisplatin-DNA adducts in carbonate buffer.

PubMed

Binter, Alexandra; Goodisman, Jerry; Dabrowiak, James C

2006-07-01

Carbonate in its various forms is an important component in blood and the cytosol. Since, under conditions that simulate therapy, carbonate reacts with cisplatin to form carbonato complexes, one of which is taken up and/or modified by the cell [C.R. Centerwall, J. Goodisman, D.J. Kerwood, J. Am. Chem. Soc., 127 (2005) 12768-12769], cisplatin-carbonato complexes may be important in the mechanism of action of cisplatin. In this report we study the binding of cisplatin to pBR322 DNA in two different buffers, using gel electrophoresis. In 23.8mM HEPES, N-(2-hydroxyethyl)-piperazine-N'-2-ethanesulfonic acid, 5mM NaCl, pH 7.4 buffer, cisplatin produces aquated species, which react with DNA to unwind supercoiled Form I DNA, increasing its mobility, and reducing the binding of ethidium to DNA. This behavior is consistent with the formation of the well-known intrastrand crosslink on DNA. In 23.8mM carbonate buffer, 5mM NaCl, pH 7.4, cisplatin forms carbonato species that produce DNA-adducts which do not significantly change supercoiling but enhance binding of ethidium to DNA. This behavior is consistent with the formation of a monofunctional cisplatin adduct on DNA. These results show that aquated cisplatin and carbonato complexes of cisplatin produce different types of lesions on DNA and they underscore the importance of carrying out binding studies with cisplatin and DNA using conditions that approximate those found in the cell.

[Energetics of complex formation of the DNA hairpin structure d(GCGAAGC) with aromatic ligands].

PubMed

Kostiukov, V V

2011-01-01

The energy contributions of various physical interactions to the total Gibbs energy of complex formation of the biologically important DNA hairpin d(GCGAAGC) with aromatic antitumor antibiotics daunomycin and novantron and the mutagens ethidium and proflavine have been calculated. It has been shown that the relatively small value of the total energy of binding of the ligands to the hairpin is the sum of components great in absolute value and different in sign. The contributions of van der Waals interactions and both intra- and intermolecular hydrogen bonds and bonds with aqueous environment have been studied. According to the calculations, the hydrophobic and van der Waals components are energetically favorable in complex formation of the ligands with the DNA pairpin d(GCGAAGC), whereas the electrostatic (with consideration of hydrogen bonds) and entropic components are unfavorable.

Intrinsic disorder in the partitioning protein KorB persists after co-operative complex formation with operator DNA and KorA.

PubMed

Hyde, Eva I; Callow, Philip; Rajasekar, Karthik V; Timmins, Peter; Patel, Trushar R; Siligardi, Giuliano; Hussain, Rohanah; White, Scott A; Thomas, Christopher M; Scott, David J

2017-08-30

The ParB protein, KorB, from the RK2 plasmid is required for DNA partitioning and transcriptional repression. It acts co-operatively with other proteins, including the repressor KorA. Like many multifunctional proteins, KorB contains regions of intrinsically disordered structure, existing in a large ensemble of interconverting conformations. Using NMR spectroscopy, circular dichroism and small-angle neutron scattering, we studied KorB selectively within its binary complexes with KorA and DNA, and within the ternary KorA/KorB/DNA complex. The bound KorB protein remains disordered with a mobile C-terminal domain and no changes in the secondary structure, but increases in the radius of gyration on complex formation. Comparison of wild-type KorB with an N-terminal deletion mutant allows a model of the ensemble average distances between the domains when bound to DNA. We propose that the positive co-operativity between KorB, KorA and DNA results from conformational restriction of KorB on binding each partner, while maintaining disorder. © 2017 The Author(s).

The interaction of E. coli integration host factor and lambda cos DNA: multiple complex formation and protein-induced bending.

PubMed Central

Kosturko, L D; Daub, E; Murialdo, H

1989-01-01

The interaction of E. coli's integration Host Factor (IHF) with fragments of lambda DNA containing the cos site has been studied by gel-mobility retardation and electron microscopy. The cos fragment used in the mobility assays is 398 bp and spans a region from 48,298 to 194 on the lambda chromosome. Several different complexes of IHF with this fragment can be distinguished by their differential mobility on polyacrylamide gels. Relative band intensities indicate that the formation of a complex between IHF and this DNA fragment has an equilibrium binding constant of the same magnitude as DNA fragments containing lambda's attP site. Gel-mobility retardation and electron microscopy have been employed to show that IHF sharply bends DNA near cos and to map the bending site. The protein-induced bend is near an intrinsic bend due to DNA sequence. The position of the bend suggests that IHF's role in lambda DNA packaging may be the enhancement of terminase binding/cos cutting by manipulating DNA structure. Images PMID:2521383

A unified view of base excision repair: lesion-dependent protein complexes regulated by post-translational modification

PubMed Central

Almeida, Karen H.; Sobol, Robert W.

2007-01-01

Base excision repair (BER) proteins act upon a significantly broad spectrum of DNA lesions that result from endogenous and exogenous sources. Multiple sub-pathways of BER (short-path or long-patch) and newly designated DNA repair pathways (e.g., SSBR and NIR) that utilize BER proteins complicate any comprehensive understanding of BER and its role in genome maintenance, chemotherapeutic response, neurodegeneration, cancer or aging. Herein, we propose a unified model of BER, comprised of three functional processes: Lesion Recognition/Strand Scission, Gap Tailoring and DNA Synthesis/Ligation, each represented by one or more multiprotein complexes and coordinated via the XRCC1/DNA Ligase III and PARP1 scaffold proteins. BER therefore may be represented by a series of repair complexes that assemble at the site of the DNA lesion and mediates repair in a coordinated fashion involving protein-protein interactions that dictate subsequent steps or sub-pathway choice. Complex formation is influenced by post-translational protein modifications that arise from the cellular state or the DNA damage response, providing an increase in specificity and efficiency to the BER pathway. In this review, we have summarized the reported BER protein-protein interactions and protein post-translational modifications and discuss the impact on DNA repair capacity and complex formation. PMID:17337257

PAM-Dependent Target DNA Recognition and Cleavage by C2c1 CRISPR-Cas Endonuclease

DOE Office of Scientific and Technical Information (OSTI.GOV)

Yang, Hui; Gao, Pu; Rajashankar, Kanagalaghatta R.

C2c1 is a newly identified guide RNA-mediated type V-B CRISPR-Cas endonuclease that site-specifically targets and cleaves both strands of target DNA. We have determined crystal structures of Alicyclobacillus acidoterrestris C2c1 (AacC2c1) bound to sgRNA as a binary complex and to target DNAs as ternary complexes, thereby capturing catalytically competent conformations of AacC2c1 with both target and non-target DNA strands independently positioned within a single RuvC catalytic pocket. Moreover, C2c1-mediated cleavage results in a staggered seven-nucleotide break of target DNA. crRNA adopts a pre-ordered five-nucleotide A-form seed sequence in the binary complex, with release of an inserted tryptophan, facilitating zippering upmore » of 20-bp guide RNA:target DNA heteroduplex on ternary complex formation. Notably, the PAM-interacting cleft adopts a “locked” conformation on ternary complex formation. Structural comparison of C2c1 ternary complexes with their Cas9 and Cpf1 counterparts highlights the diverse mechanisms adopted by these distinct CRISPR-Cas systems, thereby broadening and enhancing their applicability as genome editing tools.« less

Effect of heparin and heparan sulphate on open promoter complex formation for a simple tandem gene model using ex situ atomic force microscopy.

PubMed

Chammas, Oliver; Bonass, William A; Thomson, Neil H

2017-05-01

The influence of heparin and heparan sulphate (HepS) on the appearance and analysis of open promoter complex (RP o ) formation by E. coli RNA polymerase (RNAP) holoenzyme (σ 70 RNAP) on linear DNA using ex situ imaging by atomic force microscopy (AFM) has been investigated. Introducing heparin or HepS into the reaction mix significantly reduces non-specific interactions of the σ 70 RNAP and RNAP after RP o formation allowing for better interpretation of complexes shown within AFM images, particularly on DNA templates containing more than one promoter. Previous expectation was that negatively charged polysaccharides, often used as competitive inhibitors of σRNAP binding and RP o formation, would also inhibit binding of the DNA template to the mica support surface and thereby lower the imaging yield of active RNAP-DNA complexes. We found that the reverse of this was true, and that the yield of RP o formation detected by AFM, for a simple tandem gene model containing two λ PR promoters, increased. Moreover and unexpectedly, HepS was more efficient than heparin, with both of them having a dispersive effect on the sample, minimising unwanted RNAP-RNAP interactions as well as non-specific interactions between the RNAP and DNA template. The success of this method relied on the observation that E. coli RNAP has the highest affinity for the mica surface of all the molecular components. For our system, the affinity of the three constituent biopolymers to muscovite mica was RNAP>Heparin or HepS>DNA. While we observed that heparin and HepS can inhibit DNA binding to the mica, the presence of E. coli RNAP overcomes this effect allowing a greater yield of RP o s for AFM analysis. This method can be extended to other DNA binding proteins and enzymes, which have an affinity to mica higher than DNA, to improve sample preparation for AFM studies. Copyright © 2017 The Authors. Published by Elsevier Inc. All rights reserved.

Chemical shift changes provide evidence for overlapping single-stranded DNA- and XPA-binding sites on the 70 kDa subunit of human replication protein A.

PubMed

Daughdrill, Gary W; Buchko, Garry W; Botuyan, Maria V; Arrowsmith, Cheryl; Wold, Marc S; Kennedy, Michael A; Lowry, David F

2003-07-15

Replication protein A (RPA) is a heterotrimeric single-stranded DNA- (ssDNA) binding protein that can form a complex with the xeroderma pigmentosum group A protein (XPA). This complex can preferentially recognize UV-damaged DNA over undamaged DNA and has been implicated in the stabilization of open complex formation during nucleotide excision repair. In this report, nuclear magnetic resonance (NMR) spectroscopy was used to investigate the interaction between a fragment of the 70 kDa subunit of human RPA, residues 1-326 (hRPA70(1-326)), and a fragment of the human XPA protein, residues 98-219 (XPA-MBD). Intensity changes were observed for amide resonances in the (1)H-(15)N correlation spectrum of uniformly (15)N-labeled hRPA70(1-326) after the addition of unlabeled XPA-MBD. The intensity changes observed were restricted to an ssDNA-binding domain that is between residues 183 and 296 of the hRPA70(1-326) fragment. The hRPA70(1-326) residues with the largest resonance intensity reductions were mapped onto the structure of the ssDNA-binding domain to identify the binding surface with XPA-MBD. The XPA-MBD-binding surface showed significant overlap with an ssDNA-binding surface that was previously identified using NMR spectroscopy and X-ray crystallography. Overlapping XPA-MBD- and ssDNA-binding sites on hRPA70(1-326) suggests that a competitive binding mechanism mediates the formation of the RPA-XPA complex. To determine whether a ternary complex could form between hRPA70(1-326), XPA-MBD and ssDNA, a (1)H-(15)N correlation spectrum was acquired for uniformly (15)N-labeled hRPA70(1-326) after the simultaneous addition of unlabeled XPA-MBD and ssDNA. In this experiment, the same chemical shift perturbations were observed for hRPA70(1-326) in the presence of XPA-MBD and ssDNA as was previously observed in the presence of ssDNA alone. The ability of ssDNA to compete with XPA-MBD for an overlapping binding site on hRPA70(1-326) suggests that any complex formation between RPA and XPA that involves the interaction between XPA-MBD and hRPA70(1-326) may be modulated by ssDNA.

Chemical shift changes provide evidence for overlapping single-stranded DNA- and XPA-binding sites on the 70 kDa subunit of human replication protein A

PubMed Central

Daughdrill, Gary W.; Buchko, Garry W.; Botuyan, Maria V.; Arrowsmith, Cheryl; Wold, Marc S.; Kennedy, Michael A.; Lowry, David F.

2003-01-01

Replication protein A (RPA) is a heterotrimeric single-stranded DNA- (ssDNA) binding protein that can form a complex with the xeroderma pigmentosum group A protein (XPA). This complex can preferentially recognize UV-damaged DNA over undamaged DNA and has been implicated in the stabilization of open complex formation during nucleotide excision repair. In this report, nuclear magnetic resonance (NMR) spectroscopy was used to investigate the interaction between a fragment of the 70 kDa subunit of human RPA, residues 1–326 (hRPA701–326), and a fragment of the human XPA protein, residues 98–219 (XPA-MBD). Intensity changes were observed for amide resonances in the 1H–15N correlation spectrum of uniformly 15N-labeled hRPA701–326 after the addition of unlabeled XPA-MBD. The intensity changes observed were restricted to an ssDNA-binding domain that is between residues 183 and 296 of the hRPA701–326 fragment. The hRPA701–326 residues with the largest resonance intensity reductions were mapped onto the structure of the ssDNA-binding domain to identify the binding surface with XPA-MBD. The XPA-MBD-binding surface showed significant overlap with an ssDNA-binding surface that was previously identified using NMR spectroscopy and X-ray crystallography. Overlapping XPA-MBD- and ssDNA-binding sites on hRPA701–326 suggests that a competitive binding mechanism mediates the formation of the RPA–XPA complex. To determine whether a ternary complex could form between hRPA701–326, XPA-MBD and ssDNA, a 1H–15N correlation spectrum was acquired for uniformly 15N-labeled hRPA701–326 after the simultaneous addition of unlabeled XPA-MBD and ssDNA. In this experiment, the same chemical shift perturbations were observed for hRPA701–326 in the presence of XPA-MBD and ssDNA as was previously observed in the presence of ssDNA alone. The ability of ssDNA to compete with XPA-MBD for an overlapping binding site on hRPA701–326 suggests that any complex formation between RPA and XPA that involves the interaction between XPA-MBD and hRPA701–326 may be modulated by ssDNA. PMID:12853635

Probing the role of intercalating protein sidechains for kink formation in DNA

PubMed Central

Sandmann, Achim

2018-01-01

Protein binding can induce DNA kinks, which are for example important to enhance the specificity of the interaction and to facilitate the assembly of multi protein complexes. The respective proteins frequently exhibit amino acid sidechains that intercalate between the DNA base steps at the site of the kink. However, on a molecular level there is only little information available about the role of individual sidechains for kink formation. To unravel structural principles of protein-induced DNA kinking we have performed molecular dynamics (MD) simulations of five complexes that varied in their architecture, function, and identity of intercalated residues. Simulations were performed for the DNA complexes of wildtype proteins (Sac7d, Sox-4, CcpA, TFAM, TBP) and for mutants, in which the intercalating residues were individually or combined replaced by alanine. The work revealed that for systems with multiple intercalated residues, not all of them are necessarily required for kink formation. In some complexes (Sox-4, TBP), one of the residues proved to be essential for kink formation, whereas the second residue has only a very small effect on the magnitude of the kink. In other systems (e.g. Sac7d) each of the intercalated residues proved to be individually capable of conferring a strong kink suggesting a partially redundant role of the intercalating residues. Mutation of the key residues responsible for kinking either resulted in stable complexes with reduced kink angles or caused conformational instability as evidenced by a shift of the kink to an adjacent base step. Thus, MD simulations can help to identify the role of individual inserted residues for kinking, which is not readily apparent from an inspection of the static structures. This information might be helpful for understanding protein-DNA interactions in more detail and for designing proteins with altered DNA binding properties in the future. PMID:29432448

Probing the role of intercalating protein sidechains for kink formation in DNA.

PubMed

Sandmann, Achim; Sticht, Heinrich

2018-01-01

Protein binding can induce DNA kinks, which are for example important to enhance the specificity of the interaction and to facilitate the assembly of multi protein complexes. The respective proteins frequently exhibit amino acid sidechains that intercalate between the DNA base steps at the site of the kink. However, on a molecular level there is only little information available about the role of individual sidechains for kink formation. To unravel structural principles of protein-induced DNA kinking we have performed molecular dynamics (MD) simulations of five complexes that varied in their architecture, function, and identity of intercalated residues. Simulations were performed for the DNA complexes of wildtype proteins (Sac7d, Sox-4, CcpA, TFAM, TBP) and for mutants, in which the intercalating residues were individually or combined replaced by alanine. The work revealed that for systems with multiple intercalated residues, not all of them are necessarily required for kink formation. In some complexes (Sox-4, TBP), one of the residues proved to be essential for kink formation, whereas the second residue has only a very small effect on the magnitude of the kink. In other systems (e.g. Sac7d) each of the intercalated residues proved to be individually capable of conferring a strong kink suggesting a partially redundant role of the intercalating residues. Mutation of the key residues responsible for kinking either resulted in stable complexes with reduced kink angles or caused conformational instability as evidenced by a shift of the kink to an adjacent base step. Thus, MD simulations can help to identify the role of individual inserted residues for kinking, which is not readily apparent from an inspection of the static structures. This information might be helpful for understanding protein-DNA interactions in more detail and for designing proteins with altered DNA binding properties in the future.

DnaA protein DNA-binding domain binds to Hda protein to promote inter-AAA+ domain interaction involved in regulatory inactivation of DnaA.

PubMed

Keyamura, Kenji; Katayama, Tsutomu

2011-08-19

Chromosomal replication is initiated from the replication origin oriC in Escherichia coli by the active ATP-bound form of DnaA protein. The regulatory inactivation of DnaA (RIDA) system, a complex of the ADP-bound Hda and the DNA-loaded replicase clamp, represses extra initiations by facilitating DnaA-bound ATP hydrolysis, yielding the inactive ADP-bound form of DnaA. However, the mechanisms involved in promoting the DnaA-Hda interaction have not been determined except for the involvement of an interaction between the AAA+ domains of the two. This study revealed that DnaA Leu-422 and Pro-423 residues within DnaA domain IV, including a typical DNA-binding HTH motif, are specifically required for RIDA-dependent ATP hydrolysis in vitro and that these residues support efficient interaction with the DNA-loaded clamp·Hda complex and with Hda in vitro. Consistently, substitutions of these residues caused accumulation of ATP-bound DnaA in vivo and oriC-dependent inhibition of cell growth. Leu-422 plays a more important role in these activities than Pro-423. By contrast, neither of these residues is crucial for DNA replication from oriC, although they are highly conserved in DnaA orthologues. Structural analysis of a DnaA·Hda complex model suggested that these residues make contact with residues in the vicinity of the Hda AAA+ sensor I that participates in formation of a nucleotide-interacting surface. Together, the results show that functional DnaA-Hda interactions require a second interaction site within DnaA domain IV in addition to the AAA+ domain and suggest that these interactions are crucial for the formation of RIDA complexes that are active for DnaA-ATP hydrolysis.

DnaA Protein DNA-binding Domain Binds to Hda Protein to Promote Inter-AAA+ Domain Interaction Involved in Regulatory Inactivation of DnaA*

PubMed Central

Keyamura, Kenji; Katayama, Tsutomu

2011-01-01

Chromosomal replication is initiated from the replication origin oriC in Escherichia coli by the active ATP-bound form of DnaA protein. The regulatory inactivation of DnaA (RIDA) system, a complex of the ADP-bound Hda and the DNA-loaded replicase clamp, represses extra initiations by facilitating DnaA-bound ATP hydrolysis, yielding the inactive ADP-bound form of DnaA. However, the mechanisms involved in promoting the DnaA-Hda interaction have not been determined except for the involvement of an interaction between the AAA+ domains of the two. This study revealed that DnaA Leu-422 and Pro-423 residues within DnaA domain IV, including a typical DNA-binding HTH motif, are specifically required for RIDA-dependent ATP hydrolysis in vitro and that these residues support efficient interaction with the DNA-loaded clamp·Hda complex and with Hda in vitro. Consistently, substitutions of these residues caused accumulation of ATP-bound DnaA in vivo and oriC-dependent inhibition of cell growth. Leu-422 plays a more important role in these activities than Pro-423. By contrast, neither of these residues is crucial for DNA replication from oriC, although they are highly conserved in DnaA orthologues. Structural analysis of a DnaA·Hda complex model suggested that these residues make contact with residues in the vicinity of the Hda AAA+ sensor I that participates in formation of a nucleotide-interacting surface. Together, the results show that functional DnaA-Hda interactions require a second interaction site within DnaA domain IV in addition to the AAA+ domain and suggest that these interactions are crucial for the formation of RIDA complexes that are active for DnaA-ATP hydrolysis. PMID:21708944

Formation of Clustered DNA Damage after High-LET Irradiation: A Review

NASA Technical Reports Server (NTRS)

Hada, Megumi; Georgakilas, Alexandros G.

2008-01-01

Radiation can cause as well as cure cancer. The risk of developing radiation-induced cancer has traditionally been estimated from cancer incidence among survivors of the atomic bombs in Hiroshima and Nagasaki. These data provide the best estimate of human cancer risk over the dose range for low linear energy transfer (LET) radiations, such as X- or gamma-rays. The situation of estimating the real biological effects becomes even more difficult in the case of high LET particles encountered in space or as the result of domestic exposure to particles from radon gas emitters or other radioactive emitters like uranium-238. Complex DNA damage, i.e., the signature of high-LET radiations comprises by closely spaced DNA lesions forming a cluster of DNA damage. The two basic groups of complex DNA damage are double strand breaks (DSBs) and non-DSB oxidative clustered DNA lesions (OCDL). Theoretical analysis and experimental evidence suggest there is increased complexity and severity of complex DNA damage with increasing LET (linear energy transfer) and a high mutagenic or carcinogenic potential. Data available on the formation of clustered DNA damage (DSBs and OCDL) by high-LET radiations are often controversial suggesting a variable response to dose and type of radiation. The chemical nature and cellular repair mechanisms of complex DNA damage have been much less characterized than those of isolated DNA lesions like an oxidized base or a single strand break especially in the case of high-LET radiation. This review will focus on the induction of clustered DNA damage by high-LET radiations presenting the earlier and recent relative data.

Double-stranded DNA translocase activity of transcription factor TFIIH and the mechanism of RNA polymerase II open complex formation.

PubMed

Fishburn, James; Tomko, Eric; Galburt, Eric; Hahn, Steven

2015-03-31

Formation of the RNA polymerase II (Pol II) open complex (OC) requires DNA unwinding mediated by the transcription factor TFIIH helicase-related subunit XPB/Ssl2. Because XPB/Ssl2 binds DNA downstream from the location of DNA unwinding, it cannot function using a conventional helicase mechanism. Here we show that yeast TFIIH contains an Ssl2-dependent double-stranded DNA translocase activity. Ssl2 tracks along one DNA strand in the 5' → 3' direction, implying it uses the nontemplate promoter strand to reel downstream DNA into the Pol II cleft, creating torsional strain and leading to DNA unwinding. Analysis of the Ssl2 and DNA-dependent ATPase activity of TFIIH suggests that Ssl2 has a processivity of approximately one DNA turn, consistent with the length of DNA unwound during transcription initiation. Our results can explain why maintaining the OC requires continuous ATP hydrolysis and the function of TFIIH in promoter escape. Our results also suggest that XPB/Ssl2 uses this translocase mechanism during DNA repair rather than physically wedging open damaged DNA.

Studying of kinetics of rear earth ion (REI) nanoscale complex formation by resonant energy transfer

NASA Astrophysics Data System (ADS)

Ignatova, Tetyana; Pristinski, Denis; Rotkin, Slava V.

2011-03-01

We observed formation of nanoscale complexes between multivalent REIs (Tb and Eu) and negatively charged DNA wrapped SWNTs, ionized in the water solution. Foerster Resonance Energy Transfer (FRET) was found to be an ideal method to confirm the complex formation. Because of its high sensitivity and non-destructive characterization approach FRET can be used to trace the kinetics of the complex formation. Strong dependence of SWNT photoluminescence (PL) on the REI concentration was detected and interpreted as a competition between the REI absorption on the SWNTs and subsequent FRET enhanced PL and the SWNT agglomeration followed by PL quenching. We measured the distance between REI and SWNT which appears to be much shorter than the one from their relative concentration in solution. We speculate that Manning condensation of the REIs on the SWNT/DNA surface happens thereby significantly reducing their spacing and making FRET possible.

Quantitative Analysis of HIV-1 Preintegration Complexes

PubMed Central

Engelman, Alan; Oztop, Ilker; Vandegraaff, Nick; Raghavendra, Nidhanapati K.

2009-01-01

Retroviral replication proceeds through the formation of a provirus, an integrated DNA copy of the viral RNA genome. The linear cDNA product of reverse transcription is the integration substrate and two different integrase activities, 3′ processing and DNA strand transfer, are required for provirus formation. Integrase nicks the cDNA ends adjacent to phylogenetically-conserved CA dinucleotides during 3′ processing. After nuclear entry and locating a suitable chromatin acceptor site, integrase joins the recessed 3′-OHs to the 5′-phosphates of a double-stranded staggered cut in the DNA target. Integrase functions in the context of a large nucleoprotein complex, called the preintegration complex (PIC), and PICs are analyzed to determine levels of integrase 3′ processing and DNA strand transfer activities that occur during acute virus infection. Denatured cDNA end regions are monitored by indirect end-labeling to measure the extent of 3′ processing. Native PICs can efficiently integrate their viral cDNA into exogenously added target DNA in vitro, and Southern blotting or nested PCR assays are used to quantify the resultant DNA strand transfer activity. This study details HIV-1 infection, PIC extraction, partial purification, and quantitative analyses of integrase 3′ processing and DNA strand transfer activities. PMID:19233280

Solution Structures of 2 : 1 And 1 : 1 DNA Polymerase - DNA Complexes Probed By Ultracentrifugation And Small-Angle X-Ray Scattering

DOE Office of Scientific and Technical Information (OSTI.GOV)

Tang, K.H.; /Ohio State U.; Niebuhr, M.

2009-04-30

We report small-angle X-ray scattering (SAXS) and sedimentation velocity (SV) studies on the enzyme-DNA complexes of rat DNA polymerase {beta} (Pol {beta}) and African swine fever virus DNA polymerase X (ASFV Pol X) with one-nucleotide gapped DNA. The results indicated formation of a 2 : 1 Pol {beta}-DNA complex, whereas only 1 : 1 Pol X-DNA complex was observed. Three-dimensional structural models for the 2 : 1 Pol {beta}-DNA and 1 : 1 Pol X-DNA complexes were generated from the SAXS experimental data to correlate with the functions of the DNA polymerases. The former indicates interactions of the 8 kDamore » 5{prime}-dRP lyase domain of the second Pol {beta} molecule with the active site of the 1 : 1 Pol {beta}-DNA complex, while the latter demonstrates how ASFV Pol X binds DNA in the absence of DNA-binding motif(s). As ASFV Pol X has no 5{prime}-dRP lyase domain, it is reasonable not to form a 2 : 1 complex. Based on the enhanced activities of the 2 : 1 complex and the observation that the 8 kDa domain is not in an optimal configuration for the 5{prime}-dRP lyase reaction in the crystal structures of the closed ternary enzyme-DNA-dNTP complexes, we propose that the asymmetric 2 : 1 Pol {beta}-DNA complex enhances the function of Pol {beta}.« less

How proteins bind to DNA: target discrimination and dynamic sequence search by the telomeric protein TRF1

PubMed Central

2017-01-01

Abstract Target search as performed by DNA-binding proteins is a complex process, in which multiple factors contribute to both thermodynamic discrimination of the target sequence from overwhelmingly abundant off-target sites and kinetic acceleration of dynamic sequence interrogation. TRF1, the protein that binds to telomeric tandem repeats, faces an intriguing variant of the search problem where target sites are clustered within short fragments of chromosomal DNA. In this study, we use extensive (>0.5 ms in total) MD simulations to study the dynamical aspects of sequence-specific binding of TRF1 at both telomeric and non-cognate DNA. For the first time, we describe the spontaneous formation of a sequence-specific native protein–DNA complex in atomistic detail, and study the mechanism by which proteins avoid off-target binding while retaining high affinity for target sites. Our calculated free energy landscapes reproduce the thermodynamics of sequence-specific binding, while statistical approaches allow for a comprehensive description of intermediate stages of complex formation. PMID:28633355

3D-structured illumination microscopy reveals clustered DNA double-strand break formation in widespread γH2AX foci after high LET heavy-ion particle radiation.

PubMed

Hagiwara, Yoshihiko; Niimi, Atsuko; Isono, Mayu; Yamauchi, Motohiro; Yasuhara, Takaaki; Limsirichaikul, Siripan; Oike, Takahiro; Sato, Hiro; Held, Kathryn D; Nakano, Takashi; Shibata, Atsushi

2017-12-12

DNA double-strand breaks (DSBs) induced by ionising radiation are considered the major cause of genotoxic mutations and cell death. While DSBs are dispersed throughout chromatin after X-rays or γ-irradiation, multiple types of DNA damage including DSBs, single-strand breaks and base damage can be generated within 1-2 helical DNA turns, defined as a complex DNA lesion, after high Linear Energy Transfer (LET) particle irradiation. In addition to the formation of complex DNA lesions, recent evidence suggests that multiple DSBs can be closely generated along the tracks of high LET particle irradiation. Herein, by using three dimensional (3D)-structured illumination microscopy, we identified the formation of 3D widespread γH2AX foci after high LET carbon-ion irradiation. The large γH2AX foci in G 2 -phase cells encompassed multiple foci of replication protein A (RPA), a marker of DSBs undergoing resection during homologous recombination. Furthermore, we demonstrated by 3D analysis that the distance between two individual RPA foci within γH2AX foci was approximately 700 nm. Together, our findings suggest that high LET heavy-ion particles induce clustered DSB formation on a scale of approximately 1 μm 3 . These closely localised DSBs are considered to be a risk for the formation of chromosomal rearrangement after heavy-ion irradiation.

Silicon supported lipid-DNA thin film structures at varying temperature studied by energy dispersive X-ray diffraction and neutron reflectivity.

PubMed

Domenici, F; Castellano, C; Dell'Unto, F; Albinati, A; Congiu, A

2011-11-01

Non-viral gene transfection by means of lipid-based nanosystems, such as solid supported lipid assemblies, is often limited due to their lack of stability and the consequent loss of efficiency. Therefore not only a detailed thermo-lyotropic study of these DNA-lipid complexes is necessary to understand their interaction mechanisms, but it can also be considered as a first step in conceiving and developing new transfection biosystems. The aim of our study is a structural characterization of 1,2-dioleoyl-sn-glycero-3-phosphatidylcholine (DOPC)-dimethyl-dioctadecyl-ammonium bromide (DDAB)-DNA complex at varying temperature using the energy dispersive X-ray diffraction (EDXD) and neutron reflectivity (NR) techniques. We have shown the formation of a novel thermo-lyotropic structure of DOPC/DDAB thin film self-organized in multi-lamellar planes on (100)-oriented silicon support by spin coating, thus enlightening its ability to include DNA strands. Our NR measurements indicate that the DOPC/DDAB/DNA complex forms temperature-dependent structures. At 65°C and relative humidity of 100% DNA fragments are buried between single lamellar leaflets constituting the hydrocarbon core of the lipid bilayers. This finding supports the consistency of the hydrophobic interaction model, which implies that the coupling between lipid tails and hypo-hydrated DNA single strands could be the driving force of DNA-lipid complexation. Upon cooling to 25°C, EDXD analysis points out that full-hydrated DOPC-DDAB-DNA can switch in a different metastable complex supposed to be driven by lipid heads-DNA electrostatic interaction. Thermotropic response analysis also clarifies that DOPC has a pivotal role in promoting the formation of our observed thermophylic silicon supported lipids-DNA assembly. Copyright © 2011 Elsevier B.V. All rights reserved.

The prokaryotic enhancer binding protein NTRC has an ATPase activity which is phosphorylation and DNA dependent.

PubMed Central

Austin, S; Dixon, R

1992-01-01

The prokaryotic activator protein NTRC binds to enhancer-like elements and activates transcription in response to nitrogen limitation by catalysing open complex formation by sigma 54 RNA polymerase holoenzyme. Formation of open complexes requires the phosphorylated form of NTRC and the reaction is ATP dependent. We find that NTRC has an ATPase activity which is activated by phosphorylation and is strongly stimulated by the presence of DNA containing specific NTRC binding sites. Images PMID:1534752

DNA-RNA hybrid formation mediates RNAi-directed heterochromatin formation.

PubMed

Nakama, Mina; Kawakami, Kei; Kajitani, Takuya; Urano, Takeshi; Murakami, Yota

2012-03-01

Certain noncoding RNAs (ncRNAs) implicated in the regulation of chromatin structure associate with chromatin. During the formation of RNAi-directed heterochromatin in fission yeast, ncRNAs transcribed from heterochromatin are thought to recruit the RNAi machinery to chromatin for the formation of heterochromatin; however, the molecular details of this association are not clear. Here, using RNA immunoprecipitation assay, we showed that the heterochromatic ncRNA was associated with chromatin via the formation of a DNA-RNA hybrid and bound to the RNA-induced transcriptional silencing (RITS) complex. The presence of DNA-RNA hybrid in the cell was also confirmed by immunofluorescence analysis using anti-DNA-RNA hybrid antibody. Over-expression and depletion of RNase H in vivo decreased and increased the amount of DNA-RNA hybrid formed, respectively, and both disturbed heterochromatin. Moreover, DNA-RNA hybrid was formed on, and over-expression of RNase H inhibited the formation of, artificial heterochromatin induced by tethering of RITS to mRNA. These results indicate that heterochromatic ncRNAs are retained on chromatin via the formation of DNA-RNA hybrids and provide a platform for the RNAi-directed heterochromatin assembly and suggest that DNA-RNA hybrid formation plays a role in chromatic ncRNA function. © 2012 The Authors. Journal compilation © 2012 by the Molecular Biology Society of Japan/Blackwell Publishing Ltd.

In vitro and in vivo gene delivery using chitosan/hyaluronic acid nanoparticles: Influences of molecular mass of hyaluronic acid and lyophilization on transfection efficiency.

PubMed

Sato, Toshinori; Nakata, Mitsuhiro; Yang, Zhihong; Torizuka, Yu; Kishimoto, Satoko; Ishihara, Masayuki

2017-08-01

Lyophilization is an effective method for preserving nonviral gene vectors. To improve the stability and transgene expression of lyophilized plasmid DNA (pDNA) complexes, we coated the surfaces of pDNA/chitosan complexes with hyaluronic acid (HA) of varying molecular masses. The transgene expression of pDNA/chitosan/HA ternary complexes was characterized in vitro and in vivo. pDNA complexes were lyophilized overnight and the resultant products with spongy, porous consistencies were stored at -30, 4 or 25°C for 2 weeks. Rehydrated complexes were characterized using gel retardation assays, aiming to confirm complex formation, measure particle size and evaluate zeta potential, as well as conduct luciferase gene reporter assays. The anti-tumor effects of pDNA ternary complexes were evaluated using suicide gene (pTK) coding thymidine kinase in Huh7-implanted mice. Transfection efficiencies of pDNA/chitosan/HA ternary complexes were dependent on the average molecular masses of HA. The coating of pDNA/chitosan complexes with HA maintained the cellular transfection efficiencies of lyophilized pDNA ternary complexes. Furthermore, intratumoral injection of lyophilized, rehydrated pDNA ternary complexes into tumor-bearing mice showed a significant suppression of tumor growth. The coating of pDNA/chitosan complexes with high-molecular-weight HA augmented the stability and cellular transfection ability of the complexes after lyophilization-rehydration. Copyright © 2017 John Wiley & Sons, Ltd.

Concerted formation of macromolecular Suppressor–mutator transposition complexes

PubMed Central

Raina, Ramesh; Schläppi, Michael; Karunanandaa, Balasulojini; Elhofy, Adam; Fedoroff, Nina

1998-01-01

Transposition of the maize Suppressor–mutator (Spm) transposon requires two element-encoded proteins, TnpA and TnpD. Although there are multiple TnpA binding sites near each element end, binding of TnpA to DNA is not cooperative, and the binding affinity is not markedly affected by the number of binding sites per DNA fragment. However, intermolecular complexes form cooperatively between DNA fragments with three or more TnpA binding sites. TnpD, itself not a sequence-specific DNA-binding protein, binds to TnpA and stabilizes the TnpA–DNA complex. The high redundancy of TnpA binding sites at both element ends and the protein–protein interactions between DNA-bound TnpA complexes and between these and TnpD imply a concerted transition of the element from a linear to a protein crosslinked transposition complex within a very narrow protein concentration range. PMID:9671711

Photoactive platinum(II) complexes of nonsteroidal anti-inflammatory drug naproxen: Interaction with biological targets, antioxidant activity and cytotoxicity.

PubMed

Srivastava, Payal; Singh, Khushbu; Verma, Madhu; Sivakumar, Sri; Patra, Ashis K

2018-01-20

The effect on the therapeutic efficacy of Pt(II) complexes on combining non-steroidal anti-inflammatory drugs (NSAIDs) is an attractive strategy to circumvent chronic inflammation mediated by cancer and metastasis. Two square-planar platinum(II) complexes: [Pt(dach)(nap)Cl] (1) and [Pt(dach)(nap) 2 ] (2), where dach = (1R,2R)-dichloro(cyclohexane-1,2-diamine) and NSAID drug naproxen (nap), have been designed for studying their biological activity. The naproxen bound to the Pt(II) centre get released upon photoirradiation with low-power UV-A light as confirmed by the significant enhancement in emission intensities of the complexes. The compounds were evaluated for their photophysical properties, photostability, reactivity with 5'-guanosine monophophosphate (5'-GMP), interactions with CT-DNA and BSA, antioxidant activity and reactive oxygen species mediated photo-induced DNA damage properties. ESI-MS studies demonstrated the formation of bis-adduct with 5'-GMP and the formation of Pt II -DNA crosslinks by gel electrophoretic mobility shift assay and ITC studies. The interaction of the complexes 1 and 2 with the CT-DNA exhibits potential binding affinity (K b  ∼ 10 4  M -1 , K app ∼ 10 5  M -1 ), implying intercalation to CT-DNA through planar naphthyl ring of the complexes. Both the complexes also exhibit strong binding affinity towards BSA (K BSA ∼ 10 5  M -1 ). The complexes exhibit efficient DNA damage activity on irradiation at 365 nm via formation of singlet oxygen ( 1 O 2 ) and hydroxyl radical ( • OH) under physiological conditions. Both the complexes were cytotoxic in dark and exhibit significant enhancement of cytotoxicity upon photo-exposure against HeLa and HepG2 cancer cells giving IC 50 values ranging from 8 to 12 μM for 1 and 2. The cellular internalization data showed cytosolic and nuclear localization of the complexes in the HeLa cells. Copyright © 2017 Elsevier Masson SAS. All rights reserved.

Flexible DNA Path in the MCM Double Hexamer Loaded on DNA.

PubMed

Hizume, Kohji; Kominami, Hiroaki; Kobayashi, Kei; Yamada, Hirofumi; Araki, Hiroyuki

2017-05-16

The formation of the pre-replicative complex (pre-RC) during the G1 phase, which is also called the licensing of DNA replication, is the initial and essential step of faithful DNA replication during the subsequent S phase. It is widely accepted that in the pre-RC, double-stranded DNA passes through the holes of two ring-shaped minichromosome maintenance (MCM) 2-7 hexamers; however, the spatial organization of the DNA and proteins involved in pre-RC formation is unclear. Here we reconstituted the pre-RC from purified DNA and proteins and visualized the complex using atomic force microscopy (AFM). AFM revealed that the MCM double hexamers formed elliptical particles on DNA. Analysis of the angle of binding of DNA to the MCM double hexamer suggests that the DNA does not completely pass through both holes of the MCM hexamers, possibly because the DNA exited from the gap between Mcm2 and Mcm5. A DNA loop fastened by the MCM double hexamer was detected in pre-RC samples reconstituted from purified proteins as well as those purified from yeast cells, suggesting a higher-order architecture of the loaded MCM hexamers and DNA strands.

Electrochemical detection of sequence-specific DNA based on formation of G-quadruplex-hemin through continuous hybridization chain reaction.

PubMed

Sun, Xiaofan; Chen, Haohan; Wang, Shuling; Zhang, Yiping; Tian, Yaping; Zhou, Nandi

2018-08-27

A high-sensitive detection of sequence-specific DNA was established based on the formation of G-quadruplex-hemin complex through continuous hybridization chain reaction (HCR). Taking HIV DNA sequence as an example, a capture probe complementary to part of HIV DNA was firstly self-assembled onto the surface of Au electrode. Then a specially designed assistant probe with both terminals complementary to the target DNA and a G-quadruplex-forming sequence in the center was introduced into the detection solution. In the presence of both the target DNA and the assistant probe, the target DNA can be captured on the electrode surface and then a continuous HCR can be conducted due to the mutual recognition of the target DNA and the assistant probe, leading to the formation of a large number of G-quadruplex on the electrode surface. With the help of hemin, a pronounced electrochemical signal can be observed in differential pulse voltammetry (DPV), due to the formation of G-quadruplex-hemin complex. The peak current is linearly related with the logarithm of the concentration of the target DNA in the range from 10 fM to 10 pM. The electrochemical sensor has high selectivity to clearly discriminate single-base mismatched and three-base mismatched sequences from the original HIV DNA sequence. Moreover, the established DNA sensor was challenged by detection of HIV DNA in human serum samples, which showed the low detection limit of 6.3 fM. Thus it has great application prospect in the field of clinical diagnosis and environmental monitoring. Copyright © 2018 Elsevier B.V. All rights reserved.

Reaction mechanism of Ru(II) piano-stool complexes: umbrella sampling QM/MM MD study.

PubMed

Futera, Zdeněk; Burda, Jaroslav V

2014-07-15

Biologically relevant interactions of piano-stool ruthenium(II) complexes with ds-DNA are studied in this article by hybrid quantum mechanics-molecular mechanics (QM/MM) computational technique. The whole reaction mechanism is divided into three phases: (i) hydration of the [Ru(II) (η(6) -benzene)(en)Cl](+) complex, (ii) monoadduct formation between the resulting aqua-Ru(II) complex and N7 position of one of the guanines in the ds-DNA oligomer, and (iii) formation of the intrastrand Ru(II) bridge (cross-link) between two adjacent guanines. Free energy profiles of all the reactions are explored by QM/MM MD umbrella sampling approach where the Ru(II) complex and two guanines represent a quantum core, which is described by density functional theory methods. The combined QM/MM scheme is realized by our own software, which was developed to couple several quantum chemical programs (in this study Gaussian 09) and Amber 11 package. Calculated free energy barriers of the both ruthenium hydration and Ru(II)-N7(G) DNA binding process are in good agreement with experimentally measured rate constants. Then, this method was used to study the possibility of cross-link formation. One feasible pathway leading to Ru(II) guanine-guanine cross-link with synchronous releasing of the benzene ligand is predicted. The cross-linking is an exergonic process with the energy barrier lower than for the monoadduct reaction of Ru(II) complex with ds-DNA. Copyright © 2014 Wiley Periodicals, Inc.

Aryl-1H-imidazole[4,5f][1,10]phenanthroline Cu(II) complexes: Electrochemical and DNA interaction studies.

PubMed

Rajebhosale, Bharati S; Dongre, Shivali N; Deshpande, Sameer S; Kate, Anup N; Kumbhar, Anupa A

2017-10-01

The reaction of aryl imidazo[4,5f] [1,10]phenanthrolines with Cu(NO 3 ) 2 lead to the formation of Cu(II) complexes of the type [Cu(L)(NO 3 ) 2 ] where L=PIP, 2-(phenyl) [4,5f] imidazo phenanthroline; HPIP=2-(2-hydroxyphenyl)imidazo [4,5f] phenanthroline and NIP=2-(naphthyl) [4,5f] imidazo phenanthroline. The interaction of these complexes with calf thymus DNA has been studied using viscosity measurements, UV-visible and fluorescence spectroscopy. Chemical nuclease activity of these complexes has also been investigated. All complexes cleave DNA via oxidative pathway involving singlet oxygen. Molecular docking studies revealed that these complexes bind to DNA through minor groove. Copyright © 2017 Elsevier Inc. All rights reserved.

Characterization of active reverse transcriptase and nucleoprotein complexes of the yeast retrotransposon Ty3 in vitro.

PubMed

Cristofari, G; Gabus, C; Ficheux, D; Bona, M; Le Grice, S F; Darlix, J L

1999-12-17

Human immunodeficiency virus (HIV) and the distantly related yeast Ty3 retrotransposon encode reverse transcriptase (RT) and a nucleic acid-binding protein designated nucleocapsid protein (NCp) with either one or two zinc fingers, required for HIV-1 replication and Ty3 transposition, respectively. In vitro binding of HIV-1 NCp7 to viral 5' RNA and primer tRNA(3)(Lys) catalyzes formation of nucleoprotein complexes resembling the virion nucleocapsid. Nucleocapsid complex formation functions in viral RNA dimerization and tRNA annealing to the primer binding site (PBS). RT is recruited in these nucleoprotein complexes and synthesizes minus-strand cDNA initiated at the PBS. Recent results on yeast Ty3 have shown that the homologous NCp9 promotes annealing of primer tRNA(i)(Met) to a 5'-3' bipartite PBS, allowing RNA:tRNA dimer formation and initiation of cDNA synthesis at the 5' PBS (). To compare specific cDNA synthesis in a retrotransposon and HIV-1, we have established a Ty3 model system comprising Ty3 RNA with the 5'-3' PBS, primer tRNA(i)(Met), NCp9, and for the first time, highly purified Ty3 RT. Here we report that Ty3 RT is as active as retroviral HIV-1 or murine leukemia virus RT using a synthetic template-primer system. Moreover, and in contrast to what was found with retroviral RTs, retrotransposon Ty3 RT was unable to direct cDNA synthesis by self-priming. We also show that Ty3 nucleoprotein complexes were formed in vitro and that the N terminus of NCp9, but not the zinc finger, is required for complex formation, tRNA annealing to the PBS, RNA dimerization, and primer tRNA-directed cDNA synthesis by Ty3 RT. These results indicate that NCp9 chaperones bona fide cDNA synthesis by RT in the yeast Ty3 retrotransposon, as illustrated for NCp7 in HIV-1, reinforcing the notion that Ty3 NCp9 is an ancestor of HIV-1 NCp7.

Oligomerization of the E. coli Core RNA Polymerase: Formation of (α2ββ'ω)2–DNA Complexes and Regulation of the Oligomerization by Auxiliary Subunits

PubMed Central

Kansara, Seema G.; Sukhodolets, Maxim V.

2011-01-01

In this work, using multiple, dissimilar physico-chemical techniques, we demonstrate that the Escherichia coli RNA polymerase core enzyme obtained through a classic purification procedure forms stable (α2ββ'ω)2 complexes in the presence or absence of short DNA probes. Multiple control experiments indicate that this self-association is unlikely to be mediated by RNA polymerase-associated non-protein molecules. We show that the formation of (α2ββ'ω)2 complexes is subject to regulation by known RNA polymerase interactors, such as the auxiliary SWI/SNF subunit of RNA polymerase RapA, as well as NusA and σ70. We also demonstrate that the separation of the core RNA polymerase and RNA polymerase holoenzyme species during Mono Q chromatography is likely due to oligomerization of the core enzyme. We have analyzed the oligomeric state of the polymerase in the presence or absence of DNA, an aspect that was missing from previous studies. Importantly, our work demonstrates that RNA polymerase oligomerization is compatible with DNA binding. Through in vitro transcription and in vivo experiments (utilizing a RapAR599/Q602 mutant lacking transcription-stimulatory function), we demonstrate that the formation of tandem (α2ββ'ω)2–DNA complexes is likely functionally significant and beneficial for the transcriptional activity of the polymerase. Taken together, our findings suggest a novel structural aspect of the E. coli elongation complex. We hypothesize that transcription by tandem RNA polymerase complexes initiated at hypothetical bidirectional “origins of transcription” may explain recurring switches of the direction of transcription in bacterial genomes. PMID:21533049

Trimeric association of Hox and TALE homeodomain proteins mediates Hoxb2 hindbrain enhancer activity.

PubMed

Jacobs, Y; Schnabel, C A; Cleary, M L

1999-07-01

Pbx/exd proteins modulate the DNA binding affinities and specificities of Hox proteins and contribute to the execution of Hox-dependent developmental programs in arthropods and vertebrates. Pbx proteins also stably heterodimerize and bind DNA with Meis and Pknox1-Prep1, additional members of the TALE (three-amino-acid loop extension) superclass of homeodomain proteins that function on common genetic pathways with a subset of Hox proteins. In this study, we demonstrated that Pbx and Meis bind DNA as heterotrimeric complexes with Hoxb1 on a genetically defined Hoxb2 enhancer, r4, that mediates the cross-regulatory transcriptional effects of Hoxb1 in vivo. The DNA binding specificity of the heterotrimeric complex for r4 is mediated by a Pbx-Hox site in conjunction with a distal Meis site, which we showed to be required for ternary complex formation and Meis-enhanced transcription. Formation of heterotrimeric complexes in which all three homeodomains bind their cognate DNA sites is topologically facilitated by the ability of Pbx and Meis to interact through their amino termini and bind DNA without stringent half-site orientation and spacing requirements. Furthermore, Meis site mutation in the Hoxb2 enhancer phenocopies Pbx-Hox site mutation to abrogate enhancer-directed expression of a reporter transgene in the murine embryonic hindbrain, demonstrating that DNA binding by all three proteins is required for trimer function in vivo. Our data provide in vitro and in vivo evidence for the combinatorial regulation of Hox and TALE protein functions that are mediated, in part, by their interdependent DNA binding activities as ternary complexes. As a consequence, Hoxb1 employs Pbx and Meis-related proteins, as a pair of essential cofactors in a higher-order molecular complex, to mediate its transcriptional effects on an endogenous Hox response element.

Differential impact of ionic and coordinate covalent chromium (Cr)-DNA binding on DNA replication.

PubMed

Fornsaglio, Jamie L; O'Brien, Travis J; Patierno, Steven R

2005-11-01

The reactive species produced by the reduction of Cr(VI), particularly Cr(III), can form both ionic and coordinate covalent complexes with DNA. These Cr(III)-DNA interactions consist of Cr-DNA monoadducts, Cr-DNA ternary adducts, and Cr-DNA interstrand cross-links (Cr-ICLs), the latter of which are DNA polymerase arresting lesions (PALs). We sought to determine the impact of Cr-DNA interactions on the formation of replication blocking lesions in S. cerevisiae using a PCR-based method. We found that target sequence (TS) amplification using DNA isolated from Cr(VI)-treated yeast actually increased as a function of Cr(VI) concentration. Moreover, the enhanced TS amplification was reproduced in vitro using Cr(III)-treated DNA. In contrast, PCR amplification of TS from DNA isolated from yeast exposed to equitoxic doses of the inorganic DNA cross-linking agent cisplatin (CDDP), was decreased in a concentration-dependent manner. This paradox suggested that a specific Cr-DNA interaction, such as an ionic Cr-DNA complex, was responsible for the enhanced TS amplification, thereby masking the replication-blocking effect of certain ternary Cr-DNA adducts (i.e. interstrand cross-links). To test this possibility, we removed ionically associated Cr from the DNA using salt extraction prior to PCR analysis. This procedure obviated the increased amplification and revealed a dose-dependent decrease in TS amplification and an increase in Cr-PALs. These data from DNA analyzed ex vivo after treatment of intact cells indicate that ionic interactions of Cr with DNA result in increased DNA amplification whereas coordinate-covalent Cr-DNA complexes lead to formation of Cr-PALs. Thus, these results suggest that treatment of living cells with Cr(VI) leads to two modes of Cr-binding, which may have conflicting effects on DNA replication.

Long conducting polymer nanonecklaces with a `beads-on-a-string' morphology: DNA nanotube-template synthesis and electrical properties

NASA Astrophysics Data System (ADS)

Chen, Guofang; Mao, Chengde

2016-05-01

Complex and functional nanostructures are always desired. Herein, we present the synthesis of novel long conducting polymer nanonecklaces with a `beads-on-a-string' morphology by the DNA nanotube-template approach and in situ oxidative polymerization of the 3-methylthiophene monomer with FeCl3 as the oxidant/catalyst. The length of the nanonecklaces is up to 60 μm, and the polymer beads of around 20-25 nm in diameter are closely packed along the axis of the DNA nanotube template with a density of ca. 45 particles per μm. The formation of porous DNA nanotubes impregnated with FeCl3 was also demonstrated as intermediate nanostructures. The mechanisms for the formation of both the porous DNA nanotubes and the conducting polymer nanonecklaces are discussed in detail. The as-synthesized polymer/DNA nanonecklaces exhibit good electrical properties.Complex and functional nanostructures are always desired. Herein, we present the synthesis of novel long conducting polymer nanonecklaces with a `beads-on-a-string' morphology by the DNA nanotube-template approach and in situ oxidative polymerization of the 3-methylthiophene monomer with FeCl3 as the oxidant/catalyst. The length of the nanonecklaces is up to 60 μm, and the polymer beads of around 20-25 nm in diameter are closely packed along the axis of the DNA nanotube template with a density of ca. 45 particles per μm. The formation of porous DNA nanotubes impregnated with FeCl3 was also demonstrated as intermediate nanostructures. The mechanisms for the formation of both the porous DNA nanotubes and the conducting polymer nanonecklaces are discussed in detail. The as-synthesized polymer/DNA nanonecklaces exhibit good electrical properties. Electronic supplementary information (ESI) available. See DOI: 10.1039/c6nr01603k

Molecular dynamics simulations of DNA-polycation complexes

NASA Astrophysics Data System (ADS)

Ziebarth, Jesse; Wang, Yongmei

2008-03-01

A necessary step in the preparation of DNA for use in gene therapy is the packaging of DNA with a vector that can condense DNA and provide protection from degrading enzymes. Because of the immunoresponses caused by viral vectors, there has been interest in developing synthetic gene therapy vectors, with polycations emerging as promising candidates. Molecular dynamics simulations of the DNA duplex CGCGAATTCGCG in the presence of 20 monomer long sequences of the polycations, poly-L-lysine (PLL) and polyethyleneimine (PEI), with explicit counterions and TIP3P water, are performed to provide insight into the structure and formation of DNA polyplexes. After an initial separation of approximately 50 å, the DNA and polycation come together and form a stable complex within 10 ns. The DNA does not undergo any major structural changes upon complexation and remains in the B-form. In the formed complex, the charged amine groups of the polycation mainly interact with DNA phosphate groups, and rarely occupy electronegative sites in either the major or minor grooves. Differences between complexation with PEI and PLL will be discussed.

Dnmts and Tet target memory-associated genes after appetitive olfactory training in honey bees

PubMed Central

Biergans, Stephanie D.; Giovanni Galizia, C.; Reinhard, Judith; Claudianos, Charles

2015-01-01

DNA methylation and demethylation are epigenetic mechanisms involved in memory formation. In honey bees DNA methyltransferase (Dnmt) function is necessary for long-term memory to be stimulus specific (i.e. to reduce generalization). So far, however, it remains elusive which genes are targeted and what the time-course of DNA methylation is during memory formation. Here, we analyse how DNA methylation affects memory retention, gene expression, and differential methylation in stimulus-specific olfactory long-term memory formation. Out of 30 memory-associated genes investigated here, 9 were upregulated following Dnmt inhibition in trained bees. These included Dnmt3 suggesting a negative feedback loop for DNA methylation. Within these genes also the DNA methylation pattern changed during the first 24 hours after training. Interestingly, this was accompanied by sequential activation of the DNA methylation machinery (i.e. Dnmts and Tet). In sum, memory formation involves a temporally complex epigenetic regulation of memory-associated genes that facilitates stimulus specific long-term memory in the honey bee. PMID:26531238

3D-structured illumination microscopy reveals clustered DNA double-strand break formation in widespread γH2AX foci after high LET heavy-ion particle radiation

PubMed Central

Hagiwara, Yoshihiko; Niimi, Atsuko; Isono, Mayu; Yamauchi, Motohiro; Yasuhara, Takaaki; Limsirichaikul, Siripan; Oike, Takahiro; Sato, Hiro; Held, Kathryn D.; Nakano, Takashi; Shibata, Atsushi

2017-01-01

DNA double-strand breaks (DSBs) induced by ionising radiation are considered the major cause of genotoxic mutations and cell death. While DSBs are dispersed throughout chromatin after X-rays or γ-irradiation, multiple types of DNA damage including DSBs, single-strand breaks and base damage can be generated within 1–2 helical DNA turns, defined as a complex DNA lesion, after high Linear Energy Transfer (LET) particle irradiation. In addition to the formation of complex DNA lesions, recent evidence suggests that multiple DSBs can be closely generated along the tracks of high LET particle irradiation. Herein, by using three dimensional (3D)-structured illumination microscopy, we identified the formation of 3D widespread γH2AX foci after high LET carbon-ion irradiation. The large γH2AX foci in G2-phase cells encompassed multiple foci of replication protein A (RPA), a marker of DSBs undergoing resection during homologous recombination. Furthermore, we demonstrated by 3D analysis that the distance between two individual RPA foci within γH2AX foci was approximately 700 nm. Together, our findings suggest that high LET heavy-ion particles induce clustered DSB formation on a scale of approximately 1 μm3. These closely localised DSBs are considered to be a risk for the formation of chromosomal rearrangement after heavy-ion irradiation. PMID:29312614

Characterization of human glucocorticoid receptor complexes formed with DNA fragments containing or lacking glucocorticoid response elements

DOE Office of Scientific and Technical Information (OSTI.GOV)

Tully, D.B.; Cidlowski, J.A.

1989-03-07

Sucrose density gradient shift assays were used to study the interactions of human glucocorticoid receptors (GR) with small DNA fragments either containing or lacking glucocorticoid response element (GRE) DNA consensus sequences. When crude cytoplasmic extracts containing ({sup 3}H)triamcinolone acetonide (({sup 3}H)TA) labeled GR were incubated with unlabeled DNA under conditions of DNA excess, a GRE-containing DNA fragment obtained from the 5' long terminal repeat of mouse mammary tumor virus (MMTV LTR) formed a stable 12-16S complex with activated, but not nonactivated, ({sup 3}H)TA receptor. By contrast, if the cytosols were treated with calf thymus DNA-cellulose to deplete non-GR-DNA-binding proteins priormore » to heat activation, a smaller 7-10S complex was formed with the MMTV LTR DNA fragment. Activated ({sup 3}H)TA receptor from DNA-cellulose pretreated cytosols also interacted with two similarly sized fragments from pBR322 DNA. Stability of the complexes formed between GR and these three DNA fragments was strongly affected by even moderate alterations in either the salt concentration or the pH of the gradient buffer. Under all conditions tested, the complex formed with the MMTV LTR DNA fragment was more stable than the complexes formed with either of the pBR322 DNA fragments. Together these observations indicate that the formation of stable complexes between activated GR and isolated DNA fragments requires the presence of GRE consensus sequences in the DNA.« less

N-terminal truncations in the FhlA protein result in formate- and MoeA-independent expression of the hyc (formate hydrogenlyase) operon of Escherichia coli.

PubMed

Self, W T; Hasona, A; Shanmugam, K T

2001-11-01

The formate hydrogenlyase complex of Escherichia coli catalyses the cleavage of formate to CO2 and H2 and consists of a molybdoenzyme formate dehydrogenase-H, hydrogenase 3 and intermediate electron carriers. The structural genes of this enzyme complex are activated by the FhlA protein in the presence of both formate and molybdate; ModE-Mo serves as a secondary activator. Mutational analysis of the FhlA protein established that the unique N-terminal region of this protein was responsible for formate- and molybdenum-dependent transcriptional control of the hyc operon. Analysis of the N-terminal sequence of the FhlA protein revealed a unique motif (amino acids 7-37), which is also found in ATPases associated with several members of the ABC-type transporter family. A deletion derivative of FhlA lacking these amino acids (FhlA9-2) failed to activate the hyc operon in vivo, although the FhlA9-2 did bind to hyc promoter DNA in vitro. The ATPase activity of the FhlA9-2-DNA-formate complex was at least three times higher than that of the native protein-DNA-formate complex, and this degree of activity was achieved at a lower formate level. Extending the deletion to amino acid 117 (FhlA167) not only reversed the FhlA(-) phenotype of FhlA9-2, but also led to both molybdenum- and formate-independence. Deleting the entire N-terminal domain (between amino acids 5 and 374 of the 692 amino acid protein) also led to an effector-independent transcriptional activator (FhlA165), which had a twofold higher level of hyc operon expression than the native protein. Both FhlA165 and FhlA167 still required ModE-Mo as a secondary activator for an optimal level of hyc-lac expression. The FhlA165 protein also had a twofold higher affinity to hyc promoter DNA than the native FhlA protein, while the FhlA167 protein had a significantly lower affinity for hyc promoter DNA in vitro. Although the ATPase activity of the native protein was increased by formate, the ATPase activity of neither FhlA165 or FhlA167 responded to formate. Removal of the first 117 amino acids of the FhlA protein appears to result in a constitutive, effector-independent activation of transcription of the genes encoding the components of the formate hydrogenlyase complex. The sequence similarity to ABC-ATPases, combined with the properties of the FhlA deletion proteins, led to the proposal that the N-terminal region of the native FhlA protein interacts with formate transport proteins, both as a formate transport facilitator and as a cytoplasmic acceptor.

Ternary iron(II) complex with an emissive imidazopyridine arm from Schiff base cyclizations and its oxidative DNA cleavage activity.

PubMed

Mukherjee, Arindam; Dhar, Shanta; Nethaji, Munirathinam; Chakravarty, Akhil R

2005-01-21

The ternary iron(II) complex [Fe(L')(L")](PF6)3(1) as a synthetic model for the bleomycins, where L' and L" are formed from metal-mediated cyclizations of N,N'-(2-hydroxypropane-1,3-diyl)bis(pyridine-2-aldimine)(L), is synthesized and structurally characterized by X-ray crystallography. In the six-coordinate iron(ii) complex, ligands L' and L" show tetradentate and bidentate chelating modes of bonding. Ligand L' is formed from an intramolecular attack of the alcoholic OH group of L to one imine moiety leading to the formation of a stereochemically constrained five-membered ring. Ligand L" which is formed from an intermolecular reaction involving one imine moiety of L and pyridine-2-carbaldehyde has an emissive cationic imidazopyridine pendant arm. The complex binds to double-stranded DNA in the minor groove giving a Kapp value of 4.1 x 10(5) M(-1) and displays oxidative cleavage of supercoiled DNA in the presence of H2O2 following a hydroxyl radical pathway. The complex also shows photo-induced DNA cleavage activity on UV light exposure involving formation of singlet oxygen as the reactive species.

Splenic gene delivery system using self-assembling nano-complex with phosphatidylserine analog.

PubMed

Kurosaki, Tomoaki; Nakasone, Chihiro; Kodama, Yukinobu; Egashira, Kanoko; Harasawa, Hitomi; Muro, Takahiro; Nakagawa, Hiroo; Kitahara, Takashi; Higuchi, Norihide; Nakamura, Tadahiro; Sasaki, Hitoshi

2015-01-01

The recognition of phosphatidylserine on the erythrocyte membrane mediates erythrophagocytosis by resident spleen macrophages. The application of phosphatidylserine to a gene vector may be a novel approach for splenic drug delivery. Therefore, we chose 1,2-dioleoyl-sn-glycero-3-phospho-L-serin (DOPS) as an analogue of phosphatidylserine for splenic gene delivery of plasmid DNA (pDNA). In the present study, we successfully prepared a stable pDNA ternary complex using DOPS and polyethyleneimine (PEI) and evaluated its efficacy and safety. The pDNA/PEI complex had a positive charge and showed high transgene efficacy, although it caused cytotoxicity and agglutination. The addition of DOPS changed the ζ-potential of the pDNA/PEI complex to negative. It is known that anionic complexes are not taken up well by cells. Surprisingly, however, the pDNA/PEI/DOPS complex showed relatively high transgene efficacy in vitro. Fluorescence microscope observation revealed that the pDNA/PEI/DOPS complex internalized the cells while maintaining the complex formation. The injection of the pDNA/PEI complex killed most mice within 24 h at high doses, although all mice in the pDNA/PEI/DOPS complex group survived. The ternary complex with DOPS showed markedly better safety compared with the pDNA/PEI complex. The pDNA/PEI/DOPS complex showed high gene expression selectively in the spleen after intravenous injection into mice. Thus the ternary complex with DOPS can be used to deliver pDNA to the spleen, in which immune cells are abundant. It appears to have an excellent safety level, although further study to determine the mechanism of action is necessary.

The Mitochondrial Transcription Factor TFAM Coordinates the Assembly of Multiple DNA Molecules into Nucleoid-like Structures

PubMed Central

Kaufman, Brett A.; Durisic, Nela; Mativetsky, Jeffrey M.; Costantino, Santiago; Hancock, Mark A.; Grutter, Peter

2007-01-01

Packaging DNA into condensed structures is integral to the transmission of genomes. The mammalian mitochondrial genome (mtDNA) is a high copy, maternally inherited genome in which mutations cause a variety of multisystem disorders. In all eukaryotic cells, multiple mtDNAs are packaged with protein into spheroid bodies called nucleoids, which are the fundamental units of mtDNA segregation. The mechanism of nucleoid formation, however, remains unknown. Here, we show that the mitochondrial transcription factor TFAM, an abundant and highly conserved High Mobility Group box protein, binds DNA cooperatively with nanomolar affinity as a homodimer and that it is capable of coordinating and fully compacting several DNA molecules together to form spheroid structures. We use noncontact atomic force microscopy, which achieves near cryo-electron microscope resolution, to reveal the structural details of protein–DNA compaction intermediates. The formation of these complexes involves the bending of the DNA backbone, and DNA loop formation, followed by the filling in of proximal available DNA sites until the DNA is compacted. These results indicate that TFAM alone is sufficient to organize mitochondrial chromatin and provide a mechanism for nucleoid formation. PMID:17581862

Experimental design, modeling and optimization of polyplex formation between DNA oligonucleotides and branched polyethylenimine.

PubMed

Clima, Lilia; Ursu, Elena L; Cojocaru, Corneliu; Rotaru, Alexandru; Barboiu, Mihail; Pinteala, Mariana

2015-09-28

The complexes formed by DNA and polycations have received great attention owing to their potential application in gene therapy. In this study, the binding efficiency between double-stranded oligonucleotides (dsDNA) and branched polyethylenimine (B-PEI) has been quantified by processing of the images captured from the gel electrophoresis assays. The central composite experimental design has been employed to investigate the effects of controllable factors on the binding efficiency. On the basis of experimental data and the response surface methodology, a multivariate regression model has been constructed and statistically validated. The model has enabled us to predict the binding efficiency depending on experimental factors, such as concentrations of dsDNA and B-PEI as well as the initial pH of solution. The optimization of the binding process has been performed using simplex and gradient methods. The optimal conditions determined for polyplex formation have yielded a maximal binding efficiency close to 100%. In order to reveal the mechanism of complex formation at the atomic-scale, a molecular dynamic simulation has been carried out. According to the computation results, B-PEI amine hydrogen atoms have interacted with oxygen atoms from dsDNA phosphate groups. These interactions have led to the formation of hydrogen bonds between macromolecules, stabilizing the polyplex structure.

Rac1 Protein Signaling Is Required for DNA Damage Response Stimulated by Topoisomerase II Poisons*

PubMed Central

Huelsenbeck, Stefanie C.; Schorr, Anne; Roos, Wynand P.; Huelsenbeck, Johannes; Henninger, Christian; Kaina, Bernd; Fritz, Gerhard

2012-01-01

To investigate the potency of the topoisomerase II (topo II) poisons doxorubicin and etoposide to stimulate the DNA damage response (DDR), S139 phosphorylation of histone H2AX (γH2AX) was analyzed using rat cardiomyoblast cells (H9c2). Etoposide caused a dose-dependent increase in the γH2AX level as shown by Western blotting. By contrast, the doxorubicin response was bell-shaped with high doses failing to increase H2AX phosphorylation. Identical results were obtained by immunohistochemical analysis of γH2AX focus formation, comet assay-based DNA strand break analysis, and measuring the formation of the topo II-DNA cleavable complex. At low dose, doxorubicin activated ataxia telangiectasia mutated (ATM) but not ATM and Rad3-related (ATR). Both the lipid-lowering drug lovastatin and the Rac1-specific inhibitor NSC23766 attenuated doxorubicin- and etoposide-stimulated H2AX phosphorylation, induction of DNA strand breaks, and topo II-DNA complex formation. Lovastatin and NSC23766 acted in an additive manner. They did not attenuate doxorubicin-induced increase in p-ATM and p-Chk2 levels. DDR stimulated by topo II poisons was partially blocked by inhibition of type I p21-associated kinases. DDR evoked by the topoisomerase I poison topotecan remained unaffected by lovastatin. The data show that the mechanisms involved in DDR stimulated by topo II poisons are agent-specific with anthracyclines lacking DDR-stimulating activity at high doses. Pharmacological inhibition of Rac1 signaling counteracts doxorubicin- and etoposide-stimulated DDR by disabling the formation of the topo II-DNA cleavable complex. Based on the data we suggest that Rac1-regulated mechanisms are required for DNA damage induction and subsequent activation of the DDR following treatment with topo II but not topo I poisons. PMID:23012366

Rac1 protein signaling is required for DNA damage response stimulated by topoisomerase II poisons.

PubMed

Huelsenbeck, Stefanie C; Schorr, Anne; Roos, Wynand P; Huelsenbeck, Johannes; Henninger, Christian; Kaina, Bernd; Fritz, Gerhard

2012-11-09

To investigate the potency of the topoisomerase II (topo II) poisons doxorubicin and etoposide to stimulate the DNA damage response (DDR), S139 phosphorylation of histone H2AX (γH2AX) was analyzed using rat cardiomyoblast cells (H9c2). Etoposide caused a dose-dependent increase in the γH2AX level as shown by Western blotting. By contrast, the doxorubicin response was bell-shaped with high doses failing to increase H2AX phosphorylation. Identical results were obtained by immunohistochemical analysis of γH2AX focus formation, comet assay-based DNA strand break analysis, and measuring the formation of the topo II-DNA cleavable complex. At low dose, doxorubicin activated ataxia telangiectasia mutated (ATM) but not ATM and Rad3-related (ATR). Both the lipid-lowering drug lovastatin and the Rac1-specific inhibitor NSC23766 attenuated doxorubicin- and etoposide-stimulated H2AX phosphorylation, induction of DNA strand breaks, and topo II-DNA complex formation. Lovastatin and NSC23766 acted in an additive manner. They did not attenuate doxorubicin-induced increase in p-ATM and p-Chk2 levels. DDR stimulated by topo II poisons was partially blocked by inhibition of type I p21-associated kinases. DDR evoked by the topoisomerase I poison topotecan remained unaffected by lovastatin. The data show that the mechanisms involved in DDR stimulated by topo II poisons are agent-specific with anthracyclines lacking DDR-stimulating activity at high doses. Pharmacological inhibition of Rac1 signaling counteracts doxorubicin- and etoposide-stimulated DDR by disabling the formation of the topo II-DNA cleavable complex. Based on the data we suggest that Rac1-regulated mechanisms are required for DNA damage induction and subsequent activation of the DDR following treatment with topo II but not topo I poisons.

DNA purification by triplex-affinity capture and affinity capture electrophoresis

DOEpatents

Cantor, Charles R.; Ito, Takashi; Smith, Cassandra L.

1996-01-01

The invention provides a method for purifying or isolating double stranded DNA intact using triple helix formation. The method includes the steps of complexing an oligonucleotide and double stranded DNA to generate a triple helix and immobilization of the triple helix on a solid phase by means of a molecular recognition system such as avidin/biotin. The purified DNA is then recovered intact by treating the solid phase with a reagent that breaks the bonds between the oligonucleotide and the intact double stranded DNA while not affecting the Watson-Crick base pairs of the double helix. The present invention also provides a method for purifying or isolating double stranded DNA intact by complexing the double stranded DNA with a specific binding partner and recovering the complex during electrophoresis by immobilizing it on a solid phase trap imbedded in an electrophoretic gel.

Structure of an anti-HIV-1 hammerhead ribozyme complex with a 17-mer DNA substrate analog of HIV-1 gag RNA and a mechanism for the cleavage reaction: 750 MHz NMR and computer experiments

NASA Technical Reports Server (NTRS)

Ojha, R. P.; Dhingra, M. M.; Sarma, M. H.; Myer, Y. P.; Setlik, R. F.; Shibata, M.; Kazim, A. L.; Ornstein, R. L.; Rein, R.; Turner, C. J.;

Gigantism in a bacterium, Epulopiscium fishelsoni, correlates with complex patterns in arrangement, quantity, and segregation of DNA.

PubMed

Bresler, V; Montgomery, W L; Fishelson, L; Pollak, P E

1998-11-01

Epulopiscium fishelsoni, gut symbiont of the brown surgeonfish (Acanthurus nigrofuscus) in the Red Sea, attains a larger size than any other eubacterium, varies 10- to 20-fold in length (and >2, 000-fold in volume), and undergoes a complex daily life cycle. In early morning, nucleoids contain highly condensed DNA in elongate, chromosome-like structures which are physically separated from the general cytoplasm. Cell division involves production of two (rarely three) nucleoids within a cell, deposition of cell walls around expanded nucleoids, and emergence of daughter cells from the parent cell. Fluorescence measurements of DNA, RNA, and other cell components indicate the following. DNA quantity is proportional to cell volume over cell lengths of approximately 30 micrometers to >500 micrometers. For cells of a given size, nucleoids of cells with two nucleoids (binucleoid) contain approximately equal amounts of DNA. And each nucleoid of a binucleoid cell contains one-half the DNA of the single nucleoid in a uninucleoid cell of the same size. The life cycle involves approximately equal subdivision of DNA among daughter cells, formation of apical caps of condensed DNA from previously decondensed and diffusely distributed DNA, and "pinching" of DNA near the middle of the cell in the absence of new wall formation. Mechanisms underlying these patterns remain unclear, but formation of daughter nucleoids and cells occurs both during diurnal periods of host feeding and bacterial cell growth and during nocturnal periods of host inactivity when mean bacterial cell size declines.

The plant cell cycle: Pre-Replication complex formation and controls

PubMed Central

Brasil, Juliana Nogueira; Costa, Carinne N. Monteiro; Cabral, Luiz Mors; Ferreira, Paulo C. G.; Hemerly, Adriana S.

2017-01-01

Abstract The multiplication of cells in all living organisms requires a tight regulation of DNA replication. Several mechanisms take place to ensure that the DNA is replicated faithfully and just once per cell cycle in order to originate through mitoses two new daughter cells that contain exactly the same information from the previous one. A key control mechanism that occurs before cells enter S phase is the formation of a pre-replication complex (pre-RC) that is assembled at replication origins by the sequential association of the origin recognition complex, followed by Cdt1, Cdc6 and finally MCMs, licensing DNA to start replication. The identification of pre-RC members in all animal and plant species shows that this complex is conserved in eukaryotes and, more importantly, the differences between kingdoms might reflect their divergence in strategies on cell cycle regulation, as it must be integrated and adapted to the niche, ecosystem, and the organism peculiarities. Here, we provide an overview of the knowledge generated so far on the formation and the developmental controls of the pre-RC mechanism in plants, analyzing some particular aspects in comparison to other eukaryotes. PMID:28304073

Sequence-dependent DNA flexibility mediates DNase I cleavage.

PubMed

Heddi, Brahim; Abi-Ghanem, Josephine; Lavigne, Marc; Hartmann, Brigitte

2010-01-08

Understanding the preference of nonspecific proteins for certain DNA structural features requires an accurate description of the properties of free DNA, especially regarding their possible predisposition to adopt a conformation that favors the formation of a complex. Exploiting previous exhaustive NMR studies performed on free DNA oligomers, we investigated the molecular basis of DNase I sensitivity under conditions where DNase I binding limits the probability of cleavage. We showed that cleavage intensity was correlated with adjacent 3' phosphate linkage flexibility, monitored by (31)P chemical shifts. Examining NMR-refined DNA structures highlighted that sequence-dependent flexible phosphates were associated with large minor groove variations that may promote the affinity of DNase I, according to relevant DNA-protein complexes. In sum, this work demonstrates that specificity in DNA-DNase I interaction is mediated by DNA flexibility, which influences the induced-fit transitions required to form productive complexes.

[Molecular dynamics of immune complex of photoadduct-containing DNA with Fab-Anti-DNA antibody fragment].

PubMed

Akberova, N I; Zhmurov, A A; Nevzorova, T A; Litvinov, R I

2016-01-01

Antibodies to DNA play an important role in the pathogenesis of autoimmune diseases. The elucidation of structural mechanisms of both the antigen recognition and the interaction of anti-DNA antibodies with DNA will help to understand the role of DNA-containing immune complexes in various pathologies and can provide a basis for new treatment modalities. Moreover, the DNA-antibody complex is an analog of specific intracellular DNA-protein interactions. In this work, we used in silico molecular dynamic simulations of bimolecular complexes of the dsDNA segment containing the Fab fragment of an anti-DNA antibody to obtain the detailed thermodynamic and structural characteristics of dynamic intermolecular interactions. Using computationally modified crystal structure of the Fab-DNA complex (PDB ID: 3VW3), we studied the equilibrium molecular dynamics of the 64M-5 antibody Fab fragment associated with the dsDNA fragment containing the thymine dimer, the product of DNA photodamage. Amino acid residues that constitute paratopes and the complementary nucleotide epitopes for the Fab-DNA construct were identified. Stacking and electrostatic interactions were found to play the main role in mediating the most specific antibody-dsDNA contacts, while hydrogen bonds were less significant. These findings may shed light on the formation and properties of pathogenic anti-DNA antibodies in autoimmune diseases, such as systemic lupus erythematosus associated with skin photosensitivity and DNA photodamage.

Release of the cyano moiety in the crystal structure of N-cyanomethyl-N-(2-methoxyethyl)-daunomycin complexed with d(CGATCG).

PubMed

Saminadin, P; Dautant, A; Mondon, M; Langlois D'estaintot, B; Courseille, C; Précigoux, G

2000-01-01

Doxorubicin is among the most widely used anthracycline in cancer chemotherapy. In an attempt to avoid the cardiotoxicity and drug resistance of doxorubicin therapy, several analogues were synthesized. The cyanomorpholinyl derivative is the most cytotoxic. They differ greatly from their parent compound in their biological and pharmacological properties, inducing cross-links in drug DNA complexes. The present study concerns N-cyanomethyl-N-(2-methoxyethyl)-daunomycin (CMDa), a synthetic analogue of cyanomorpholino-daunomycin. Compared to doxorubicin, CMDa displays a cytotoxic activity on L1210 leukemia cells at higher concentration but is effective on doxorubicin resistant cells. The results of fluorescence quenching experiments as well as the melting temperature (DeltaTm = 7.5 degrees C) studies are consistent with a drug molecule which intercalates between the DNA base pairs and stabilizes the DNA double helix. The crystal structure of CMDa complexed to the hexanucleotide d(CGATCG) has been determined at 1.5 A resolution. The complex crystallizes in the space group P41212 and is similar to other anthracycline-hexanucleotide complexes. In the crystal state, the observed densities indicate the formation of N-hydroxymethyl-N-(2-methoxyethyl)-daunomycin (HMDa) with the release of the cyano moiety without DNA alkylation. The formation of this degradation compound is discussed in relation with other drug modifications when binding to DNA. Comparison with two other drug-DNA crystal structures suggests a correlation between a slight change in DNA conformation and the nature of the amino sugar substituents at the N3' position located in the minor groove.

Protective role of quercetin against copper(II)-induced oxidative stress: A spectroscopic, theoretical and DNA damage study.

PubMed

Jomova, Klaudia; Lawson, Michael; Drostinova, Lenka; Lauro, Peter; Poprac, Patrik; Brezova, Vlasta; Michalik, Martin; Lukes, Vladimir; Valko, Marian

2017-12-01

The radical scavenging and metal chelating properties of flavonoids indicate that they may play a protective role in diseases with perturbed metal homeostasis such as Alzheimer's disease. In this work we investigated the effect of the coordination of quercetin to copper(II) in view of the formation of ROS in Cu-catalyzed Fenton reaction. ABTS and DPPH assays confirmed that the copper(II)-quercetin complex exhibits a stronger radical scavenging activity than does quercetin alone. EPR spin trapping experiments have shown that chelation of quercetin to copper significantly suppressed the formation of hydroxyl radicals in the Cu(II)-Fenton reaction. DNA damage experiments revealed a protective effect for quercetin, but only at higher stoichiometric ratios of quercetin relative to copper. DNA protective effect of quercetin against ROS attack was described by two mechanisms. The first mechanism lies in suppressed formation of ROS due to the decreased catalytic action of copper in the Fenton reaction, as a consequence of its chelation and direct scavenging of ROS by free quercetin. Since the Cu-quercetin complex intercalates into DNA, the second mechanism was attributed to a suppressed intercalating ability of the Cu-quercetin complex due to the mildly intercalating free quercetin into DNA, thus creating a protective wall against stronger intercalators. Copyright © 2017 Elsevier Ltd. All rights reserved.

Downstream DNA Tension Regulates the Stability of the T7 RNA Polymerase Initiation Complex

PubMed Central

Skinner, Gary M.; Kalafut, Bennett S.; Visscher, Koen

2011-01-01

Gene transcription by the enzyme RNA polymerase is tightly regulated. In many cases, such as in the lac operon in Escherichia coli, this regulation is achieved through the action of protein factors on DNA. Because DNA is an elastic polymer, its response to enzymatic processing can lead to mechanical perturbations (e.g., linear stretching and supercoiling) that can affect the operation of other DNA processing complexes acting elsewhere on the same substrate molecule. Using an optical-tweezers assay, we measured the binding kinetics between single molecules of bacteriophage T7 RNA polymerase and DNA, as a function of tension. We found that increasing DNA tension under conditions that favor formation of the open complex results in destabilization of the preinitiation complex. Furthermore, with zero ribonucleotides present, when the closed complex is favored, we find reduced tension sensitivity, implying that it is predominantly the open complex that is sensitive. This result strongly supports the “scrunching” model for T7 transcription initiation, as the applied tension acts against the movement of the DNA into the scrunched state, and introduces linear DNA tension as a potential regulatory quantity for transcription initiation. PMID:21320448

Understanding the mechanism of DNA deactivation in ion therapy of cancer cells: hydrogen peroxide action*

NASA Astrophysics Data System (ADS)

Piatnytskyi, Dmytro V.; Zdorevskyi, Oleksiy O.; Perepelytsya, Sergiy M.; Volkov, Sergey N.

2015-11-01

Changes in the medium of biological cells under ion beam irradiation has been considered as a possible cause of cell function disruption in the living body. The interaction of hydrogen peroxide, a long-lived molecular product of water radiolysis, with active sites of DNA macromolecule was studied, and the formation of stable DNA-peroxide complexes was considered. The phosphate groups of the macromolecule backbone were picked out among the atomic groups of DNA double helix as a probable target for interaction with hydrogen peroxide molecules. Complexes consisting of combinations including: the DNA phosphate group, H2O2 and H2O molecules, and Na+ counterion, were considered. The counterions have been taken into consideration insofar as under the natural conditions they neutralise DNA sugar-phosphate backbone. The energy of the complexes have been determined by considering the electrostatic and the Van der Waals interactions within the framework of atom-atom potential functions. As a result, the stability of various configurations of molecular complexes was estimated. It was shown that DNA phosphate groups and counterions can form stable complexes with hydrogen peroxide molecules, which are as stable as the complexes with water molecules. It has been demonstrated that the formation of stable complexes of H2O2-Na+-PO4- may be detected experimentally by observing specific vibrations in the low-frequency Raman spectra. The interaction of H2O2 molecule with phosphate group of the double helix backbone can disrupt DNA biological function and induce the deactivation of the cell genetic apparatus. Thus, the production of hydrogen peroxide molecules in the nucleus of living cells can be considered as an additional mechanism by which high-energy ion beams destroy tumour cells during ion beam therapy. Contribution to the Topical Issue "COST Action Nano-IBCT: Nano-scale Processes Behind Ion-Beam Cancer Therapy", edited by Andrey Solov'yov, Nigel Mason, Gustavo García, Eugene Surdutovich.

Structural and mechanistic insights into Mcm2-7 double-hexamer assembly and function

DOE PAGES

Sun, Jingchuan; Li, Huilin; Fernandez-Cid, Alejandra; ...

2014-10-15

Eukaryotic cells license each DNA replication origin during G1 phase by assembling a prereplication complex that contains a Mcm2–7 (minichromosome maintenance proteins 2–7) double hexamer. During S phase, each Mcm2–7 hexamer forms the core of a replicative DNA helicase. However, the mechanisms of origin licensing and helicase activation are poorly understood. The helicase loaders ORC–Cdc6 function to recruit a single Cdt1–Mcm2–7 heptamer to replication origins prior to Cdt1 release and ORC–Cdc6–Mcm2–7 complex formation, but how the second Mcm2–7 hexamer is recruited to promote double-hexamer formation is not well understood. Here, structural evidence for intermediates consisting of an ORC–Cdc6–Mcm2–7 complex andmore » an ORC–Cdc6–Mcm2–7–Mcm2–7 complex are reported, which together provide new insights into DNA licensing. Detailed structural analysis of the loaded Mcm2–7 double-hexamer complex demonstrates that the two hexamers are interlocked and misaligned along the DNA axis and lack ATP hydrolysis activity that is essential for DNA helicase activity. Moreover, we show that the head-to-head juxtaposition of the Mcm2–7 double hexamer generates a new protein interaction surface that creates a multisubunit-binding site for an S-phase protein kinase that is known to activate DNA replication. The data suggest how the double hexamer is assembled and how helicase activity is regulated during DNA licensing, with implications for cell cycle control of DNA replication and genome stability.« less

Studies on the formation and stability of triplex DNA using fluorescence correlation spectroscopy.

PubMed

Hu, Hongyan; Huang, Xiangyi; Ren, Jicun

2016-05-01

Triplex DNA has become one of the most useful recognition motifs in the design of new molecular biology tools, therapeutic agents and sophisticated DNA-based nanomaterials because of its direct recognition of natural double-stranded DNA. In this paper, we developed a sensitive and microscale method to study the formation and stability characterization of triplex DNA using fluorescence correlation spectroscopy (FCS). The principle of this method is mainly based on the excellent capacity of FCS for sensitively distinguishing between free single-strand DNA (ssDNA) fluorescent probes and fluorescent probe-double-strand DNA (dsDNA) hybridized complexes. First, we systematically investigated the experimental conditions of triplex DNA formation. Then, we evaluated the equilibrium association constants (K(a)) under different ssDNA probe lengths, composition and pH. Finally, we used FCS to measure the hybridization fraction of a 20-mer perfectly matched ssDNA probe and three single-base mismatched ssDNA probes with 146-mer dsDNA. Our data illustrated that FCS is a useful tool for the direct determination of the thermodynamic parameters of triplex DNA formation and discrimination of a single-base mismatch of triplex DNA without denaturation. Compared with current methods, our method is characterized by high sensitivity, good universality and small sample and reagent requirements. More importantly, our method has the potential to become a platform for triplex DNA research in vitro. Copyright © 2015 John Wiley & Sons, Ltd.

Two modes of longe-range orientation of DNA bases realized upon compaction.

PubMed Central

Yevdokimov YuM; Salyanov, V I; Berg, H

1981-01-01

Formation of compact particles from linear DNA-anthracycline complexes is accompanied by appearance of intense bands in the CD spectra in the region of absorption of DNA bases (UV-region) and in the region of absorption of anthracycline chromophores (visible region). The intense (positive or negative) bands in the region of anthracycline absorption demonstrate an ordered helical location of anthracycline molecules on the DNA template. This fact, in its turn, is related to formation of the DNA superstructure in PEG-containing water-salt solutions with a long-range orientation of nitrogen bases. Possible types of DNA superstructures and the relation between the local- and the long-range order of bases in the DNA superstructure are discussed. PMID:6938929

DNA assisted self-assembly of PAMAM dendrimers.

PubMed

Mandal, Taraknath; Kumar, Mattaparthi Venkata Satish; Maiti, Prabal K

2014-10-09

We report DNA assisted self-assembly of polyamidoamine (PAMAM) dendrimers using all atom Molecular Dynamics (MD) simulations and present a molecular level picture of a DNA-linked PAMAM dendrimer nanocluster, which was first experimentally reported by Choi et al. (Nano Lett., 2004, 4, 391-397). We have used single stranded DNA (ssDNA) to direct the self-assembly process. To explore the effect of pH on this mechanism, we have used both the protonated (low pH) and nonprotonated (high pH) dendrimers. In all cases studied here, we observe that the DNA strand on one dendrimer unit drives self-assembly as it binds to the complementary DNA strand present on the other dendrimer unit, leading to the formation of a DNA-linked dendrimer dimeric complex. However, this binding process strongly depends on the charge of the dendrimer and length of the ssDNA. We observe that the complex with a nonprotonated dendrimer can maintain a DNA length dependent inter-dendrimer distance. In contrast, for complexes with a protonated dendrimer, the inter-dendrimer distance is independent of the DNA length. We attribute this observation to the electrostatic complexation of a negatively charged DNA strand with the positively charged protonated dendrimer.

Lanthanoplatins: emissive Eu(iii) and Tb(iii) complexes staining nucleoli targeted through Pt-DNA crosslinking.

PubMed

Singh, Khushbu; Singh, Swati; Srivastava, Payal; Sivakumar, Sri; Patra, Ashis K

2017-06-01

Two highly luminescent water-soluble heterometallic LnPt 2 complexes, [{cis-PtCl(NH 3 ) 2 } 2 Ln(L)(H 2 O)](NO 3 ) 2 (Ln = Eu (1), Tb (2)), have been designed for their selective nucleoli staining through formation of Pt-DNA crosslinks. The complexes showed significant cellular uptake and distinctive nucleoli localization through intrinsic emission from Eu III or Tb III observed through confocal fluorescence microscopy.

Stimulation of ribosomal RNA gene promoter by transcription factor Sp1 involves active DNA demethylation by Gadd45-NER pathway.

PubMed

Rajput, Pallavi; Pandey, Vijaya; Kumar, Vijay

2016-08-01

The well-studied Pol II transcription factor Sp1 has not been investigated for its regulatory role in rDNA transcription. Here, we show that Sp1 bound to specific sites on rDNA and localized into the nucleoli during the G1 phase of cell cycle to activate rDNA transcription. It facilitated the recruitment of Pol I pre-initiation complex and impeded the binding of nucleolar remodeling complex (NoRC) to rDNA resulting in the formation of euchromatin active state. More importantly, Sp1 also orchestrated the site-specific binding of Gadd45a-nucleotide excision repair (NER) complex resulting in active demethylation and transcriptional activation of rDNA. Interestingly, knockdown of Sp1 impaired rDNA transcription due to reduced engagement of the Gadd45a-NER complex and hypermethylation of rDNA. Thus, the present study unveils a novel role of Sp1 in rDNA transcription involving promoter demethylation. Copyright © 2016 Elsevier B.V. All rights reserved.

Molecular dynamics simulations show altered secondary structure of clawless in binary complex with DNA providing insights into aristaless-clawless-DNA ternary complex formation.

PubMed

Kachhap, Sangita; Priyadarshini, Pragya; Singh, Balvinder

2017-05-01

Aristaless (Al) and clawless (Cll) homeodomains that are involved in leg development in Drosophila melanogaster are known to bind cooperatively to 5'-(T/C)TAATTAA(T/A)(T/A)G-3' DNA sequence, but the mechanism of their binding to DNA is unknown. Molecular dynamics (MD) studies have been carried out on binary, ternary, and reconstructed protein-DNA complexes involving Al, Cll, and DNA along with binding free energy analysis of these complexes. Analysis of MD trajectories of Cll-3A01, binary complex reveals that C-terminal end of helixIII of Cll, unwind in the absence of Al and remains so in reconstructed ternary complex, Cll-3A01-Al. In addition, this change in secondary structure of Cll does not allow it to form protein-protein interactions with Al in the ternary reconstructed complex. However, secondary structure of Cll and its interactions are maintained in other reconstructed ternary complex, Al-3A01-Cll where Cll binds to Al-3A01, binary complex to form ternary complex. These interactions as observed during MD simulations compare well with those observed in ternary crystal structure. Thus, this study highlights the role of helixIII of Cll and protein-protein interactions while proposing likely mechanism of recognition in ternary complex, Al-Cll-DNA.

DNA purification by triplex-affinity capture and affinity capture electrophoresis

DOEpatents

Cantor, C.R.; Ito, Takashi; Smith, C.L.

1996-01-09

The invention provides a method for purifying or isolating double stranded DNA intact using triple helix formation. The method includes the steps of complexing an oligonucleotide and double stranded DNA to generate a triple helix and immobilization of the triple helix on a solid phase by means of a molecular recognition system such as avidin/biotin. The purified DNA is then recovered intact by treating the solid phase with a reagent that breaks the bonds between the oligonucleotide and the intact double stranded DNA while not affecting the Watson-Crick base pairs of the double helix. The present invention also provides a method for purifying or isolating double stranded DNA intact by complexing the double stranded DNA with a specific binding partner and recovering the complex during electrophoresis by immobilizing it on a solid phase trap imbedded in an electrophoretic gel. 6 figs.

THE OPEP COARSE-GRAINED PROTEIN MODEL: FROM SINGLE MOLECULES, AMYLOID FORMATION, ROLE OF MACROMOLECULAR CROWDING AND HYDRODYNAMICS TO RNA/DNA COMPLEXES

PubMed Central

Sterpone, Fabio; Melchionna, Simone; Tuffery, Pierre; Pasquali, Samuela; Mousseau, Normand; Cragnolini, Tristan; Chebaro, Yassmine; Saint-Pierre, Jean-Francois; Kalimeri, Maria; Barducci, Alessandro; Laurin, Yohan; Tek, Alex; Baaden, Marc; Nguyen, Phuong Hoang; Derreumaux, Philippe

2015-01-01

The OPEP coarse-grained protein model has been applied to a wide range of applications since its first release 15 years ago. The model, which combines energetic and structural accuracy and chemical specificity, allows studying single protein properties, DNA/RNA complexes, amyloid fibril formation and protein suspensions in a crowded environment. Here we first review the current state of the model and the most exciting applications using advanced conformational sampling methods. We then present the current limitations and a perspective on the on-going developments. PMID:24759934

A dual switch controls bacterial enhancer-dependent transcription

PubMed Central

Wiesler, Simone C.; Burrows, Patricia C.; Buck, Martin

2012-01-01

Bacterial RNA polymerases (RNAPs) are targets for antibiotics. Myxopyronin binds to the RNAP switch regions to block structural rearrangements needed for formation of open promoter complexes. Bacterial RNAPs containing the major variant σ54 factor are activated by enhancer-binding proteins (bEBPs) and transcribe genes whose products are needed in pathogenicity and stress responses. We show that (i) enhancer-dependent RNAPs help Escherichia coli to survive in the presence of myxopyronin, (ii) enhancer-dependent RNAPs partially resist inhibition by myxopyronin and (iii) ATP hydrolysis catalysed by bEBPs is obligatory for functional interaction of the RNAP switch regions with the transcription start site. We demonstrate that enhancer-dependent promoters contain two barriers to full DNA opening, allowing tight regulation of transcription initiation. bEBPs engage in a dual switch to (i) allow propagation of nucleated DNA melting from an upstream DNA fork junction and (ii) complete the formation of the transcription bubble and downstream DNA fork junction at the RNA synthesis start site, resulting in switch region-dependent RNAP clamp closure and open promoter complex formation. PMID:22965125

Gigantism in a Bacterium, Epulopiscium fishelsoni, Correlates with Complex Patterns in Arrangement, Quantity, and Segregation of DNA

PubMed Central

Bresler, V.; Montgomery, W. L.; Fishelson, L.; Pollak, P. E.

1998-01-01

Epulopiscium fishelsoni, gut symbiont of the brown surgeonfish (Acanthurus nigrofuscus) in the Red Sea, attains a larger size than any other eubacterium, varies 10- to 20-fold in length (and >2,000-fold in volume), and undergoes a complex daily life cycle. In early morning, nucleoids contain highly condensed DNA in elongate, chromosome-like structures which are physically separated from the general cytoplasm. Cell division involves production of two (rarely three) nucleoids within a cell, deposition of cell walls around expanded nucleoids, and emergence of daughter cells from the parent cell. Fluorescence measurements of DNA, RNA, and other cell components indicate the following. DNA quantity is proportional to cell volume over cell lengths of ∼30 μm to >500 μm. For cells of a given size, nucleoids of cells with two nucleoids (binucleoid) contain approximately equal amounts of DNA. And each nucleoid of a binucleoid cell contains one-half the DNA of the single nucleoid in a uninucleoid cell of the same size. The life cycle involves approximately equal subdivision of DNA among daughter cells, formation of apical caps of condensed DNA from previously decondensed and diffusely distributed DNA, and “pinching” of DNA near the middle of the cell in the absence of new wall formation. Mechanisms underlying these patterns remain unclear, but formation of daughter nucleoids and cells occurs both during diurnal periods of host feeding and bacterial cell growth and during nocturnal periods of host inactivity when mean bacterial cell size declines. PMID:9791108

Trimeric Association of Hox and TALE Homeodomain Proteins Mediates Hoxb2 Hindbrain Enhancer Activity

PubMed Central

Jacobs, Yakop; Schnabel, Catherine A.; Cleary, Michael L.

1999-01-01

Pbx/exd proteins modulate the DNA binding affinities and specificities of Hox proteins and contribute to the execution of Hox-dependent developmental programs in arthropods and vertebrates. Pbx proteins also stably heterodimerize and bind DNA with Meis and Pknox1-Prep1, additional members of the TALE (three-amino-acid loop extension) superclass of homeodomain proteins that function on common genetic pathways with a subset of Hox proteins. In this study, we demonstrated that Pbx and Meis bind DNA as heterotrimeric complexes with Hoxb1 on a genetically defined Hoxb2 enhancer, r4, that mediates the cross-regulatory transcriptional effects of Hoxb1 in vivo. The DNA binding specificity of the heterotrimeric complex for r4 is mediated by a Pbx-Hox site in conjunction with a distal Meis site, which we showed to be required for ternary complex formation and Meis-enhanced transcription. Formation of heterotrimeric complexes in which all three homeodomains bind their cognate DNA sites is topologically facilitated by the ability of Pbx and Meis to interact through their amino termini and bind DNA without stringent half-site orientation and spacing requirements. Furthermore, Meis site mutation in the Hoxb2 enhancer phenocopies Pbx-Hox site mutation to abrogate enhancer-directed expression of a reporter transgene in the murine embryonic hindbrain, demonstrating that DNA binding by all three proteins is required for trimer function in vivo. Our data provide in vitro and in vivo evidence for the combinatorial regulation of Hox and TALE protein functions that are mediated, in part, by their interdependent DNA binding activities as ternary complexes. As a consequence, Hoxb1 employs Pbx and Meis-related proteins, as a pair of essential cofactors in a higher-order molecular complex, to mediate its transcriptional effects on an endogenous Hox response element. PMID:10373562

RPA homologs and ssDNA processing during meiotic recombination.

PubMed

Ribeiro, Jonathan; Abby, Emilie; Livera, Gabriel; Martini, Emmanuelle

2016-06-01

Meiotic homologous recombination is a specialized process that involves homologous chromosome pairing and strand exchange to guarantee proper chromosome segregation and genetic diversity. The formation and repair of DNA double-strand breaks (DSBs) during meiotic recombination differs from those during mitotic recombination in that the homologous chromosome rather than the sister chromatid is the preferred repair template. The processing of single-stranded DNA (ssDNA) formed on intermediate recombination structures is central to driving the specific outcomes of DSB repair during meiosis. Replication protein A (RPA) is the main ssDNA-binding protein complex involved in DNA metabolism. However, the existence of RPA orthologs in plants and the recent discovery of meiosis specific with OB domains (MEIOB), a widely conserved meiosis-specific RPA1 paralog, strongly suggest that multiple RPA complexes evolved and specialized to subdivide their roles during DNA metabolism. Here we review ssDNA formation and maturation during mitotic and meiotic recombination underlying the meiotic specific features. We describe and discuss the existence and properties of MEIOB and multiple RPA subunits in plants and highlight how they can provide meiosis-specific fates to ssDNA processing during homologous recombination. Understanding the functions of these RPA homologs and how they interact with the canonical RPA subunits is of major interest in the fields of meiosis and DNA repair.

Exploring protein-DNA interactions in 3D using in situ construction, manipulation, and visualization of individual DNA-dumbbells with optical traps, microfluidics, and fluorescence microscopy

PubMed Central

Forget, Anthony L.; Dombrowski, Christopher C.; Amitani, Ichiro; Kowalczykowski, Stephen C.

2015-01-01

In this Protocol, we describe a procedure to generate ‘DNA-dumbbells’ — single molecules of DNA with a microscopic bead attached at each end — and techniques for manipulating individual DNA-dumbbells. We also detail the design and fabrication of a microfluidic device (flow cell) used in conjunction with dual optical trapping to manipulate DNA-dumbbells and to visualize individual protein–DNA complexes by single-molecule epifluorescence microscopy. Our design of the flow cell enables the rapid movement of trapped molecules between laminar flow channels and a flow-free ‘reservoir’. The reservoir provides the means to examine formation of DNA–protein complexes in solution in the absence of external flow forces, while still maintaining a predetermined end-to-end extension of the DNA. These features facilitate examination of the role of three-dimensional DNA conformation and dynamics in protein–DNA interactions. Preparation of flow cells and reagents requires two days each; in situ DNA-dumbbell assembly and imaging of single protein–DNA complexes requires another day. PMID:23411634

[Mechanistic modelling allows to assess pathways of DNA lesion interactions underlying chromosome aberration formation].

PubMed

Eĭdel'man, Iu A; Slanina, S V; Sal'nikov, I V; Andreev, S G

2012-12-01

The knowledge of radiation-induced chromosomal aberration (CA) mechanisms is required in many fields of radiation genetics, radiation biology, biodosimetry, etc. However, these mechanisms are yet to be quantitatively characterised. One of the reasons is that the relationships between primary lesions of DNA/chromatin/chromosomes and dose-response curves for CA are unknown because the pathways of lesion interactions in an interphase nucleus are currently inaccessible for direct experimental observation. This article aims for the comparative analysis of two principally different scenarios of formation of simple and complex interchromosomal exchange aberrations: by lesion interactions at chromosome territories' surface vs. in the whole space of the nucleus. The analysis was based on quantitative mechanistic modelling of different levels of structures and processes involved in CA formation: chromosome structure in an interphase nucleus, induction, repair and interactions of DNA lesions. It was shown that the restricted diffusion of chromosomal loci, predicted by computational modelling of chromosome organization, results in lesion interactions in the whole space of the nucleus being impossible. At the same time, predicted features of subchromosomal dynamics agrees well with in vivo observations and does not contradict the mechanism of CA formation at the surface of chromosome territories. On the other hand, the "surface mechanism" of CA formation, despite having certain qualities, proved to be insufficient to explain high frequency of complex exchange aberrations observed by mFISH technique. The alternative mechanism, CA formation on nuclear centres is expected to be sufficient to explain frequent complex exchanges.

Neutral Porphyrin Derivative Exerts Anticancer Activity by Targeting Cellular Topoisomerase I (Top1) and Promotes Apoptotic Cell Death without Stabilizing Top1-DNA Cleavage Complexes

PubMed Central

2017-01-01

Camptothecin (CPT) selectively traps topoisomerase 1-DNA cleavable complexes (Top1cc) to promote anticancer activity. Here, we report the design and synthesis of a new class of neutral porphyrin derivative 5,10-bis(4-carboxyphenyl)-15, 20-bis(4-dimethylaminophenyl)porphyrin (compound 8) as a potent catalytic inhibitor of human Top1. In contrast to CPT, compound 8 reversibly binds with the free enzyme and inhibits the formation of Top1cc and promotes reversal of the preformed Top1cc with CPT. Compound 8 induced inhibition of Top1cc formation in live cells was substantiated by fluorescence recovery after photobleaching (FRAP) assays. We established that MCF7 cells treated with compound 8 trigger proteasome-mediated Top1 degradation, accumulate higher levels of reactive oxygen species (ROS), PARP1 cleavage, oxidative DNA fragmentation, and stimulate apoptotic cell death without stabilizing apoptotic Top1-DNA cleavage complexes. Finally, compound 8 shows anticancer activity by targeting cellular Top1 and preventing the enzyme from directly participating in the apoptotic process. PMID:29290109

Dynamics of water around the complex structures formed between the KH domains of far upstream element binding protein and single-stranded DNA molecules

DOE Office of Scientific and Technical Information (OSTI.GOV)

Chakraborty, Kaushik; Bandyopadhyay, Sanjoy, E-mail: sanjoy@chem.iitkgp.ernet.in

2015-07-28

Single-stranded DNA (ss-DNA) binding proteins specifically bind to the single-stranded regions of the DNA and protect it from premature annealing, thereby stabilizing the DNA structure. We have carried out atomistic molecular dynamics simulations of the aqueous solutions of two DNA binding K homology (KH) domains (KH3 and KH4) of the far upstream element binding protein complexed with two short ss-DNA segments. Attempts have been made to explore the influence of the formation of such complex structures on the microscopic dynamics and hydrogen bond properties of the interfacial water molecules. It is found that the water molecules involved in bridging themore » ss-DNA segments and the protein domains form a highly constrained thin layer with extremely retarded mobility. These water molecules play important roles in freezing the conformational oscillations of the ss-DNA oligomers and thereby forming rigid complex structures. Further, it is demonstrated that the effect of complexation on the slow long-time relaxations of hydrogen bonds at the interface is correlated with hindered motions of the surrounding water molecules. Importantly, it is observed that the highly restricted motions of the water molecules bridging the protein and the DNA components in the complexed forms originate from more frequent hydrogen bond reformations.« less

Attenuation of drug-stimulated topoisomerase II-DNA cleavable complex formation in wild-type HL-60 cells treated with an intracellular calcium buffer is correlated with decreased cytotoxicity and site-specific hypophosphorylation of topoisomerase IIalpha.

PubMed Central

Aoyama, M; Grabowski, D R; Dubyak, G R; Constantinou, A I; Rybicki, L A; Bukowski, R M; Ganapathi, M K; Hickson, I D; Ganapathi, R

1998-01-01

Topoisomerase II (topo II), an essential enzyme for cell viability, is also the target for clinically important anti-neoplastic agents that stimulate topo II-mediated DNA scission. The role of alterations in topo IIalpha phosphorylation and its effect on drug-induced DNA damage and cytotoxicity were investigated. Following loading of HL-60 cells with the calcium buffer 1, 2-bis-(o-aminophenoxy)ethane-N,N,N',N'-tetra-acetic acid tetra(acetoxymethyl) ester (BAPTA-AM), which abrogates intracellular Ca2+ transients, a significant decrease in etoposide (VP-16)- or amsacrine (m-AMSA)-stabilized topo II-DNA cleavable complex formation and a corresponding decrease in cytotoxicity was observed. In a cell-free system, nuclear extracts from BAPTA-AM-treated cells exhibited markedly less activity when assayed for VP-16-stabilized topo II-DNA complex formation, but not decatenation of kinetoplast DNA. In contrast, the loading of HL-60 cells with N,N,N', N'-tetrakis-(2-pyridyl)ethylenediamine (TPEN), which binds heavy metals without disturbing calcium or magnesium concentrations, did not significantly affect VP-16-stimulated topo II-DNA cleavable complex formation or cytotoxicity. In HL-60 cells the accumulation of BAPTA, but not TPEN, also led to the hypophosphorylation of topo IIalpha. Tryptic phosphopeptide mapping of topo IIalpha protein from HL-60 cells revealed: (a) eight major phosphorylation sites in untreated cells; (b) hypophosphorylation of two out of eight sites in BAPTA-AM-treated cells; and (c) hypophosphorylation of between two and four out of eight sites in topo II-poison-resistant HL-60 cells. The two hypophosphorylated sites present following BAPTA-AM treatment of wild-type cells were identical with the hypophosphorylated sites in the resistant cells, but were not the same as the sites that are substrates for casein kinase II [Wells, Addison, Fry, Ganapathi and Hickson (1994) J. Biol. Chem. 269, 29746-29751]. In summary, changes in intracellular Ca2+ transients that lead to the site-specific hypophosphorylation of topo IIalpha are possibly involved in regulating the DNA damage caused by and the cytotoxic potential of topo II poisons. PMID:9841887

Biophysical and transfection studies of the diC(14)-amidine/DNA complex.

PubMed Central

Cherezov, Vadim; Qiu, Hong; Pector, Veronique; Vandenbranden, Michel; Ruysschaert, Jean-Marie; Caffrey, Martin

2002-01-01

Liposomes of the synthetic cationic lipid, N-t-butyl-N'-tetradecylamino-propionamidine (diC(14)-amidine), efficiently ports DNA into mammalian cells in the absence of other (neutral) lipids. The compositional simplicity of this transfection mix makes it attractive from a formulation perspective. We have used low- and wide-angle x-ray diffraction and polarized light microscopy to characterize the thermotropic phase behavior and microstructure of diC(14)-amidine and of the lipid/DNA (circular plasmid, 5.4 kb) complex with a view to understanding the structure of the complex and its role in transfection. Upon heating, the lipid in buffer undergoes a lamellar crystalline (L(c), d(001) = 41.7 A)-to-lamellar liquid crystal (L(c)(alpha), d(001) depends on hydration and T) transition at approximately 40 degrees C. Sonicated lipid vesicles with a reported transition temperature of approximately 23 degrees C complex with DNA. Complex formation is complete at a DNA/lipid mole ratio (rho) of 0.8. Adding DNA to the lipid causes d(001) of the multilayered complex to drop from 52 to 49 A as rho rises from 0.03 to 1.64. The minimal DNA-DNA duplex separation observed is 26 A, consistent with the close packing of B-DNA. Lipid bilayers in the complex undergo a lamellar gel (L(c)(beta))-to-L(c)(alpha) (superscript c refers to complex) transition at approximately 23 degrees C. Transfection efficiency was maximized at rho = 0.4. The structure and transfection data combined suggest that densely packaged DNA in a net positively charged complex is essential for transfection. PMID:12023234

Preparation of bio-deep eutectic solvent triggered cephalopod shaped silver chloride-DNA hybrid material having antibacterial and bactericidal activity.

PubMed

Bhatt, Jitkumar; Mondal, Dibyendu; Bhojani, Gopal; Chatterjee, Shruti; Prasad, Kamalesh

2015-11-01

2.5% w/w DNA (Salmon testes) was solubilized in a bio-deep eutectic solvent [(bio-DES), obtained by the complexation of choline chloride and ethylene glycol at 1:2 molar ratio] containing 1% w/w of silver chloride (AgCl) to yield a AgCl decorated DNA based hybrid material. Concentration dependent formation of AgCl crystals in the DES was observed and upon interaction with DNA it gave formation of a cephalopod shaped hybrid material. DNA was found to maintain its chemical and structural stability in the material. Further, AgCl microstructures were found to have orderly self assembled on the DNA helices indicating the electrostatic interaction between Ag(+) and phosphate side chain of DNA as a driving force for the formation of the material with ordered microstructural distribution of AgCl. Furthermore, the functionalized material exhibited excellent antibacterial and bactericidal activity against both Gram negative and Gram positive pathogenic bacteria. Copyright © 2015 Elsevier B.V. All rights reserved.

[DNA complexes, formed on aqueous phase surfaces: new planar polymeric and composite nanostructures].

PubMed

Antipina, M N; Gaĭnutdinov, R V; Rakhnianskaia, A A; Sergeev-Cherenkov, A N; Tolstikhina, A L; Iurova, T V; Kislov, V V; Khomutov, G B

2003-01-01

The formation of DNA complexes with Langmuir monolayers of the cationic lipid octadecylamine (ODA) and the new amphiphilic polycation poly-4-vinylpyridine with 16% of cetylpyridinium groups (PVP-16) on the surface of an aqueous solution of native DNA of low ionic strength was studied. Topographic images of Langmuir-Blodgett films of DNA/ODA and DNA/PVP-16 complexes applied to micaceous substrates were investigated by the method of atomic force microscopy. It was found that films of the amphiphilic polycation have an ordered planar polycrystalline structure. The morphology of planar DNA complexes with the amphiphilic cation substantially depended on the incubation time and the phase state of the monolayer on the surface of the aqueous DNA solution. Complex structures and individual DNA molecules were observed on the surface of the amphiphilic monolayer. Along with quasi-linear individual bound DNA molecules, characteristic extended net-like structures and quasi-circular toroidal condensed conformations of planar DNA complexes were detected. Mono- and multilayer films of DNA/PVP-16 complexes were used as templates and nanoreactors for the synthesis of inorganic nanostructures via the binding of metal cations from the solution and subsequent generation of the inorganic phase. As a result, ultrathin polymeric composite films with integrated DNA building blocks and quasi-linear arrays of inorganic semiconductor (CdS) and iron oxide nanoparticles and nanowires were obtained. The nanostructures obtained were characterized by scanning probe microscopy and transmission electron microscopy techniques. The methods developed are promising for investigating the mechanisms of structural organization and transformation in DNA and polyelectrolyte complexes at the gas-liquid interface and for the design of new extremely thin highly ordered planar polymeric and composite materials, films, and coatings with controlled ultrastructure for applications in nanoelectronics and nanobiotechnology.

How a low-fidelity DNA polymerase chooses non-Watson-Crick from Watson-Crick incorporation.

PubMed

Wu, Wen-Jin; Su, Mei-I; Wu, Jian-Li; Kumar, Sandeep; Lim, Liang-Hin; Wang, Chun-Wei Eric; Nelissen, Frank H T; Chen, Ming-Chuan Chad; Doreleijers, Jurgen F; Wijmenga, Sybren S; Tsai, Ming-Daw

2014-04-02

A dogma for DNA polymerase catalysis is that the enzyme binds DNA first, followed by MgdNTP. This mechanism contributes to the selection of correct dNTP by Watson-Crick base pairing, but it cannot explain how low-fidelity DNA polymerases overcome Watson-Crick base pairing to catalyze non-Watson-Crick dNTP incorporation. DNA polymerase X from the deadly African swine fever virus (Pol X) is a half-sized repair polymerase that catalyzes efficient dG:dGTP incorporation in addition to correct repair. Here we report the use of solution structures of Pol X in the free, binary (Pol X:MgdGTP), and ternary (Pol X:DNA:MgdGTP with dG:dGTP non-Watson-Crick pairing) forms, along with functional analyses, to show that Pol X uses multiple unprecedented strategies to achieve the mutagenic dG:dGTP incorporation. Unlike high fidelity polymerases, Pol X can prebind purine MgdNTP tightly and undergo a specific conformational change in the absence of DNA. The prebound MgdGTP assumes an unusual syn conformation stabilized by partial ring stacking with His115. Upon binding of a gapped DNA, also with a unique mechanism involving primarily helix αE, the prebound syn-dGTP forms a Hoogsteen base pair with the template anti-dG. Interestingly, while Pol X prebinds MgdCTP weakly, the correct dG:dCTP ternary complex is readily formed in the presence of DNA. H115A mutation disrupted MgdGTP binding and dG:dGTP ternary complex formation but not dG:dCTP ternary complex formation. The results demonstrate the first solution structural view of DNA polymerase catalysis, a unique DNA binding mode, and a novel mechanism for non-Watson-Crick incorporation by a low-fidelity DNA polymerase.

An anti-DNA antibody prefers damaged dsDNA over native.

PubMed

Akberova, N I; Zhmurov, A A; Nevzorova, T A; Litvinov, R I

2017-01-01

DNA-protein interactions, including DNA-antibody complexes, have both fundamental and practical significance. In particular, antibodies against double-stranded DNA play an important role in the pathogenesis of autoimmune diseases. Elucidation of structural mechanisms of an antigen recognition and interaction of anti-DNA antibodies provides a basis for understanding the role of DNA-containing immune complexes in human pathologies and for new treatments. Here we used Molecular Dynamic simulations of bimolecular complexes of a segment of dsDNA with a monoclonal anti-DNA antibody's Fab-fragment to obtain detailed structural and physical characteristics of the dynamic intermolecular interactions. Using a computationally modified crystal structure of a Fab-DNA complex (PDB: 3VW3), we studied in silico equilibrium Molecular Dynamics of the Fab-fragment associated with two homologous dsDNA fragments, containing or not containing dimerized thymine, a product of DNA photodamage. The Fab-fragment interactions with the thymine dimer-containing DNA was thermodynamically more stable than with the native DNA. The amino acid residues constituting a paratope and the complementary nucleotide epitopes for both Fab-DNA constructs were identified. Stacking and electrostatic interactions were shown to play the main role in the antibody-dsDNA contacts, while hydrogen bonds were less significant. The aggregate of data show that the chemically modified dsDNA (containing a covalent thymine dimer) has a higher affinity toward the antibody and forms a stronger immune complex. These findings provide a mechanistic insight into formation and properties of the pathogenic anti-DNA antibodies in autoimmune diseases, such as systemic lupus erythematosus, associated with skin photosensibilization and DNA photodamage.

DNA adduct formation among workers in a Thai industrial estate and nearby residents.

PubMed

Peluso, Marco; Srivatanakul, Petcharin; Munnia, Armelle; Jedpiyawongse, Adisorn; Meunier, Aurelie; Sangrajrang, Suleeporn; Piro, Sara; Ceppi, Marcello; Boffetta, Paolo

2008-01-25

The genotoxic effects of air pollutant exposures have been studied in people living and working in Map Ta Phut, Rayong province, Thailand, a site where is located the Map Ta Phut Industrial Estate (MIE) one of the largest steel, refinery and petrochemical complex in the South-Eastern Asia. This was done by the conduction of a transversal study aimed to compare the prevalence of bulky DNA adducts in groups of subjects experiencing various degree of air pollution. DNA adduct analysis was performed in the leukocytes of 201 volunteers by the (32)P-postlabelling assay: 79 were workers in the MIE complex, including 24 refinery workers, 40 steel workers and 15 tinplate workers, 72 were people residing downwind in the MIE area and 50 were residents in a control district of the same Rayong province but without industrial exposures. The groups of workers were analyzed separately to evaluate if DNA adduct formation differs by the type of industry. The levels of bulky DNA adducts were 1.17+/-0.17 (SE) adducts/10(8) nucleotides in refinery workers, 1.19+/-0.19 (SE) in steel workers, 0.87+/-0.17 (SE) in tinplate workers, 0.85+/-0.07 (SE) in MIE residents and 0.53+/-0.05 (SE) in district controls. No effects of smoking habits on DNA adducts was found. The multivariate regression analysis shows that the levels of DNA adducts were significantly increased among the individuals living near the MIE industrial complex in respect to those resident in a control district (p<0.05). In the groups of occupationally exposed workers, the highest levels of DNA adducts were found among the workers experiencing an occupational exposure to polycyclic aromatic hydrocarbons, e.g. the steel factory and refinery workers. When we have evaluated if the levels of DNA adducts of the PAH exposed workers were different from those of the MIE residents, a statistical significantly difference was found (p<0.05). Our present study indicates that people living near point sources of industrial air pollution can experiment an excess of DNA adduct formation. The emissions from the MIE complex are the main source of air pollution in this area and can be the cause of such increment in the levels of DNA damage.

Self-recognition of the racemic ligand in the formation of homochiral dinuclear V(V) complex: In vitro anticancer activity, DNA and HSA interaction.

PubMed

Kazemi, Zahra; Amiri Rudbari, Hadi; Mirkhani, Valiollah; Sahihi, Mehdi; Moghadam, Majid; Tangestaninejad, Shahram; Mohammadpoor-Baltork, Iraj; Kajani, Abolghasem Abbasi; Azimi, Gholamhassan

2017-07-28

The reaction of a racemic mixture of Schiff base tridentate ligand with vanadium(V) affords homochiral vanadium complex, (VO(R-L)) 2 O and (VO(S-L)) 2 O due to ligand "self-recognition" process. The formation of homochiral vanadium complex was confirmed by 1 H NMR, 13 C NMR and X-ray diffraction. The HSA- and DNA-binding of the resultant complex is assessed by absorption, fluorescence and circular dichroism (CD) spectroscopy methods. Based on the results, the HSA- and DNA-binding constant, K b , were found to be 8.0 × 10 4 and 1.9 × 10 5  M -1 , respectively. Interestingly, in vitro cytotoxicity assay revealed the potent anticancer activity of this complex on two prevalent cancer cell lines of MCF-7 (IC50 value of 14 μM) and HeLa (IC50 value of 36 μM), with considerably low toxicity on normal human fibroblast cells. The maximum cell mortality of 12.3% obtained after 48 h incubation of fibroblast cells with 100 μM of the complex. Additionally, the specific DNA- and HSA-binding was also shown using molecular docking method. The synthesized complex displayed high potential for biomedical applications especially for development of novel and efficient anticancer agents. Copyright © 2017 Elsevier Masson SAS. All rights reserved.

Nucleosome Positioning and Epigenetics

NASA Astrophysics Data System (ADS)

Schwab, David; Bruinsma, Robijn

2008-03-01

The role of chromatin structure in gene regulation has recently taken center stage in the field of epigenetics, phenomena that change the phenotype without changing the DNA sequence. Recent work has also shown that nucleosomes, a complex of DNA wrapped around a histone octamer, experience a sequence dependent energy landscape due to the variation in DNA bend stiffness with sequence composition. In this talk, we consider the role nucleosome positioning might play in the formation of heterochromatin, a compact form of DNA generically responsible for gene silencing. In particular, we discuss how different patterns of nucleosome positions, periodic or random, could either facilitate or suppress heterochromatin stability and formation.

Polyplex formation between four-arm poly(ethylene oxide)-b-poly(2-(diethylamino)ethyl methacrylate) and plasmid DNA in gene delivery.

PubMed

He, E; Yue, C Y; Simeon, F; Zhou, L H; Too, H P; Tam, K C

2009-12-01

Amphiphilic polyelectrolytes comprising cationic and uncharged hydrophilic segments condensed negatively charged DNA to form a core-shell structure stabilized by a layer of hydrophilic corona chains. At physiological pH, four-arm star-shaped poly(ethylene oxide)-b-poly(2-(diethylamino)ethyl methacrylate) (four-arm PEO-b-PDEAEMA) block copolymer possessed positively charged amine groups that interacted with negatively charged plasmid DNA to form polymer/DNA complexes. The mechanism and physicochemical properties of the complex formation were investigated at varying molar ratio of amine groups on polymer chains and phosphate group on plasmid DNA segments (N/P ratio). The capability of the star block copolymer to condense DNA was demonstrated through gel electrophoresis and ethidium bromide exclusion assay. In the absence of salt, the hydrodynamic radius of polyplexes was about 94 nm at low polymer/DNA ratio, and it decreased to about 34 nm at large N/P ratios, forming a compact spherical structure with a weighted average molecular weight of 4.39 +/- 0.22 x 10(6) g/mol. Approximately 15 polymeric chains were required to condense a plasmid DNA. The addition of monovalent salt to the polyplexes significantly altered the size of the complexes, which would have an impact on cell transfection. Because of the electrostatic interaction induced by the diffusion of small ions, the polyplex increased in size to about 53 nm with a less compact structure. In vitro cytotoxicty of polymer and polymer/pDNA complexes were evaluated, and the polyplexes exhibited low toxicity at low N/P ratios. At N/P ratio of 4.5, the four-arm PEO-b-PDEAEMA showed the highest level of transfection in Neuro-2A cells. These observations showed that the star-shaped multi-arm polymers offers interesting properties in self-association and condensation ability for plasmid DNA and can serve as a nonviral DNA delivery system. Copyright 2008 Wiley Periodicals, Inc.

High Throughput Measurement of Extracellular DNA Release and Quantitative NET Formation in Human Neutrophils In Vitro.

PubMed

Sil, Payel; Yoo, Dae-Goon; Floyd, Madison; Gingerich, Aaron; Rada, Balazs

2016-06-18

Neutrophil granulocytes are the most abundant leukocytes in the human blood. Neutrophils are the first to arrive at the site of infection. Neutrophils developed several antimicrobial mechanisms including phagocytosis, degranulation and formation of neutrophil extracellular traps (NETs). NETs consist of a DNA scaffold decorated with histones and several granule markers including myeloperoxidase (MPO) and human neutrophil elastase (HNE). NET release is an active process involving characteristic morphological changes of neutrophils leading to expulsion of their DNA into the extracellular space. NETs are essential to fight microbes, but uncontrolled release of NETs has been associated with several disorders. To learn more about the clinical relevance and the mechanism of NET formation, there is a need to have reliable tools capable of NET quantitation. Here three methods are presented that can assess NET release from human neutrophils in vitro. The first one is a high throughput assay to measure extracellular DNA release from human neutrophils using a membrane impermeable DNA-binding dye. In addition, two other methods are described capable of quantitating NET formation by measuring levels of NET-specific MPO-DNA and HNE-DNA complexes. These microplate-based methods in combination provide great tools to efficiently study the mechanism and regulation of NET formation of human neutrophils.

Coordination properties of tridentate (N,O,O) heterocyclic alcohol (PDC) with Cu(II). Mixed ligand complex formation reactions of Cu(II) with PDC and some bio-relevant ligands.

PubMed

El-Sherif, Ahmed A; Shoukry, Mohamed M

2007-03-01

The formation equilibria of copper(II) complexes and the ternary complexes Cu(PDC)L (PDC=2,6-bis-(hydroxymethyl)-pyridine, HL=amino acid, amides or DNA constituents) have been investigated. Ternary complexes are formed by a simultaneous mechanism. The results showed the formation of Cu(PDC)L, Cu(PDC, H(-1))(L) and Cu(PDC, H(-2))(L) complexes. The concentration distribution of the complexes in solution is evaluated as a function of pH. The effect of dioxane as a solvent on the protonation constant of PDC and the formation constants of Cu(II) complexes are discussed. The thermodynamic parameters DeltaH degrees and DeltaS degrees calculated from the temperature dependence of the equilibrium constants are investigated.

Mechanism of the formation of the RecA-ssDNA nucleoprotein filament structure: a coarse-grained approach.

PubMed

Mukherjee, Goutam; Pal, Arumay; Levy, Yaakov

2017-11-21

In prokaryotes, the RecA protein catalyzes the repair and strand exchange of double-stranded DNA. RecA binds to single-stranded DNA (ssDNA) and forms a presynaptic complex in which the protein polymerizes around the ssDNA to form a right-handed helical nucleoprotein filament structure. In the present work, the mechanism for the formation of the RecA-ssDNA filament structure is modeled using coarse-grained molecular dynamics simulations. Information from the X-ray structure was used to model the protein itself but not its interactions; the interactions between the protein and the ssDNA were modeled solely by electrostatic, aromatic, and repulsive energies. For the present study, the monomeric, dimeric, and trimeric units of RecA and 4, 8, and 11 NT-long ssDNA, respectively, were studied. Our results indicate that monomeric RecA is not sufficient for nucleoprotein filament formation; rather, dimeric RecA is the elementary binding unit, with higher multimeric units of RecA facilitating filament formation. Our results reveal that loop region flexibility at the primary binding site of RecA is essential for it to bind the incoming ssDNA, that the aromatic residues present in the loop region play an important role in ssDNA binding, and that ATP may play a role in guiding the ssDNA by changing the electrostatic potential of the RecA protein.

An experimental double-blind irradiation study of a novel topical product (TPF 50) compared to other topical products with DNA repair enzymes, antioxidants, and growth factors with sunscreens: implications for preventing skin aging and cancer.

PubMed

Emanuele, Enzo; Spencer, James M; Braun, Martin

2014-03-01

The exposure to ultraviolet radiation (UVR) is a major risk factor for skin aging and the development of non-melanoma skin cancer (NMSC). Although traditional sunscreens remain the mainstay for the prevention of UVR-induced skin damage, they cannot ensure a complete protection against the whole spectrum of molecular lesions associated with UVR exposure. The formation of helix-distorting photoproducts such as cyclobutane pyrimidine dimers (CPD), as well as oxidative damage to DNA bases, including the formation of 8-oxo-7,8-dihydro-2'-deoxyguanosine (8OHdG) are among the key DNA lesions associated with photoaging and tumorigenesis. Besides DNA lesions, UVR-induced formation of free radicals can result in protein carbonylation (PC), a major form of irreversible protein damage that inactivates their biological function. This study compares a complex novel topical product (TPF50) consisting of three actives, ie, 1) traditional physical sunscreens (SPF 50), 2) a liposome-encapsulated DNA repair enzymes complex (photolyase, endonuclease, and 8-oxoguanine glycosylase [OGG1]), and 3) a potent antioxidant complex (carnosine, arazine, ergothionine) to existing products. Specifically, we assessed the ability of TFP50 vs those of DNA repair and antioxidant and growth factor topical products used with SPF 50 sunscreens in preventing CPD, 8OHdG, and PC formation in human skin biopsies after experimental irradiations. In head-to-head comparison studies, TPF50 showed the best efficacy in reducing all of the three molecular markers. The results indicated that the three TPF50 components had a synergistic effect in reducing CPD and PC, but not 8OHdG. Taken together, our results indicate that TPF50 improves the genomic and proteomic integrity of skin cells after repeated exposure to UVR, ultimately reducing the risk of skin aging and NMSC.

Towards a DNA Nanoprocessor: Reusable Tile-Integrated DNA Circuits.

PubMed

Gerasimova, Yulia V; Kolpashchikov, Dmitry M

2016-08-22

Modern electronic microprocessors use semiconductor logic gates organized on a silicon chip to enable efficient inter-gate communication. Here, arrays of communicating DNA logic gates integrated on a single DNA tile were designed and used to process nucleic acid inputs in a reusable format. Our results lay the foundation for the development of a DNA nanoprocessor, a small and biocompatible device capable of performing complex analyses of DNA and RNA inputs. © 2016 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

Interaction of an Fe derivative of TMAP (Fe(TMAP)OAc) with DNA in comparison with free-base TMAP.

PubMed

Ghaderi, Masoumeh; Bathaie, S Zahra; Saboury, Ali-Akbar; Sharghi, Hashem; Tangestaninejad, Shahram

2007-07-01

We investigated the interaction of meso-tetrakis (N-para-methylanilium) porphyrin (TMAP) in its free base and Fe(II) form (Fe(TMAP)OAc) as a new derivative, with high molecular weight DNA at different ionic strengths, using various spectroscopic methods and microcalorimetry. The data obtained by spectrophotometery, circular dichroism (CD), fluorescence quenching and resonance light scattering (RLS) have demonstrated that TMAP association with DNA is via outside binding with self-stacking manner, which is accompanied with the "end-on" type complex formation in low ionic strength. However, in the case of Fe(TMAP)OAc, predominant mode of interaction is groove binding and after increasing in DNA concentration, unstable stacking-type aggregates are formed. In addition, isothermal titration calorimetric measurements have indicated the exothermic process of porphyrins binding to DNA, but the exothermisity in metal derivative of porphyrin is less than the free base. It confirmed the formation of a more organized aggregate of TMAP on DNA surface. Interactions of both porphyrins with DNA show high sensitivity to ionic strength. By addition of salt, the downfield CD signal of TMAP aggregates is shifted to a higher wavelength, which indicates some changes in the aggregates position. In the case of Fe(TMAP)OAc, addition of salt leads to changes in the mode of binding from groove binding to outside binding with self-stacking, which is accompanied with major changes in CD spectra, possibly indicating the formation of "face-on" type complex.

The Fission Yeast Minichromosome Maintenance (MCM)-binding Protein (MCM-BP), Mcb1, Regulates MCM Function during Prereplicative Complex Formation in DNA Replication*

PubMed Central

Santosa, Venny; Martha, Sabrina; Hirose, Noriaki; Tanaka, Katsunori

2013-01-01

The minichromosome maintenance (MCM) complex is a replicative helicase, which is essential for chromosome DNA replication. In recent years, the identification of a novel MCM-binding protein (MCM-BP) in most eukaryotes has led to numerous studies investigating its function and its relationship to the MCM complex. However, the mechanisms by which MCM-BP functions and associates with MCM complexes are not well understood; in addition, the functional role of MCM-BP remains controversial and may vary between model organisms. The present study aims to elucidate the nature and biological function of the MCM-BP ortholog, Mcb1, in fission yeast. The Mcb1 protein continuously interacts with MCM proteins during the cell cycle in vivo and can interact with any individual MCM subunit in vitro. To understand the detailed characteristics of mcb1+, two temperature-sensitive mcb1 gene mutants (mcb1ts) were isolated. Extensive genetic analysis showed that the mcb1ts mutants were suppressed by a mcm5+ multicopy plasmid and displayed synthetic defects with many S-phase-related gene mutants. Moreover, cyclin-dependent kinase modulation by Cig2 repression or Rum1 overproduction suppressed the mcb1ts mutants, suggesting the involvement of Mcb1 in pre-RC formation during DNA replication. These data are consistent with the observation that Mcm7 loading onto replication origins is reduced and S-phase progression is delayed in mcb1ts mutants. Furthermore, the mcb1ts mutation led to the redistribution of MCM subunits to the cytoplasm, and this redistribution was dependent on an active nuclear export system. These results strongly suggest that Mcb1 promotes efficient pre-RC formation during DNA replication by regulating the MCM complex. PMID:23322785

The fission yeast minichromosome maintenance (MCM)-binding protein (MCM-BP), Mcb1, regulates MCM function during prereplicative complex formation in DNA replication.

PubMed

Santosa, Venny; Martha, Sabrina; Hirose, Noriaki; Tanaka, Katsunori

2013-03-08

The minichromosome maintenance (MCM) complex is a replicative helicase, which is essential for chromosome DNA replication. In recent years, the identification of a novel MCM-binding protein (MCM-BP) in most eukaryotes has led to numerous studies investigating its function and its relationship to the MCM complex. However, the mechanisms by which MCM-BP functions and associates with MCM complexes are not well understood; in addition, the functional role of MCM-BP remains controversial and may vary between model organisms. The present study aims to elucidate the nature and biological function of the MCM-BP ortholog, Mcb1, in fission yeast. The Mcb1 protein continuously interacts with MCM proteins during the cell cycle in vivo and can interact with any individual MCM subunit in vitro. To understand the detailed characteristics of mcb1(+), two temperature-sensitive mcb1 gene mutants (mcb1(ts)) were isolated. Extensive genetic analysis showed that the mcb1(ts) mutants were suppressed by a mcm5(+) multicopy plasmid and displayed synthetic defects with many S-phase-related gene mutants. Moreover, cyclin-dependent kinase modulation by Cig2 repression or Rum1 overproduction suppressed the mcb1(ts) mutants, suggesting the involvement of Mcb1 in pre-RC formation during DNA replication. These data are consistent with the observation that Mcm7 loading onto replication origins is reduced and S-phase progression is delayed in mcb1(ts) mutants. Furthermore, the mcb1(ts) mutation led to the redistribution of MCM subunits to the cytoplasm, and this redistribution was dependent on an active nuclear export system. These results strongly suggest that Mcb1 promotes efficient pre-RC formation during DNA replication by regulating the MCM complex.

Effect of uv light on formation and synthetic capacity of DNA-membrane complexes from T7-infected cells of E. coli

DOE Office of Scientific and Technical Information (OSTI.GOV)

Wintermantel, G.

1974-02-01

The action of uv light on the attachment of T7-DNA to the bacterial cell membrane, and the RNA- and DNA-synthesizing activity of the DNA-membrane complex, were investigated. In E. coli H560 cells infected by uv-irradiated T7 phages, the amount of membrane-associated parental /sup 32/P-labeled T7-DNA was determined. To measure the RNA- and DNA-synthesizing activity, the isolated DNA- membrane complexes were incubated with an equimolar mixture of the corresponding four nucleoside triphosphates. The uv-sensitivities of the functions tested were calculated from the slopes of the dose-effect curves. In relation to the uv- sensitivity for plaque-forming ability of the T7bacteriophages they amountmore » to 0.07 ( plus or minus 0.01); 0.15 ( plus or minus 0.01); and 0.56 ( plus or minus 0.04), respectively. (auth)« less

RNF168 forms a functional complex with RAD6 during the DNA damage response

PubMed Central

Liu, Chao; Wang, Degui; Wu, Jiaxue; Keller, Jennifer; Ma, Teng; Yu, Xiaochun

2013-01-01

Summary Protein ubiquitination plays an important role in initiating the DNA damage response. Following DNA damage, E2 ubiquitin conjugating enzymes are crucial for catalyzing substrate ubiquitination that recruits downstream DNA repair factors to DNA lesions. To identify novel E2 conjugating enzymes important for initiating the DNA-damage-induced ubiquitination cascade, we screened most of the known E2 enzymes and found that RAD6A and RAD6B function together with RNF168 in the ionizing radiation (IR)-induced DNA damage response. Similarly to RNF168-deficient cells, RAD6A- or RAD6B-deficient cells exhibit a reduction in DNA-damage-induced protein ubiquitination. Correspondingly, DNA-damage-induced foci formation of DNA damage repair proteins, such as BRCA1 and 53BP1, is impaired in the absence of RAD6A or RAD6B. Moreover, the RNF168–RAD6 complex targeted histone H1.2 for ubiquitination in vitro and regulated DNA-damage-induced histone H1.2 ubiquitination in vivo. Collectively, these data demonstrate that RNF168, in complex with RAD6A or RAD6B, is activated in the DNA-damage-induced protein ubiquitination cascade. PMID:23525009

Simocyclinone D8, an inhibitor of DNA gyrase with a novel mode of action.

PubMed

Flatman, Ruth H; Howells, Alison J; Heide, Lutz; Fiedler, Hans-Peter; Maxwell, Anthony

2005-03-01

We have characterized the interaction of a new class of antibiotics, simocyclinones, with bacterial DNA gyrase. Even though their structures include an aminocoumarin moiety, a key feature of novobiocin, coumermycin A(1), and clorobiocin, which also target gyrase, simocyclinones behave strikingly differently from these compounds. Simocyclinone D8 is a potent inhibitor of gyrase supercoiling, with a 50% inhibitory concentration lower than that of novobiocin. However, it does not competitively inhibit the DNA-independent ATPase reaction of GyrB, which is characteristic of other aminocoumarins. Simocyclinone D8 also inhibits DNA relaxation by gyrase but does not stimulate cleavage complex formation, unlike quinolones, the other major class of gyrase inhibitors; instead, it abrogates both Ca(2+)- and quinolone-induced cleavage complex formation. Binding studies suggest that simocyclinone D8 interacts with the N-terminal domain of GyrA. Taken together, our results demonstrate that simocyclinones inhibit an early step of the gyrase catalytic cycle by preventing binding of the enzyme to DNA. This is a novel mechanism for a gyrase inhibitor and presents new possibilities for antibacterial drug development.

Real-time observation of DNA recognition and rejection by the RNA-guided endonuclease Cas9.

PubMed

Singh, Digvijay; Sternberg, Samuel H; Fei, Jingyi; Doudna, Jennifer A; Ha, Taekjip

2016-09-14

Binding specificity of Cas9-guide RNA complexes to DNA is important for genome-engineering applications; however, how mismatches influence target recognition/rejection kinetics is not well understood. Here we used single-molecule FRET to probe real-time interactions between Cas9-RNA and DNA targets. The bimolecular association rate is only weakly dependent on sequence; however, the dissociation rate greatly increases from <0.006 s(-1) to >2 s(-1) upon introduction of mismatches proximal to protospacer-adjacent motif (PAM), demonstrating that mismatches encountered early during heteroduplex formation induce rapid rejection of off-target DNA. In contrast, PAM-distal mismatches up to 11 base pairs in length, which prevent DNA cleavage, still allow formation of a stable complex (dissociation rate <0.006 s(-1)), suggesting that extremely slow rejection could sequester Cas9-RNA, increasing the Cas9 expression level necessary for genome-editing, thereby aggravating off-target effects. We also observed at least two different bound FRET states that may represent distinct steps in target search and proofreading.

Electrochemical, spectroscopic, and theoretical studies on the interaction between azathioprine and DNA.

PubMed

Jalali, Fahimeh; Rasaee, Gelareh

2015-11-01

Possible interaction between immunosuppressive drug, azathioprine, and calf thymus DNA was explored by cyclic voltammetry, spectrophotometry, competitive spectrofluorimetry, circular dichroism spectroscopy (CD), and viscosity measurements. Cyclic voltammetry showed negative shift in the reduction peak of azathioprine in the presence of DNA, and large decrease in peak current, referring to the predominance of electrostatic forces. The binding constant was calculated to be 1.22×10(3)M(-1). Absorption hyperchromism without shift in wavelength was observed when DNA was added to azathioprine solution. Competitive fluorescence experiments were conducted by using Hoechst 33258 and methylene blue as probes for minor groove and intercalation binding modes, respectively. The studies showed that azathioprine could release Hoechst 33258, while negligible effect was detected in the case of methylene blue. Stern-Volmer quenching constant (KSV) and complex formation constant (Kf) were obtained from the fluorescence measurements to be 7.6×10(3)M(-1) and 7.76×10(4)M(-1), respectively, at 298K. Enthalpy and entropy changes during the interaction between azathioprine and DNA were calculated from Van't Hoff plot (ΔH=-20.2kJmol(-1); ΔS=26.11Jmol(-1)K(-1) at 298K) which showed an exothermic spontaneous reaction, and involvement of electrostatic forces in the complex formation with DNA. Moreover, circular dichroism studies revealed that azathioprine induced detectable changes in the negative band of DNA spectrum. Viscosity of DNA solution decreased in the presence of azathioprine, showed a non-intercalative mode of interaction. Finally, molecular docking calculations showed that in the lowest energy level of drug-DNA complex, azathioprine approaches the minor grooves of DNA. Copyright © 2015 Elsevier B.V. All rights reserved.

Positive regulation of meiotic DNA double-strand break formation by activation of the DNA damage checkpoint kinase Mec1(ATR).

PubMed

Gray, Stephen; Allison, Rachal M; Garcia, Valerie; Goldman, Alastair S H; Neale, Matthew J

2013-07-31

During meiosis, formation and repair of programmed DNA double-strand breaks (DSBs) create genetic exchange between homologous chromosomes-a process that is critical for reductional meiotic chromosome segregation and the production of genetically diverse sexually reproducing populations. Meiotic DSB formation is a complex process, requiring numerous proteins, of which Spo11 is the evolutionarily conserved catalytic subunit. Precisely how Spo11 and its accessory proteins function or are regulated is unclear. Here, we use Saccharomyces cerevisiae to reveal that meiotic DSB formation is modulated by the Mec1(ATR) branch of the DNA damage signalling cascade, promoting DSB formation when Spo11-mediated catalysis is compromised. Activation of the positive feedback pathway correlates with the formation of single-stranded DNA (ssDNA) recombination intermediates and activation of the downstream kinase, Mek1. We show that the requirement for checkpoint activation can be rescued by prolonging meiotic prophase by deleting the NDT80 transcription factor, and that even transient prophase arrest caused by Ndt80 depletion is sufficient to restore meiotic spore viability in checkpoint mutants. Our observations are unexpected given recent reports that the complementary kinase pathway Tel1(ATM) acts to inhibit DSB formation. We propose that such antagonistic regulation of DSB formation by Mec1 and Tel1 creates a regulatory mechanism, where the absolute frequency of DSBs is maintained at a level optimal for genetic exchange and efficient chromosome segregation.

Entropy-driven one-step formation of Phi29 pRNA 3WJ from three RNA fragments.

PubMed

Binzel, Daniel W; Khisamutdinov, Emil F; Guo, Peixuan

2014-04-15

The emerging field of RNA nanotechnology necessitates creation of functional RNA nanoparticles but has been limited by particle instability. It has been shown that the three-way junction of bacteriophage phi29 motor pRNA has unusual stability and can self-assemble from three fragments with high efficiency. It is generally believed that RNA and DNA folding is energy landscape-dependent, and the folding of RNA is driven by enthalpy. Here we examine the thermodynamic characteristics of the 3WJ components as 2'-fluoro RNA, DNA, and RNA. It was seen that the three fragments existed either in 3WJ complex or as monomers, with the intermediate of dimers almost undetectable. It seems that the three fragments can lead to the formation of the 3WJ complex efficiently within a rapid time. A low dissociation constant (apparent KD) of 11.4 nM was determined for RNA, inclusion of 2'-F pyrimidines strengthened the KD to 4.5 nM, and substitution of DNA weakened it to 47.7 nM. The ΔG°37, were -36, -28, and -15 kcal/mol for 3WJ2'-F, 3WJRNA, and 3WJDNA, respectively. It is found that the formation of the three-component complex was governed by entropy, instead of enthalpy, as usually found in RNA complexes. Here entropy-driven is referring to a dominating entropic contribution to the increased stability of the 3WJ(2'-F and 3WJ(RNA) compared to the 3WJ(DNA,) instead of referring to the absolute role or total energy governing 3WJ folding. [corrected].

A Monofunctional Platinum Complex Coordinated to a Rhodium Metalloinsertor Selectively Binds Mismatched DNA in the Minor Groove

PubMed Central

Weidmann, Alyson G.; Barton, Jacqueline K.

2015-01-01

We report the synthesis and characterization of a bimetallic complex derived from a new family of potent and selective metalloinsertors containing an unusual Rh—O axial coordination. This complex incorporates a monofunctional platinum center containing only one labile site for coordination to DNA, rather than two, and coordinates DNA non-classically through adduct formation in the minor groove. This conjugate displays bifunctional, interdependent binding of mismatched DNA via metalloinsertion at a mismatch as well as covalent platinum binding. DNA sequencing experiments revealed that the preferred site of platinum coordination is not the traditional N7-guanine site in the major groove, but rather N3-adenine in the minor groove. The complex also displays enhanced cytotoxicity in mismatch repair-deficient and mismatch repair-proficient human colorectal carcinoma cell lines compared to the chemotherapeutic cisplatin, and triggers cell death via an apoptotic pathway, rather than the necrotic pathway induced by rhodium metalloinsertors. PMID:26397309

Assembly of Slx4 signaling complexes behind DNA replication forks.

PubMed

Balint, Attila; Kim, TaeHyung; Gallo, David; Cussiol, Jose Renato; Bastos de Oliveira, Francisco M; Yimit, Askar; Ou, Jiongwen; Nakato, Ryuichiro; Gurevich, Alexey; Shirahige, Katsuhiko; Smolka, Marcus B; Zhang, Zhaolei; Brown, Grant W

2015-08-13

Obstructions to replication fork progression, referred to collectively as DNA replication stress, challenge genome stability. In Saccharomyces cerevisiae, cells lacking RTT107 or SLX4 show genome instability and sensitivity to DNA replication stress and are defective in the completion of DNA replication during recovery from replication stress. We demonstrate that Slx4 is recruited to chromatin behind stressed replication forks, in a region that is spatially distinct from that occupied by the replication machinery. Slx4 complex formation is nucleated by Mec1 phosphorylation of histone H2A, which is recognized by the constitutive Slx4 binding partner Rtt107. Slx4 is essential for recruiting the Mec1 activator Dpb11 behind stressed replication forks, and Slx4 complexes are important for full activity of Mec1. We propose that Slx4 complexes promote robust checkpoint signaling by Mec1 by stably recruiting Dpb11 within a discrete domain behind the replication fork, during DNA replication stress. © 2015 The Authors.

A monofunctional platinum complex coordinated to a rhodium metalloinsertor selectively binds mismatched DNA in the minor groove.

PubMed

Weidmann, Alyson G; Barton, Jacqueline K

2015-10-05

We report the synthesis and characterization of a bimetallic complex derived from a new family of potent and selective metalloinsertors containing an unusual Rh-O axial coordination. This complex incorporates a monofunctional platinum center containing only one labile site for coordination to DNA, rather than two, and coordinates DNA nonclassically through adduct formation in the minor groove. This conjugate displays bifunctional, interdependent binding of mismatched DNA via metalloinsertion at a mismatch as well as covalent platinum binding. DNA sequencing experiments revealed that the preferred site of platinum coordination is not the traditional N7-guanine site in the major groove, but rather N3-adenine in the minor groove. The complex also displays enhanced cytotoxicity in mismatch repair-deficient and mismatch repair-proficient human colorectal carcinoma cell lines compared to the chemotherapeutic cisplatin, and it triggers cell death via an apoptotic pathway, rather than the necrotic pathway induced by rhodium metalloinsertors.

ITS all right mama: investigating the formation of chimeric sequences in the ITS2 region by DNA metabarcoding analyses of fungal mock communities of different complexities.

PubMed

Bjørnsgaard Aas, Anders; Davey, Marie Louise; Kauserud, Håvard

2017-07-01

The formation of chimeric sequences can create significant methodological bias in PCR-based DNA metabarcoding analyses. During mixed-template amplification of barcoding regions, chimera formation is frequent and well documented. However, profiling of fungal communities typically uses the more variable rDNA region ITS. Due to a larger research community, tools for chimera detection have been developed mainly for the 16S/18S markers. However, these tools are widely applied to the ITS region without verification of their performance. We examined the rate of chimera formation during amplification and 454 sequencing of the ITS2 region from fungal mock communities of different complexities. We evaluated the chimera detecting ability of two common chimera-checking algorithms: perseus and uchime. Large proportions of the chimeras reported were false positives. No false negatives were found in the data set. Verified chimeras accounted for only 0.2% of the total ITS2 reads, which is considerably less than what is typically reported in 16S and 18S metabarcoding analyses. Verified chimeric 'parent sequences' had significantly higher per cent identity to one another than to random members of the mock communities. Community complexity increased the rate of chimera formation. GC content was higher around the verified chimeric break points, potentially facilitating chimera formation through base pair mismatching in the neighbouring regions of high similarity in the chimeric region. We conclude that the hypervariable nature of the ITS region seems to buffer the rate of chimera formation in comparison with other, less variable barcoding regions, due to shorter regions of high sequence similarity. © 2016 John Wiley & Sons Ltd.

DNA condensing effects and sequence selectivity of DNA binding of antitumor noncovalent polynuclear platinum complexes.

PubMed

Malina, Jaroslav; Farrell, Nicholas P; Brabec, Viktor

2014-02-03

The noncovalent analogues of antitumor polynuclear platinum complexes represent a structurally discrete class of platinum drugs. Their chemical and biological properties differ significantly from those of most platinum chemotherapeutics, which bind to DNA in a covalent manner by formation of Pt-DNA adducts. In spite of the fact that these noncovalent polynuclear platinum complexes contain no leaving groups, they have been shown to bind to DNA with high affinity. We report here on the DNA condensation properties of a series of noncovalent analogues of antitumor polynuclear platinum complexes described by biophysical and biochemical methods. The results demonstrate that these polynuclear platinum compounds are capable of inducing DNA condensation at more than 1 order of magnitude lower concentrations than conventional spermine. Atomic force microscopy studies of DNA condensation confined to a mica substrate have revealed that the DNA morphologies become more compact with increasing concentration of the platinum complexes. Moreover, we also found that the noncovalent polynuclear platinum complex [{Pt(NH3)3}2-μ-{trans-Pt(NH3)2(NH2(CH2)6NH2)2}](6+) (TriplatinNC-A) binds to DNA in a sequence-dependent manner, namely, to A/T-rich sequences and A-tract regions, and that noncovalent polynuclear platinum complexes protect DNA from enzymatic cleavage by DNase I. The results suggest that mechanisms of antitumor and cytotoxic activities of these complexes may be associated with their unique ability to condense DNA along with their sequence-specific DNA binding. Owing to their high cellular accumulation, it is also reasonable to suggest that their mechanism of action is based on the competition with naturally occurring DNA condensing agents, such as polyamines spermine, spermidine, and putrescine, for intracellular binding sites, resulting in the disturbance of the correct binding of regulatory proteins initiating the onset of apoptosis.

Mechanism of opening a sliding clamp

PubMed Central

Douma, Lauren G.; Yu, Kevin K.; England, Jennifer K.

2017-01-01

Abstract Clamp loaders load ring-shaped sliding clamps onto DNA where the clamps serve as processivity factors for DNA polymerases. In the first stage of clamp loading, clamp loaders bind and stabilize clamps in an open conformation, and in the second stage, clamp loaders place the open clamps around DNA so that the clamps encircle DNA. Here, the mechanism of the initial clamp opening stage is investigated. Mutations were introduced into the Escherichia coli β-sliding clamp that destabilize the dimer interface to determine whether the formation of an open clamp loader–clamp complex is dependent on spontaneous clamp opening events. In other work, we showed that mutation of a positively charged Arg residue at the β-dimer interface and high NaCl concentrations destabilize the clamp, but neither facilitates the formation of an open clamp loader–clamp complex in experiments presented here. Clamp opening reactions could be fit to a minimal three-step ‘bind-open-lock’ model in which the clamp loader binds a closed clamp, the clamp opens, and subsequent conformational rearrangements ‘lock’ the clamp loader–clamp complex in a stable open conformation. Our results support a model in which the E. coli clamp loader actively opens the β-sliding clamp. PMID:28973453

Catalysis of a 1,3-dipolar reaction by distorted DNA incorporating a heterobimetallic platinum(ii) and copper(ii) complex.

PubMed

Rivilla, Iván; de Cózar, Abel; Schäfer, Thomas; Hernandez, Frank J; Bittner, Alexander M; Eleta-Lopez, Aitziber; Aboudzadeh, Ali; Santos, José I; Miranda, José I; Cossío, Fernando P

2017-10-01

A novel catalytic system based on covalently modified DNA is described. This catalyst promotes 1,3-dipolar reactions between azomethine ylides and maleimides. The catalytic system is based on the distortion of the double helix of DNA by means of the formation of Pt(ii) adducts with guanine units. This distortion, similar to that generated in the interaction of DNA with platinum chemotherapeutic drugs, generates active sites that can accommodate N -metallated azomethine ylides. The proposed reaction mechanism, based on QM(DFT)/MM calculations, is compatible with thermally allowed concerted (but asynchronous) [π4s + π2s] mechanisms leading to the exclusive formation of racemic endo -cycloadducts.

Light amplification and lasing from dyes doped in DNA-complex thin films prepared by soaking method

NASA Astrophysics Data System (ADS)

Kawabe, Yutaka; Suzuki, Takemasa; Iisaka, You

2014-08-01

An alternative fabrication method for dye-doped DNA-surfactant complex films was developed and amplified spontaneous emission (ASE) and lasing under low energy optical pumping were demonstrated. In this new preparation technique, thin DNA-cethyltrimethylammonium (CTMA) complex films made by a spin coating method were stained with a hemicyanine dye by soaking them in acetone solution of the dye for one day. Molar ratio of the dye to DNA base pair for the final products was estimated to be 0.2, the value was much higher than those achieved via usual mixing method. ASE threshold value under pumping of a pulsed frequency-doubled YAG laser was about 0.3 mJ/cm2. Laser emission was also attained under the excitation with two interfering beams forming a dynamic grating of gain coefficient. Durability test indicated that 70% of their initial performance was maintained after 1 hour of continuous pumping. The technique was applied to water soluble dyes because the DNA complex was insoluble to water as well as acetone. We employed anionic Eosin Y dye, succeeding in sample formation and ASE emission. Different types of surfactants were also complexed with DNA, showing variation of emission peak wavelength. These results give a clue about the structure of the complex or interaction modes between DNA and surfactants, strongly suggesting that dye molecules are not intercalated into nor bound to DNA double strand directly, but are incorporated in the complex system via ion-exchange process or aggregating with cationic surfactants.

Structure analysis of FAAP24 reveals single-stranded DNA-binding activity and domain functions in DNA damage response

PubMed Central

Wang, Yucai; Han, Xiao; Wu, Fangming; Leung, Justin W; Lowery, Megan G; Do, Huong; Chen, Junjie; Shi, Chaowei; Tian, Changlin; Li, Lei; Gong, Weimin

2013-01-01

The FANCM/FAAP24 heterodimer has distinct functions in protecting cells from complex DNA lesions such as interstrand crosslinks. These functions rely on the biochemical activity of FANCM/FAAP24 to recognize and bind to damaged DNA or stalled replication forks. However, the DNA-binding activity of this complex was not clearly defined. We investigated how FAAP24 contributes to the DNA-interacting functions of the FANCM/FAAP24 complex by acquiring the N-terminal and C-terminal solution structures of human FAAP24. Modeling of the FAAP24 structure indicates that FAAP24 may possess a high affinity toward single-stranded DNA (ssDNA). Testing of various FAAP24 mutations in vitro and in vivo validated this prediction derived from structural analyses. We found that the DNA-binding and FANCM-interacting functions of FAAP24, although both require the C-terminal (HhH)2 domain, can be distinguished by segregation-of-function mutations. These results demonstrate dual roles of FAAP24 in DNA damage response against crosslinking lesions, one through the formation of FANCM/FAAP24 heterodimer and the other via its ssDNA-binding activity required in optimized checkpoint activation. PMID:23999858

Polyclonal antibodies against a structure mimicking the covalent linkage unit between picornavirus RNA and VPg: an immunochemical study.

PubMed

Ivanova, O A; Venyaminova, A G; Repkova, M N; Drygin, Yu F

2005-09-01

We propose that therapy of patients with anticancer drugs that poison DNA topoisomerases induces formation of covalent complexes of cellular RNAs and DNA topoisomerases. The appearance of these complexes can be detected with antibodies against a synthetic hapten mimicking the covalent linkage unit Tyr-pU(p) of picornavirus RNA and VPg. We synthesized hapten [N(Ac),CO(NH2)]Tyr-(5 P --> O)Up-O-(CH2)6NH2, conjugated it with BSA, and immunized rabbits with the antigen obtained. The raised polyclonal antibodies were purified by successive affinity chromatography on BSA-Sepharose and hapten-Sepharose columns. Target antibodies recognized hapten and encephalomyocarditis virus RNA-VPg complex specifically as found using the dot-immunogold method. We believe that these antibodies might be useful to study mechanism of picorna and similar virus RNA synthesis. The discovery and qualitative determination of the cellular RNA-DNA topoisomerases covalent complexes with these antibodies might be useful to monitor therapy efficacy by drugs "freezing" dead-end complexes of DNA topoisomerases and nucleic acids and to understand the mechanism of DNA topoisomerase poisoning in situ.

DNA Polymerase α Subunit Residues and Interactions Required for Efficient Initiation Complex Formation Identified by a Genetic Selection.

PubMed

Lindow, Janet C; Dohrmann, Paul R; McHenry, Charles S

2015-07-03

Biophysical and structural studies have defined many of the interactions that occur between individual components or subassemblies of the bacterial replicase, DNA polymerase III holoenzyme (Pol III HE). Here, we extended our knowledge of residues and interactions that are important for the first step of the replicase reaction: the ATP-dependent formation of an initiation complex between the Pol III HE and primed DNA. We exploited a genetic selection using a dominant negative variant of the polymerase catalytic subunit that can effectively compete with wild-type Pol III α and form initiation complexes, but cannot elongate. Suppression of the dominant negative phenotype was achieved by secondary mutations that were ineffective in initiation complex formation. The corresponding proteins were purified and characterized. One class of mutant mapped to the PHP domain of Pol III α, ablating interaction with the ϵ proofreading subunit and distorting the polymerase active site in the adjacent polymerase domain. Another class of mutation, found near the C terminus, interfered with τ binding. A third class mapped within the known β-binding domain, decreasing interaction with the β2 processivity factor. Surprisingly, mutations within the β binding domain also ablated interaction with τ, suggesting a larger τ binding site than previously recognized. © 2015 by The American Society for Biochemistry and Molecular Biology, Inc.

Detection of damaged DNA bases by DNA glycosylase enzymes.

PubMed

Friedman, Joshua I; Stivers, James T

2010-06-22

A fundamental and shared process in all forms of life is the use of DNA glycosylase enzymes to excise rare damaged bases from genomic DNA. Without such enzymes, the highly ordered primary sequences of genes would rapidly deteriorate. Recent structural and biophysical studies are beginning to reveal a fascinating multistep mechanism for damaged base detection that begins with short-range sliding of the glycosylase along the DNA chain in a distinct conformation we call the search complex (SC). Sliding is frequently punctuated by the formation of a transient "interrogation" complex (IC) where the enzyme extrahelically inspects both normal and damaged bases in an exosite pocket that is distant from the active site. When normal bases are presented in the exosite, the IC rapidly collapses back to the SC, while a damaged base will efficiently partition forward into the active site to form the catalytically competent excision complex (EC). Here we review the unique problems associated with enzymatic detection of rare damaged DNA bases in the genome and emphasize how each complex must have specific dynamic properties that are tuned to optimize the rate and efficiency of damage site location.

Mapping of interaction domains between human repair proteins ERCC1 and XPF.

PubMed

de Laat, W L; Sijbers, A M; Odijk, H; Jaspers, N G; Hoeijmakers, J H

1998-09-15

ERCC1-XPF is a heterodimeric protein complexinvolved in nucleotide excision repair and recombinational processes. Like its homologous complex in Saccharomyces cerevisiae , Rad10-Rad1, it acts as a structure-specific DNA endonuclease, cleaving at duplex-single-stranded DNA junctions. In repair, ERCC1-XPF and Rad10-Rad1 make an incision on the the 5'-side of the lesion. No humans with a defect in the ERCC1 subunit of this protein complex have been identified and ERCC1-deficient mice suffer from severe developmental problems and signs of premature aging on top of a repair-deficient phenotype. Xeroderma pigmentosum group F patients carry mutations in the XPF subunit and generally show the clinical symptoms of mild DNA repair deficiency. All XP-F patients examined demonstrate reduced levels of XPF and ERCC1 protein, suggesting that proper complex formation is required for stability of the two proteins. To better understand the molecular and clinical consequences of mutations in the ERCC1-XPF complex, we decided to map the interaction domains between the two subunits. The XPF-binding domain comprises C-terminal residues 224-297 of ERCC1. Intriguingly, this domain resides outside the region of homology with its yeast Rad10 counterpart. The ERCC1-binding domain in XPF maps to C-terminal residues 814-905. ERCC1-XPF complex formation is established by a direct interaction between these two binding domains. A mutation from an XP-F patient that alters the ERCC1-binding domain in XPF indeed affects complex formation with ERCC1.

Mapping of interaction domains between human repair proteins ERCC1 and XPF.

PubMed Central

de Laat, W L; Sijbers, A M; Odijk, H; Jaspers, N G; Hoeijmakers, J H

1998-01-01

ERCC1-XPF is a heterodimeric protein complexinvolved in nucleotide excision repair and recombinational processes. Like its homologous complex in Saccharomyces cerevisiae , Rad10-Rad1, it acts as a structure-specific DNA endonuclease, cleaving at duplex-single-stranded DNA junctions. In repair, ERCC1-XPF and Rad10-Rad1 make an incision on the the 5'-side of the lesion. No humans with a defect in the ERCC1 subunit of this protein complex have been identified and ERCC1-deficient mice suffer from severe developmental problems and signs of premature aging on top of a repair-deficient phenotype. Xeroderma pigmentosum group F patients carry mutations in the XPF subunit and generally show the clinical symptoms of mild DNA repair deficiency. All XP-F patients examined demonstrate reduced levels of XPF and ERCC1 protein, suggesting that proper complex formation is required for stability of the two proteins. To better understand the molecular and clinical consequences of mutations in the ERCC1-XPF complex, we decided to map the interaction domains between the two subunits. The XPF-binding domain comprises C-terminal residues 224-297 of ERCC1. Intriguingly, this domain resides outside the region of homology with its yeast Rad10 counterpart. The ERCC1-binding domain in XPF maps to C-terminal residues 814-905. ERCC1-XPF complex formation is established by a direct interaction between these two binding domains. A mutation from an XP-F patient that alters the ERCC1-binding domain in XPF indeed affects complex formation with ERCC1. PMID:9722633

Interactions of tetracationic porphyrins with DNA and their effects on DNA cleavage

NASA Astrophysics Data System (ADS)

Lebedeva, Natalya Sh.; Yurina, Elena S.; Gubarev, Yury A.; Syrbu, Sergey A.

2018-06-01

The interaction of tetracationic porphyrins with DNA was studied using UV-Vis absorption, fluorescence spectroscopy and viscometry, and the particle sizes were determined. Аs cationic porphyrins, two isomer porphyrins, 3,3‧,3″,3‴-(5,10,15,20-Porphyrintetrayl)tetrakis(1-methylpyridinium) (TMPyP3) and 4,4‧,4″,4‴-(5,10,15,20-Porphyrintetrayl)tetrakis(1-methylpyridinium) (TMPyP4), were studied. They differ in the position of NCH3+ group in phenyl ring of the porphyrins and hence, in degree of freedom of rotation of the phenyl rings about the central macrocycle. It was found that intercalated complexes are formed at DNA/porphyrin molar ratios (R) of 2.2 and 3.9 for TMPyP3 и TMPyP4, respectively. Decreasing R up to 0.4 and 0.8 for TMPyP3 и TMPyP4, respectively, leads mainly to formation of outside complexes due to π-π stacking between the porphyrin chromophores interacting electrostatically with phosphate framework of DNA. Each type of the obtained complexes was characterized using Scatchard approach. It was ascertained that the affinity of TMPyP4 to DNA is stronger than TMPyP3, meanwhile the wedge effect of the latter is higher. The differences between the porphyrin isomers become more evident at irradiation of their complexes with DNA. It was established that irradiation of the intercalated complexes results in DNA fragmentation. In the case of TMPyP4, DNA fragments of different size are formed. The irradiation of the outside DNA/porphyrin complexes leads to cleavage of DNA (TMPyP3 and TMPyP4) and partial destruction of the complex due to photolysis of the porphyrin (TMPyP3).

Destabilization of the PCNA trimer mediated by its interaction with the NEIL1 DNA glycosylase

DOE Office of Scientific and Technical Information (OSTI.GOV)

Prakash, Aishwarya; Moharana, Kedar; Wallace, Susan S.

The base excision repair (BER) pathway repairs oxidized lesions in the DNA that result from reactive oxygen species generated in cells. If left unrepaired, these damaged DNA bases can disrupt cellular processes such as replication. NEIL1 is one of the 11 human DNA glycosylases that catalyze the first step of the BER pathway, i.e. recognition and excision of DNA lesions. NEIL1 interacts with essential replication proteins such as the ring-shaped homotrimeric proliferating cellular nuclear antigen (PCNA). We isolated a complex formed between NEIL1 and PCNA (±DNA) using size exclusion chromatography (SEC). This interaction was confirmed using native gel electrophoresis andmore » mass spectrometry. Stokes radii measured by SEC hinted that PCNA in complex with NEIL1 (±DNA) was no longer a trimer. Height measurements and images obtained by atomic force microscopy also demonstrated the dissociation of the PCNA homotrimer in the presence of NEIL1 and DNA, while small-angle X-ray scattering analysis confirmed the NEIL1 mediated PCNA trimer dissociation and formation of a 1:1:1 NEIL1-DNA-PCNA(monomer) complex. Furthermore, ab initio shape reconstruction provides insights into the solution structure of this previously unreported complex. Together, these data point to a potential mechanistic switch between replication and BER.« less

Adsorption of Nucleic Acid/Protein Supramolecular Complexes on Goethite: The Influence of Solution Interactions on Behavior at the Solution-Mineral Interface

NASA Astrophysics Data System (ADS)

Schmidt, M.; Martinez, C. E.

2017-12-01

Adsorption of biomolecule rich supramolecular complexes onto mineral surfaces plays an important role in the development of organo-mineral associations in soils. In this study, a series of supramolecular complexes of a model nucleic acid (deoxyribonucleic acid (DNA)) and protein (bovine serum albumin (BSA)) are synthesized, characterized and exposed to goethite to probe their adsorption behavior. To synthesize DNA/BSA complexes, a fixed DNA concentration (0.1 mg/mL) was mixed with a range of BSA concentrations (0.025-0.5 mg/mL) in 5 mM KCl at pH=5.0. Circular dichroism spectroscopy demonstrates strong, cooperative, Hill-type binding between DNA and BSA (Ka= 4.74 x 105 M-1) with DNA saturation achieved when BSA concentration reaches 0.4 mg/mL. Dynamic light scattering measurements of DNA/BSA complexes suggest binding accompanies disruption of DNA-DNA intermolecular electrostatic repulsion, resulting in a decrease of the DNA slow relaxation mode with increasing amount of BSA. Zeta potential measurements show increasing amounts of BSA lead to a reduction of negative charge on DNA/BSA complexes, in line with light scattering results. In situ attenuated total reflectance Fourier transform infrared spectroscopic studies of adsorption of DNA/BSA complexes onto goethite show that complexation of BSA with DNA appears to hinder direct coordination of DNA backbone phosphodiester groups with goethite, relative to DNA by itself. Furthermore, increasing amount of BSA (up to 0.4 mg/mL) in DNA/BSA complexes enhances DNA adsorption, possibly as a result of reduced repulsion between adsorbed DNA helices. When BSA concentration exceeds 0.4 mg/mL, a decrease in adsorbed DNA is observed. We hypothesize that this discrepancy in behavior between systems with BSA concentrations below and above saturation of DNA is caused by initial fast adsorption of loosely associated BSA on goethite, restricting access to goethite surface sites. Overall, these results highlight the impact of solution interaction between biomolecules on subsequent behavior at mineral surfaces. This work represents a bridge between model experiments with individual biomolecules and more complex natural systems, yielding a fundamental viewpoint of the formation of organo-mineral associations in soils.

EM structure of a helicase-loader complex depicting a 6:2 binding sub-stoichiometry from Geobacillus kaustophilus HTA426

DOE Office of Scientific and Technical Information (OSTI.GOV)

Lin, Yen-Chen; Naveen, Vankadari; Molecular Cell Biology, Taiwan International Graduate Program, Institute of Molecular Biology, Academia Sinica, and Graduate Institute of Life Sciences, National Defense Medical Center, Taipei, Taiwan

During DNA replication, bacterial helicase is recruited as a complex in association with loader proteins to unwind the parental duplex. Previous structural studies have reported saturated 6:6 helicase-loader complexes with different conformations. However, structural information on the sub-stoichiometric conformations of these previously-documented helicase-loader complexes remains elusive. Here, with the aid of single particle electron-microscopy (EM) image reconstruction, we present the Geobacillus kaustophilus HTA426 helicase-loader (DnaC-DnaI) complex with a 6:2 binding stoichiometry in the presence of ATPγS. In the 19 Å resolution EM map, the undistorted and unopened helicase ring holds a robust loader density above the C-terminal RecA-like domain. Meanwhile, themore » path of the central DNA binding channel appears to be obstructed by the reconstructed loader density, implying its potential role as a checkpoint conformation to prevent the loading of immature complex onto DNA. Our data also reveals that the bound nucleotides and the consequently induced conformational changes in the helicase hexamer are essential for active association with loader proteins. These observations provide fundamental insights into the formation of the helicase-loader complex in bacteria that regulates the DNA replication process. - Highlights: • Helicase-loader complex structure with 6:2 sub-stoichiometry is resolved by EM. • Helicase hexamer in 6:2 sub-stoichiometry is constricted and un-opened. • 6:2 binding ratio of helicase-loader complex could act as a DNA loading checkpoint. • Nucleotides stabilize helicase-loader complex at low protein concentrations.« less

Rapid colorimetric assay for detection of Listeria monocytogenes in food samples using LAMP formation of DNA concatemers and gold nanoparticle-DNA probe complex

NASA Astrophysics Data System (ADS)

Wachiralurpan, Sirirat; Sriyapai, Thayat; Areekit, Supatra; Sriyapai, Pichapak; Augkarawaritsawong, Suphitcha; Santiwatanakul, Somchai; Chansiri, Kosum

2018-04-01

ABSTRACT Listeria monocytogenes is a major foodborne pathogen of global health concern. Herein, the rapid diagnosis of L. monocytogenes has been achieved using loop-mediated isothermal amplification (LAMP) based on the phosphatidylcholine-phospholipase C gene (plcB). Colorimetric detection was then performed through the formation of DNA concatemers and a gold nanoparticle/DNA probe complex (GNP/DNA probe). The overall detection process was accomplished within approximately 1 h with no need for complicated equipment. The limits of detection for L. monocytogenes in the forms of purified genomic DNA and pure culture were 800 fg and 2.82 CFU mL-1, respectively. No cross reactions were observed from closely related bacteria species. The LAMP-GNP/DNA probe assay was applied to the detection of 200 raw chicken meat samples and compared to routine standard methods. The data revealed that the specificity, sensitivity and accuracy were 100%, 90.20% and 97.50%, respectively. The present assay was 100% in conformity with LAMP-agarose gel electrophoresis assay. Five samples that were negative by both assays appeared to have the pathogen at below the level of detection. The assay can be applied as a rapid direct screening method for L. monocytogenes.

[Single-molecule detection and characterization of DNA replication based on DNA origami].

PubMed

Wang, Qi; Fan, Youjie; Li, Bin

2014-08-01

To investigate single-molecule detection and characterization of DNA replication. Single-stranded DNA (ssDNA) as the template of DNA replication was attached to DNA origami by a hybridization reaction based on the complementary base-pairing principle. DNA replication catalyzed by E.coli DNA polymerase I Klenow Fragment (KF) was detected using atomic force microscopy (AFM). The height variations between the ssDNA and the double-stranded DNA (dsDNA), the distribution of KF during DNA replication and biotin-streptavidin (BA) complexes on the DNA strand after replication were detected. Agarose gel electrophoresis was employed to analyze the changes in the DNA after replication. The designed ssDNA could be anchored on the target positions of over 50% of the DNA origami. The KF was capable of binding to the ssDNA fixed on DNA origami and performing its catalytic activities, and was finally dissociated from the DNA after replication. The height of DNA strand increased by about 0.7 nm after replication. The addition of streptavidin also resulted in an DNA height increase to about 4.9 nm due to the formation of BA complexes on the biotinylated dsDNA. The resulting dsDNA and BA complex were subsequently confirmed by agarose gel electrophoresis. The combination of AFM and DNA origami allows detection and characterization of DNA replication at the single molecule level, and this approach provides better insights into the mechanism of DNA polymerase and the factors affecting DNA replication.

Efficient interrupting skills of amino acid metallointercalators with DNA at physiological pH: Evaluation of biological assays

NASA Astrophysics Data System (ADS)

Raman, Natarajan; Selvaganapathy, Muthusamy; Radhakrishnan, Srinivasan

2014-06-01

The 4-aminoantipyrine derivatives (sbnd NO2, sbnd OCH3) and their mixed-ligand complexes with amino acids have been synthesized and investigated for their binding with CT DNA using UV-visible spectroscopy, cyclic voltammetry, and viscosity measurements under physiological conditions of pH (stomach 4.7; blood 7.4). The results from all techniques i.e. binding constant (Kb), and free energy change (ΔG) were in good agreement and inferred spontaneous compound-DNA complexes formation via intercalation. Among all the compounds 1 and 4 showed comparatively greater binding at pH 7.4 as evident from its greater Kb values. All the complexes exhibit oxidative cleavage of supercoiled (SC) pBR322 plasmid DNA in the presence of H2O2 as an activator. It is remarkable that at 25 μM concentration 1 and 4 completely degrade SC DNA into undetectable minor fragments and thus they act as efficient chemical nucleases. Among the new complexes, complexes 1 and 4 have highest potential against all the microorganisms tested. The results of the above biological experiments also reveal that the choice of different metal ions has little influence on the DNA binding, DNA cleavage and antimicrobial assay.

DNA-lipid complexes: stability of honeycomb-like and spaghetti-like structures.

PubMed Central

May, S; Ben-Shaul, A

1997-01-01

A molecular level theory is presented for the thermodynamic stability of two (similar) types of structural complexes formed by (either single strand or supercoiled) DNA and cationic liposomes, both involving a monolayer-coated DNA as the central structural unit. In the "spaghetti" complex the central unit is surrounded by another, oppositely curved, monolayer, thus forming a bilayer mantle. The "honeycomb" complex is a bundle of hexagonally packed DNA-monolayer units. The formation free energy of these complexes, starting from a planar cationic/neutral lipid bilayer and bare DNA, is expressed as a sum of electrostatic, bending, mixing, and (for the honeycomb) chain frustration contributions. The electrostatic free energy is calculated using the Poisson-Boltzmann equation. The bending energy of the mixed lipid layers is treated in the quadratic curvature approximation with composition-dependent bending rigidity and spontaneous curvature. Ideal lipid mixing is assumed within each lipid monolayer. We found that the most stable monolayer-coated DNA units are formed when the charged/neutral lipid composition corresponds (nearly) to charge neutralization; the optimal monolayer radius corresponds to close DNA-monolayer contact. These conclusions are also valid for the honeycomb complex, as the chain frustration energy is found to be negligible. Typically, the stabilization energies for these structures are on the order of 1 k(B)T/A of DNA length, reflecting mainly the balance between the electrostatic and bending energies. The spaghetti complexes are less stable due to the additional bending energy of the external monolayer. A thermodynamic analysis is presented for calculating the equilibrium lipid compositions when the complexes coexist with excess bilayer. PMID:9370436

Gene Transfer in Eukaryotic Cells Using Activated Dendrimers

NASA Astrophysics Data System (ADS)

Dennig, Jörg

Gene transfer into eukaryotic cells plays an important role in cell biology. Over the last 30 years a number of transfection methods have been developed to mediate gene transfer into eukaryotic cells. Classical methods include co-precipitation of DNA with calcium phosphate, charge-dependent precipitation of DNA with DEAE-dextran, electroporation of nucleic acids, and formation of transfection complexes between DNA and cationic liposomes. Gene transfer technologies based on activated PAMAM-dendrimers provide another class of transfection reagents. PAMAM-dendrimers are highly branched, spherical molecules. Activation of newly synthesized dendrimers involves hydrolytic removal of some of the branches, and results in a molecule with a higher degree of flexibility. Activated dendrimers assemble DNA into compact structures via charge interactions. Activated dendrimer - DNA complexes bind to the cell membrane of eukaryotic cells, and are transported into the cell by non-specific endocytosis. A structural model of the activated dendrimer - DNA complex and a potential mechanism for its uptake into cells will be discussed.

Transfer Hydrogenation and Antiproliferative Activity of Tethered Half-Sandwich Organoruthenium Catalysts

PubMed Central

2018-01-01

We report the synthesis and characterization of four neutral organometallic tethered complexes, [Ru(η6-Ph(CH2)3-ethylenediamine-N-R)Cl], where R = methanesulfonyl (Ms, 1), toluenesulfonyl (Ts, 2), 4-trifluoromethylbenzenesulfonyl (Tf, 3), and 4-nitrobenzenesulfonyl (Nb, 4), including their X-ray crystal structures. These complexes exhibit moderate antiproliferative activity toward human ovarian, lung, hepatocellular, and breast cancer cell lines. Complex 2 in particular exhibits a low cross-resistance with cisplatin. The complexes show potent catalytic activity in the transfer hydrogenation of NAD+ to NADH with formate as hydride donor in aqueous solution (310 K, pH 7). Substituents on the chelated ligand decreased the turnover frequency in the order Nb > Tf > Ts > Ms. An enhancement of antiproliferative activity (up to 22%) was observed on coadministration with nontoxic concentrations of sodium formate (0.5–2 mM). Complex 2 binds to nucleobase guanine (9-EtG), but DNA appears not to be the target, as little binding to calf thymus DNA or bacterial plasmid DNA was observed. In addition, complex 2 reacts rapidly with glutathione (GSH), which might hamper transfer hydrogenation reactions in cells. Complex 2 induced a dose-dependent G1 cell cycle arrest after 24 h exposure in A2780 human ovarian cancer cells while promoting an increase in reactive oxygen species (ROS), which is likely to contribute to its antiproliferative activity.

Histone H3 phosphorylation near the nucleosome dyad alters chromatin structure

PubMed Central

North, Justin A.; Šimon, Marek; Ferdinand, Michelle B.; Shoffner, Matthew A.; Picking, Jonathan W.; Howard, Cecil J.; Mooney, Alex M.; van Noort, John; Poirier, Michael G.; Ottesen, Jennifer J.

2014-01-01

Nucleosomes contain ∼146 bp of DNA wrapped around a histone protein octamer that controls DNA accessibility to transcription and repair complexes. Posttranslational modification (PTM) of histone proteins regulates nucleosome function. To date, only modest changes in nucleosome structure have been directly attributed to histone PTMs. Histone residue H3(T118) is located near the nucleosome dyad and can be phosphorylated. This PTM destabilizes nucleosomes and is implicated in the regulation of transcription and repair. Here, we report gel electrophoretic mobility, sucrose gradient sedimentation, thermal disassembly, micrococcal nuclease digestion and atomic force microscopy measurements of two DNA–histone complexes that are structurally distinct from nucleosomes. We find that H3(T118ph) facilitates the formation of a nucleosome duplex with two DNA molecules wrapped around two histone octamers, and an altosome complex that contains one DNA molecule wrapped around two histone octamers. The nucleosome duplex complex forms within short ∼150 bp DNA molecules, whereas altosomes require at least ∼250 bp of DNA and form repeatedly along 3000 bp DNA molecules. These results are the first report of a histone PTM significantly altering the nucleosome structure. PMID:24561803

Functional requirements of AID's higher order structures and their interaction with RNA-binding proteins.

PubMed

Mondal, Samiran; Begum, Nasim A; Hu, Wenjun; Honjo, Tasuku

2016-03-15

Activation-induced cytidine deaminase (AID) is essential for the somatic hypermutation (SHM) and class-switch recombination (CSR) of Ig genes. Although both the N and C termini of AID have unique functions in DNA cleavage and recombination, respectively, during SHM and CSR, their molecular mechanisms are poorly understood. Using a bimolecular fluorescence complementation (BiFC) assay combined with glycerol gradient fractionation, we revealed that the AID C terminus is required for a stable dimer formation. Furthermore, AID monomers and dimers form complexes with distinct heterogeneous nuclear ribonucleoproteins (hnRNPs). AID monomers associate with DNA cleavage cofactor hnRNP K whereas AID dimers associate with recombination cofactors hnRNP L, hnRNP U, and Serpine mRNA-binding protein 1. All of these AID/ribonucleoprotein associations are RNA-dependent. We propose that AID's structure-specific cofactor complex formations differentially contribute to its DNA-cleavage and recombination functions.

Functional requirements of AID’s higher order structures and their interaction with RNA-binding proteins

PubMed Central

Mondal, Samiran; Begum, Nasim A.; Hu, Wenjun; Honjo, Tasuku

2016-01-01

Activation-induced cytidine deaminase (AID) is essential for the somatic hypermutation (SHM) and class-switch recombination (CSR) of Ig genes. Although both the N and C termini of AID have unique functions in DNA cleavage and recombination, respectively, during SHM and CSR, their molecular mechanisms are poorly understood. Using a bimolecular fluorescence complementation (BiFC) assay combined with glycerol gradient fractionation, we revealed that the AID C terminus is required for a stable dimer formation. Furthermore, AID monomers and dimers form complexes with distinct heterogeneous nuclear ribonucleoproteins (hnRNPs). AID monomers associate with DNA cleavage cofactor hnRNP K whereas AID dimers associate with recombination cofactors hnRNP L, hnRNP U, and Serpine mRNA-binding protein 1. All of these AID/ribonucleoprotein associations are RNA-dependent. We propose that AID’s structure-specific cofactor complex formations differentially contribute to its DNA-cleavage and recombination functions. PMID:26929374

Perspectives of ruthenium(ii) polyazaaromatic photo-oxidizing complexes photoreactive towards tryptophan-containing peptides and derivatives.

PubMed

Estalayo-Adrián, S; Garnir, K; Moucheron, C

2018-01-04

Ru II polyazaaromatic complexes have been studied with the aim of developing molecular tools for DNA and oligonucleotides. In this context, Ru II -TAP (TAP = 1,4,5,8-tetraazaphenanthrene) complexes have been developed as specific photoreagents targeting the genetic material. The advantage of such compounds is due to the formation of photo-addition products between the Ru-TAP complex and the biomolecule, originating from a photo-induced electron transfer process that takes place between the excited Ru-TAP complex and guanine (G) bases of DNA. This photo-addition has been more recently extended to amino acids in view of applications involving peptides, such as inhibition or photocontrol of proteins. More particularly, tryptophan (Trp) and Trp-containing peptides are also able to be photo-oxidized by Ru II -TAP complexes, leading to the formation of photo-addition products. This mini review focuses on recent advances in the search for Ru II polyazaaromatic photo-oxidizing complexes of interest as molecular tools and photoreagents for Trp-containing peptides and proteins. Different possible future directions in this field are also discussed.

Eukaryotic ribosome display with in situ DNA recovery.

PubMed

He, Mingyue; Edwards, Bryan M; Kastelic, Damjana; Taussig, Michael J

2012-01-01

Ribosome display is a cell-free display technology for in vitro selection and optimisation of proteins from large diversified libraries. It operates through the formation of stable protein-ribosome-mRNA (PRM) complexes and selection of ligand-binding proteins, followed by DNA recovery from the selected genetic information. Both prokaryotic and eukaryotic ribosome display systems have been developed. In this chapter, we describe the eukaryotic rabbit reticulocyte method in which a distinct in situ single-primer RT-PCR procedure is used to recover DNA from the selected PRM complexes without the need for prior disruption of the ribosome.

Re-wiring of energy metabolism promotes viability during hyperreplication stress in E. coli

PubMed Central

Campion, Christopher; Weimann, Allan

2017-01-01

Chromosome replication in Escherichia coli is initiated by DnaA. DnaA binds ATP which is essential for formation of a DnaA-oriC nucleoprotein complex that promotes strand opening, helicase loading and replisome assembly. Following initiation, DnaAATP is converted to DnaAADP primarily by the Regulatory Inactivation of DnaA process (RIDA). In RIDA deficient cells, DnaAATP accumulates leading to uncontrolled initiation of replication and cell death by accumulation of DNA strand breaks. Mutations that suppress RIDA deficiency either dampen overinitiation or permit growth despite overinitiation. We characterize mutations of the last group that have in common that distinct metabolic routes are rewired resulting in the redirection of electron flow towards the cytochrome bd-1. We propose a model where cytochrome bd-1 lowers the formation of reactive oxygen species and hence oxidative damage to the DNA in general. This increases the processivity of replication forks generated by overinitiation to a level that sustains viability. PMID:28129339

Double-strand DNA-templated formation of copper nanoparticles as fluorescent probe for label-free aptamer sensor.

PubMed

Zhou, Zhixue; Du, Yan; Dong, Shaojun

2011-07-01

Double-strand DNA (dsDNA) can act as an efficient template for the formation of copper nanoparticles (Cu NPs) at low concentration of CuSO(4), and the formed Cu NPs have excellent fluorescence, whereas a single-strand DNA (ssDNA) template does not support Cu NPs' formation. This property of dsDNA-Cu NPs makes it suitable for DNA sensing. However, exploration of dsDNA-Cu NPs applied in biological analysis is still at an early stage. In this regard, we report herein for the first time a sensitive, cost-effective, and simple aptamer sensor (aptasensor) using dsDNA-Cu NPs as fluorescent probe. The design consists of a dsDNA with reporter DNA (here, aptamer) as template for the formation of Cu NPs, and the formed dsDNA-Cu NPs show high fluorescence. Using adenosine triphosphate (ATP) as a model analyte, the introduction of ATP triggers the structure switching of reporter DNA to form aptamer-ATP complex, causing the destruction of the double helix and thus no formation of the Cu NPs, resulting in low fluorescence. The preferable linear range (0.05-500 μM), sensitivity (LOD 28 nM), and simplicity for the detection of ATP indicate that dsDNA-Cu NPs may have great prospects in the field of biological analysis. We also use this novel fluorescent probe to determine ATP in 1% human serum and human adenocarcinoma HeLa cells. The dsDNA-Cu NPs probes provide recovery of 104-108% in 1% human serum and a prominent fluorescent signal is obtained in cellular ATP assay, revealing the practicality of using dsDNA-Cu NPs for the determination of ATP in real samples. Besides, this design is simply based on nucleic acid hybridization, so it can be generally applied to other aptamers for label-free detection of a broad range of analytes. Successful detection of cocaine with detection limit of 0.1 μM demonstrates its potential to be a general method.

Elongator complex is critical for cell cycle progression and leaf patterning in Arabidopsis.

PubMed

Xu, Deyang; Huang, Weihua; Li, Yang; Wang, Hua; Huang, Hai; Cui, Xiaofeng

2012-03-01

The mitotic cell cycle in higher eukaryotes is of pivotal importance for organ growth and development. Here, we report that Elongator, an evolutionarily conserved histone acetyltransferase complex, acts as an important regulator of mitotic cell cycle to promote leaf patterning in Arabidopsis. Mutations in genes encoding Elongator subunits resulted in aberrant cell cycle progression, and the altered cell division affects leaf polarity formation. The defective cell cycle progression is caused by aberrant DNA replication and increased DNA damage, which activate the DNA replication checkpoint to arrest the cell cycle. Elongator interacts with proliferating cell nuclear antigen (PCNA) and is required for efficient histone 3 (H3) and H4 acetylation coupled with DNA replication. Levels of chromatin-bound H3K56Ac and H4K5Ac known to associate with replicons during DNA replication were reduced in the mutants of both Elongator and chromatin assembly factor 1 (CAF-1), another protein complex that physically interacts with PCNA for DNA replication-coupled chromatin assembly. Disruptions of CAF-1 also led to severe leaf polarity defects, which indicated that Elongator and CAF-1 act, at least partially, in the same pathway to promote cell cycle progression. Collectively, our results demonstrate that Elongator is an important regulator of mitotic cell cycle, and the Elongator pathway plays critical roles in promoting leaf polarity formation. © 2011 The Authors. The Plant Journal © 2011 Blackwell Publishing Ltd.

Analysis of DNA interactions using single-molecule force spectroscopy.

PubMed

Ritzefeld, Markus; Walhorn, Volker; Anselmetti, Dario; Sewald, Norbert

2013-06-01

Protein-DNA interactions are involved in many biochemical pathways and determine the fate of the corresponding cell. Qualitative and quantitative investigations on these recognition and binding processes are of key importance for an improved understanding of biochemical processes and also for systems biology. This review article focusses on atomic force microscopy (AFM)-based single-molecule force spectroscopy and its application to the quantification of forces and binding mechanisms that lead to the formation of protein-DNA complexes. AFM and dynamic force spectroscopy are exciting tools that allow for quantitative analysis of biomolecular interactions. Besides an overview on the method and the most important immobilization approaches, the physical basics of the data evaluation is described. Recent applications of AFM-based force spectroscopy to investigate DNA intercalation, complexes involving DNA aptamers and peptide- and protein-DNA interactions are given.

Genetic variations in the DNA replication origins of human papillomavirus family correlate with their oncogenic potential.

PubMed

Yilmaz, Gulden; Biswas-Fiss, Esther E; Biswas, Subhasis B

2018-04-01

Human papillomaviruses (HPVs) encompass a large family of viruses that range from benign to highly carcinogenic. The crucial differences between benign and carcinogenic types of HPV remain unknown, except that the two HPV types differ in the frequency of DNA replication. We have systematically analyzed the mechanism of HPV DNA replication initiation in low-risk and high-risk HPVs. Our results demonstrate that HPV-encoded E2 initiator protein and its four binding sites in the replication origin play pivotal roles in determining the destiny of the HPV-infected cell. We have identified strain-specific single nucleotide variations in E2 binding sites found only in the high-risk HPVs. We have demonstrated that these variations result in attenuated formation of the E2-DNA complex. E2 binding to these sites is linked to the activation of the DNA replication origin as well as initiation of DNA replication. Both electrophoretic mobility shift assay and atomic force microscopy studies demonstrated that binding of E2 from either low- or high-risk HPVs with variant binding sequences lacked multimeric E2-DNA complex formation in vitro. These results provided a molecular basis of differential DNA replication in the two types of HPVs and pointed to a correlation with the development of cancer. Copyright © 2017. Published by Elsevier B.V.

Study of DNA-emodin interaction by FTIR and UV-vis spectroscopy.

PubMed

Saito, Samuel T; Silva, Givaldo; Pungartnik, Cristina; Brendel, Martin

2012-06-04

Emodin, a plant- and fungus-derived anthraquinone, exerts genotoxic and antioxidative effects and shows promise in antitumor and antibacterial therapies. The aim of this study was to examine the molecular interactions of emodin with DNA in aqueous solution at physiological pH using spectroscopic methods. Fourier Transform Infrared (FTIR) Spectroscopy and UV absorption spectra were used to determine the structural features, the binding mode and the association constants. Our UV-spectroscopic results indicate that emodin interacts with DNA by intercalation and by external binding. FTIR results suggest that emodin interaction occurs preferably via adenine and thymine base pairs and also weakly with the phosphate backbone of the DNA double helix. The binding constant for emodin-DNA complex formation is estimated to be K=5.59×10(3)M(-1). No significant changes of DNA conformation were observed upon emodin-DNA complexation. Copyright © 2012 Elsevier B.V. All rights reserved.

Electrostatically assembled dendrimer complex with a high-affinity protein binder for targeted gene delivery.

PubMed

Kim, Jong-Won; Lee, Joong-Jae; Choi, Joon Sig; Kim, Hak-Sung

2018-06-10

Although a variety of non-viral gene delivery systems have been developed, they still suffer from low efficiency and specificity. Herein, we present the assembly of a dendrimer complex comprising a DNA cargo and a targeting moiety as a new format for targeted gene delivery. A PAMAM dendrimer modified with histidine and arginine (HR-dendrimer) was used to enhance the endosomal escape and transfection efficiency. An EGFR-specific repebody, composed of leucine-rich repeat (LRR) modules, was employed as a targeting moiety. A polyanionic peptide was genetically fused to the repebody, followed by incubation with an HR-dendrimer and a DNA cargo to assemble the dendrimer complex through an electrostatic interaction. The resulting dendrimer complex was shown to deliver a DNA cargo with high efficiency in a receptor-specific manner. An analysis using a confocal microscope confirmed the internalization of the dendrimer complex and subsequent dissociation of a DNA cargo from the complex. The present approach can be broadly used in a targeted gene delivery in many areas. Copyright © 2018 Elsevier B.V. All rights reserved.

On the binding of indeno[1,2-c]isoquinolines in the DNA-topoisomerase I cleavage complex.

PubMed

Xiao, Xiangshu; Antony, Smitha; Pommier, Yves; Cushman, Mark

2005-05-05

An ab initio quantum mechanics calculation is reported which predicts the orientation of indenoisoquinoline 4 in the ternary cleavage complex formed from DNA and topoisomerase I (top1). The results of this calculation are consistent with the hypothetical structures previously proposed for the indenoisoquinoline-DNA-top1 ternary complexes based on molecular modeling, the crystal structure of a recently reported ternary complex, and the biological results obtained with a pair of diaminoalkyl-substituted indenoisoquinoline enantiomers. The results of these studies indicate that the pi-pi stacking interactions between the indenoisoquinolines and the neighboring DNA base pairs play a major role in determining binding orientation. The calculation of the electrostatic potential surface maps of the indenoisoquinolines and the adjacent DNA base pairs shows electrostatic complementarity in the observed binding orientation, leading to the conclusion that electrostatic attraction between the intercalators and the base pairs in the cleavage complex plays a major stabilizing role. On the other hand, the calculation of LUMO and HOMO energies of indenoisoquinoline 13b and neighboring DNA base pairs in conjunction with NBO analysis indicates that charge transfer complex formation plays a relatively minor role in stabilizing the ternary complexes derived from indenoisoquinolines, DNA, and top1. The results of these studies are important in understanding the existing structure-activity relationships for the indenoisoquinolines as top1 inhibitors and as anticancer agents, and they will be important in the future design of indenoisoquinoline-based top1 inhibitors.

AgI -Induced Switching of DNA Binding Modes via Formation of a Supramolecular Metallacycle.

PubMed

Basak, Shibaji; Léon, J Christian; Ferranco, Annaleizle; Sharma, Renu; Hebenbrock, Marian; Lough, Alan; Müller, Jens; Kraatz, Heinz-Bernhard

2018-03-12

The histidine derivative L1 of the DNA intercalator naphthalenediimide (NDI) forms a triangular Ag I complex (C2). The interactions of L1 and of C2 with DNA were studied by circular dichroism (CD) and UV/Vis spectroscopy and by viscosity studies. Different binding modes were observed for L1 and for C2, as the Ag I complex C2 is too large in size to act as an intercalator. If Ag I is added to the NDI molecule that is already intercalated into a duplex, higher order complexes are formed within the DNA duplex and cause disruptions in the helical duplex structure, which leads to a significant decrease in the characteristic CD features of B-DNA. Thus, via addition of a metal we show how a classic and well-known organic intercalator unit can be turned into a partial metallo insertor. We also show how electrochemical impedance spectroscopy (EIS) can be used to probe DNA binding modes on DNA films that are immobilized on gold surfaces. © 2018 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim.

Sequential and ordered assembly of a large DNA repair complex on undamaged chromatin

PubMed Central

Ziani, Salim; Nagy, Zita; Alekseev, Sergey; Soutoglou, Evi; Egly, Jean-Marc

2014-01-01

In nucleotide excision repair (NER), damage recognition by XPC-hHR23b is described as a critical step in the formation of the preincision complex (PInC) further composed of TFIIH, XPA, RPA, XPG, and ERCC1-XPF. To obtain new molecular insights into the assembly of the PInC, we analyzed its formation independently of DNA damage by using the lactose operator/repressor reporter system. We observed a sequential and ordered self-assembly of the PInC operating upon immobilization of individual NER factors on undamaged chromatin and mimicking that functioning on a bona fide NER substrate. We also revealed that the recruitment of the TFIIH subunit TTDA, involved in trichothiodystrophy group A disorder (TTD-A), was key in the completion of the PInC. TTDA recruits XPA through its first 15 amino acids, depleted in some TTD-A patients. More generally, these results show that proteins forming large nuclear complexes can be recruited sequentially on chromatin in the absence of their natural DNA target and with no reciprocity in their recruitment. PMID:25154395

Study of intermolecular contacts in the proline-rich homeodomain (PRH)-DNA complex using molecular dynamics simulations.

PubMed

Jalili, Seifollah; Karami, Leila

2012-03-01

The proline-rich homeodomain (PRH)-DNA complex consists of a protein with 60 residues and a 13-base-pair DNA. The PRH protein is a transcription factor that plays a key role in the regulation of gene expression. PRH is a significant member of the Q50 class of homeodomain proteins. The homeodomain section of PRH is essential for binding to DNA and mediates sequence-specific DNA binding. Three 20-ns molecular dynamics (MD) simulations (free protein, free DNA and protein-DNA complex) in explicit solvent water were performed to elucidate the intermolecular contacts in the PRH-DNA complex and the role of dynamics of water molecules forming water-mediated contacts. The simulation provides a detailed explanation of the trajectory of hydration water molecules. The simulations show that some water molecules in the protein-DNA interface exchange with bulk waters. The simulation identifies that most of the contacts consisted of direct interactions between the protein and DNA including specific and non-specific contacts, but several water-mediated polar contacts were also observed. The specific interaction between Gln50 and C18 and water-mediated hydrogen bond between Gln50 and T7 were found to be present during almost the entire time of the simulation. These results show good consistency with experimental and previous computational studies. Structural properties such as root-mean-square deviations (RMSD), root-mean-square fluctuations (RMSF) and secondary structure were also analyzed as a function of time. Analyses of the trajectories showed that the dynamic fluctuations of both the protein and the DNA were lowered by the complex formation.

The mechanism of a nuclear pore assembly: a molecular biophysics view.

PubMed

Kuvichkin, Vasily V

2011-06-01

The basic problem of nuclear pore assembly is the big perinuclear space that must be overcome for nuclear membrane fusion and pore creation. Our investigations of ternary complexes: DNA-PC liposomes-Mg²⁺, and modern conceptions of nuclear pore structure allowed us to introduce a new mechanism of nuclear pore assembly. DNA-induced fusion of liposomes (membrane vesicles) with a single-lipid bilayer or two closely located nuclear membranes is considered. After such fusion on the lipid bilayer surface, traces of a complex of ssDNA with lipids were revealed. At fusion of two identical small liposomes (membrane vesicles) < 100 nm in diameter, a "big" liposome (vesicle) with ssDNA on the vesicle equator is formed. ssDNA occurrence on liposome surface gives a biphasic character to the fusion kinetics. The "big" membrane vesicle surrounded by ssDNA is the base of nuclear pore assembly. Its contact with the nuclear envelope leads to fast fusion of half of the vesicles with one nuclear membrane; then ensues a fusion delay when ssDNA reaches the membrane. The next step is to turn inside out the second vesicle half and its fusion to other nuclear membrane. A hole is formed between the two membranes, and nucleoporins begin pore complex assembly around the ssDNA. The surface tension of vesicles and nuclear membranes along with the kinetic energy of a liquid inside a vesicle play the main roles in this process. Special cases of nuclear pore formation are considered: pore formation on both nuclear envelope sides, the difference of pores formed in various cell-cycle phases and linear nuclear pore clusters.

Energetics of drug-DNA interactions.

PubMed

Chaires, J B

1997-01-01

Understanding the thermodynamics of drug binding to DNA is of both practical and fundamental interest. The practical interest lies in the contribution that thermodynamics can make to the rational design process for the development of new DNA targeted drugs. Thermodynamics offer key insights into the molecular forces that drive complex formation that cannot be obtained by structural or computational studies alone. The fundamental interest in these interactions lies in what they can reveal about the general problems of parsing and predicting ligand binding free energies. For these problems, drug-DNA interactions offer several distinct advantages, among them being that the structures of many drug-DNA complexes are known at high resolution and that such structures reveal that in many cases the drug acts as a rigid body, with little conformational change upon binding. Complete thermodynamic profiles (delta G, delta H, delta S, delta Cp) for numerous drug-DNA interactions have been obtained, with the help of high-sensitivity microcalorimetry. The purpose of this article is to offer a perspective on the interpretation of these thermodynamics parameters, and in particular how they might be correlated with known structural features. Obligatory conformational changes in the DNA to accommodate intercalators and the loss of translational and rotational freedom upon complex formation both present unfavorable free energy barriers for binding. Such barriers must be overcome by favorable free energy contributions from the hydrophobic transfer of ligand from solution into the binding site, polyelectrolyte contributions from coupled ion release, and molecular interactions (hydrogen and ionic bonds, van der Waals interactions) that form within the binding site. Theoretical and semiempirical tools that allow estimates of these contributions to be made will be discussed, and their use in dissecting experimental data illustrated. This process, even at the current level of approximation, can shed considerable light on the drug-DNA binding process.

NOTCH1 Inhibits Activation of ATM by Impairing the Formation of an ATM-FOXO3a-KAT5/Tip60 Complex.

PubMed

Adamowicz, Marek; Vermezovic, Jelena; d'Adda di Fagagna, Fabrizio

2016-08-23

The DNA damage response (DDR) signal transduction pathway is responsible for sensing DNA damage and further relaying this signal into the cell. ATM is an apical DDR kinase that orchestrates the activation and the recruitment of downstream DDR factors to induce cell-cycle arrest and repair. We have previously shown that NOTCH1 inhibits ATM activation upon DNA damage, but the underlying mechanism remains unclear. Here, we show that NOTCH1 does not impair ATM recruitment to DNA double-strand breaks (DSBs). Rather, NOTCH1 prevents binding of FOXO3a and KAT5/Tip60 to ATM through a mechanism in which NOTCH1 competes with FOXO3a for ATM binding. Lack of FOXO3a binding to ATM leads to the loss of KAT5/Tip60 association with ATM. Moreover, expression of NOTCH1 or depletion of ATM impairs the formation of the FOXO3a-KAT5/Tip60 protein complex. Finally, we show that pharmacological induction of FOXO3a nuclear localization sensitizes NOTCH1-driven cancers to DNA-damage-induced cell death. Copyright © 2016 The Authors. Published by Elsevier Inc. All rights reserved.

Kinetics of Mismatch Formation opposite Lesions by the Replicative DNA Polymerase from Bacteriophage RB69

DOE Office of Scientific and Technical Information (OSTI.GOV)

Hogg, Matthew; Rudnicki, Jean; Midkiff, John

2010-04-12

The fidelity of DNA replication is under constant threat from the formation of lesions within the genome. Oxidation of DNA bases leads to the formation of altered DNA bases such as 8-oxo-7,8-dihydroguanine, commonly called 8-oxoG, and 2-hydroxyadenenine, or 2-OHA. In this work we have examined the incorporation kinetics opposite these two oxidatively derived lesions as well as an abasic site analogue by the replicative DNA polymerase from bacteriophage RB69. We compared the kinetic parameters for both wild type and the low fidelity L561A variant. While nucleotide incorporation rates (k{sub pol}) were generally higher for the variant, the presence of amore » lesion in the templating position reduced the ability of both the wild-type and variant DNA polymerases to form ternary enzyme-DNA-dNTP complexes. Thus, the L561A substitution does not significantly affect the ability of the RB69 DNA polymerase to recognize damaged DNA; instead, the mutation increases the probability that nucleotide incorporation will occur. We have also solved the crystal structure of the L561A variant forming an 8-oxoG {center_dot} dATP mispair and show that the propensity for forming this mispair depends on an enlarged polymerase active site.« less

Highly Conductive Thin Uniform Gold-Coated DNA Nanowires.

PubMed

Stern, Avigail; Eidelshtein, Gennady; Zhuravel, Roman; Livshits, Gideon I; Rotem, Dvir; Kotlyar, Alexander; Porath, Danny

2018-06-01

Over the past decades, DNA, the carrier of genetic information, has been used by researchers as a structural template material. Watson-Crick base pairing enables the formation of complex 2D and 3D structures from DNA through self-assembly. Various methods have been developed to functionalize these structures for numerous utilities. Metallization of DNA has attracted much attention as a means of forming conductive nanostructures. Nevertheless, most of the metallized DNA wires reported so far suffer from irregularity and lack of end-to-end electrical connectivity. An effective technique for formation of thin gold-coated DNA wires that overcomes these drawbacks is developed and presented here. A conductive atomic force microscopy setup, which is suitable for measuring tens to thousands of nanometer long molecules and wires, is used to characterize these DNA-based nanowires. The wires reported here are the narrowest gold-coated DNA wires that display long-range conductivity. The measurements presented show that the conductivity is limited by defects, and that thicker gold coating reduces the number of defects and increases the conductive length. This preparation method enables the formation of molecular wires with dimensions and uniformity that are much more suitable for DNA-based molecular electronics. © 2018 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

Cdt1p, through its interaction with Mcm6p, is required for the formation, nuclear accumulation and chromatin loading of the MCM complex.

PubMed

Wu, Rentian; Wang, Jiafeng; Liang, Chun

2012-01-01

Regulation of DNA replication initiation is essential for the faithful inheritance of genetic information. Replication initiation is a multi-step process involving many factors including ORC, Cdt1p, Mcm2-7p and other proteins that bind to replication origins to form a pre-replicative complex (pre-RC). As a prerequisite for pre-RC assembly, Cdt1p and the Mcm2-7p heterohexameric complex accumulate in the nucleus in G1 phase in an interdependent manner in budding yeast. However, the nature of this interdependence is not clear, nor is it known whether Cdt1p is required for the assembly of the MCM complex. In this study, we provide the first evidence that Cdt1p, through its interaction with Mcm6p with the C-terminal regions of the two proteins, is crucial for the formation of the MCM complex in both the cytoplasm and nucleoplasm. We demonstrate that disruption of the interaction between Cdt1p and Mcm6p prevents the formation of the MCM complex, excludes Mcm2-7p from the nucleus, and inhibits pre-RC assembly and DNA replication. Our findings suggest a function for Cdt1p in promoting the assembly of the MCM complex and maintaining its integrity by interacting with Mcm6p.

DNA - peptide polyelectrolyte complexes: Phase control by hybridization

NASA Astrophysics Data System (ADS)

Vieregg, Jeffrey; Lueckheide, Michael; Marciel, Amanda; Leon, Lorraine; Tirrell, Matthew

DNA is one of the most highly-charged molecules known, and interacts strongly with charged molecules in the cell. Condensation of long double-stranded DNA is one of the classic problems of biophysics, but the polyelectrolyte behavior of short and/or single-stranded nucleic acids has attracted far less study despite its importance for both biological and engineered systems. We report here studies of DNA oligonucleotides complexed with cationic peptides and polyamines. As seen previously for longer sequences, double-stranded oligonucleotides form solid precipitates, but single-stranded oligonucleotides instead undergo liquid-liquid phase separation to form coacervate droplets. Complexed oligonucleotides remain competent for hybridization, and display sequence-dependent environmental response. We observe similar behavior for RNA oligonucleotides, and methylphosphonate substitution of the DNA backbone indicates that nucleic acid charge density controls whether liquid or solid complexes are formed. Liquid-liquid phase separations of this type have been implicated in formation of membraneless organelles in vivo, and have been suggested as protocells in early life scenarios; oligonucleotides offer an excellent method to probe the physics controlling these phenomena.

Multiple conformational states of DnaA protein regulate its interaction with DnaA boxes in the initiation of DNA replication.

PubMed

Patel, Meera J; Bhatia, Lavesh; Yilmaz, Gulden; Biswas-Fiss, Esther E; Biswas, Subhasis B

2017-09-01

DnaA protein is the initiator of genomic DNA replication in prokaryotes. It binds to specific DNA sequences in the origin of DNA replication and unwinds small AT-rich sequences downstream for the assembly of the replisome. The mechanism of activation of DnaA that enables it to bind and organize the origin DNA and leads to replication initiation remains unclear. In this study, we have developed double-labeled fluorescent DnaA probes to analyze conformational states of DnaA protein upon binding DNA, nucleotide, and Soj sporulation protein using Fluorescence Resonance Energy Transfer (FRET). Our studies demonstrate that DnaA protein undergoes large conformational changes upon binding to substrates and there are multiple distinct conformational states that enable it to initiate DNA replication. DnaA protein adopted a relaxed conformation by expanding ~15Å upon binding ATP and DNA to form the ATP·DnaA·DNA complex. Hydrolysis of bound ATP to ADP led to a contraction of DnaA within the complex. The relaxed conformation of DnaA is likely required for the formation of the multi-protein ATP·DnaA·DNA complex. In the initiation of sporulation, Soj binding to DnaA prevented relaxation of its conformation. Soj·ADP appeared to block the activation of DnaA, suggesting a mechanism for Soj·ADP in switching initiation of DNA replication to sporulation. Our studies demonstrate that multiple conformational states of DnaA protein regulate its binding to DNA in the initiation of DNA replication. Copyright © 2017 Elsevier B.V. All rights reserved.

Formation of A Wrapped DNA-Protein Interface: Expermental Characterization and Analysis of the Large Contributions of Ions and Water to the Thermodynamics of Binding IHF to H′DNA

PubMed Central

Vander Meulen, Kirk A.; Saecker, Ruth M.; Record, M. Thomas

2008-01-01

To characterize driving forces and driven processes in formation of a large-interface, wrapped protein-DNA complex analogous to the nucleosome, we have investigated the thermodynamics of binding the 34 bp H′ DNA sequence to the E. coli DNA-remodeling protein Integration Host Factor (IHF). Isothermal titration calorimetry (ITC) and fluorescence resonance energy transfer (FRET) are applied to determine effects of salt concentration (KCl, KF, KGlutamate (KGlu)), and of the excluded solute glycine betaine, on the binding thermodynamics at 20°C. Both the binding constant Kobs and enthalpy ΔH°obs depend strongly on [salt] and anion identity. Formation of the wrapped complex is enthalpy-driven, especially at low [salt] (e.g. ΔH°obs = −20.2 kcal · mol−1 in 0.04 M KCl). ΔH°obs increases linearly with [salt] with a slope (dΔH°obs/d[salt]) which is much larger in KCl (38 ± 3 kcal · mol−1M−1) than in KF or KGlu (average 11 ± 2 kcal · mol−1M−1). At 0.33 M [salt], Kobs is approximately 30-fold larger in KGlu or KF than in KCl, and the [salt] derivative SKobs = dlnKobs/dln[salt] is almost twice as large in magnitude in KCl (−8.8 ± 0.7) as in KF or KGlu (average −4.7 ± 0.6). A novel analysis of the large effects of anion identity on Kobs, SKobs and on ΔH°obs dissects coulombic, Hofmeister and osmotic contributions to these quantities. This analysis attributes anion-specific differences in Kobs, SKobs and ΔH°obs to (i) displacement of a large number of waters of hydration (estimated to be 1.0 (± 0.2) × 103) from the 5340 Å2 of IHF and H′ DNA surface buried in complex formation, and (ii) significant local exclusion of F− and Glu− from this hydration water, relative to the situation with Cl−, which we propose is randomly distributed. To quantify net water release from anionic surface (22% of the surface buried in complexation, mostly from DNA phosphates), we determined the stabilizing effect of glycine betaine (GB) on Kobs: dlnKobs/d[GB] = 2.7 ± 0.4 at constant KCl activity, indicating the net release of 150 H2O from anionic surface. PMID:18237740

NF-κB mediates Gadd45β expression and DNA demethylation in the hippocampus during fear memory formation.

PubMed

Jarome, Timothy J; Butler, Anderson A; Nichols, Jessica N; Pacheco, Natasha L; Lubin, Farah D

2015-01-01

Gadd45-mediated DNA demethylation mechanisms have been implicated in the process of memory formation. However, the transcriptional mechanisms involved in the regulation of Gadd45 gene expression during memory formation remain unexplored. NF-κB (nuclear factor kappa-light-chain-enhancer of activated B cells) controls transcription of genes in neurons and is a critical regulator of synaptic plasticity and memory formation. In silico analysis revealed several NF-κB (p65/RelA and cRel) consensus sequences within the Gadd45β gene promoter. Whether NF-κB activity regulates Gadd45 expression and associated DNA demethylation in neurons during memory formation is unknown. Here, we found that learning in a fear conditioning paradigm increased Gadd45β gene expression and brain-derivedneurotrophic factor (BDNF) DNA demethylation in area CA1 of the hippocampus, both of which were prevented with pharmacological inhibition of NF-κB activity. Further experiments found that conditional mutations in p65/RelA impaired fear memory formation but did not alter changes in Gadd45β expression. The learning-induced increases in Gadd45β mRNA levels, Gadd45β binding at the BDNF gene and BDNF DNA demethylation were blocked in area CA1 of the c-rel knockout mice. Additionally, local siRNA-mediated knockdown of c-rel in area CA1 prevented fear conditioning-induced increases in Gadd45β expression and BDNF DNA demethylation, suggesting that c-Rel containing NF-κB transcription factor complex is responsible for Gadd45β regulation during memory formation. Together, these results support a novel transcriptional role for NF-κB in regulation of Gadd45β expression and DNA demethylation in hippocampal neurons during fear memory.

Molecular interactions of orthologues of floral homeotic proteins from the gymnosperm Gnetum gnemon provide a clue to the evolutionary origin of 'floral quartets'.

PubMed

Wang, Yong-Qiang; Melzer, Rainer; Theissen, Günter

2010-10-01

Several lines of evidence suggest that the identity of floral organs in angiosperms is specified by multimeric transcription factor complexes composed of MADS-domain proteins. These bind to specific cis-regulatory elements ('CArG-boxes') of their target genes involving DNA-loop formation, thus constituting 'floral quartets'. Gymnosperms, angiosperms' closest relatives, contain orthologues of floral homeotic genes, but when and how the interactions constituting floral quartets were established during evolution has remained unknown. We have comprehensively studied the dimerization and DNA-binding of several classes of MADS-domain proteins from the gymnosperm Gnetum gnemon. Determination of protein-protein and protein-DNA interactions by yeast two-hybrid, in vitro pull-down and electrophoretic mobility shift assays revealed complex patterns of homo- and heterodimerization among orthologues of floral homeotic class B, class C and class E proteins and B(sister) proteins. Using DNase I footprint assays we demonstrate that both orthologues of class B with C proteins, and orthologues of class C proteins alone, but not orthologues of class B proteins alone can loop DNA in floral quartet-like complexes. This is in contrast to class B and class C proteins from angiosperms, which require other factors such as class E floral homeotic proteins to 'glue' them together in multimeric complexes. Our findings suggest that the evolutionary origin of floral quartet formation is based on the interaction of different DNA-bound homodimers, does not depend on class E proteins, and predates the origin of angiosperms. © 2010 The Authors. Journal compilation © 2010 Blackwell Publishing Ltd.

Detection of Damaged DNA Bases by DNA Glycosylase Enzymes†

PubMed Central

Friedman, Joshua I.; Stivers, James T.

2010-01-01

A fundamental and shared process in all forms of life is the use of DNA glycosylase enzymes to excise rare damaged bases from genomic DNA. Without such enzymes, the highly-ordered primary sequences of genes would rapidly deteriorate. Recent structural and biophysical studies are beginning to reveal a fascinating multistep mechanism for damaged base detection that begins with short-range sliding of the glycosylase along the DNA chain in a distinct conformation we refer to as the search complex (SC). Sliding is frequently punctuated by the formation of a transient “interrogation” complex (IC) where the enzyme extrahelically inspects both normal and damaged bases in an exosite pocket that is distant from the active site. When normal bases are presented in the exosite, the IC rapidly collapses back to the SC, while a damaged base will efficiently partition forward into the active site to form the catalytically competent excision complex (EC). Here we review the unique problems associated with enzymatic detection of rare damaged DNA bases in the genome, and emphasize how each complex must have specific dynamic properties that are tuned to optimize the rate and efficiency of damage site location. PMID:20469926

Synthesis, characterization, redox behavior, DNA and protein binding and antibacterial activity studies of ruthenium(II) complexes of bidentate schiff bases.

PubMed

Paul, Hena; Sen, Buddhadeb; Mondal, Tapan Kumar; Chattopadhyay, Pabitra

2017-08-03

Two new ruthenium(II) complexes of Schiff base ligands (L) derived from cinnamaldehyde and ethylenediamine formulated as [Ru(L)(bpy) 2 ](ClO 4 ) 2 , where L 1 = N,N'-bis(4-nitrocinnamald-ehyde)ethylenediamine and L 2 = N,N'-bis(2-nitrocinnamaldehyde)-ethylenediamine for complex 1 and 2, respectively, were isolated in pure form. The complexes were characterized by physicochemical and spectroscopic methods. The electrochemical behavior of the complexes showed the Ru(III)/Ru(II) couple at different potentials with quasi-reversible voltammograms. The interaction of the complexes with calf thymus DNA (CT-DNA) using absorption, emission spectral studies and electrochemical techniques have been used to determine the binding constant, K b and the linear Stern-Volmer quenching constant, K SV . The results indicate that the ruthenium(II) complexes interact with CT-DNA strongly in a groove binding mode. The interactions of bovine serum albumin (BSA) with the complexes were also investigated with the help of absorption and fluorescence spectroscopy tools. Absorption spectroscopy proved the formation of a ground state BSA-[Ru(L)(bpy) 2 ](ClO 4 ) 2 complex. The antibacterial study showed that the Ru(II) complexes (1 and 2) have better activity than the standard antibiotics but weak activity than the ligands.

Relationship between DNA damage response, initiated by camptothecin or oxidative stress, and DNA replication, analyzed by quantitative 3D image analysis.

PubMed

Berniak, K; Rybak, P; Bernas, T; Zarębski, M; Biela, E; Zhao, H; Darzynkiewicz, Z; Dobrucki, J W

2013-10-01

A method of quantitative analysis of spatial (3D) relationship between discrete nuclear events detected by confocal microscopy is described and applied in analysis of a dependence between sites of DNA damage signaling (γH2AX foci) and DNA replication (EdU incorporation) in cells subjected to treatments with camptothecin (Cpt) or hydrogen peroxide (H2O2). Cpt induces γH2AX foci, likely reporting formation of DNA double-strand breaks (DSBs), almost exclusively at sites of DNA replication. This finding is consistent with the known mechanism of induction of DSBs by DNA topoisomerase I (topo1) inhibitors at the sites of collisions of the moving replication forks with topo1-DNA "cleavable complexes" stabilized by Cpt. Whereas an increased level of H2AX histone phosphorylation is seen in S-phase of cells subjected to H2O2, only a minor proportion of γH2AX foci coincide with DNA replication sites. Thus, the increased level of H2AX phosphorylation induced by H2O2 is not a direct consequence of formation of DNA lesions at the sites of moving DNA replication forks. These data suggest that oxidative stress induced by H2O2 and formation of the primary H2O2-induced lesions (8-oxo-7,8-dihydroguanosine) inhibits replication globally and triggers formation of γH2AX at various distances from replication forks. Quantitative analysis of a frequency of DNA replication sites and γH2AX foci suggests also that stalling of replicating forks by Cpt leads to activation of new DNA replication origins. © 2013 International Society for Advancement of Cytometry. Copyright © 2013 International Society for Advancement of Cytometry.

Serine hydroxymethyltransferase anchors de novo thymidylate synthesis pathway to nuclear lamina for DNA synthesis.

PubMed

Anderson, Donald D; Woeller, Collynn F; Chiang, En-Pei; Shane, Barry; Stover, Patrick J

2012-03-02

The de novo thymidylate biosynthetic pathway in mammalian cells translocates to the nucleus for DNA replication and repair and consists of the enzymes serine hydroxymethyltransferase 1 and 2α (SHMT1 and SHMT2α), thymidylate synthase, and dihydrofolate reductase. In this study, we demonstrate that this pathway forms a multienzyme complex that is associated with the nuclear lamina. SHMT1 or SHMT2α is required for co-localization of dihydrofolate reductase, SHMT, and thymidylate synthase to the nuclear lamina, indicating that SHMT serves as scaffold protein that is essential for complex formation. The metabolic complex is enriched at sites of DNA replication initiation and associated with proliferating cell nuclear antigen and other components of the DNA replication machinery. These data provide a mechanism for previous studies demonstrating that SHMT expression is rate-limiting for de novo thymidylate synthesis and indicate that de novo thymidylate biosynthesis occurs at replication forks.

Mechanism of Promoter Melting by the Xeroderma Pigmentosum Complementation Group B Helicase of Transcription Factor IIH Revealed by Protein-DNA Photo-Cross-Linking

PubMed Central

Douziech, Maxime; Coin, Frédéric; Chipoulet, Jean-Marc; Arai, Yoko; Ohkuma, Yoshiaki; Egly, Jean-Marc; Coulombe, Benoit

2000-01-01

The p89/xeroderma pigmentosum complementation group B (XPB) ATPase-helicase of transcription factor IIH (TFIIH) is essential for promoter melting prior to transcription initiation by RNA polymerase II (RNAPII). By studying the topological organization of the initiation complex using site-specific protein-DNA photo-cross-linking, we have shown that p89/XPB makes promoter contacts both upstream and downstream of the initiation site. The upstream contact, which is in the region where promoter melting occurs (positions −9 to +2), requires tight DNA wrapping around RNAPII. The addition of hydrolyzable ATP tethers the template strand at positions −5 and +1 to RNAPII subunits. A mutation in p89/XPB found in a xeroderma pigmentosum patient impairs the ability of TFIIH to associate correctly with the complex and thereby melt promoter DNA. A model for open complex formation is proposed. PMID:11027286

Structural impact of complete CpG methylation within target DNA on specific complex formation of the inducible transcription factor Egr-1.

PubMed

Zandarashvili, Levani; White, Mark A; Esadze, Alexandre; Iwahara, Junji

2015-07-08

The inducible transcription factor Egr-1 binds specifically to 9-bp target sequences containing two CpG sites that can potentially be methylated at four cytosine bases. Although it appears that complete CpG methylation would make an unfavorable steric clash in the previous crystal structures of the complexes with unmethylated or partially methylated DNA, our affinity data suggest that DNA recognition by Egr-1 is insensitive to CpG methylation. We have determined, at a 1.4-Å resolution, the crystal structure of the Egr-1 zinc-finger complex with completely methylated target DNA. Structural comparison of the three different methylation states reveals why Egr-1 can recognize the target sequences regardless of CpG methylation. Copyright © 2015 Federation of European Biochemical Societies. Published by Elsevier B.V. All rights reserved.

BLM and RMI1 alleviate RPA inhibition of TopoIIIα decatenase activity.

PubMed

Yang, Jay; Bachrati, Csanad Z; Hickson, Ian D; Brown, Grant W

2012-01-01

RPA is a single-stranded DNA binding protein that physically associates with the BLM complex. RPA stimulates BLM helicase activity as well as the double Holliday junction dissolution activity of the BLM-topoisomerase IIIα complex. We investigated the effect of RPA on the ssDNA decatenase activity of topoisomerase IIIα. We found that RPA and other ssDNA binding proteins inhibit decatenation by topoisomerase IIIα. Complex formation between BLM, TopoIIIα, and RMI1 ablates inhibition of decatenation by ssDNA binding proteins. Together, these data indicate that inhibition by RPA does not involve species-specific interactions between RPA and BLM-TopoIIIα-RMI1, which contrasts with RPA modulation of double Holliday junction dissolution. We propose that topoisomerase IIIα and RPA compete to bind to single-stranded regions of catenanes. Interactions with BLM and RMI1 enhance toposiomerase IIIα activity, promoting decatenation in the presence of RPA.

UVA irradiation of BrU-substituted DNA in the presence of Hoechst 33258.

PubMed

Saha, Abhijit; Kizaki, Seiichiro; Han, Ji Hoon; Yu, Zutao; Sugiyama, Hiroshi

2018-01-01

Given that our knowledge of DNA repair is limited because of the complexity of the DNA system, a technique called UVA micro-irradiation has been developed that can be used to visualize the recruitment of DNA repair proteins at double-strand break (DSB) sites. Interestingly, Hoechst 33258 was used under micro-irradiation to sensitize 5-bromouracil ( Br U)-labelled DNA, causing efficient DSBs. However, the molecular basis of DSB formation under UVA micro-irradiation remains unknown. Herein, we investigated the mechanism of DSB formation under UVA micro-irradiation conditions. Our results suggest that the generation of a uracil-5-yl radical through electron transfer from Hoechst 33258 to Br U caused DNA cleavage preferentially at self-complementary 5'-AA Br U Br U-3' sequences to induce DSB. We also investigated the DNA cleavage in the context of the nucleosome to gain a better understanding of UVA micro-irradiation in a cell-like model. We found that DNA cleavage occurred in both core and linker DNA regions although its efficiency reduced in core DNA. Copyright © 2017 Elsevier Ltd. All rights reserved.

Determinants for Tight and Selective Binding of a Medicinal Dicarbene Gold(I) Complex to a Telomeric DNA G-Quadruplex: a Joint ESI MS and XRD Investigation.

PubMed

Bazzicalupi, Carla; Ferraroni, Marta; Papi, Francesco; Massai, Lara; Bertrand, Benoît; Messori, Luigi; Gratteri, Paola; Casini, Angela

2016-03-18

The dicarbene gold(I) complex [Au(9-methylcaffein-8-ylidene)2 ]BF4 is an exceptional organometallic compound of profound interest as a prospective anticancer agent. This gold(I) complex was previously reported to be highly cytotoxic toward various cancer cell lines in vitro and behaves as a selective G-quadruplex stabilizer. Interactions of the gold complex with various telomeric DNA models have been analyzed by a combined ESI MS and X-ray diffraction (XRD) approach. ESI MS measurements confirmed formation of stable adducts between the intact gold(I) complex and Tel 23 DNA sequence. The crystal structure of the adduct formed between [Au(9-methylcaffein-8-ylidene)2 ](+) and Tel 23 DNA G-quadruplex was solved. Tel 23 maintains a characteristic propeller conformation while binding three gold(I) dicarbene moieties at two distinct sites. Stacking interactions appear to drive noncovalent binding of the gold(I) complex. The structural basis for tight gold(I) complex/G-quadruplex recognition and its selectivity are described. © 2016 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

Genetics Home Reference: Björnstad syndrome

MedlinePlus

... species, which are harmful molecules that can damage DNA and tissues. BCS1L gene mutations involved in Björnstad syndrome alter the BCS1L protein and impair its ability to aid in complex III formation. The resulting decrease in complex III activity reduces ...

DNA-mediated association of two histone-bound complexes of yeast Chromatin Assembly Factor-1 (CAF-1) drives tetrasome assembly in the wake of DNA replication.

PubMed

Mattiroli, Francesca; Gu, Yajie; Yadav, Tejas; Balsbaugh, Jeremy L; Harris, Michael R; Findlay, Eileen S; Liu, Yang; Radebaugh, Catherine A; Stargell, Laurie A; Ahn, Natalie G; Whitehouse, Iestyn; Luger, Karolin

2017-03-18

Nucleosome assembly in the wake of DNA replication is a key process that regulates cell identity and survival. Chromatin assembly factor 1 (CAF-1) is a H3-H4 histone chaperone that associates with the replisome and orchestrates chromatin assembly following DNA synthesis. Little is known about the mechanism and structure of this key complex. Here we investigate the CAF-1•H3-H4 binding mode and the mechanism of nucleosome assembly. We show that yeast CAF-1 binding to a H3-H4 dimer activates the Cac1 winged helix domain interaction with DNA. This drives the formation of a transient CAF-1•histone•DNA intermediate containing two CAF-1 complexes, each associated with one H3-H4 dimer. Here, the (H3-H4) 2 tetramer is formed and deposited onto DNA. Our work elucidates the molecular mechanism for histone deposition by CAF-1, a reaction that has remained elusive for other histone chaperones, and it advances our understanding of how nucleosomes and their epigenetic information are maintained through DNA replication.

Structural insights into the histone H1-nucleosome complex

PubMed Central

Zhou, Bing-Rui; Feng, Hanqiao; Kato, Hidenori; Dai, Liang; Yang, Yuedong; Zhou, Yaoqi; Bai, Yawen

2013-01-01

Linker H1 histones facilitate formation of higher-order chromatin structures and play important roles in various cell functions. Despite several decades of effort, the structural basis of how H1 interacts with the nucleosome remains elusive. Here, we investigated Drosophila H1 in complex with the nucleosome, using solution nuclear magnetic resonance spectroscopy and other biophysical methods. We found that the globular domain of H1 bridges the nucleosome core and one 10-base pair linker DNA asymmetrically, with its α3 helix facing the nucleosomal DNA near the dyad axis. Two short regions in the C-terminal tail of H1 and the C-terminal tail of one of the two H2A histones are also involved in the formation of the H1–nucleosome complex. Our results lead to a residue-specific structural model for the globular domain of the Drosophila H1 in complex with the nucleosome, which is different from all previous experiment-based models and has implications for chromatin dynamics in vivo. PMID:24218562

Functional Architecture of T7 RNA Polymerase Transcription Complexes

PubMed Central

Nayak, Dhananjaya; Guo, Qing; Sousa, Rui

2007-01-01

Summary T7 RNA polymerase is the best-characterized member of a widespread family of single-subunit RNA polymerases. Crystal structures of T7 RNA polymerase initiation and elongation complexes have provided a wealth of detailed information on RNA polymerase interactions with the promoter and transcription bubble, but the absence of DNA downstream of the melted region of the template in the initiation complex structure, and the absence of DNA upstream of the transcription bubble in the elongation complex structure means that our picture of the functional architecture of T7 RNA polymerase transcription complexes remains incomplete. Here we use the site-specifically tethered chemical nucleases and functional characterization of directed T7 RNAP mutants to both reveal the architecture of the duplex DNA that flanks the transcription bubble in the T7 RNAP initiation and elongation complexes, and to define the function of the interactions made by these duplex elements. We find that downstream duplex interactions made with a cluster of lysines (K711/K713/K714) are present during both elongation and initiation where they contribute to stabilizing a bend in the downstream DNA that is important for promoter opening. The upstream DNA in the elongation complex is also found to be sharply bent at the upstream edge of the transcription bubble, thereby allowing formation of upstream duplex:polymerase interactions that contribute to elongation complex stability. PMID:17580086

A novel activity of HMG domains: promotion of the triple-stranded complex formation between DNA containing (GGA/TCC)11 and d(GGA)11 oligonucleotides.

PubMed Central

Suda, T; Mishima, Y; Takayanagi, K; Asakura, H; Odani, S; Kominami, R

1996-01-01

The high mobility group protein (HMG)-box is a DNA-binding domain found in many proteins that bind preferentially to DNA of irregular structures in a sequence-independent manner and can bend the DNA. We show here that GST-fusion proteins of HMG domains from HMG1 and HMG2 promote a triple-stranded complex formation between DNA containing the (GGA/TCC)11 repeat and oligonucleotides of d(GGA)11 probably due to G:G base pairing. The activity is to reduce association time and requirements of Mg2+ and oligonucleotide concentrations. The HMG box of SRY, the protein determining male-sex differentiation, also has the activity, suggesting that it is not restricted to the HMG-box domains derived from HMG1/2 but is common to those from other members of the HMG-box family of proteins. Interestingly, the box-AB and box-B of HMG1 bend DNA containing the repeat, but SRY fails to bend in a circularization assay. The difference suggests that the two activities of association-promotion and DNA bending are distinct. These results suggest that the HMG-box domain has a novel activity of promoting the association between GGA repeats which might be involved in higher-order architecture of chromatin. PMID:8972860

Structure and interactions of the Bacillus subtilis sporulation inhibitor of DNA replication, SirA, with domain I of DnaA

PubMed Central

Jameson, Katie H; Rostami, Nadia; Fogg, Mark J; Turkenburg, Johan P; Grahl, Anne; Murray, Heath; Wilkinson, Anthony J

2014-01-01

Chromosome copy number in cells is controlled so that the frequency of initiation of DNA replication matches that of cell division. In bacteria, this is achieved through regulation of the interaction between the initiator protein DnaA and specific DNA elements arrayed at the origin of replication. DnaA assembles at the origin and promotes DNA unwinding and the assembly of a replication initiation complex. SirA is a DnaA-interacting protein that inhibits initiation of replication in diploid Bacillus subtilis cells committed to the developmental pathway leading to formation of a dormant spore. Here we present the crystal structure of SirA in complex with the N-terminal domain of DnaA revealing a heterodimeric complex. The interacting surfaces of both proteins are α-helical with predominantly apolar side-chains packing in a hydrophobic interface. Site-directed mutagenesis experiments confirm the importance of this interface for the interaction of the two proteins in vitro and in vivo. Localization of GFP–SirA indicates that the protein accumulates at the replisome in sporulating cells, likely through a direct interaction with DnaA. The SirA interacting surface of DnaA corresponds closely to the HobA-interacting surface of DnaA from Helicobacter pylori even though HobA is an activator of DnaA and SirA is an inhibitor. PMID:25041308

Molecular dynamics studies on the DNA-binding process of ERG.

PubMed

Beuerle, Matthias G; Dufton, Neil P; Randi, Anna M; Gould, Ian R

2016-11-15

The ETS family of transcription factors regulate gene targets by binding to a core GGAA DNA-sequence. The ETS factor ERG is required for homeostasis and lineage-specific functions in endothelial cells, some subset of haemopoietic cells and chondrocytes; its ectopic expression is linked to oncogenesis in multiple tissues. To date details of the DNA-binding process of ERG including DNA-sequence recognition outside the core GGAA-sequence are largely unknown. We combined available structural and experimental data to perform molecular dynamics simulations to study the DNA-binding process of ERG. In particular we were able to reproduce the ERG DNA-complex with a DNA-binding simulation starting in an unbound configuration with a final root-mean-square-deviation (RMSD) of 2.1 Å to the core ETS domain DNA-complex crystal structure. This allowed us to elucidate the relevance of amino acids involved in the formation of the ERG DNA-complex and to identify Arg385 as a novel key residue in the DNA-binding process. Moreover we were able to show that water-mediated hydrogen bonds are present between ERG and DNA in our simulations and that those interactions have the potential to achieve sequence recognition outside the GGAA core DNA-sequence. The methodology employed in this study shows the promising capabilities of modern molecular dynamics simulations in the field of protein DNA-interactions.

M-DNA is stabilised in G•C tracts or by incorporation of 5-fluorouracil

PubMed Central

Wood, David O.; Dinsmore, Michael J.; Bare, Grant A.; Lee, Jeremy S.

2002-01-01

M-DNA is a complex between the divalent metal ions Zn2+, Ni2+ and Co2+ and duplex DNA which forms at a pH of ∼8.5. The stability and formation of M-DNA was monitored with an ethidium fluorescence assay in order to assess the relationship between pH, metal ion concentration, DNA concentration and the base composition. The dismutation of calf thymus DNA exhibits hysteresis with the formation of M-DNA occurring at a higher pH than the reconversion of M-DNA back to B-DNA. Hysteresis is most prominent with the Ni form of M-DNA where complete reconversion to B-DNA takes several hours even in the presence of EDTA. Increasing the DNA concentration leads to an increase in the metal ion concentration required for M-DNA formation. Both poly(dG)•poly(dC) and poly(dA)•poly(dT) formed M-DNA more readily than the corresponding mixed sequence DNAs. For poly(dG)•(poly(dC) M-DNA formation was observed at pH 7.4 with 0.5 mM ZnCl2. Modified bases were incorporated into a 500 bp fragment of phage λ DNA by polymerase chain reaction. DNAs in which guanine was replaced with hypoxanthine or thymine with 5-fluorouracil formed M-DNA at pHs below 8 whereas substitutions such as 2-aminoadenine and 5-methylcytosine had little effect. Poly[d(A5FU)] also formed a very stable M-DNA duplex as judged from Tm measurements. It is evident that the lower the pKa of the imino proton of the base, the lower the pH at which M-DNA will form; a finding that is consistent with the replacement of the imino proton with the metal ion. PMID:12000844

Cooperative Assembly of Co-Smad4 MH1 with R-Smad1/3 MH1 on DNA: A Molecular Dynamics Simulation Study

PubMed Central

Wang, Guihong; Li, Chaoqun; Wang, Yan; Chen, Guangju

2013-01-01

Background Smads, the homologs of Sma and MAD proteins, play a key role in gene expression regulation in the transforming growth factor-β (TGF-β) signaling pathway. Recent experimental studies have revealed that Smad4/R-Smad heterodimers bound on DNA are energetically more favorable than homodimeric R-Smad/R-Smad complexes bound on DNA, which indicates that Smad4 might act as binding vehicle to cooperatively assemble with activated R-Smads on DNA in the nucleus. However, the details of interaction mechanism for cooperative recruitment of Smad4 protein to R-Smad proteins on DNA, and allosteric communication between the Smad4-DNA and R-Smad-DNA interfaces via DNA mediating are not yet clear so far. Methodology In the present work, we have constructed a series of Smadn+DNA+Smadn (n = 1, 3, 4) models and carried out molecular dynamics simulations, free energy calculations and DNA dynamics analysis for them to study the interaction properties of Smadn (n = 1, 3, 4) with DNA molecule. Results The results revealed that the binding of Smad4 protein to DNA molecule facilitates energetically the formation of the heteromeric Smad4+DNA+Smad1/3 complex by increasing the affinity of Smad1/3 with DNA molecule. Further investigations through the residue/base motion correlation and DNA dynamics analyses predicted that the binding of Smad4 protein to DNA molecule in the heteromeric Smad4+DNA+Smad1/3 model induces an allosteric communication from the Smad4-DNA interface to Smad1/Smad3-DNA interface via DNA base-pair helical motions, surface conformation changes and new hydrogen bond formations. The present work theoretically explains the mechanism of cooperative recruitment of Smad4 protein to Smad1/3 protein via DNA-mediated indirect readout mode in the nucleus. PMID:23326519

Effect of DNA-CTMA complex on optical properties of LDS 821 dye

NASA Astrophysics Data System (ADS)

Udayan, Sony; Ramachandran, Vijesh Kavumoottil; Sebastian, Mathew; Chandran, Pradeep; Nampoori, Vadakkedath Parameswaran Narayanan; Thomas, Sheenu

2017-07-01

We have investigated the fluorescence behavior of LDS 821 dye (Styryl 9 M) with deoxyribonucleic acid attached with cetyltrimethyl-ammonium (DNA-CTMA). Optical absorption studies confirm the intercalation of the dye molecules with DNA-CTMA. Fluorescence studies show an enhancement of fluorescence intensity of dye with DNA-CTMA, which suggest the reduction of TICT states of the dye molecule. The FWHM of the fluorescence spectrum increases from 95 nm to 161 nm indicating the formation of new energy levels when DNA-CTMA forms a complex with LDS 821 dye. Fluorescence lifetime measurements shows that lifetime of LDS 821 varies from 507ps to 953 ps with the addition of DNA-CTMA, which also confirms the deactivation of TICT states of dye molecule. Results show that the incorporation of DNA-CTMA with LDS 821 dye improves the optical characteristics of LDS 821 dye and therefore, can be used as a good fluorescence probe for DNA visualization as well as in lasing applications.

The optical biomedical sensors for DNA detection and imaging based on two-photon excited luminescent styryl dyes: phototoxic influence on the DNA

NASA Astrophysics Data System (ADS)

Yashchuk, Valeriy M.; Kudrya, Vladislav Yu.; Losytskyy, Mykhaylo Yu.; Tokar, Valentyna P.; Yarmoluk, Sergiy M.; Dmytruk, Igor M.; Prokopets, Vadym M.; Kovalska, Vladyslava B.; Balanda, Anatoliy O.; Kryvorotenko, Dmytro V.; Ogul'chansky, Tymish Yu.

2007-06-01

The optical absorption, fluorescence and phosphorescence of the novel styryl dyes developed for the fluorescent detection of DNA were investigated. The energy structures of dye molecules as well as spectral manifestations of the dyes aggregate formation and interaction with DNA were studied. The dramatic increase (up to 1000 times) of the fluorescence intensity of dyes in the presence of DNA was observed. The photostability and phototoxic influence on the DNA of several styryl dyes were studied by analyzing absorption, fluorescence and phosphorescence spectra of these dyes and dye-DNA systems. Changes of the optical density value of dye-DNA solutions caused by the visible light irradiation were fixed in the wavelength regions of the DNA absorption and of the dye absorption. Fluorescence emission of dye-DNA complexes upon two-photon excitation (TPE) at wavelength 1064 nm with the 20 ns pulsed YAG: Nd3+ laser and at 840 nm with the 90 fs pulsed Ti:sapphire laser was registered. The values of two-photon absorption cross-sections of dye-DNA complexes were evaluated.

DNA Repair by DNA: The UV1C DNAzyme Catalyzes Photoreactivation of Cyclobutane Thymine Dimers in DNA More Effectively than Their de Novo Formation.

PubMed

Barlev, Adam; Sekhon, Gurpreet S; Bennet, Andrew J; Sen, Dipankar

2016-11-01

UV1C, a 42-nt DNA oligonucleotide, is a deoxyribozyme (DNAzyme) that optimally uses 305 nm wavelength light to catalyze photoreactivation of a cyclobutane thymine dimer placed within a gapped, unnatural DNA substrate, TDP. Herein we show that UV1C is also capable of photoreactivating thymine dimers within an authentic single-stranded DNA substrate, LDP. This bona fide UV1C substrate enables, for the first time, investigation of whether UV1C catalyzes only photoreactivation or also the de novo formation of thymine dimers. Single-turnover experiments carried out with LDP and UV1C, relative to control experiments with LDP alone in single-stranded and double-stranded contexts, show that while UV1C does modestly promote thymine dimer formation, its major activity is indeed photoreactivation. Distinct photostationary states are reached for LDP in its three contexts: as a single strand, as a constituent of a double-helix, and as a 1:1 complex with UV1C. The above results on the cofactor-independent photoreactivation capabilities of a catalytic DNA reinforce a series of recent, unexpected reports that purely nucleotide-based photoreactivation is also operational within conventional double-helical DNA.

Cationic micelles self-assembled from cholesterol-conjugated oligopeptides as an efficient gene delivery vector.

PubMed

Guo, Xin Dong; Tandiono, Fanny; Wiradharma, Nikken; Khor, Dingyue; Tan, Chuan Guan; Khan, Majad; Qian, Yu; Yang, Yi-Yan

2008-12-01

Cholesterol-conjugated H(5)R(10) and H(10)R(10) oligopeptides (HR15-Chol and HR20-Chol) were designed and synthesized. These amphiphilic oligopeptides were able to self-assemble into cationic micelles in aqueous solution at low concentrations, and their critical micelle concentrations in sodium acetate buffer (20mM, pH 5.0) were 17.8 and 28.2mg/L respectively. The micelle formation was further evidenced via SEM and dynamic light scattering analyses. The average hydrodynamic size of HR15-Chol and HR20-Chol micelles was about 425 and 435 nM in diameter with zeta potential of 64 and 66 mV respectively. The formation of micelles increased local concentration of cationic charge, leading to higher DNA binding efficiency as compared to the control peptides HR15 and HR20. The minimum size observed for HR15-Chol/DNA and HR20-Chol/DNA complexes was about 175-176 nM, and the maximum zeta potential was around 61-62 mV. In comparison, HR15 and HR20 formed DNA complexes with a similar size but significantly lower zeta potential (i.e. about 31-40 mV). In particular, after being challenged by DMEM medium, the size of peptide/DNA complexes was increased significantly and their surface charge was neutralized. Nevertheless, the size of the micelle/DNA complexes formed from HR15-Chol and HR20-Chol was still about 200 nM with positive charge of around 20 mV at high N/P ratios. The micelles induced much higher overall gene expression (i.e. luciferase expression) levels than the peptides in both HepG2 and HEK293 cell lines. Increasing the histidine residue from 0 to 5 to 10 further increased gene expression efficiency. In particular, HR20-Chol micelles yielded 95% GFP-positive HepG2 cells at N/P 50, much higher than that induced by PEI at its optimal N/P ratio (i.e. 10), which was 6.8%. In 4T1 cells, HR20-Chol induced 2 times higher luciferase expression level than PEI at their optimal N/P ratios. Moreover, HR20-Chol micelle/DNA complexes were less cytotoxic than PEI/DNA complexes. These micelles may be a promising carrier for delivery of therapeutic genes.

M-DNA: a self-assembling molecular wire for nanoelectronics and biosensing.

PubMed

Wettig, Shawn D; Li, Chen-Zhong; Long, Yi-Tao; Kraatz, Heinz-Bernhard; Lee, Jeremy S

2003-01-01

M-DNA is a complex between divalent metal ions such as Zn2+ and duplex DNA which forms at pH 8.5. Unlike B-DNA, M-DNA does not bind ethidium so that M-DNA formation can be monitored conveniently by an ethidium fluorescence assay. M-DNA was shown to be a better conductor than B-DNA by fluorometric measurements of electron transport in donor-acceptor labelled duplexes; by direct conductivity measurements of M-DNA bound between gold electrodes and by cyclic voltammetric studies on ferrocene labelled duplexes attached to gold microelectrodes. As is the case with B-DNA, M-DNA can self-assemble into a variety of structures and is anticipated to find widespread use in nanoelectronics and biosensing.

PNA binding to the non-template DNA strand interferes with transcription, suggesting a blockage mechanism mediated by R-loop formation.

PubMed

Belotserkovskii, Boris P; Hanawalt, Philip C

2015-11-01

Peptide Nucleic Acids (PNAs) are artificial DNA mimics with superior nucleic acid binding capabilities. T7 RNA polymerase (T7 RNAP) transcription upon encountering PNA bound to the non-template DNA strand was studied in vitro. A characteristic pattern of blockage signals was observed, extending downstream from the PNA binding site, similar to that produced by G-rich homopurine-homopyrimidine (hPu-hPy) sequences and likely caused by R-loop formation. Since blocked transcription complexes in association with stable R-loops may interfere with replication and in some cases trigger apoptosis, targeted R-loop formation might be employed to inactivate selected cells, such as those in tumors, based upon their unique complement of expressed genes. © 2014 The Authors. Molecular Carcinogenesis published by Wiley Periodicals, Inc.

A new class of antibacterials, the imidazopyrazinones, reveal structural transitions involved in DNA gyrase poisoning and mechanisms of resistance.

PubMed

Germe, Thomas; Vörös, Judit; Jeannot, Frederic; Taillier, Thomas; Stavenger, Robert A; Bacqué, Eric; Maxwell, Anthony; Bax, Benjamin D

2018-05-04

Imidazopyrazinones (IPYs) are a new class of compounds that target bacterial topoisomerases as a basis for their antibacterial activity. We have characterized the mechanism of these compounds through structural/mechanistic studies showing they bind and stabilize a cleavage complex between DNA gyrase and DNA ('poisoning') in an analogous fashion to fluoroquinolones, but without the requirement for the water-metal-ion bridge. Biochemical experiments and structural studies of cleavage complexes of IPYs compared with an uncleaved gyrase-DNA complex, reveal conformational transitions coupled to DNA cleavage at the DNA gate. These involve movement at the GyrA interface and tilting of the TOPRIM domains toward the scissile phosphate coupled to capture of the catalytic metal ion. Our experiments show that these structural transitions are involved generally in poisoning of gyrase by therapeutic compounds and resemble those undergone by the enzyme during its adenosine triphosphate-coupled strand-passage cycle. In addition to resistance mutations affecting residues that directly interact with the compounds, we characterized a mutant (D82N) that inhibits formation of the cleavage complex by the unpoisoned enzyme. The D82N mutant appears to act by stabilizing the binary conformation of DNA gyrase with uncleaved DNA without direct interaction with the compounds. This provides general insight into the resistance mechanisms to antibiotics targeting bacterial type II topoisomerases.

A new class of antibacterials, the imidazopyrazinones, reveal structural transitions involved in DNA gyrase poisoning and mechanisms of resistance

PubMed Central

Germe, Thomas; Vörös, Judit; Jeannot, Frederic; Taillier, Thomas; Stavenger, Robert A; Bacqué, Eric; Bax, Benjamin D

2018-01-01

Abstract Imidazopyrazinones (IPYs) are a new class of compounds that target bacterial topoisomerases as a basis for their antibacterial activity. We have characterized the mechanism of these compounds through structural/mechanistic studies showing they bind and stabilize a cleavage complex between DNA gyrase and DNA (‘poisoning’) in an analogous fashion to fluoroquinolones, but without the requirement for the water–metal–ion bridge. Biochemical experiments and structural studies of cleavage complexes of IPYs compared with an uncleaved gyrase–DNA complex, reveal conformational transitions coupled to DNA cleavage at the DNA gate. These involve movement at the GyrA interface and tilting of the TOPRIM domains toward the scissile phosphate coupled to capture of the catalytic metal ion. Our experiments show that these structural transitions are involved generally in poisoning of gyrase by therapeutic compounds and resemble those undergone by the enzyme during its adenosine triphosphate-coupled strand-passage cycle. In addition to resistance mutations affecting residues that directly interact with the compounds, we characterized a mutant (D82N) that inhibits formation of the cleavage complex by the unpoisoned enzyme. The D82N mutant appears to act by stabilizing the binary conformation of DNA gyrase with uncleaved DNA without direct interaction with the compounds. This provides general insight into the resistance mechanisms to antibiotics targeting bacterial type II topoisomerases. PMID:29538767

Mechanism of duplex DNA destabilization by RNA-guided Cas9 nuclease during target interrogation

PubMed Central

Mekler, Vladimir; Minakhin, Leonid; Severinov, Konstantin

2017-01-01

The prokaryotic clustered regularly interspaced short palindromic repeats (CRISPR)-associated 9 (Cas9) endonuclease cleaves double-stranded DNA sequences specified by guide RNA molecules and flanked by a protospacer adjacent motif (PAM) and is widely used for genome editing in various organisms. The RNA-programmed Cas9 locates the target site by scanning genomic DNA. We sought to elucidate the mechanism of initial DNA interrogation steps that precede the pairing of target DNA with guide RNA. Using fluorometric and biochemical assays, we studied Cas9/guide RNA complexes with model DNA substrates that mimicked early intermediates on the pathway to the final Cas9/guide RNA–DNA complex. The results show that Cas9/guide RNA binding to PAM favors separation of a few PAM-proximal protospacer base pairs allowing initial target interrogation by guide RNA. The duplex destabilization is mediated, in part, by Cas9/guide RNA affinity for unpaired segments of nontarget strand DNA close to PAM. Furthermore, our data indicate that the entry of double-stranded DNA beyond a short threshold distance from PAM into the Cas9/single-guide RNA (sgRNA) interior is hindered. We suggest that the interactions unfavorable for duplex DNA binding promote DNA bending in the PAM-proximal region during early steps of Cas9/guide RNA–DNA complex formation, thus additionally destabilizing the protospacer duplex. The mechanism that emerges from our analysis explains how the Cas9/sgRNA complex is able to locate the correct target sequence efficiently while interrogating numerous nontarget sequences associated with correct PAMs. PMID:28484024

Mechanism of duplex DNA destabilization by RNA-guided Cas9 nuclease during target interrogation.

PubMed

Mekler, Vladimir; Minakhin, Leonid; Severinov, Konstantin

2017-05-23

The prokaryotic clustered regularly interspaced short palindromic repeats (CRISPR)-associated 9 (Cas9) endonuclease cleaves double-stranded DNA sequences specified by guide RNA molecules and flanked by a protospacer adjacent motif (PAM) and is widely used for genome editing in various organisms. The RNA-programmed Cas9 locates the target site by scanning genomic DNA. We sought to elucidate the mechanism of initial DNA interrogation steps that precede the pairing of target DNA with guide RNA. Using fluorometric and biochemical assays, we studied Cas9/guide RNA complexes with model DNA substrates that mimicked early intermediates on the pathway to the final Cas9/guide RNA-DNA complex. The results show that Cas9/guide RNA binding to PAM favors separation of a few PAM-proximal protospacer base pairs allowing initial target interrogation by guide RNA. The duplex destabilization is mediated, in part, by Cas9/guide RNA affinity for unpaired segments of nontarget strand DNA close to PAM. Furthermore, our data indicate that the entry of double-stranded DNA beyond a short threshold distance from PAM into the Cas9/single-guide RNA (sgRNA) interior is hindered. We suggest that the interactions unfavorable for duplex DNA binding promote DNA bending in the PAM-proximal region during early steps of Cas9/guide RNA-DNA complex formation, thus additionally destabilizing the protospacer duplex. The mechanism that emerges from our analysis explains how the Cas9/sgRNA complex is able to locate the correct target sequence efficiently while interrogating numerous nontarget sequences associated with correct PAMs.

Lipid based drug delivery systems: Kinetics by SANS

NASA Astrophysics Data System (ADS)

Uhríková, D.; Teixeira, J.; Hubčík, L.; Búcsi, A.; Kondela, T.; Murugova, T.; Ivankov, O. I.

2017-05-01

N,N-dimethyldodecylamine-N-oxide (C12NO) is a surfactant that may exist either in a neutral or protonated form depending on the pH of aqueous solutions. Using small angle X-ray diffraction (SAXD) we demonstrate structural responsivity of C12NO/dioleoylphospha-tidylethanolamine (DOPE)/DNA complexes designed as pH sensitive gene delivery vectors. Small angle neutron scattering (SANS) was employed to follow kinetics of C12NO protonization and DNA binding into C12NO/DOPE/DNA complexes in solution of 150 mM NaCl at acidic condition. SANS data analyzed using paracrystal lamellar model show the formation of complexes with stacking up to ∼32 bilayers, spacing ∼ 62 Å, and lipid bilayer thickness ∼37 Å in 3 minutes after changing pH from 7 to 4. Subsequent structural reorganization of the complexes was observed along 90 minutes of SANS mesurements.

Conformational heterogeneity and bubble dynamics in single bacterial transcription initiation complexes

PubMed Central

Duchi, Diego; Gryte, Kristofer; Robb, Nicole C; Morichaud, Zakia; Sheppard, Carol; Wigneshweraraj, Sivaramesh

2018-01-01

Abstract Transcription initiation is a major step in gene regulation for all organisms. In bacteria, the promoter DNA is first recognized by RNA polymerase (RNAP) to yield an initial closed complex. This complex subsequently undergoes conformational changes resulting in DNA strand separation to form a transcription bubble and an RNAP-promoter open complex; however, the series and sequence of conformational changes, and the factors that influence them are unclear. To address the conformational landscape and transitions in transcription initiation, we applied single-molecule Förster resonance energy transfer (smFRET) on immobilized Escherichia coli transcription open complexes. Our results revealed the existence of two stable states within RNAP–DNA complexes in which the promoter DNA appears to adopt closed and partially open conformations, and we observed large-scale transitions in which the transcription bubble fluctuated between open and closed states; these transitions, which occur roughly on the 0.1 s timescale, are distinct from the millisecond-timescale dynamics previously observed within diffusing open complexes. Mutational studies indicated that the σ70 region 3.2 of the RNAP significantly affected the bubble dynamics. Our results have implications for many steps of transcription initiation, and support a bend-load-open model for the sequence of transitions leading to bubble opening during open complex formation. PMID:29177430

FISH-aimed karyotyping and characterization of Renner complexes in permanent heterozygote Rhoeo spathacea.

PubMed

Golczyk, Hieronim; Hasterok, Robert; Joachimiak, Andrzej J

2005-02-01

Fluorescence in situ hybridization (FISH) using 25S rDNA, 5S rDNA, and telomere sequences as probes was carried out in the complex permanent heterozygote Rhoeo spathacea. Telomere sites were exclusively terminal. All 10 25S rDNA loci were located distally and appeared transcriptionally active after silver staining. Six distal and 2 interstitial 5S rDNA sites were detected; 2 of the distal sites strictly colocalized with 25S rDNA loci. The 2 intercalary 5S rDNA loci occurred in short arms of 2 chromosomes that conjoined at meiosis. Chromosomes differed as to the amount of AT-rich centric heterochromatin, suggesting involvement of pericentromeric regions in translocations. The possibility of Robertsonian-like rearrangements was discussed. Double target FISH with ribosomal probes along with DAPI fluorescence gave the basis for full chromosome identification in mitosis. The 2 Renner complexes are structurally balanced, both having 5 25S and 4 5S rDNA sites. Centromere clustering, telomere association, a high number of NOR sites, and a strong tendency for formation of joint nucleoli contribute to the preservation of highly polarized Rabl arrangement at interphase. These findings were discussed in relation to meiotic catenation in Rhoeo.

Modeling DNA bubble formation at the atomic scale

DOE Office of Scientific and Technical Information (OSTI.GOV)

Beleva, V; Rasmussen, K. O.; Garcia, A. E.

We describe the fluctuations of double stranded DNA molecules using a minimalist Go model over a wide range of temperatures. Minimalist models allow us to describe, at the atomic level, the opening and formation of bubbles in DNA double helices. This model includes all the geometrical constraints in helix melting imposed by the 3D structure of the molecule. The DNA forms melted bubbles within double helices. These bubbles form and break as a function of time. The equilibrium average number of broken base pairs shows a sharp change as a function of T. We observe a temperature profile of sequencemore » dependent bubble formation similar to those measured by Zeng et al. Long nuclei acid molecules melt partially through the formations of bubbles. It is known that CG rich sequences melt at higher temperatures than AT rich sequences. The melting temperature, however, is not solely determined by the CG content, but by the sequence through base stacking and solvent interactions. Recently, models that incorporate the sequence and nonlinear dynamics of DNA double strands have shown that DNA exhibits a very rich dynamics. Recent extensions of the Bishop-Peyrard model show that fluctuations in the DNA structure lead to opening in localized regions, and that these regions in the DNA are associated with transcription initiation sites. 1D and 2D models of DNA may contain enough information about stacking and base pairing interactions, but lack the coupling between twisting, bending and base pair opening imposed by the double helical structure of DNA that all atom models easily describe. However, the complexity of the energy function used in all atom simulations (including solvent, ions, etc) does not allow for the description of DNA folding/unfolding events that occur in the microsecond time scale.« less

Two-stage DNA compaction induced by silver ions suggests a cooperative binding mechanism

NASA Astrophysics Data System (ADS)

Jiang, Wen-Yan; Ran, Shi-Yong

2018-05-01

The interaction between silver ions and DNA plays an important role in the therapeutic use of silver ions and in related technologies such as DNA sensors. However, the underlying mechanism has not been fully understood. In this study, the dynamics of Ag+-DNA interaction at a single-molecule level was studied using magnetic tweezers. AgNO3 solutions with concentrations ranging from 1 μM to 20 μM led to a 1.4-1.8 μm decrease in length of a single λ-DNA molecule, indicating that Ag+ has a strong binding with DNA, causing the DNA conformational change. The compaction process comprises one linear declining stage and another sigmoid-shaped stage, which can be attributed to the interaction mechanism. Considering the cooperative effect, the sigmoid trend was well explained using a phenomenological model. By contrast, addition of silver nanoparticle solution induced no detectable transition of DNA. The dependence of the interaction on ionic strength and DNA concentration was examined via morphology characterization and particle size distribution measurement. The size of the Ag+-DNA complex decreased with an increase in Ag+ ionic strength ranging from 1 μM to 1 mM. Morphology characterization confirmed that silver ions induced DNA to adopt a compacted globular conformation. At a fixed [AgNO3]:[DNA base pairs] ratio, increasing DNA concentration led to increased sizes of the complexes. Intermolecular interaction is believed to affect the Ag+-DNA complex formation to a large extent.

Condensin suppresses recombination and regulates double-strand break processing at the repetitive ribosomal DNA array to ensure proper chromosome segregation during meiosis in budding yeast

PubMed Central

Li, Ping; Jin, Hui; Yu, Hong-Guo

2014-01-01

During meiosis, homologues are linked by crossover, which is required for bipolar chromosome orientation before chromosome segregation at anaphase I. The repetitive ribosomal DNA (rDNA) array, however, undergoes little or no meiotic recombination. Hyperrecombination can cause chromosome missegregation and rDNA copy number instability. We report here that condensin, a conserved protein complex required for chromosome organization, regulates double-strand break (DSB) formation and repair at the rDNA gene cluster during meiosis in budding yeast. Condensin is highly enriched at the rDNA region during prophase I, released at the prophase I/metaphase I transition, and reassociates with rDNA before anaphase I onset. We show that condensin plays a dual role in maintaining rDNA stability: it suppresses the formation of Spo11-mediated rDNA breaks, and it promotes DSB processing to ensure proper chromosome segregation. Condensin is unnecessary for the export of rDNA breaks outside the nucleolus but required for timely repair of meiotic DSBs. Our work reveals that condensin coordinates meiotic recombination with chromosome segregation at the repetitive rDNA sequence, thereby maintaining genome integrity. PMID:25103240

Self-assembly programming of DNA polyominoes.

PubMed

Ong, Hui San; Syafiq-Rahim, Mohd; Kasim, Noor Hayaty Abu; Firdaus-Raih, Mohd; Ramlan, Effirul Ikhwan

2016-10-20

Fabrication of functional DNA nanostructures operating at a cellular level has been accomplished through molecular programming techniques such as DNA origami and single-stranded tiles (SST). During implementation, restrictive and constraint dependent designs are enforced to ensure conformity is attainable. We propose a concept of DNA polyominoes that promotes flexibility in molecular programming. The fabrication of complex structures is achieved through self-assembly of distinct heterogeneous shapes (i.e., self-organised optimisation among competing DNA basic shapes) with total flexibility during the design and assembly phases. In this study, the plausibility of the approach is validated using the formation of multiple 3×4 DNA network fabricated from five basic DNA shapes with distinct configurations (monomino, tromino and tetrominoes). Computational tools to aid the design of compatible DNA shapes and the structure assembly assessment are presented. The formations of the desired structures were validated using Atomic Force Microscopy (AFM) imagery. Five 3×4 DNA networks were successfully constructed using combinatorics of these five distinct DNA heterogeneous shapes. Our findings revealed that the construction of DNA supra-structures could be achieved using a more natural-like orchestration as compared to the rigid and restrictive conventional approaches adopted previously. Copyright © 2016 Elsevier B.V. All rights reserved.

Surface salt bridges modulate DNA wrapping by the type II DNA-binding protein TF1.

PubMed

Grove, Anne

2003-07-29

The histone-like protein HU is involved in compaction of the bacterial genome. Up to 37 bp of DNA may be wrapped about some HU homologues in a process that has been proposed to depend on a linked disruption of surface salt bridges that liberates cationic side chains for interaction with the DNA. Despite significant sequence conservation between HU homologues, binding sites from 9 to 37 bp have been reported. TF1, an HU homologue that is encoded by Bacillus subtilis bacteriophage SPO1, has nM affinity for 37 bp preferred sites in DNA with 5-hydroxymethyluracil (hmU) in place of thymine. On the basis of electrophoretic mobility shift assays, we show that TF1-DNA complex formation is associated with a net release of only approximately 0.5 cations. The structure of TF1 suggests that Asp13 can form a dehydrated surface salt bridge with Lys23; substitution of Asp13 with Ala increases the net release of cations to approximately 1. These data are consistent with complex formation linked to disruption of surface salt bridges. Substitution of Glu90 with Ala, which would expose Lys87 predicted to contact DNA immediately distal to a proline-mediated DNA kink, causes an increase in affinity and an abrogation of the preference for hmU-containing DNA. We propose that hmU preference is due to finely tuned interactions at the sites of kinking that expose a differential flexibility of hmU- and T-containing DNA. Our data further suggest that the difference in binding site size for HU homologues is based on a differential ability to stabilize the DNA kinks.

Role of advanced glycation on aggregation and DNA binding properties of α-synuclein.

PubMed

Padmaraju, Vasudevaraju; Bhaskar, Jamuna J; Prasada Rao, Ummiti J S; Salimath, Paramahans V; Rao, K S

2011-01-01

Parkinson's disease (PD) is a neurodegenerative disease with multiple etiologies. Advanced glycation end products (AGEs) accumulate in the aging brain and could be one of the reasons for age-related diseases like PD. Oxidative stress also leads to the formation of AGEs and may be involved in neurodegeneration by altering the properties of proteins. α-Synuclein is involved in pathogenesis of PD and there are limited studies on the role of AGE-α-synuclein in neurodegeneration. We studied the aggregation and DNA binding ability of AGE-α-synuclein in vitro. α-Synuclein is glycated using methylglyoxal and formation of AGE-α-synuclein is characterized using fluorescence studies, intrinsic tyrosine fluorescence, and fructosamine estimation. The results indicated that AGE-α-synuclein aggregates into smaller globular-like aggregates compared to fibrils formed with native α-synuclein. Further, it is found that AGE-α-synuclein induced conformational changes in scDNA from B-form to B-C-A mixed conformation. Additionally, AGE-α-synuclein altered DNA integrity as evidenced by the melting temperature, ethidium bromide, and DNAse I sensitivity studies. AGE-α-synuclein converted biphasic Tm to higher monophasic Tm. The Tm of AGE-α-synuclein-scDNA complex is more than that of native α-synuclein-scDNA complex, indicating that AGE-α-synuclein stabilized the uncoiled scDNA. AGE-α-synuclein could stabilize the uncoiled scDNA, as shown by the decrease in the number of ethidium bromide binding molecules per base pair of DNA. DNAse I sensitive studies indicated that both AGE-α-synuclein-scDNA and α-synuclein-scDNA are resistant to DNAse I digestion. The relevance of these findings to neuronal cell death is discussed.

The role of extracellular DNA in uranium precipitation and biomineralisation.

PubMed

Hufton, Joseph; Harding, John H; Romero-González, Maria E

2016-10-26

Bacterial extra polymeric substances (EPS) have been associated with the extracellular precipitation of uranium. Here we report findings on the biomineralisation of uranium, with extracellular DNA (eDNA) used as a model biomolecule representative of EPS. The complexation and precipitation of eDNA with uranium were investigated as a function of pH, ionic strength and varying concentrations of reactants. The role of phosphate moieties in the biomineralisation mechanism was studied by enzymatically releasing phosphate (ePO 4 ) from eDNA compared to abiotic phosphate (aPO 4 ). The eDNA-uranium precipitates and uranium minerals obtained were characterised by Attenuated Total Reflectance-Fourier Transform Infrared (ATR-FT-IR) spectroscopy, Scanning Electron Microscopy-Energy Dispersive X-Ray analysis (SEM-EDX), X-Ray Powder Diffraction (XRD) and X-Ray Photoelectron Spectroscopy (XPS). ATR-FT-IR showed that at pH 5, the eDNA-uranium precipitation mechanism was predominantly mediated by interactions with phosphate moieties from eDNA. At pH 2, the uranium interactions with eDNA occur mainly through phosphate. The solubility equilibrium was dependent on pH with the formation of precipitate reduced as the pH increased. The XRD data confirmed the formation of a uranium phosphate precipitate when synthesised using ePO 4 . XPS and SEM-EDX studies showed the incorporation of carbon and nitrogen groups from the enzymatic orthophosphate hydrolysis on the obtained precipitated. These results suggested that the removal of uranium from solution occurs via two mechanisms: complexation by eDNA molecules and precipitation of a uranium phosphate mineral of the type (UO 2 HPO 4 )·xH 2 O by enzymatic orthophosphate hydrolysis. This demonstrated that eDNA from bacterial EPS is a key contributor to uranium biomineralisation.

[The study of complex-formation of DNA with the antimicrobial drug decamethoxine].

PubMed

Sorokin, V A; Blagoĭ, Iu P; Valeev, V A; Gladchenko, G O; Sukhodub, L F; Volianskiĭ, Iu L

1990-01-01

The interaction of effective antibacterial drug decametoxyn with natural DNA was studied by UV-spectroscopy. Decametoxyn shows a specificity to nucleotides: it decreases the cooperativity of melting and the thermal stability of DNA parts enriched by AT pairs. The characteristics of the helix-coil transition on the DNA parts enriched by GC-pairs are invariable. Interaction with AT-pairs results in their partial or complete melting at room temperature, followed by intermolecule aggregation. Interacting with phosphates decametoxyn manifests itself not as a dication but as two single-charged ions.

Binding regularities in complexes of transcription factors with operator DNA: homeodomain family.

PubMed

Chirgadze, Yu N; Zheltukhin, E I; Polozov, R V; Sivozhelezov, V S; Ivanov, V V

2009-06-01

In order to disclose general regularities of binding in homeodomain-DNA complexes we considered five of them and extended the observed regularities over the entire homeodomain family. The five complexes have been selected by similarity of protein structures and patterns of contacting residues. Their long range interactions and interfaces were compared. The long-range stage of the recognition process was characterized by electrostatic potentials about 5 Angstrom away from molecular surfaces of protein or DNA. For proteins, clear positive potential is displayed only at the side contacting the DNA. The double-chained DNA molecule displays a rather strong negative potential, especially in their grooves. Thus, a functional role of electrostatics is a guiding of the protein into the DNA major groove, so the protein and DNA could form a loose non-specific complex. At the close-range stage, neutralization of the phosphate charges by positively charged residues is necessary for decreasing the strong electrostatic potential of DNA, allowing nucleotide bases to participate in the formation of protein-DNA atomic contacts in the interface. The recognizing alpha-helix of protein was shown to form both invariant and variable groups of contacts with DNA by means of certain specific side groups. The invariant contacts included highly specific protein-DNA hydrogen bonds between asparagine and adenine, nonpolar contacts of hydrophobic amino acids serving as a stereochemical barrier for fixing the protein factor on DNA, and an interface cluster of water molecules providing local conformational mobility necessary for the dissociation process. There is a unique water molecule within the interface that is conservative and located at the interface center. Invariant contacts of the proteins are mostly formed with the TAAT motif of the promoter DNA forward strand. While the invariant contacts specify the family of homeodomains, the variable contacts that are formed with the reverse strand of DNA provide specificity of individual complexes within the homeodomain family.

Comparing anterior and posterior Hox complex formation reveals guidelines for predicting cis-regulatory elements

PubMed Central

Uhl, Juli D.; Cook, Tiffany A.; Gebelein, Brian

2010-01-01

Hox transcription factors specify numerous cell fates along the anterior-posterior axis by regulating the expression of downstream target genes. While expression analysis has uncovered large numbers of de-regulated genes in cells with altered Hox activity, determining which are direct versus indirect targets has remained a significant challenge. Here, we characterize the DNA binding activity of Hox transcription factor complexes on eight experimentally verified cis-regulatory elements. Hox factors regulate the activity of each element by forming protein complexes with two cofactor proteins, Extradenticle (Exd) and Homothorax (Hth). Using comparative DNA binding assays, we found that a number of flexible arrangements of Hox, Exd, and Hth binding sites mediate cooperative transcription factor complexes. Moreover, analysis of a Distal-less regulatory element (DMXR) that is repressed by abdominal Hox factors revealed that suboptimal binding sites can be combined to form high affinity transcription complexes. Lastly, we determined that the anterior Hox factors are more dependent upon Exd and Hth for complex formation than posterior Hox factors. Based upon these findings, we suggest a general set of guidelines to serve as a basis for designing bioinformatics algorithms aimed at identifying Hox regulatory elements using the wealth of recently sequenced genomes. PMID:20398649

Cationic polymers for DNA origami coating - examining their binding efficiency and tuning the enzymatic reaction rates

NASA Astrophysics Data System (ADS)

Kiviaho, Jenny K.; Linko, Veikko; Ora, Ari; Tiainen, Tony; Järvihaavisto, Erika; Mikkilä, Joona; Tenhu, Heikki; Nonappa, Affc; Kostiainen, Mauri A.

2016-06-01

DNA origamis are fully tailored, programmable, biocompatible and readily functionalizable nanostructures that provide an excellent foundation for the development of sophisticated drug-delivery systems. However, the DNA origami objects suffer from certain drawbacks such as low cell-transfection rates and low stability. A great deal of studies on polymer-based transfection agents, mainly focusing on polyplex formation and toxicity, exists. In this study, the electrostatic binding between a brick-like DNA origami and cationic block-copolymers was explored. The effect of the polymer structure on the binding was investigated and the toxicity of the polymer-origami complexes evaluated. The study shows that all of the analyzed polymers had a suitable binding efficiency irrespective of the block structure. It was also observed that the toxicity of polymer-origami complexes was insignificant at the biologically relevant concentration levels. Besides brick-like DNA origamis, tubular origami carriers equipped with enzymes were also coated with the polymers. By adjusting the amount of cationic polymers that cover the DNA structures, we showed that it is possible to control the enzyme kinetics of the complexes. This work gives a starting point for further development of biocompatible and effective polycation-based block copolymers that can be used in coating different DNA origami nanostructures for various bioapplications.DNA origamis are fully tailored, programmable, biocompatible and readily functionalizable nanostructures that provide an excellent foundation for the development of sophisticated drug-delivery systems. However, the DNA origami objects suffer from certain drawbacks such as low cell-transfection rates and low stability. A great deal of studies on polymer-based transfection agents, mainly focusing on polyplex formation and toxicity, exists. In this study, the electrostatic binding between a brick-like DNA origami and cationic block-copolymers was explored. The effect of the polymer structure on the binding was investigated and the toxicity of the polymer-origami complexes evaluated. The study shows that all of the analyzed polymers had a suitable binding efficiency irrespective of the block structure. It was also observed that the toxicity of polymer-origami complexes was insignificant at the biologically relevant concentration levels. Besides brick-like DNA origamis, tubular origami carriers equipped with enzymes were also coated with the polymers. By adjusting the amount of cationic polymers that cover the DNA structures, we showed that it is possible to control the enzyme kinetics of the complexes. This work gives a starting point for further development of biocompatible and effective polycation-based block copolymers that can be used in coating different DNA origami nanostructures for various bioapplications. Electronic supplementary information (ESI) available: Details of materials, syntheses of the polymers, fabrication and purification of DNA origamis, luminescence decay assays, agarose gel electrophoresis, ethidium bromide displacement assay, MTT assay and TEM characterization. See DOI: 10.1039/c5nr08355a

Conformational heterogeneity in the Hsp70 chaperone-substrate ensemble identified from analysis of NMR-detected titration data.

PubMed

Sekhar, Ashok; Nagesh, Jayashree; Rosenzweig, Rina; Kay, Lewis E

2017-11-01

The Hsp70 chaperone system plays a critical role in cellular homeostasis by binding to client protein molecules. We have recently shown by methyl-TROSY NMR methods that the Escherichia coli Hsp70, DnaK, can form multiple bound complexes with a small client protein, hTRF1. In an effort to characterize the interactions further we report here the results of an NMR-based titration study of hTRF1 and DnaK, where both molecular components are monitored simultaneously, leading to a binding model. A central finding is the formation of a previously undetected 3:1 hTRF1-DnaK complex, suggesting that under heat shock conditions, DnaK might be able to protect cytosolic proteins whose net concentrations would exceed that of the chaperone. Moreover, these results provide new insight into the heterogeneous ensemble of complexes formed by DnaK chaperones and further emphasize the unique role of NMR spectroscopy in obtaining information about individual events in a complex binding scheme by exploiting a large number of probes that report uniquely on distinct binding processes. © 2017 The Protein Society.

Emerging players in the initiation of eukaryotic DNA replication

PubMed Central

2012-01-01

Faithful duplication of the genome in eukaryotes requires ordered assembly of a multi-protein complex called the pre-replicative complex (pre-RC) prior to S phase; transition to the pre-initiation complex (pre-IC) at the beginning of DNA replication; coordinated progression of the replisome during S phase; and well-controlled regulation of replication licensing to prevent re-replication. These events are achieved by the formation of distinct protein complexes that form in a cell cycle-dependent manner. Several components of the pre-RC and pre-IC are highly conserved across all examined eukaryotic species. Many of these proteins, in addition to their bona fide roles in DNA replication are also required for other cell cycle events including heterochromatin organization, chromosome segregation and centrosome biology. As the complexity of the genome increases dramatically from yeast to human, additional proteins have been identified in higher eukaryotes that dictate replication initiation, progression and licensing. In this review, we discuss the newly discovered components and their roles in cell cycle progression. PMID:23075259

Autonomous replication of nucleic acids by polymerization/nicking enzyme/DNAzyme cascades for the amplified detection of DNA and the aptamer-cocaine complex.

PubMed

Wang, Fuan; Freage, Lina; Orbach, Ron; Willner, Itamar

2013-09-03

The progressive development of amplified DNA sensors and aptasensors using replication/nicking enzymes/DNAzyme machineries is described. The sensing platforms are based on the tailoring of a DNA template on which the recognition of the target DNA or the formation of the aptamer-substrate complex trigger on the autonomous isothermal replication/nicking processes and the displacement of a Mg(2+)-dependent DNAzyme that catalyzes the generation of a fluorophore-labeled nucleic acid acting as readout signal for the analyses. Three different DNA sensing configurations are described, where in the ultimate configuration the target sequence is incorporated into a nucleic acid blocker structure associated with the sensing template. The target-triggered isothermal autonomous replication/nicking process on the modified template results in the formation of the Mg(2+)-dependent DNAzyme tethered to a free strand consisting of the target sequence. This activates additional template units for the nucleic acid self-replication process, resulting in the ultrasensitive detection of the target DNA (detection limit 1 aM). Similarly, amplified aptamer-based sensing platforms for cocaine are developed along these concepts. The modification of the cocaine-detection template by the addition of a nucleic acid sequence that enables the autonomous secondary coupled activation of a polymerization/nicking machinery and DNAzyme generation path leads to an improved analysis of cocaine (detection limit 10 nM).

Genotype Specification Language.

PubMed

Wilson, Erin H; Sagawa, Shiori; Weis, James W; Schubert, Max G; Bissell, Michael; Hawthorne, Brian; Reeves, Christopher D; Dean, Jed; Platt, Darren

2016-06-17

We describe here the Genotype Specification Language (GSL), a language that facilitates the rapid design of large and complex DNA constructs used to engineer genomes. The GSL compiler implements a high-level language based on traditional genetic notation, as well as a set of low-level DNA manipulation primitives. The language allows facile incorporation of parts from a library of cloned DNA constructs and from the "natural" library of parts in fully sequenced and annotated genomes. GSL was designed to engage genetic engineers in their native language while providing a framework for higher level abstract tooling. To this end we define four language levels, Level 0 (literal DNA sequence) through Level 3, with increasing abstraction of part selection and construction paths. GSL targets an intermediate language based on DNA slices that translates efficiently into a wide range of final output formats, such as FASTA and GenBank, and includes formats that specify instructions and materials such as oligonucleotide primers to allow the physical construction of the GSL designs by individual strain engineers or an automated DNA assembly core facility.

Transcription-Replication Conflict Orientation Modulates R-Loop Levels and Activates Distinct DNA Damage Responses.

PubMed

Hamperl, Stephan; Bocek, Michael J; Saldivar, Joshua C; Swigut, Tomek; Cimprich, Karlene A

2017-08-10

Conflicts between transcription and replication are a potent source of DNA damage. Co-transcriptional R-loops could aggravate such conflicts by creating an additional barrier to replication fork progression. Here, we use a defined episomal system to investigate how conflict orientation and R-loop formation influence genome stability in human cells. R-loops, but not normal transcription complexes, induce DNA breaks and orientation-specific DNA damage responses during conflicts with replication forks. Unexpectedly, the replisome acts as an orientation-dependent regulator of R-loop levels, reducing R-loops in the co-directional (CD) orientation but promoting their formation in the head-on (HO) orientation. Replication stress and deregulated origin firing increase the number of HO collisions leading to genome-destabilizing R-loops. Our findings connect DNA replication to R-loop homeostasis and suggest a mechanistic basis for genome instability resulting from deregulated DNA replication, observed in cancer and other disease states. Copyright © 2017 Elsevier Inc. All rights reserved.

Hybridization behavior of mixed DNA/alkylthiol monolayers on gold: characterization by surface plasmon resonance and 32P radiometric assay.

PubMed

Gong, Ping; Lee, Chi-Ying; Gamble, Lara J; Castner, David G; Grainger, David W

2006-05-15

Nucleic acid assay from a complex biological milieu is attractive but currently difficult and far from routine. In this study, DNA hybridization from serum dilutions into mixed DNA/mercaptoundecanol (MCU) adlayers on gold was monitored by surface plasmon resonance (SPR). Immobilized DNA probe and hybridized target densities on these surfaces were quantified using 32P-radiometric assays as a function of MCU diluent exposure. SPR surface capture results correlated with radiometric analysis for hybridization performance, demonstrating a maximum DNA hybridization on DNA/MCU mixed adlayers. The maximum target surface capture produced by MCU addition to the DNA probe layer correlates with structural and conformational data on identical mixed DNA/MCU adlayers on gold derived from XPS, NEXAFS, and fluorescence intensity measurements reported in a related study (Lee, C.-Y.; Gong, P.; Harbers, G. M.; Grainger, D. W.; Castner, D. G.; Gamble, L. J. Anal. Chem. 2006, 78, 3316-3325.). MCU addition into the DNA adlayer on gold also improved surface resistance to both nonspecific DNA and serum protein adsorption. Target DNA hybridization from serum dilutions was monitored with SPR on the optimally mixed DNA/MCU adlayers. Both hybridization kinetics and efficiency were strongly affected by nonspecific protein adsorption from a complex milieu even at a minimal serum concentration (e.g., 1%). No target hybridization was detected in SPR assays from serum concentrations above 30%, indicating nonspecific protein adsorption interference of DNA capture and hybridization from complex milieu. Removal of nonsignal proteins from nucleic acid targets prior to assay represents a significant issue for direct sample-to-assay nucleic acid diagnostics from food, blood, tissue, PCR mixtures, and many other biologically complex sample formats.

Reversed-phase ion-pair liquid chromatography method for purification of duplex DNA with single base pair resolution

PubMed Central

Wysoczynski, Christina L.; Roemer, Sarah C.; Dostal, Vishantie; Barkley, Robert M.; Churchill, Mair E. A.; Malarkey, Christopher S.

2013-01-01

Obtaining quantities of highly pure duplex DNA is a bottleneck in the biophysical analysis of protein–DNA complexes. In traditional DNA purification methods, the individual cognate DNA strands are purified separately before annealing to form DNA duplexes. This approach works well for palindromic sequences, in which top and bottom strands are identical and duplex formation is typically complete. However, in cases where the DNA is non-palindromic, excess of single-stranded DNA must be removed through additional purification steps to prevent it from interfering in further experiments. Here we describe and apply a novel reversed-phase ion-pair liquid chromatography purification method for double-stranded DNA ranging in lengths from 17 to 51 bp. Both palindromic and non-palindromic DNA can be readily purified. This method has the unique ability to separate blunt double-stranded DNA from pre-attenuated (n-1, n-2, etc) synthesis products, and from DNA duplexes with single base pair overhangs. Additionally, palindromic DNA sequences with only minor differences in the central spacer sequence of the DNA can be separated, and the purified DNA is suitable for co-crystallization of protein–DNA complexes. Thus, double-stranded ion-pair liquid chromatography is a useful approach for duplex DNA purification for many applications. PMID:24013567

Synthesis, physicochemical studies, embryos toxicity and DNA interaction of some new Iron(II) Schiff base amino acid complexes

NASA Astrophysics Data System (ADS)

Abdel-Rahman, Laila H.; El-Khatib, Rafat M.; Nassr, Lobna A. E.; Abu-Dief, Ahmed M.

2013-05-01

New Fe(II) Schiff base amino acid complexes derived from the condensation of o-hydroxynaphthaldehyde with L-alanine, L-phenylalanine, L-aspartic acid, L-histidine and L-arginine were synthesized and characterized by elemental analysis, IR, electronic spectra, and conductance measurements. The stoichiometry and the stability constants of the complexes were determined spectrophotometrically. The investigated Schiff bases exhibited tridentate coordination mode with the general formulae [Fe(HL)2]·nH2O for all amino acids except L-histidine. But in case of L-histidine, the ligand acts as tetradentate ([FeL(H2O)2]·2H2O), where HL = mono anion and L = dianion of the ligand. The structure of the prepared complexes is suggested to be octahedral. The prepared complexes were tested for their toxicity on chick embryos and found to be safe until a concentration of 100 μg/egg with full embryos formation. The interaction between CT-DNA and the investigated complexes were followed by spectrophotometry and viscosity measurements. It was found that, the prepared complexes bind to DNA via classical intercalative mode and showed a different DNA cleavage activity with the sequence: nhi > nari > nali > nasi > nphali. The thermodynamic Profile of the binding of nphali complex and CT-DNA was constructed by analyzing the experimental data of absorption titration and UV melting studies with the McGhee equation, van't Hoff's equation, and the Gibbs-Helmholtz equation.

New Insights into the Mechanism Underlying the Synergistic Action of Ionizing Radiation With Platinum Chemotherapeutic Drugs: The Role of Low-Energy Electrons

DOE Office of Scientific and Technical Information (OSTI.GOV)

Rezaee, Mohammad, E-mail: Mohammad.Rezaee@USherbrooke.ca; Hunting, Darel John; Sanche, Léon

Purpose: To investigate the efficiencies of platinum chemotherapeutic drugs (Pt-drugs) in the sensitization of DNA to the direct effects of ionizing radiation and to determine the role of low-energy electrons (LEEs) in this process. Methods and Materials: Complexes of supercoiled plasmid DNA covalently bound to either cisplatin, carboplatin, or oxaliplatin were prepared in different molar ratios. Solid films of DNA and DNA modified by Pt-drugs were irradiated with either 10-KeV or 10-eV electrons. Damages to DNA were quantified by gel electrophoresis, and the yields for damage formation were obtained from exposure–response curves. Results: The presence of an average of 2more » Pt-drug–DNA adducts (Pt-adducts) in 3199-bp plasmid DNA increases the probability of a double-strand break by factors of 3.1, 2.5, and 2.4 for carboplatin, cisplatin, and oxaliplatin, respectively. Electrons with energies of 10 eV and 10 KeV interact with Pt-adducts to preferentially enhance the formation of cluster lesions. The maximum increase in radiosensitivity per Pt-adduct is found at ratios up to 3.1 × 10{sup −4} Pt-adducts per nucleotide, which is equivalent to an average of 2 adducts per plasmid. Carboplatin and oxaliplatin show higher efficiencies than cisplatin in the radiosensitization of DNA. Because carboplatin and cisplatin give rise to identical reactive species that attach to DNA, carboplatin must be considered as a better radiosensitizer for equal numbers of Pt-adducts. Conclusion: Platinum chemotherapeutic drugs preferentially enhance the formation of cluster damage to DNA induced by the direct effect of ionizing radiation, and LEEs are the main species responsible for such an enhancement via the formation of electron resonances.« less

New insights into the mechanism underlying the synergistic action of ionizing radiation with platinum chemotherapeutic drugs: the role of low-energy electrons.

PubMed

Rezaee, Mohammad; Hunting, Darel John; Sanche, Léon

2013-11-15

To investigate the efficiencies of platinum chemotherapeutic drugs (Pt-drugs) in the sensitization of DNA to the direct effects of ionizing radiation and to determine the role of low-energy electrons (LEEs) in this process. Complexes of supercoiled plasmid DNA covalently bound to either cisplatin, carboplatin, or oxaliplatin were prepared in different molar ratios. Solid films of DNA and DNA modified by Pt-drugs were irradiated with either 10-KeV or 10-eV electrons. Damages to DNA were quantified by gel electrophoresis, and the yields for damage formation were obtained from exposure-response curves. The presence of an average of 2 Pt-drug-DNA adducts (Pt-adducts) in 3199-bp plasmid DNA increases the probability of a double-strand break by factors of 3.1, 2.5, and 2.4 for carboplatin, cisplatin, and oxaliplatin, respectively. Electrons with energies of 10 eV and 10 KeV interact with Pt-adducts to preferentially enhance the formation of cluster lesions. The maximum increase in radiosensitivity per Pt-adduct is found at ratios up to 3.1×10(-4) Pt-adducts per nucleotide, which is equivalent to an average of 2 adducts per plasmid. Carboplatin and oxaliplatin show higher efficiencies than cisplatin in the radiosensitization of DNA. Because carboplatin and cisplatin give rise to identical reactive species that attach to DNA, carboplatin must be considered as a better radiosensitizer for equal numbers of Pt-adducts. Platinum chemotherapeutic drugs preferentially enhance the formation of cluster damage to DNA induced by the direct effect of ionizing radiation, and LEEs are the main species responsible for such an enhancement via the formation of electron resonances. Copyright © 2013 Elsevier Inc. All rights reserved.

The phase behavior of cationic lipid-DNA complexes.

PubMed Central

May, S; Harries, D; Ben-Shaul, A

2000-01-01

We present a theoretical analysis of the phase behavior of solutions containing DNA, cationic lipids, and nonionic (helper) lipids. Our model allows for five possible structures, treated as incompressible macroscopic phases: two lipid-DNA composite (lipoplex) phases, namely, the lamellar (L(alpha)(C)) and hexagonal (H(II)(C)) complexes; two binary (cationic/neutral) lipid phases, that is, the bilayer (L(alpha)) and inverse-hexagonal (H(II)) structures, and uncomplexed DNA. The free energy of the four lipid-containing phases is expressed as a sum of composition-dependent electrostatic, elastic, and mixing terms. The electrostatic free energies of all phases are calculated based on Poisson-Boltzmann theory. The phase diagram of the system is evaluated by minimizing the total free energy of the three-component mixture with respect to all the compositional degrees of freedom. We show that the phase behavior, in particular the preferred lipid-DNA complex geometry, is governed by a subtle interplay between the electrostatic, elastic, and mixing terms, which depend, in turn, on the lipid composition and lipid/DNA ratio. Detailed calculations are presented for three prototypical systems, exhibiting markedly different phase behaviors. The simplest mixture corresponds to a rigid planar membrane as the lipid source, in which case, only lamellar complexes appear in solution. When the membranes are "soft" (i.e., low bending modulus) the system exhibits the formation of both lamellar and hexagonal complexes, sometimes coexisting with each other, and with pure lipid or DNA phases. The last system corresponds to a lipid mixture involving helper lipids with strong propensity toward the inverse-hexagonal phase. Here, again, the phase diagram is rather complex, revealing a multitude of phase transitions and coexistences. Lamellar and hexagonal complexes appear, sometimes together, in different regions of the phase diagram. PMID:10733951

Development of a slow non-viral DNA release system from PDLLA scaffolds fabricated using a supercritical CO2 technique.

PubMed

Heyde, Mieke; Partridge, Kris A; Howdle, Steven M; Oreffo, Richard O C; Garnett, Martin C; Shakesheff, Kevin M

2007-10-15

Polyamidoamine polymers (PAA) comprising methylene-bisacrylamide/dimethylethylene-diamine monomers were synthesized, complexed with DNA and incorporated into porous P(DL)LA scaffolds by using a supercritical CO(2) (scCO(2)) technique. Scaffolds were made in a dry state consequently there was a need to lyophilize the complexes. A statistically significant reduction of the transfection efficiency was observed in the absence of trehalose when compared to the original complex after freeze-drying. Increasing concentrations (0-10% w/v) of trehalose were added to the complex prior to freeze-drying. Structure dependent differences in DNA binding were evaluated by gel electrophoresis and thermal transition analysis. TEM and PCS showed aggregate formation after freeze-drying without trehalose. Scaffolds were characterized by pore sizes of 173 +/- 73 microm and a porosity of 71%. The transfection potential of the released DNA was investigated by seeding scaffolds with A549 cells and following firefly luciferase as a marker gene after 48 h exposure. Low but continuous levels of transfection were observed for PAA complexes during a 60-day study. Complexes made with Lipofectaminetrade mark gave initially higher levels of DNA release but no further expression was seen after 40 days. Uncomplexed DNA showed background levels of transfection. Culturing cells on 3D scaffolds showed a benefit in retention of transfection activity with time compared to 2D controls. Transfection levels could be increased when cells were grown in OptiMEM. This study demonstrated that PAA/DNA complexes incorporated into a P(DL)LA scaffold made by using scCO(2) processing exhibited a slow release and extended gene expression profile. Copyright 2007 Wiley Periodicals, Inc.

Addition of DNA to CrVI and Cytochrome b5 Containing Proteoliposomes Leads to Generation of DNA Strand Breaks and CrIII Complexes

PubMed Central

Borthiry, Griselda R.; Antholine, William E.; Myers, Judith M.; Myers, Charles R.

2009-01-01

Chromium (Cr) is a cytotoxic metal that can be associated with a variety of types of DNA damage, including Cr-DNA adducts and strand breaks. Prior studies with purified human cytochrome b5 and NADPH :P450 reductase in reconstituted proteoliposomes (PLs) demonstrated rapid reduction of CrVI (hexavalent chromium, as CrO42− ), and the generation of CrV, superoxide (O2·−) , and hydroxyl radical (HO˙). Studies reported here examined the potential for the species produced by this system to interact with DNA. Strand breaks of purified plasmid DNA increased over time aerobically, but were not observed in the absence of O2. CrV is formed under both conditions, so the breaks are not mediated directly by CrV. The aerobic strand breaks were significantly prevented by catalase and EtOH, but not by the metal chelator diethylenetriaminepentaacetic acid (DTPA), suggesting that they are largely due to HO˙ from Cr-mediated redox cycling. EPR was used to assess the formation of Cr-DNA complexes. Following a 10-min incubation of PLs, CrO42− , and plasmid DNA, intense EPR signals at g = 5.7and g = 5.0 were observed. These signals are attributed to specific CrIII complexes with large zero field splitting (ZFS). Without DNA, the signals in the g = 5 region were weak. The large ZFS signals were not seen, when CrIIICl3 was incubated with DNA, suggesting that the CrIII–DNA interactions are different when generated by the PLs. After 24 h, a broad signal at g = 2 is attributed to CrIII complexes with a small ZFS. This g = 2 signal was observed without DNA, but it was different from that seen with plasmid. It is concluded that EPR can detect specific CrIII complexes that depend on the presence of plasmid DNA and the manner in which the CrIII is formed. PMID:18729091

Disruption of Higher Order DNA Structures in Friedreich’s Ataxia (GAA)n Repeats by PNA or LNA Targeting

PubMed Central

Bergquist, Helen; Rocha, Cristina S. J.; Álvarez-Asencio, Rubén; Nguyen, Chi-Hung; Rutland, Mark. W.; Smith, C. I. Edvard; Good, Liam; Nielsen, Peter E.; Zain, Rula

2016-01-01

Expansion of (GAA)n repeats in the first intron of the Frataxin gene is associated with reduced mRNA and protein levels and the development of Friedreich’s ataxia. (GAA)n expansions form non-canonical structures, including intramolecular triplex (H-DNA), and R-loops and are associated with epigenetic modifications. With the aim of interfering with higher order H-DNA (like) DNA structures within pathological (GAA)n expansions, we examined sequence-specific interaction of peptide nucleic acid (PNA) with (GAA)n repeats of different lengths (short: n=9, medium: n=75 or long: n=115) by chemical probing of triple helical and single stranded regions. We found that a triplex structure (H-DNA) forms at GAA repeats of different lengths; however, single stranded regions were not detected within the medium size pathological repeat, suggesting the presence of a more complex structure. Furthermore, (GAA)4-PNA binding of the repeat abolished all detectable triplex DNA structures, whereas (CTT)5-PNA did not. We present evidence that (GAA)4-PNA can invade the DNA at the repeat region by binding the DNA CTT strand, thereby preventing non-canonical-DNA formation, and that triplex invasion complexes by (CTT)5-PNA form at the GAA repeats. Locked nucleic acid (LNA) oligonucleotides also inhibited triplex formation at GAA repeat expansions, and atomic force microscopy analysis showed significant relaxation of plasmid morphology in the presence of GAA-LNA. Thus, by inhibiting disease related higher order DNA structures in the Frataxin gene, such PNA and LNA oligomers may have potential for discovery of drugs aiming at recovering Frataxin expression. PMID:27846236

Osmotically Induced Reversible Transitions in Lipid-DNA Mesophases

PubMed Central

Danino, Dganit; Kesselman, Ellina; Saper, Gadiel; Petrache, Horia I.; Harries, Daniel

2009-01-01

We follow the effect of osmotic pressure on isoelectric complexes that self-assemble from mixtures of DNA and mixed neutral and cationic lipids. Using small angle x-ray diffraction and freeze-fracture cryo-electron microscopy, we find that lamellar complexes known to form in aqueous solutions can reversibly transition to hexagonal mesophases under high enough osmotic stress exerted by adding a neutral polymer. Using molecular spacings derived from x-ray diffraction, we estimate the reversible osmotic pressure-volume (Π-V) work needed to induce this transition. We find that the transition free energy is comparable to the work required to elastically bend lipid layers around DNA. Consistent with this, the required work is significantly lowered by an addition of hexanol, which is known to soften lipid bilayers. Our findings not only help to resolve the free-energy contributions associated with lipid-DNA complex formation, but they also demonstrate the importance that osmotic stress can have to the macromolecular phase geometry in realistic biological environments. PMID:19348739

The structure of SV40 large T hexameric helicase in complex with AT-rich origin DNA

PubMed Central

Gai, Dahai; Wang, Damian; Li, Shu-Xing; Chen, Xiaojiang S

2016-01-01

DNA replication is a fundamental biological process. The initial step in eukaryotic DNA replication is the assembly of the pre-initiation complex, including the formation of two head-to-head hexameric helicases around the replication origin. How these hexameric helicases interact with their origin dsDNA remains unknown. Here, we report the co-crystal structure of the SV40 Large-T Antigen (LT) hexameric helicase bound to its origin dsDNA. The structure shows that the six subunits form a near-planar ring that interacts with the origin, so that each subunit makes unique contacts with the DNA. The origin dsDNA inside the narrower AAA+ domain channel shows partial melting due to the compression of the two phosphate backbones, forcing Watson-Crick base-pairs within the duplex to flip outward. This structure provides the first snapshot of a hexameric helicase binding to origin dsDNA, and suggests a possible mechanism of origin melting by LT during SV40 replication in eukaryotic cells. DOI: http://dx.doi.org/10.7554/eLife.18129.001 PMID:27921994

Elongator complex influences telomeric gene silencing and DNA damage response by its role in wobble uridine tRNA modification.

PubMed

Chen, Changchun; Huang, Bo; Eliasson, Mattias; Rydén, Patrik; Byström, Anders S

2011-09-01

Elongator complex is required for formation of the side chains at position 5 of modified nucleosides 5-carbamoylmethyluridine (ncm⁵U₃₄), 5-methoxycarbonylmethyluridine (mcm⁵U₃₄), and 5-methoxycarbonylmethyl-2-thiouridine (mcm⁵s²U₃₄) at wobble position in tRNA. These modified nucleosides are important for efficient decoding during translation. In a recent publication, Elongator complex was implicated to participate in telomeric gene silencing and DNA damage response by interacting with proliferating cell nuclear antigen (PCNA). Here we show that elevated levels of tRNA(Lys)(s²UUU), tRNA(Gln)(s²UUG), and tRNA(Glu)(s²UUC), which in a wild-type background contain the mcm⁵s²U nucleoside at position 34, suppress the defects in telomeric gene silencing and DNA damage response observed in the Elongator mutants. We also found that the reported differences in telomeric gene silencing and DNA damage response of various elp3 alleles correlated with the levels of modified nucleosides at U₃₄. Defects in telomeric gene silencing and DNA damage response are also observed in strains with the tuc2Δ mutation, which abolish the formation of the 2-thio group of the mcm⁵s²U nucleoside in tRNA(Lys)(mcm⁵s²UUU), tRNA(Gln)(mcm⁵s²UUG), and tRNA(Glu)(mcm⁵s²UUC). These observations show that Elongator complex does not directly participate in telomeric gene silencing and DNA damage response, but rather that modified nucleosides at U₃₄ are important for efficient expression of gene products involved in these processes. Consistent with this notion, we found that expression of Sir4, a silent information regulator required for assembly of silent chromatin at telomeres, was decreased in the elp3Δ mutants.

Elongator Complex Influences Telomeric Gene Silencing and DNA Damage Response by Its Role in Wobble Uridine tRNA Modification

PubMed Central

Chen, Changchun; Huang, Bo; Eliasson, Mattias; Rydén, Patrik; Byström, Anders S.

2011-01-01

Elongator complex is required for formation of the side chains at position 5 of modified nucleosides 5-carbamoylmethyluridine (ncm5U34), 5-methoxycarbonylmethyluridine (mcm5U34), and 5-methoxycarbonylmethyl-2-thiouridine (mcm5s2U34) at wobble position in tRNA. These modified nucleosides are important for efficient decoding during translation. In a recent publication, Elongator complex was implicated to participate in telomeric gene silencing and DNA damage response by interacting with proliferating cell nuclear antigen (PCNA). Here we show that elevated levels of tRNALys s2 UUU, tRNAGln s2 UUG, and tRNAGlu s2 UUC, which in a wild-type background contain the mcm5s2U nucleoside at position 34, suppress the defects in telomeric gene silencing and DNA damage response observed in the Elongator mutants. We also found that the reported differences in telomeric gene silencing and DNA damage response of various elp3 alleles correlated with the levels of modified nucleosides at U34. Defects in telomeric gene silencing and DNA damage response are also observed in strains with the tuc2Δ mutation, which abolish the formation of the 2-thio group of the mcm5s2U nucleoside in tRNALys mcm5s2UUU, tRNAGln mcm5s2UUG, and tRNAGlu mcm5s2UUC. These observations show that Elongator complex does not directly participate in telomeric gene silencing and DNA damage response, but rather that modified nucleosides at U34 are important for efficient expression of gene products involved in these processes. Consistent with this notion, we found that expression of Sir4, a silent information regulator required for assembly of silent chromatin at telomeres, was decreased in the elp3Δ mutants. PMID:21912530

Protein associations in DnaA-ATP hydrolysis mediated by the Hda-replicase clamp complex.

PubMed

Su'etsugu, Masayuki; Shimuta, Toh-Ru; Ishida, Takuma; Kawakami, Hironori; Katayama, Tsutomu

2005-02-25

In Escherichia coli, the activity of ATP-bound DnaA protein in initiating chromosomal replication is negatively controlled in a replication-coordinated manner. The RIDA (regulatory inactivation of DnaA) system promotes DnaA-ATP hydrolysis to produce the inactivated form DnaA-ADP in a manner depending on the Hda protein and the DNA-loaded form of the beta-sliding clamp, a subunit of the replicase holoenzyme. A highly functional form of Hda was purified and shown to form a homodimer in solution, and two Hda dimers were found to associate with a single clamp molecule. Purified mutant Hda proteins were used in a staged in vitro RIDA system followed by a pull-down assay to show that Hda-clamp binding is a prerequisite for DnaA-ATP hydrolysis and that binding is mediated by an Hda N-terminal motif. Arg(168) in the AAA(+) Box VII motif of Hda plays a role in stable homodimer formation and in DnaA-ATP hydrolysis, but not in clamp binding. Furthermore, the DnaA N-terminal domain is required for the functional interaction of DnaA with the Hda-clamp complex. Single cells contain approximately 50 Hda dimers, consistent with the results of in vitro experiments. These findings and the features of AAA(+) proteins, including DnaA, suggest the following model. DnaA-ATP is hydrolyzed at a binding interface between the AAA(+) domains of DnaA and Hda; the DnaA N-terminal domain supports this interaction; and the interaction of DnaA-ATP with the Hda-clamp complex occurs in a catalytic mode.

The distribution of DNA damage is defined by region-specific susceptibility to DNA damage formation rather than repair differences.

PubMed

Strand, Janne M; Scheffler, Katja; Bjørås, Magnar; Eide, Lars

2014-06-01

The cellular genomes are continuously damaged by reactive oxygen species (ROS) from aerobic processes. The impact of DNA damage depends on the specific site as well as the cellular state. The steady-state level of DNA damage is the net result of continuous formation and subsequent repair, but it is unknown to what extent heterogeneous damage distribution is caused by variations in formation or repair of DNA damage. Here, we used a restriction enzyme/qPCR based method to analyze DNA damage in promoter and coding regions of four nuclear genes: the two house-keeping genes Gadph and Tbp, and the Ndufa9 and Ndufs2 genes encoding mitochondrial complex I subunits, as well as mt-Rnr1 encoded by mitochondrial DNA (mtDNA). The distribution of steady-state levels of damage varied in a site-specific manner. Oxidative stress induced damage in nDNA to a similar extent in promoter and coding regions, and more so in mtDNA. The subsequent removal of damage from nDNA was efficient and comparable with recovery times depending on the initial damage load, while repair of mtDNA was delayed with subsequently slower repair rate. The repair was furthermore found to be independent of transcription or the transcription-coupled repair factor CSB, but dependent on cellular ATP. Our results demonstrate that the capacity to repair DNA is sufficient to remove exogenously induced damage. Thus, we conclude that the heterogeneous steady-state level of DNA damage in promoters and coding regions is caused by site-specific DNA damage/modifications that take place under normal metabolism. Copyright © 2014 Elsevier B.V. All rights reserved.

Compensatory functions and interdependency of the DNA-binding domain of BRCA2 with the BRCA1-PALB2-BRCA2 complex.

PubMed

Al Abo, Muthana; Dejsuphong, Donniphat; Hirota, Kouji; Yonetani, Yasukazu; Yamazoe, Mitsuyoshi; Kurumizaka, Hitoshi; Takeda, Shunichi

2014-02-01

BRCA1, BRCA2, and PALB2 are key players in cellular tolerance to chemotherapeutic agents, including camptothecin, cisplatin, and PARP inhibitor. The N-terminal segment of BRCA2 interacts with PALB2, thus contributing to the formation of the BRCA1-PALB2-BRCA2 complex. To understand the role played by BRCA2 in this complex, we deleted its N-terminal segment and generated BRCA2(Δ)(N) mutant cells. Although previous studies have suggested that BRCA1-PALB2 plays a role in the recruitment of BRCA2 to DNA-damage sites, BRCA2(Δ)(N) mutant cells displayed a considerably milder phenotype than did BRCA2(-/-) null-deficient cells. We hypothesized that the DNA-binding domain (DBD) of BRCA2 might compensate for a defect in BRCA2(ΔN) that prevented stable interaction with PALB2. To test this hypothesis, we disrupted the DBD of BRCA2 in wild-type and BRCA2(Δ)(N) cells. Remarkably, although the resulting BRCA2(Δ)(DBD) cells displayed a moderate phenotype, the BRCA2(Δ)(N+ΔDBD) cells displayed a very severe phenotype, as did the BRCA2(-/-) cells, suggesting that the N-terminal segment and the DBD play a substantially overlapping role in the functionality of BRCA2. We also showed that the formation of both the BRCA1-PALB2-BRCA2 complex and the DBD is required for efficient recruitment of BRCA2 to DNA-damage sites. Our study revealed the essential role played by both the BRCA1-PALB2-BRCA2 complex and the DBD in the functionality of BRCA2, as each can compensate for the other in the recruitment of BRCA2 to DNA-damage sites. This knowledge adds to our ability to accurately predict the efficacy of antimalignant therapies for patients carrying mutations in the BRCA2 gene.

Analysis of Oligonucleotide DNA Binding and Sedimentation Properties of Montmorillonite Clay Using Ultraviolet Light Spectroscopy

PubMed Central

Beall, Gary W.; Sowersby, Drew S.; Roberts, Rachel D.; Robson, Michael H.; Lewis, L. Kevin

2009-01-01

Smectite clays such as montmorillonite form complexes with a variety of biomolecules, including the nucleic acids DNA and RNA. Most previous studies of DNA adsorption onto clay have relied upon spectrophotometric analysis after separation of free nucleic acids from bound complexes by centrifugation. In the current work we demonstrate that such studies produce a consistent error due to (a) incomplete sedimentation of montmorillonite and (b) strong absorbance of the remaining clay at 260 nm. Clay sedimentation efficiency was strongly dependent upon cation concentration (Na+ or Mg2+) and on the level of dispersion of the original suspension. An improved clay:DNA adsorption assay was developed and utilized to assess the impact of metal counterions on binding of single-stranded DNA to montmorillonite. X-ray diffraction demonstrated, for the first time, formation of intercalated structures consistent with orientation of the DNA strands parallel to the clay surface. Observed gallery spacings were found to closely match values calculated utilizing atomistic modeling techniques. PMID:19061334

Structurally Ordered Nanowire Formation from Co-Assembly of DNA Origami and Collagen-Mimetic Peptides

DOE Office of Scientific and Technical Information (OSTI.GOV)

Jiang, Tao; Meyer, Travis A.; Modlin, Charles

In this paper, we describe the co-assembly of two different building units: collagen-mimetic peptides and DNA origami. Two peptides CP ++ and sCP ++ are designed with a sequence comprising a central block (Pro-Hyp-Gly) and two positively charged domains (Pro-Arg-Gly) at both N- and C-termini. Co-assembly of peptides and DNA origami two-layer (TL) nanosheets affords the formation of one-dimensional nanowires with repeating periodicity of similar to 10 nm. Structural analyses suggest a face-to-face stacking of DNA nanosheets with peptides aligned perpendicularly to the sheet surfaces. We demonstrate the potential of selective peptide-DNA association between face-to-face and edge-to-edge packing by tailoringmore » the size of DNA nanostructures. Finally, this study presents an attractive strategy to create hybrid biomolecular assemblies from peptide and DNA-based building blocks that takes advantage of the intrinsic chemical and physical properties of the respective components to encode structural and, potentially, functional complexity within readily accessible biomimetic materials.« less

Structurally Ordered Nanowire Formation from Co-Assembly of DNA Origami and Collagen-Mimetic Peptides

DOE PAGES

Jiang, Tao; Meyer, Travis A.; Modlin, Charles; ...

2017-09-26

In this paper, we describe the co-assembly of two different building units: collagen-mimetic peptides and DNA origami. Two peptides CP ++ and sCP ++ are designed with a sequence comprising a central block (Pro-Hyp-Gly) and two positively charged domains (Pro-Arg-Gly) at both N- and C-termini. Co-assembly of peptides and DNA origami two-layer (TL) nanosheets affords the formation of one-dimensional nanowires with repeating periodicity of similar to 10 nm. Structural analyses suggest a face-to-face stacking of DNA nanosheets with peptides aligned perpendicularly to the sheet surfaces. We demonstrate the potential of selective peptide-DNA association between face-to-face and edge-to-edge packing by tailoringmore » the size of DNA nanostructures. Finally, this study presents an attractive strategy to create hybrid biomolecular assemblies from peptide and DNA-based building blocks that takes advantage of the intrinsic chemical and physical properties of the respective components to encode structural and, potentially, functional complexity within readily accessible biomimetic materials.« less

Differential Deformability of the DNA Minor Groove and Altered BI/BII Backbone Conformational Equilibrium by the Monovalent Ions Li+, Na+, K+ and Rb+ via Water-Mediated Hydrogen Bonding

PubMed Central

Savelyev, Alexey; MacKerell, Alexander D.

2015-01-01

Recently, we reported the differential impact of the monovalent cations Li+, Na+, K+ and Rb+ on DNA conformational properties. These were identified from variations in the calculated solution-state X-ray DNA spectra as a function of the ion type in the solvation buffer in MD simulations using our recently developed polarizable force field based on the classical Drude oscillator. Changes in the DNA structure were found to mainly involve variations in the minor groove width. Because minor groove dimensions vary significantly in protein-DNA complexes and have been shown to play a critical role in both specific and nonspecific DNA readout, understanding the origins of the observed differential DNA modulation by the first-group monovalent ions is of great biological importance. In the present study we show that the primary microscopic mechanism for the phenomenon is the formation of the water-mediated hydrogen bonds between solvated cations located inside the minor groove and simultaneously to two DNA strands, a process whose intensity and impact on DNA structure depends on both the type of the ion and DNA sequence. Additionally, it is shown that formation of such ion-DNA hydrogen bond complexes appreciably modulates the conformation of the backbone by increasing the population of the BII substate. Notably, the differential impact of the ions on DNA conformational behavior is only predicted by the Drude polarizable model for DNA, with virtually no effect observed from MD simulations utilizing the additive CHARMM36 model. Analysis of dipole moments of the water shows the Drude SWM4 model to possess high sensitivity to changes in the local environment, which indicates the important role of electronic polarization in the salt-dependent conformational properties. This also suggests that inclusion of polarization effects is required to model even relatively simple biological systems such as DNA in various ionic solutions. PMID:26575937

Novel One-Tube-One-Step Real-Time Methodology for Rapid Transcriptomic Biomarker Detection: Signal Amplification by Ternary Initiation Complexes.

PubMed

Fujita, Hiroto; Kataoka, Yuka; Tobita, Seiji; Kuwahara, Masayasu; Sugimoto, Naoki

2016-07-19

We have developed a novel RNA detection method, termed signal amplification by ternary initiation complexes (SATIC), in which an analyte sample is simply mixed with the relevant reagents and allowed to stand for a short time under isothermal conditions (37 °C). The advantage of the technique is that there is no requirement for (i) heat annealing, (ii) thermal cycling during the reaction, (iii) a reverse transcription step, or (iv) enzymatic or mechanical fragmentation of the target RNA. SATIC involves the formation of a ternary initiation complex between the target RNA, a circular DNA template, and a DNA primer, followed by rolling circle amplification (RCA) to generate multiple copies of G-quadruplex (G4) on a long DNA strand like beads on a string. The G4s can be specifically fluorescence-stained with N(3)-hydroxyethyl thioflavin T (ThT-HE), which emits weakly with single- and double-stranded RNA/DNA but strongly with parallel G4s. An improved dual SATIC system, which involves the formation of two different ternary initiation complexes in the RCA process, exhibited a wide quantitative detection range of 1-5000 pM. Furthermore, this enabled visual observation-based RNA detection, which is more rapid and convenient than conventional isothermal methods, such as reverse transcription-loop-mediated isothermal amplification, signal mediated amplification of RNA technology, and RNA-primed rolling circle amplification. Thus, SATIC methodology may serve as an on-site and real-time measurement technique for transcriptomic biomarkers for various diseases.

UV damage-specific DNA-binding protein in xeroderma pigmentosum complementation group E

DOE Office of Scientific and Technical Information (OSTI.GOV)

Kataoka, H.; Fujiwara, Y.

1991-03-29

The gel mobility shift assay method revealed a specifically ultraviolet (UV) damage recognizing, DNA-binding protein in nuclear extracts of normal human cells. The resulted DNA/protein complexes caused the two retarded mobility shifts. Four xeroderma pigmentosum complementation group E (XPE) fibroblast strains derived from unrelated Japanese families were not deficient in such a DNA damage recognition/binding protein because of the normal complex formation and gel mobility shifts, although we confirmed the reported lack of the protein in the European XPE (XP2RO and XP3RO) cells. Thus, the absence of this binding protein is not always commonly observed in all the XPE strains,more » and the partially repair-deficient and intermediately UV-hypersensitive phenotype of XPE cells are much similar whether or not they lack the protein.« less

Molecular mechanism of DNA association with single-stranded DNA binding protein

PubMed Central

Maffeo, Christopher

2017-01-01

Abstract During DNA replication, the single-stranded DNA binding protein (SSB) wraps single-stranded DNA (ssDNA) with high affinity to protect it from degradation and prevent secondary structure formation. Although SSB binds ssDNA tightly, it can be repositioned along ssDNA to follow the advancement of the replication fork. Using all-atom molecular dynamics simulations, we characterized the molecular mechanism of ssDNA association with SSB. Placed in solution, ssDNA–SSB assemblies were observed to change their structure spontaneously; such structural changes were suppressed in the crystallographic environment. Repeat simulations of the SSB–ssDNA complex under mechanical tension revealed a multitude of possible pathways for ssDNA to come off SSB punctuated by prolonged arrests at reproducible sites at the SSB surface. Ensemble simulations of spontaneous association of short ssDNA fragments with SSB detailed a three-dimensional map of local affinity to DNA; the equilibrium amount of ssDNA bound to SSB was found to depend on the electrolyte concentration but not on the presence of the acidic tips of the SSB tails. Spontaneous formation of ssDNA bulges and their diffusive motion along SSB surface was directly observed in multiple 10-µs-long simulations. Such reptation-like motion was confined by DNA binding to high-affinity spots, suggesting a two-step mechanism for SSB diffusion. PMID:29059392

New Insights into the Mechanism Underlying the Synergistic Action of Ionizing Radiation with Platinum Chemotherapeutic Drugs: The Role of Low-Energy Electrons

PubMed Central

Rezaee, Mohammad; Hunting, Darel John; Sanche, Léon

2013-01-01

Purpose To investigate the efficiencies of platinum chemotherapeutic drugs (Pt-drugs) in the sensitization of DNA to the direct effects of ionizing radiation and to determine the role of low-energy electrons (LEEs) in this process. Methods and Materials Complexes of supercoiled plasmid DNA covalently bound to either cisplatin, carboplatin or oxaliplatin were prepared in different molar ratios. Solid films of DNA and DNA modified by Pt-drugs were irradiated with either 10-KeV or 10-eV electrons. DNA damages were quantified by gel electrophoresis, and the yields for damage formation were obtained from exposure-response curves. Results The presence of an average of two Pt-adducts in 3199-bp plasmid DNA increases the probability of a double-strand break by factors of 3.1, 2.5 and 2.4 for carboplatin, cisplatin and oxaliplatin, respectively. Electrons with energies of 10-eV and 10-KeV interact with Pt-adducts to preferentially enhance the formation of cluster lesions. The maximum increase in radiosensitivity per Pt-adduct is found at ratios up to 3.1 × 10−4 Pt-adducts per nucleotide which is equivalent to an average of two adducts per plasmid. Carboplatin and oxaliplatin show higher efficiencies than cisplatin in the radiosensitization of DNA. Since carboplatin and cisplatin give rise to identical reactive species which attach to DNA, carboplatin must be considered as a better radiosensitizers for equal number of Pt-adducts. Conclusion Pt-drugs preferentially enhance the formation of cluster damage to DNA induced by the direct effect of ionizing radiation and LEEs are the main species responsible for such an enhancement via the formation of electron resonances. PMID:23910707

Electrochemical study of the interaction between dsDNA and copper(I) using carbon paste and hanging mercury drop electrode.

PubMed

Stanić, Z; Girousi, S

2008-06-30

The interaction of copper(I) with double-stranded (ds) calf thymus DNA was studied in solution and at the electrode surface by means of transfer voltammetry using a carbon paste electrode (CPE) as working electrode in 0.2 M acetate buffer solution (pH 5.0). As a result of the interaction of Cu(I) between the base pairs of the dsDNA, the characteristic peaks of dsDNA, due to the oxidation of guanine and adenine, increased and after a certain concentration of Cu(I) a new peak at +1.37 V appeared, probably due to the formation of a purine-Cu(I) complex (dsDNA-Cu(I) complex). Accordingly, the interaction of copper(I) with calf thymus dsDNA was studied in solution as well as at the electrode surface using hanging mercury drop electrode (HMDE) by means of alternating current voltammetry (AC voltammetry) in 0.3 M NaCl and 50 mM sodium phosphate buffer (pH 8.5) as supporting electrolyte. Its interaction with DNA is shown to be time dependent. Significant changes in the characteristic peaks of dsDNA were observed after addition of higher concentration of Cu(I) to a solution containing dsDNA, as a result of the interaction between Cu(I) and dsDNA. All the experimental results indicate that Cu(I) can bind to DNA by electrostatic binding and form an association complex.

Probing the structure of RecA-DNA filaments. Advantages of a fluorescent guanine analog.

PubMed

Singleton, Scott F; Roca, Alberto I; Lee, Andrew M; Xiao, Jie

2007-04-23

The RecA protein of Escherichia coli plays a crucial roles in DNA recombination and repair, as well as various aspects of bacterial pathogenicity. The formation of a RecA-ATP-ssDNA complex initiates all RecA activities and yet a complete structural and mechanistic description of this filament has remained elusive. An analysis of RecA-DNA interactions was performed using fluorescently labeled oligonucleotides. A direct comparison was made between fluorescein and several fluorescent nucleosides. The fluorescent guanine analog 6-methylisoxanthopterin (6MI) demonstrated significant advantages over the other fluorophores and represents an important new tool for characterizing RecA-DNA interactions.

Sufficient Amounts of Functional HOP2/MND1 Complex Promote Interhomolog DNA Repair but Are Dispensable for Intersister DNA Repair during Meiosis in Arabidopsis[W

PubMed Central

Uanschou, Clemens; Ronceret, Arnaud; Von Harder, Mona; De Muyt, Arnaud; Vezon, Daniel; Pereira, Lucie; Chelysheva, Liudmila; Kobayashi, Wataru; Kurumizaka, Hitoshi; Schlögelhofer, Peter; Grelon, Mathilde

2013-01-01

During meiosis, homologous recombination (HR) is essential to repair programmed DNA double-strand breaks (DSBs), and a dedicated protein machinery ensures that the homologous chromosome is favored over the nearby sister chromatid as a repair template. The HOMOLOGOUS-PAIRING PROTEIN2/MEIOTIC NUCLEAR DIVISION PROTEIN1 (HOP2/MND1) protein complex has been identified as a crucial factor of meiotic HR in Arabidopsis thaliana, since loss of either MND1 or HOP2 results in failure of DNA repair. We isolated two mutant alleles of HOP2 (hop2-2 and hop2-3) that retained the capacity to repair meiotic DSBs via the sister chromatid but failed to use the homologous chromosome. We show that in these alleles, the recombinases RADIATION SENSITIVE51 (RAD51) and DISRUPTED MEIOTIC cDNA1 (DMC1) are loaded, but only the intersister DNA repair pathway is activated. The hop2-2 phenotype is correlated with a decrease in HOP2/MND1 complex abundance. In hop2-3, a truncated HOP2 protein is produced that retains its ability to bind to DMC1 and DNA but forms less stable complexes with MND1 and fails to efficiently stimulate DMC1-driven D-loop formation. Genetic analyses demonstrated that in the absence of DMC1, HOP2/MND1 is dispensable for RAD51-mediated intersister DNA repair, while in the presence of DMC1, a minimal amount of functional HOP2/MND1 is essential to drive intersister DNA repair. PMID:24363313

Determination for Enterobacter cloacae based on a europium ternary complex labeled DNA probe

NASA Astrophysics Data System (ADS)

He, Hui; Niu, Cheng-Gang; Zeng, Guang-Ming; Ruan, Min; Qin, Pin-Zhu; Liu, Jing

2011-11-01

The fast detection and accurate diagnosis of the prevalent pathogenic bacteria is very important for the treatment of disease. Nowadays, fluorescence techniques are important tools for diagnosis. A two-probe tandem DNA hybridization assay was designed for the detection of Enterobacter cloacae based on time-resolved fluorescence. In this work, the authors synthesized a novel europium ternary complex Eu(TTA) 3(5-NH 2-phen) with intense luminescence, high fluorescence quantum yield and long lifetime before. We developed a method based on this europium complex for the specific detection of original extracted DNA from E. cloacae. In the hybridization assay format, the reporter probe was labeled with Eu(TTA) 3(5-NH 2-phen) on the 5'-terminus, and the capture probe capture probe was covalent immobilized on the surface of the glutaraldehyde treated glass slides. The original extracted DNA of samples was directly used without any DNA purification and amplification. The detection was conducted by monitoring the fluorescence intensity from the glass surface after DNA hybridization. The detection limit of the DNA was 5 × 10 -10 mol L -1. The results of the present work proved that this new approach was easy to operate with high sensitivity and specificity. It could be conducted as a powerful tool for the detection of pathogen microorganisms in the environment.

Cationic polymers for DNA origami coating - examining their binding efficiency and tuning the enzymatic reaction rates.

PubMed

Kiviaho, Jenny K; Linko, Veikko; Ora, Ari; Tiainen, Tony; Järvihaavisto, Erika; Mikkilä, Joona; Tenhu, Heikki; Nonappa; Kostiainen, Mauri A

2016-06-02

DNA origamis are fully tailored, programmable, biocompatible and readily functionalizable nanostructures that provide an excellent foundation for the development of sophisticated drug-delivery systems. However, the DNA origami objects suffer from certain drawbacks such as low cell-transfection rates and low stability. A great deal of studies on polymer-based transfection agents, mainly focusing on polyplex formation and toxicity, exists. In this study, the electrostatic binding between a brick-like DNA origami and cationic block-copolymers was explored. The effect of the polymer structure on the binding was investigated and the toxicity of the polymer-origami complexes evaluated. The study shows that all of the analyzed polymers had a suitable binding efficiency irrespective of the block structure. It was also observed that the toxicity of polymer-origami complexes was insignificant at the biologically relevant concentration levels. Besides brick-like DNA origamis, tubular origami carriers equipped with enzymes were also coated with the polymers. By adjusting the amount of cationic polymers that cover the DNA structures, we showed that it is possible to control the enzyme kinetics of the complexes. This work gives a starting point for further development of biocompatible and effective polycation-based block copolymers that can be used in coating different DNA origami nanostructures for various bioapplications.

One-milliliter wet-digestion for inductively coupled plasma mass spectrometry (ICP-MS): determination of platinum-DNA adducts in cells treated with platinum(II) complexes.

PubMed

Yamada, Kanae; Kato, Naoyuki; Takagi, Akimitsu; Koi, Minoru; Hemmi, Hiromichi

2005-08-01

Platinum (Pt)-DNA adducts formed by the anti-tumor agent cisplatin are recognized by the DNA mismatch repair (MMR) system. To investigate the involvement of MMR proteins including hMLH1 in the removal of these adducts, we developed a mL-scale wet-digestion method for inductively coupled plasma mass spectrometry (ICP-MS). The detection limit was 0.01 ng mL(-1) Pt, which corresponded to 2 pg Pt/microg DNA when 10 microg of DNA was used. The mean relative errors were 5.4% or better for a dynamic range of 0.01-10 ng mL(-1) Pt. DNA (approximately 500 microg) had no matrix effect. To improve the accuracy, DNA preparations were treated with ribonuclease and the apparent reduction in the concentration of Pt was corrected using cellular DNA levels, which were determined with Hoechst 33258. No significant differences were observed, in terms of the formation of Pt-DNA adducts or their removal over 6 h, between hMLH1-deficient HCT116 cells, a human colorectal cancer cell line, and hMLH1-complemented HCT116+ch3 cells (n=5; P>0.05), indicating that the hMLH1-dependent DNA repair systems contribute to neither the formation nor the removal of the adducts at detectable levels. In addition, approximately 19% of the adducts were removed within 6 h in both cell lines. A time course analysis (~24 h) suggested that the removal of cisplatin-generated Pt-DNA adducts follows first-order kinetics (t(1/2)=32 h). The amount of Pt-DNA adduct formed by oxaliplatin in 1 h was 56% (ratio of means) of that generated by an equimolar concentration of cisplatin in HCT116. The proposed procedure could be useful for determining Pt-DNA adducts formed by Pt(II) complexes.

Influence of chitosan structure on the formation and stability of DNA-chitosan polyelectrolyte complexes.

PubMed

Strand, Sabina P; Danielsen, Signe; Christensen, Bjørn E; Vårum, Kjell M

2005-01-01

The interactions between DNA and chitosans varying in fractional content of acetylated units (FA), degree of polymerization (DP), and degree of ionization were investigated by several techniques, including an ethidium bromide (EtBr) fluorescence assay, gel retardation, atomic force microscopy, and dynamic and electrophoretic light scattering. The charge density of the chitosan and the number of charges per chain were found to be the dominating factors for the structure and stability of DNA-chitosan complexes. All high molecular weight chitosans condensed DNA into physically stable polyplexes; however, the properties of the complexes were strongly dependent on FA, and thereby the charge density of chitosan. By employing fully charged oligomers of constant charge density, it was shown that the complexation of DNA and stability of the polyplexes is governed by the number of cationic residues per chain. A minimum of 6-9 positive charges appeared necessary to provide interaction strength comparable to that of polycations. In contrast, further increase in the number of charges above 9 did not increase the apparent binding affinity as judged from the EtBr displacement assay. The chitosan oligomers exhibited a pH-dependent interaction with DNA, reflecting the number of ionized amino groups. The complexation of DNA and the stability of oligomer-based polyplexes became reduced above pH 7.4. Such pH-dependent dissociation of polyplexes around the physiological pH is highly relevant in gene delivery applications and might be one of the reasons for the high transfection activity of oligomer-based polyplexes observed.

Complex formation equilibria of binary and ternary complexes involving 3,3-bis(1-methylimidazol-2yl)propionic acid and bio-relevant ligands as 1-aminocyclopropane carboxylic acid with reference to plant hormone

NASA Astrophysics Data System (ADS)

Shoukry, Mohamed M.; Hassan, Safaa S.

2014-01-01

The formation equilibria for the binary complexes of Cu(II) with 1-aminocyclopropane carboxylic acid (ACC) and 3,3-bis(1-methylimidazol-2-yl)propionic acid (BIMP) were investigated. ACC and BIMP form the complexes 1 1 0, 1 2 0 and 1 1 -1. The ternary complexes of Cu(II) with BIMP and biorelevant ligands as some selected amino acids, peptides and DNA constituents are formed in a stepwise mechanism. The stability constants of the complexes formed were determined and their distribution diagrams were evaluated. The kinetics of hydrolysis of glycine methyl ester in presence of [Cu(BIMP)]+ was investigated by pH-stat technique and the mechanism was discussed.

Unraveling DNA dynamics using atomic force microscopy.

PubMed

Suzuki, Yuki; Yoshikawa, Yuko; Yoshimura, Shige H; Yoshikawa, Kenichi; Takeyasu, Kunio

2011-01-01

The elucidation of structure-function relationships of biological samples has become important issue in post-genomic researches. In order to unveil the molecular mechanisms controlling gene regulations, it is essential to understand the interplay between fundamental DNA properties and the dynamics of the entire molecule. The wide range of applicability of atomic force microscopy (AFM) has allowed us to extract physicochemical properties of DNA and DNA-protein complexes, as well as to determine their topographical information. Here, we review how AFM techniques have been utilized to study DNA and DNA-protein complexes and what types of analyses have accelerated the understanding of the DNA dynamics. We begin by illustrating the application of AFM to investigate the fundamental feature of DNA molecules; topological transition of DNA, length dependent properties of DNA molecules, flexibility of double-stranded DNA, and capability of the formation of non-Watson-Crick base pairing. These properties of DNA are critical for the DNA folding and enzymatic reactions. The technical advancement in the time-resolution of AFM and sample preparation methods enabled visual analysis of DNA-protein interactions at sub-second time region. DNA tension-dependent enzymatic reaction and DNA looping dynamics by restriction enzymes were examined at a nanoscale in physiological environments. Contribution of physical properties of DNA to dynamics of nucleosomes and transition of the higher-order structure of reconstituted chromatin are also reviewed. Copyright © 2011 John Wiley & Sons, Inc.

Characterization of the NTPR and BD1 interacting domains of the human PICH-BEND3 complex.

PubMed

Pitchai, Ganesha P; Hickson, Ian D; Streicher, Werner; Montoya, Guillermo; Mesa, Pablo

2016-08-01

Chromosome integrity depends on DNA structure-specific processing complexes that resolve DNA entanglement between sister chromatids. If left unresolved, these entanglements can generate either chromatin bridging or ultrafine DNA bridging in the anaphase of mitosis. These bridge structures are defined by the presence of the PICH protein, which interacts with the BEND3 protein in mitosis. To obtain structural insights into PICH-BEND3 complex formation at the atomic level, their respective NTPR and BD1 domains were cloned, overexpressed and crystallized using 1.56 M ammonium sulfate as a precipitant at pH 7.0. The protein complex readily formed large hexagonal crystals belonging to space group P6122, with unit-cell parameters a = b = 47.28, c = 431.58 Å and with one heterodimer in the asymmetric unit. A complete multiwavelength anomalous dispersion (MAD) data set extending to 2.2 Å resolution was collected from a selenomethionine-labelled crystal at the Swiss Light Source.

Reconstitution of the yeast RNA polymerase III transcription system with all recombinant factors.

PubMed

Ducrot, Cécile; Lefebvre, Olivier; Landrieux, Emilie; Guirouilh-Barbat, Josée; Sentenac, André; Acker, Joel

2006-04-28

Transcription factor TFIIIC is a multisubunit complex required for promoter recognition and transcriptional activation of class III genes. We describe here the reconstitution of complete recombinant yeast TFIIIC and the molecular characterization of its two DNA-binding domains, tauA and tauB, using the baculovirus expression system. The B block-binding module, rtauB, was reconstituted with rtau138, rtau91, and rtau60 subunits. rtau131, rtau95, and rtau55 formed also a stable complex, rtauA, that displayed nonspecific DNA binding activity. Recombinant rTFIIIC was functionally equivalent to purified yeast TFIIIC, suggesting that the six recombinant subunits are necessary and sufficient to reconstitute a transcriptionally active TFIIIC complex. The formation and the properties of rTFIIIC-DNA complexes were affected by dephosphorylation treatments. The combination of complete recombinant rTFIIIC and rTFIIIB directed a low level of basal transcription, much weaker than with the crude B'' fraction, suggesting the existence of auxiliary factors that could modulate the yeast RNA polymerase III transcription system.

A ternary AppA-PpsR-DNA complex mediates light regulation of photosynthesis-related gene expression.

PubMed

Winkler, Andreas; Heintz, Udo; Lindner, Robert; Reinstein, Jochen; Shoeman, Robert L; Schlichting, Ilme

2013-07-01

The anoxygenic phototrophic bacterium Rhodobacter sphaeroides uses different energy sources, depending on environmental conditions including aerobic respiration or, in the absence of oxygen, photosynthesis. Photosynthetic genes are repressed at high oxygen tension, but at intermediate levels their partial expression prepares the bacterium for using light energy. Illumination, however, enhances repression under semiaerobic conditions. Here, we describe molecular details of two proteins mediating oxygen and light control of photosynthesis-gene expression: the light-sensing antirepressor AppA and the transcriptional repressor PpsR. Our crystal structures of both proteins and their complex and hydrogen/deuterium-exchange data show that light activation of AppA-PpsR2 affects the PpsR effector region within the complex. DNA binding studies demonstrate the formation of a light-sensitive ternary AppA-PpsR-DNA complex. We discuss implications of these results for regulation by light and oxygen, highlighting new insights into blue light-mediated signal transduction.

Synthesis, characterization, crystal structure, DNA- and HSA-binding studies of a dinuclear Schiff base Zn(II) complex derived from 2-hydroxynaphtaldehyde and 2-picolylamine

NASA Astrophysics Data System (ADS)

Kazemi, Zahra; Rudbari, Hadi Amiri; Mirkhani, Valiollah; Sahihi, Mehdi; Moghadam, Majid; Tangestaninejad, Sharam; Mohammadpoor-Baltork, Iraj

2015-09-01

A tridentate Schiff base ligand NNO donor (HL: 1-((E)-((pyridin-2-yl)methylimino)methyl)naphthalen-2-ol was synthesized from condensation of 2-hydroxynaphtaldehyde and 2-picolylamine. Zinc complex, Zn2L2(NO3)2, was prepared from reaction of Zn(NO3)2 and HL at ambient temperature. The ligand and complex were characterized by FT-IR, 1H NMR, 13C NMR and elemental analysis (CHN). Furthermore, the structure of dinuclear Zn(II) complex was determined by single crystal X-ray analysis. The complex, Zn2L2(NO3)2, is centrosymmetric dimer in which deprotonated phenolates bridge the two Zn(II) atoms and link the two halves of the dimer. In the structure, Zinc(II) ions have a highly distorted six-coordinate structure bonded to two oxygen atoms from a bidentate nitrate group, the pyridine nitrogen, an amine nitrogen and phenolate oxygens. The interaction of dinuclear Zn(II) complex with fish sperm DNA (FS-DNA) and HSA was investigated under physiological conditions using fluorescence quenching, UV-Vis spectroscopy, molecular dynamics simulation and molecular docking methods. The estimated binding constants for the DNA-complex and HSA-complex were (3.60 ± 0.18) × 104 M-1 and (1.35 ± 0.24) × 104 M-1, respectively. The distance between dinuclear Zn(II) complex and HSA was obtained based on the Förster's theory of non-radiative energy transfer. Molecular docking studies revealed the binding of dinuclear Zn(II) complex to the major groove of FS-DNA and IIA site of protein by formation of hydrogen bond, π-cation and hydrophobic interactions.

Pt(IV) complexes as prodrugs for cisplatin.

PubMed

Shi, Yi; Liu, Shu-An; Kerwood, Deborah J; Goodisman, Jerry; Dabrowiak, James C

2012-02-01

The antitumor effects of platinum(IV) complexes, considered prodrugs for cisplatin, are believed to be due to biological reduction of Pt(IV) to Pt(II), with the reduction products binding to DNA and other cellular targets. In this work we used pBR322 DNA to capture the products of reduction of oxoplatin, c,t,c-[PtCl(2)(OH)(2)(NH(3))(2)], 3, and a carboxylate-modified analog, c,t,c-[PtCl(2)(OH)(O(2)CCH(2)CH(2)CO(2)H)(NH(3))(2)], 4, by ascorbic acid (AsA) or glutathione (GSH). Since carbonate plays a significant role in the speciation of platinum complexes in solution, we also investigated the effects of carbonate on the reduction/DNA-binding process. In pH 7.4 buffer in the absence of carbonate, both 3 and 4 are reduced by AsA to cisplatin (confirmed using ((195))Pt NMR), which binds to and unwinds closed circular DNA in a manner consistent with the formation of the well-known 1, 2 intrastrand DNA crosslink. However, when GSH is used as the reducing agent for 3 and 4, ((195))Pt NMR shows that cisplatin is not produced in the reaction medium. Although the Pt(II) products bind to closed circular DNA, their effect on the mobility of Form I DNA is different from that produced by cisplatin. When physiological carbonate is present in the reduction medium, ((13))C NMR shows that Pt(II) carbonato complexes form which block or impede platinum binding to DNA. The results of the study vis-à-vis the ability of the Pt(IV) complexes to act as prodrugs for cisplatin are discussed. Copyright © 2011 Elsevier Inc. All rights reserved.

The G-quadruplex DNA stabilizing drug pyridostatin promotes DNA damage and downregulates transcription of Brca1 in neurons.

PubMed

Moruno-Manchon, Jose F; Koellhoffer, Edward C; Gopakumar, Jayakrishnan; Hambarde, Shashank; Kim, Nayun; McCullough, Louise D; Tsvetkov, Andrey S

2017-09-12

The G-quadruplex is a non-canonical DNA secondary structure formed by four DNA strands containing multiple runs of guanines. G-quadruplexes play important roles in DNA recombination, replication, telomere maintenance, and regulation of transcription. Small molecules that stabilize the G-quadruplexes alter gene expression in cancer cells. Here, we hypothesized that the G-quadruplexes regulate transcription in neurons. We discovered that pyridostatin, a small molecule that specifically stabilizes G-quadruplex DNA complexes, induced neurotoxicity and promoted the formation of DNA double-strand breaks (DSBs) in cultured neurons. We also found that pyridostatin downregulated transcription of the Brca1 gene, a gene that is critical for DSB repair. Importantly, in an in vitro gel shift assay, we discovered that an antibody specific to the G-quadruplex structure binds to a synthetic oligonucleotide, which corresponds to the first putative G-quadruplex in the Brca1 gene promoter. Our results suggest that the G-quadruplex complexes regulate transcription in neurons. Studying the G-quadruplexes could represent a new avenue for neurodegeneration and brain aging research.

Fanconi anemia proteins FANCD2 and FANCI exhibit different DNA damage responses during S-phase

PubMed Central

Sareen, Archana; Chaudhury, Indrajit; Adams, Nicole; Sobeck, Alexandra

2012-01-01

Fanconi anemia (FA) pathway members, FANCD2 and FANCI, contribute to the repair of replication-stalling DNA lesions. FA pathway activation relies on phosphorylation of FANCI by the ataxia telangiectasia and Rad3-related (ATR) kinase, followed by monoubiquitination of FANCD2 and FANCI by the FA core complex. FANCD2 and FANCI are thought to form a functional heterodimer during DNA repair, but it is unclear how dimer formation is regulated or what the functions of the FANCD2–FANCI complex versus the monomeric proteins are. We show that the FANCD2–FANCI complex forms independently of ATR and FA core complex, and represents the inactive form of both proteins. DNA damage-induced FA pathway activation triggers dissociation of FANCD2 from FANCI. Dissociation coincides with FANCD2 monoubiquitination, which significantly precedes monoubiquitination of FANCI; moreover, monoubiquitination responses of FANCD2 and FANCI exhibit distinct DNA substrate specificities. A phosphodead FANCI mutant fails to dissociate from FANCD2, whereas phosphomimetic FANCI cannot interact with FANCD2, indicating that FANCI phosphorylation is the molecular trigger for FANCD2–FANCI dissociation. Following dissociation, FANCD2 binds replicating chromatin prior to—and independently of—FANCI. Moreover, the concentration of chromatin-bound FANCD2 exceeds that of FANCI throughout replication. Our results suggest that FANCD2 and FANCI function separately at consecutive steps during DNA repair in S-phase. PMID:22753026

DNA condensation by partially acetylated poly(amido amine) dendrimers: effects of dendrimer charge density on complex formation.

PubMed

Yu, Shi; Li, Ming-Hsin; Choi, Seok Ki; Baker, James R; Larson, Ronald G

2013-09-03

The ability of poly(amido amine) (or PAMAM) dendrimers to condense semiflexible dsDNA and penetrate cell membranes gives them great potential in gene therapy and drug delivery but their high positive surface charge makes them cytotoxic. Here, we describe the effects of partial neutralization by acetylation on DNA condensation using light scattering, circular dichroism, and single molecule imaging of dendrimer-DNA complexes combed onto surfaces and tethered to those surfaces under flow. We find that DNA can be condensed by generation-five (G5) dendrimers even when the surface charges are more than 65% neutralized, but that such dendrimers bind negligibly when an end-tethered DNA is stretched in flow. We also find that when fully charged dendrimers are introduced by flow to end-tethered DNA, all DNA molecules become equally highly coated with dendrimers at a rate that becomes very fast at high dendrimer concentration, and that dendrimers remain bound during subsequent flow of dendrimer-free buffer. These results suggest that the presence of dendrimer-free DNA coexisting with dendrimer-bound DNA after bulk mixing of the two in solution may result from diffusion-limited irreversible dendrimer-DNA binding, rather than, or in addition to, the previously proposed cooperative binding mechanism of dendrimers to DNA.

A unique enhancer boundary complex on the mouse ribosomal RNA genes persists after loss of Rrn3 or UBF and the inactivation of RNA polymerase I transcription

PubMed Central

Stefanovsky, Victor Y.; Tremblay, Michel G.; Lindsay, Helen; Robinson, Mark D.

2017-01-01

Transcription of the several hundred of mouse and human Ribosomal RNA (rRNA) genes accounts for the majority of RNA synthesis in the cell nucleus and is the determinant of cytoplasmic ribosome abundance, a key factor in regulating gene expression. The rRNA genes, referred to globally as the rDNA, are clustered as direct repeats at the Nucleolar Organiser Regions, NORs, of several chromosomes, and in many cells the active repeats are transcribed at near saturation levels. The rDNA is also a hotspot of recombination and chromosome breakage, and hence understanding its control has broad importance. Despite the need for a high level of rDNA transcription, typically only a fraction of the rDNA is transcriptionally active, and some NORs are permanently silenced by CpG methylation. Various chromatin-remodelling complexes have been implicated in counteracting silencing to maintain rDNA activity. However, the chromatin structure of the active rDNA fraction is still far from clear. Here we have combined a high-resolution ChIP-Seq protocol with conditional inactivation of key basal factors to better understand what determines active rDNA chromatin. The data resolve questions concerning the interdependence of the basal transcription factors, show that preinitiation complex formation is driven by the architectural factor UBF (UBTF) independently of transcription, and that RPI termination and release corresponds with the site of TTF1 binding. They further reveal the existence of an asymmetric Enhancer Boundary Complex formed by CTCF and Cohesin and flanked upstream by phased nucleosomes and downstream by an arrested RNA Polymerase I complex. We find that the Enhancer Boundary Complex is the only site of active histone modification in the 45kbp rDNA repeat. Strikingly, it not only delimits each functional rRNA gene, but also is stably maintained after gene inactivation and the re-establishment of surrounding repressive chromatin. Our data define a poised state of rDNA chromatin and place the Enhancer Boundary Complex as the likely entry point for chromatin remodelling complexes. PMID:28715449

A unique enhancer boundary complex on the mouse ribosomal RNA genes persists after loss of Rrn3 or UBF and the inactivation of RNA polymerase I transcription.

PubMed

Herdman, Chelsea; Mars, Jean-Clement; Stefanovsky, Victor Y; Tremblay, Michel G; Sabourin-Felix, Marianne; Lindsay, Helen; Robinson, Mark D; Moss, Tom

2017-07-01

Transcription of the several hundred of mouse and human Ribosomal RNA (rRNA) genes accounts for the majority of RNA synthesis in the cell nucleus and is the determinant of cytoplasmic ribosome abundance, a key factor in regulating gene expression. The rRNA genes, referred to globally as the rDNA, are clustered as direct repeats at the Nucleolar Organiser Regions, NORs, of several chromosomes, and in many cells the active repeats are transcribed at near saturation levels. The rDNA is also a hotspot of recombination and chromosome breakage, and hence understanding its control has broad importance. Despite the need for a high level of rDNA transcription, typically only a fraction of the rDNA is transcriptionally active, and some NORs are permanently silenced by CpG methylation. Various chromatin-remodelling complexes have been implicated in counteracting silencing to maintain rDNA activity. However, the chromatin structure of the active rDNA fraction is still far from clear. Here we have combined a high-resolution ChIP-Seq protocol with conditional inactivation of key basal factors to better understand what determines active rDNA chromatin. The data resolve questions concerning the interdependence of the basal transcription factors, show that preinitiation complex formation is driven by the architectural factor UBF (UBTF) independently of transcription, and that RPI termination and release corresponds with the site of TTF1 binding. They further reveal the existence of an asymmetric Enhancer Boundary Complex formed by CTCF and Cohesin and flanked upstream by phased nucleosomes and downstream by an arrested RNA Polymerase I complex. We find that the Enhancer Boundary Complex is the only site of active histone modification in the 45kbp rDNA repeat. Strikingly, it not only delimits each functional rRNA gene, but also is stably maintained after gene inactivation and the re-establishment of surrounding repressive chromatin. Our data define a poised state of rDNA chromatin and place the Enhancer Boundary Complex as the likely entry point for chromatin remodelling complexes.

Interaction investigations of HipA binding to HipB dimer and HipB dimer + DNA complex: a molecular dynamics simulation study.

PubMed

Li, Chaoqun; Wang, Yaru; Wang, Yan; Chen, Guangju

2013-11-01

We carried out molecular dynamics simulations and free energy calculations for a series of ternary and diplex models for the HipA protein, HipB dimer, and DNA molecule to address the mechanism of HipA sequestration and the binding order of events from apo HipB/HipA to 2HipA + HipB dimer + DNA complex. The results revealed that the combination of DNA with the HipB dimer is energetically favorable for the combination of HipB dimer with HipA protein. The binding of DNA to HipB dimer induces a long-range allosteric communication from the HipB2 -DNA interface to the HipA-HipB2 interface, which involves the closeness of α1 helices of HipB dimer to HipA protein and formations of extra hydrogen bonds in the HipA-HipB2 interface through the extension of α2/3 helices in the HipB dimer. These simulated results suggested that the DNA molecule, as a regulative media, modulates the HipB dimer conformation, consequently increasing the interactions of HipB dimer with the HipA proteins, which explains the mechanism of HipA sequestration reported by the previous experiment. Simultaneously, these simulations also explored that the thermodynamic binding order in a simulated physiological environment, that is, the HipB dimer first bind to DNA to form HipB dimer + DNA complex, then capturing strongly the HipA proteins to form a ternary complex, 2HipA + HipB dimer + DNA, for sequestrating HipA in the nucleoid. Copyright © 2013 John Wiley & Sons, Ltd.

Electrostatic control of DNA intersegmental translocation by the ETS transcription factor ETV6.

PubMed

Vo, Tam; Wang, Shuo; Poon, Gregory M K; Wilson, W David

2017-08-11

To find their DNA target sites in complex solution environments containing excess heterogeneous DNA, sequence-specific DNA-binding proteins execute various translocation mechanisms known collectively as facilitated diffusion. For proteins harboring a single DNA contact surface, long-range translocation occurs by jumping between widely spaced DNA segments. We have configured biosensor-based surface plasmon resonance to directly measure the affinity and kinetics of this intersegmental jumping by the ETS-family transcription factor ETS variant 6 (ETV6). To isolate intersegmental target binding in a functionally defined manner, we pre-equilibrated ETV6 with excess salmon sperm DNA, a heterogeneous polymer, before exposing the nonspecifically bound protein to immobilized oligomeric DNA harboring a high-affinity ETV6 site. In this way, the mechanism of ETV6-target association could be toggled electrostatically through varying NaCl concentration in the bulk solution. Direct measurements of association and dissociation kinetics of the site-specific complex indicated that 1) freely diffusive binding by ETV6 proceeds through a nonspecific-like intermediate, 2) intersegmental jumping is rate-limited by dissociation from the nonspecific polymer, and 3) dissociation of the specific complex is independent of the history of complex formation. These results show that target searches by proteins with an ETS domain, such as ETV6, whose single DNA-binding domain cannot contact both source and destination sites simultaneously, are nonetheless strongly modulated by intersegmental jumping in heterogeneous site environments. Our findings establish biosensors as a general technique for directly and specifically measuring target site search by DNA-binding proteins via intersegmental translocation. © 2017 by The American Society for Biochemistry and Molecular Biology, Inc.

DNA double strand breaks induced by the indirect effect of radiation are more efficiently repaired by non-homologous end joining compared to homologous recombination repair.

PubMed

Bajinskis, Ainars; Natarajan, Adayapalam T; Erixon, Klaus; Harms-Ringdahl, Mats

2013-08-30

The aim of this study was to investigate the relative involvement of three major DNA repair pathways, i.e., non-homologous end joining (NHEJ), homologous recombination (HRR) and base excision (BER) in repair of DNA lesions of different complexity induced by low- or high-LET radiation with emphasis on the contribution of the indirect effect of radiation for these radiation qualities. A panel of DNA repair-deficient CHO cell lines was irradiated by (137)Cs γ-rays or radon progeny α-particles. Irradiation was also performed in the presence of 2M DMSO to reduce the indirect effect of radiation and the complexity of the DNA damage formed. Clonogenic survival and micronucleus assays were used to estimate efficiencies of the different repair pathways for DNA damages produced by direct and indirect effects. Removal of the indirect effect of low-LET radiation by DMSO increased clonogenic survival and decreased MN formation for all cell lines investigated. A direct contribution of the indirect effect of radiation to DNA base damage was suggested by the significant protection by DMSO seen for the BER deficient cell line. Lesions formed by the indirect effect are more readily repaired by the NHEJ pathway than by HRR after irradiation with γ-rays or α-particles as evaluated by cell survival and the yields of MN. The results obtained with BER- and NHEJ-deficient cells suggest that the indirect effect of radiation contributes significantly to the formation of repair substrates for these pathways. Copyright © 2013 Elsevier B.V. All rights reserved.

Visualization of complex DNA double-strand breaks in a tumor treated with carbon ion radiotherapy

PubMed Central

Oike, Takahiro; Niimi, Atsuko; Okonogi, Noriyuki; Murata, Kazutoshi; Matsumura, Akihiko; Noda, Shin-Ei; Kobayashi, Daijiro; Iwanaga, Mototaro; Tsuchida, Keisuke; Kanai, Tatsuaki; Ohno, Tatsuya; Shibata, Atsushi; Nakano, Takashi

2016-01-01

Carbon ion radiotherapy shows great potential as a cure for X-ray-resistant tumors. Basic research suggests that the strong cell-killing effect induced by carbon ions is based on their ability to cause complex DNA double-strand breaks (DSBs). However, evidence supporting the formation of complex DSBs in actual patients is lacking. Here, we used advanced high-resolution microscopy with deconvolution to show that complex DSBs are formed in a human tumor clinically treated with carbon ion radiotherapy, but not in a tumor treated with X-ray radiotherapy. Furthermore, analysis using a physics model suggested that the complexity of radiotherapy-induced DSBs is related to linear energy transfer, which is much higher for carbon ion beams than for X-rays. Visualization of complex DSBs in clinical specimens will help us to understand the anti-tumor effects of carbon ion radiotherapy. PMID:26925533

NF-κB regulates DNA double-strand break repair in conjunction with BRCA1-CtIP complexes.

PubMed

Volcic, Meta; Karl, Sabine; Baumann, Bernd; Salles, Daniela; Daniel, Peter; Fulda, Simone; Wiesmüller, Lisa

2012-01-01

NF-κB is involved in immune responses, inflammation, oncogenesis, cell proliferation and apoptosis. Even though NF-κB can be activated by DNA damage via Ataxia telangiectasia-mutated (ATM) signalling, little was known about an involvement in DNA repair. In this work, we dissected distinct DNA double-strand break (DSB) repair mechanisms revealing a stimulatory role of NF-κB in homologous recombination (HR). This effect was independent of chromatin context, cell cycle distribution or cross-talk with p53. It was not mediated by the transcriptional NF-κB targets Bcl2, BAX or Ku70, known for their dual roles in apoptosis and DSB repair. A contribution by Bcl-xL was abrogated when caspases were inhibited. Notably, HR induction by NF-κB required the targets ATM and BRCA2. Additionally, we provide evidence that NF-κB interacts with CtIP-BRCA1 complexes and promotes BRCA1 stabilization, and thereby contributes to HR induction. Immunofluorescence analysis revealed accelerated formation of replication protein A (RPA) and Rad51 foci upon NF-κB activation indicating HR stimulation through DSB resection by the interacting CtIP-BRCA1 complex and Rad51 filament formation. Taken together, these results define multiple NF-κB-dependent mechanisms regulating HR induction, and thereby providing a novel intriguing explanation for both NF-κB-mediated resistance to chemo- and radiotherapies as well as for the sensitization by pharmaceutical intervention of NF-κB activation.

Characterizing DNA Repair Processes at Transient and Long-lasting Double-strand DNA Breaks by Immunofluorescence Microscopy.

PubMed

Murthy, Vaibhav; Dacus, Dalton; Gamez, Monica; Hu, Changkun; Wendel, Sebastian O; Snow, Jazmine; Kahn, Andrew; Walterhouse, Stephen H; Wallace, Nicholas A

2018-06-08

The repair of double-stranded breaks (DSBs) in DNA is a highly coordinated process, necessitating the formation and resolution of multi-protein repair complexes. This process is regulated by a myriad of proteins that promote the association and disassociation of proteins to these lesions. Thanks in large part to the ability to perform functional screens of a vast library of proteins, there is a greater appreciation of the genes necessary for the double-strand DNA break repair. Often knockout or chemical inhibitor screens identify proteins involved in repair processes by using increased toxicity as a marker for a protein that is required for DSB repair. Although useful for identifying novel cellular proteins involved in maintaining genome fidelity, functional analysis requires the determination of whether the protein of interest promotes localization, formation, or resolution of repair complexes. The accumulation of repair proteins can be readily detected as distinct nuclear foci by immunofluorescence microscopy. Thus, association and disassociation of these proteins at sites of DNA damage can be accessed by observing these nuclear foci at representative intervals after the induction of double-strand DNA breaks. This approach can also identify mis-localized repair factor proteins, if repair defects do not simultaneously occur with incomplete delays in repair. In this scenario, long-lasting double-strand DNA breaks can be engineered by expressing a rare cutting endonuclease (e.g., I-SceI) in cells where the recognition site for the said enzyme has been integrated into the cellular genome. The resulting lesion is particularly hard to resolve as faithful repair will reintroduce the enzyme's recognition site, prompting another round of cleavage. As a result, differences in the kinetics of repair are eliminated. If repair complexes are not formed, localization has been impeded. This protocol describes the methodology necessary to identify changes in repair kinetics as well as repair protein localization.

Mechanism for verification of mismatched and homoduplex DNAs by nucleotides-bound MutS analyzed by molecular dynamics simulations.

PubMed

Ishida, Hisashi; Matsumoto, Atsushi

2016-09-01

In order to understand how MutS recognizes mismatched DNA and induces the reaction of DNA repair using ATP, the dynamics of the complexes of MutS (bound to the ADP and ATP nucleotides, or not) and DNA (with mismatched and matched base-pairs) were investigated using molecular dynamics simulations. As for DNA, the structure of the base-pairs of the homoduplex DNA which interacted with the DNA recognition site of MutS was intermittently disturbed, indicating that the homoduplex DNA was unstable. As for MutS, the disordered loops in the ATPase domains, which are considered to be necessary for the induction of DNA repair, were close to (away from) the nucleotide-binding sites in the ATPase domains when the nucleotides were (not) bound to MutS. This indicates that the ATPase domains changed their structural stability upon ATP binding using the disordered loop. Conformational analysis by principal component analysis showed that the nucleotide binding changed modes which have structurally solid ATPase domains and the large bending motion of the DNA from higher to lower frequencies. In the MutS-mismatched DNA complex bound to two nucleotides, the bending motion of the DNA at low frequency modes may play a role in triggering the formation of the sliding clamp for the following DNA-repair reaction step. Moreover, MM-PBSA/GBSA showed that the MutS-homoduplex DNA complex bound to two nucleotides was unstable because of the unfavorable interactions between MutS and DNA. This would trigger the ATP hydrolysis or separation of MutS and DNA to continue searching for mismatch base-pairs. Proteins 2016; 84:1287-1303. © 2016 Wiley Periodicals, Inc. © 2016 Wiley Periodicals, Inc.

Changes in solvation during DNA binding and cleavage are critical to altered specificity of the EcoRI endonuclease

PubMed Central

Robinson, Clifford R.; Sligar, Stephen G.

1998-01-01

Restriction endonucleases such as EcoRI bind and cleave DNA with great specificity and represent a paradigm for protein–DNA interactions and molecular recognition. Using osmotic pressure to induce water release, we demonstrate the participation of bound waters in the sequence discrimination of substrate DNA by EcoRI. Changes in solvation can play a critical role in directing sequence-specific DNA binding by EcoRI and are also crucial in assisting site discrimination during catalysis. By measuring the volume change for complex formation, we show that at the cognate sequence (GAATTC) EcoRI binding releases about 70 fewer water molecules than binding at an alternate DNA sequence (TAATTC), which differs by a single base pair. EcoRI complexation with nonspecific DNA releases substantially less water than either of these specific complexes. In cognate substrates (GAATTC) kcat decreases as osmotic pressure is increased, indicating the binding of about 30 water molecules accompanies the cleavage reaction. For the alternate substrate (TAATTC), release of about 40 water molecules accompanies the reaction, indicated by a dramatic acceleration of the rate when osmotic pressure is raised. These large differences in solvation effects demonstrate that water molecules can be key players in the molecular recognition process during both association and catalytic phases of the EcoRI reaction, acting to change the specificity of the enzyme. For both the protein–DNA complex and the transition state, there may be substantial conformational differences between cognate and alternate sites, accompanied by significant alterations in hydration and solvent accessibility. PMID:9482860

Phosphodiester Cleavage in Ribonuclease H Occurs via an Associative Two-Metal-Aided Catalytic Mechanism

PubMed Central

De Vivo, Marco; Dal Peraro, Matteo; Klein, Michael L.

2009-01-01

Ribonuclease H (RNase H) belongs to the nucleotidyl-transferase (NT) superfamily and hydrolyzes the phosphodiester linkages that form the backbone of the RNA strand in RNA·DNA hybrids. This enzyme is implicated in replication initiation and DNA topology restoration and represents a very promising target for anti-HIV drug design. Structural information has been provided by high-resolution crystal structures of the complex RNase H/RNA·DNA from Bacillus halodurans (Bh), which reveals that two metal ions are required for formation of a catalytic active complex. Here, we use classical force field-based and quantum mechanics/molecular mechanics calculations for modeling the nucleotidyl transfer reaction in RNase H, clarifying the role of the metal ions and the nature of the nucleophile (water versus hydroxide ion). During the catalysis, the two metal ions act cooperatively, facilitating nucleophile formation and stabilizing both transition state and leaving group. Importantly, the two Mg2+ metals also support the formation of a meta-stable phosphorane intermediate along the reaction, which resembles the phosphorane intermediate structure obtained only in the debated β-phosphoglucomutase crystal. The nucleophile formation (i.e., water deprotonation) can be achieved in situ, after migration of one proton from the water to the scissile phosphate in the transition state. This proton transfer is actually mediated by solvation water molecules. Due to the highly conserved nature of the enzymatic bimetal motif, these results might also be relevant for structurally similar enzymes belonging to the NT superfamily. PMID:18662000

Replication Protein A2c Coupled with Replication Protein A1c Regulates Crossover Formation during Meiosis in Rice[C][W][OPEN

PubMed Central

Li, Xingwang; Chang, Yuxiao; Xin, Xiaodong; Zhu, Chunmei; Li, Xianghua; Higgins, James D.; Wu, Changyin

2013-01-01

Replication protein A (RPA) is a conserved heterotrimeric protein complex comprising RPA1, RPA2, and RPA3 subunits involved in multiple DNA metabolism pathways attributable to its single-stranded DNA binding property. Unlike other species possessing a single RPA2 gene, rice (Oryza sativa) possesses three RPA2 paralogs, but their functions remain unclear. In this study, we identified RPA2c, a rice gene preferentially expressed during meiosis. A T-DNA insertional mutant (rpa2c) exhibited reduced bivalent formation, leading to chromosome nondisjunction. In rpa2c, chiasma frequency is reduced by ∼78% compared with the wild type and is accompanied by loss of the obligate chiasma. The residual ∼22% chiasmata fit a Poisson distribution, suggesting loss of crossover control. RPA2c colocalized with the meiotic cohesion subunit REC8 and the axis-associated protein PAIR2. Localization of REC8 was necessary for loading of RPA2c to the chromosomes. In addition, RPA2c partially colocalized with MER3 during late leptotene, thus indicating that RPA2c is required for class I crossover formation at a late stage of homologous recombination. Furthermore, we identified RPA1c, an RPA1 subunit with nearly overlapping distribution to RPA2c, required for ∼79% of chiasmata formation. Our results demonstrate that an RPA complex comprising RPA2c and RPA1c is required to promote meiotic crossovers in rice. PMID:24122830

Unrepaired DNA damage in macrophages causes elevation of particulate matter- induced airway inflammatory response.

PubMed

Luo, Man; Bao, Zhengqiang; Xu, Feng; Wang, Xiaohui; Li, Fei; Li, Wen; Chen, Zhihua; Ying, Songmin; Shen, Huahao

2018-04-14

The inflammatory cascade can be initiated with the recognition of damaged DNA. Macrophages play an essential role in particulate matter (PM)-induced airway inflammation. In this study, we aim to explore the PM induced DNA damage response of macrophages and its function in airway inflammation. The DNA damage response and inflammatory response were assessed using bone marrow-derived macrophages following PM treatment and mouse model instilled intratracheally with PM. We found that PM induced significant DNA damage both in vitro and in vivo and simultaneously triggered a rapid DNA damage response, represented by nuclear RPA, 53BP1 and γH2AX foci formation. Genetic ablation or chemical inhibition of the DNA damage response sensor amplified the production of cytokines including Cxcl1, Cxcl2 and Ifn-γ after PM stimulation in bone marrow-derived macrophages. Similar to that seen in vitro , mice with myeloid-specific deletion of RAD50 showed higher levels of airway inflammation in response to the PM challenge, suggesting a protective role of DNA damage sensor during inflammation. These data demonstrate that PM exposure induces DNA damage and activation of DNA damage response sensor MRN complex in macrophages. Disruption of MRN complex lead to persistent, unrepaired DNA damage that causes elevated inflammatory response.

Topological Behavior of Plasmid DNA

PubMed Central

Higgins, N. Patrick; Vologodskii, Alexander V.

2015-01-01

The discovery of the B-form structure of DNA by Watson and Crick led to an explosion of research on nucleic acids in the fields of biochemistry, biophysics, and genetics. Powerful techniques were developed to reveal a myriad of different structural conformations that change B-DNA as it is transcribed, replicated, and recombined and as sister chromosomes are moved into new daughter cell compartments during cell division. This article links the original discoveries of superhelical structure and molecular topology to non-B form DNA structure and contemporary biochemical and biophysical techniques. The emphasis is on the power of plasmids for studying DNA structure and function. The conditions that trigger the formation of alternative DNA structures such as left-handed Z-DNA, inter- and intra-molecular triplexes, triple-stranded DNA, and linked catenanes and hemicatenanes are explained. The DNA dynamics and topological issues are detailed for stalled replication forks and for torsional and structural changes on DNA in front of and behind a transcription complex and a replisome. The complex and interconnected roles of topoisomerases and abundant small nucleoid association proteins are explained. And methods are described for comparing in vivo and in vitro reactions to probe and understand the temporal pathways of DNA and chromosome chemistry that occur inside living cells. PMID:26104708

Regulated Eukaryotic DNA Replication Origin Firing with Purified Proteins

PubMed Central

Yeeles, Joseph T.P.; Deegan, Tom D.; Janska, Agnieszka; Early, Anne; Diffley, John F. X.

2016-01-01

Eukaryotic cells initiate DNA replication from multiple origins, which must be tightly regulated to promote precise genome duplication in every cell cycle. To accomplish this, initiation is partitioned into two temporally discrete steps: a double hexameric MCM complex is first loaded at replication origins during G1 phase, and then converted to the active CMG (Cdc45, MCM, GINS) helicase during S phase. Here we describe the reconstitution of budding yeast DNA replication initiation with 16 purified replication factors, made from 42 polypeptides. Origin-dependent initiation recapitulates regulation seen in vivo. Cyclin dependent kinase (CDK) inhibits MCM loading by phosphorylating the origin recognition complex (ORC) and promotes CMG formation by phosphorylating Sld2 and Sld3. Dbf4 dependent kinase (DDK) promotes replication by phosphorylating MCM, and can act either before or after CDK. These experiments define the minimum complement of proteins, protein kinase substrates and co-factors required for regulated eukaryotic DNA replication. PMID:25739503

Regulated eukaryotic DNA replication origin firing with purified proteins.

PubMed

Yeeles, Joseph T P; Deegan, Tom D; Janska, Agnieszka; Early, Anne; Diffley, John F X

2015-03-26

Eukaryotic cells initiate DNA replication from multiple origins, which must be tightly regulated to promote precise genome duplication in every cell cycle. To accomplish this, initiation is partitioned into two temporally discrete steps: a double hexameric minichromosome maintenance (MCM) complex is first loaded at replication origins during G1 phase, and then converted to the active CMG (Cdc45-MCM-GINS) helicase during S phase. Here we describe the reconstitution of budding yeast DNA replication initiation with 16 purified replication factors, made from 42 polypeptides. Origin-dependent initiation recapitulates regulation seen in vivo. Cyclin-dependent kinase (CDK) inhibits MCM loading by phosphorylating the origin recognition complex (ORC) and promotes CMG formation by phosphorylating Sld2 and Sld3. Dbf4-dependent kinase (DDK) promotes replication by phosphorylating MCM, and can act either before or after CDK. These experiments define the minimum complement of proteins, protein kinase substrates and co-factors required for regulated eukaryotic DNA replication.

Mediator and Cohesin Connect Gene Expression and Chromatin Architecture

PubMed Central

Kagey, Michael H.; Newman, Jamie J.; Bilodeau, Steve; Zhan, Ye; Orlando, David A.; van Berkum, Nynke L.; Ebmeier, Christopher C.; Goossens, Jesse; Rahl, Peter B.; Levine, Stuart S.; Taatjes, Dylan J.; Dekker, Job; Young, Richard A.

2010-01-01

Summary Transcription factors control cell specific gene expression programs through interactions with diverse coactivators and the transcription apparatus. Gene activation may involve DNA loop formation between enhancer-bound transcription factors and the transcription apparatus at the core promoter, but this process is not well understood. We report here that Mediator and Cohesin physically and functionally connect the enhancers and core promoters of active genes in embryonic stem cells. Mediator, a transcriptional coactivator, forms a complex with Cohesin, which can form rings that connect two DNA segments. The Cohesin loading factor Nipbl is associated with Mediator/Cohesin complexes, providing a means to load Cohesin at promoters. DNA looping is observed between the enhancers and promoters occupied by Mediator and Cohesin. Mediator and Cohesin occupy different promoters in different cells, thus generating cell-type specific DNA loops linked to the gene expression program of each cell. PMID:20720539

SAD-3, a Putative Helicase Required for Meiotic Silencing by Unpaired DNA, Interacts with Other Components of the Silencing Machinery

PubMed Central

Hammond, Thomas M.; Xiao, Hua; Boone, Erin C.; Perdue, Tony D.; Pukkila, Patricia J.; Shiu, Patrick K. T.

2011-01-01

In Neurospora crassa, genes lacking a pairing partner during meiosis are suppressed by a process known as meiotic silencing by unpaired DNA (MSUD). To identify novel MSUD components, we have developed a high-throughput reverse-genetic screen for use with the N. crassa knockout library. Here we describe the screening method and the characterization of a gene (sad-3) subsequently discovered. SAD-3 is a putative helicase required for MSUD and sexual spore production. It exists in a complex with other known MSUD proteins in the perinuclear region, a center for meiotic silencing activity. Orthologs of SAD-3 include Schizosaccharomyces pombe Hrr1, a helicase required for RNAi-induced heterochromatin formation. Both SAD-3 and Hrr1 interact with an RNA-directed RNA polymerase and an Argonaute, suggesting that certain aspects of silencing complex formation may be conserved between the two fungal species. PMID:22384347

Antimicrobial enzymes: an emerging strategy to fight microbes and microbial biofilms.

PubMed

Thallinger, Barbara; Prasetyo, Endry N; Nyanhongo, Gibson S; Guebitz, Georg M

2013-01-01

With the increasing prevalence of antibiotic resistance, antimicrobial enzymes aimed at the disruption of bacterial cellular machinery and biofilm formation are under intense investigation. Several enzyme-based products have already been commercialized for application in the healthcare, food and biomedical industries. Successful removal of complex biofilms requires the use of multi-enzyme formulations that contain enzymes capable of degrading microbial DNA, polysaccharides, proteins and quorum-sensing molecules. The inclusion of anti-quorum sensing enzymes prevents biofilm reformation. The development of effective complex enzyme formulations is urgently needed to deal with the problems associated with biofilm formation in manufacturing, environmental protection and healthcare settings. Nevertheless, advances in synthetic biology, enzyme engineering and whole DNA-Sequencing technologies show great potential to facilitate the development of more effective antimicrobial and anti-biofilm enzymes. Copyright © 2013 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

The mismatch repair and meiotic recombination endonuclease Mlh1-Mlh3 is activated by polymer formation and can cleave DNA substrates in trans

PubMed Central

Manhart, Carol M.; Ni, Xiaodan; White, Martin A.; Ortega, Joaquin; Surtees, Jennifer A.

2017-01-01

Crossing over between homologs is initiated in meiotic prophase by the formation of DNA double-strand breaks that occur throughout the genome. In the major interference-responsive crossover pathway in baker’s yeast, these breaks are resected to form 3' single-strand tails that participate in a homology search, ultimately forming double Holliday junctions (dHJs) that primarily include both homologs. These dHJs are resolved by endonuclease activity to form exclusively crossovers, which are critical for proper homolog segregation in Meiosis I. Recent genetic, biochemical, and molecular studies in yeast are consistent with the hypothesis of Mlh1-Mlh3 DNA mismatch repair complex acting as the major endonuclease activity that resolves dHJs into crossovers. However, the mechanism by which the Mlh1-Mlh3 endonuclease is activated is unknown. Here, we provide evidence that Mlh1-Mlh3 does not behave like a structure-specific endonuclease but forms polymers required to generate nicks in DNA. This conclusion is supported by DNA binding studies performed with different-sized substrates that contain or lack polymerization barriers and endonuclease assays performed with varying ratios of endonuclease-deficient and endonuclease-proficient Mlh1-Mlh3. In addition, Mlh1-Mlh3 can generate religatable double-strand breaks and form an active nucleoprotein complex that can nick DNA substrates in trans. Together these observations argue that Mlh1-Mlh3 may not act like a canonical, RuvC-like Holliday junction resolvase and support a novel model in which Mlh1-Mlh3 is loaded onto DNA to form an activated polymer that cleaves DNA. PMID:28453523

The mismatch repair and meiotic recombination endonuclease Mlh1-Mlh3 is activated by polymer formation and can cleave DNA substrates in trans.

PubMed

Manhart, Carol M; Ni, Xiaodan; White, Martin A; Ortega, Joaquin; Surtees, Jennifer A; Alani, Eric

2017-04-01

Crossing over between homologs is initiated in meiotic prophase by the formation of DNA double-strand breaks that occur throughout the genome. In the major interference-responsive crossover pathway in baker's yeast, these breaks are resected to form 3' single-strand tails that participate in a homology search, ultimately forming double Holliday junctions (dHJs) that primarily include both homologs. These dHJs are resolved by endonuclease activity to form exclusively crossovers, which are critical for proper homolog segregation in Meiosis I. Recent genetic, biochemical, and molecular studies in yeast are consistent with the hypothesis of Mlh1-Mlh3 DNA mismatch repair complex acting as the major endonuclease activity that resolves dHJs into crossovers. However, the mechanism by which the Mlh1-Mlh3 endonuclease is activated is unknown. Here, we provide evidence that Mlh1-Mlh3 does not behave like a structure-specific endonuclease but forms polymers required to generate nicks in DNA. This conclusion is supported by DNA binding studies performed with different-sized substrates that contain or lack polymerization barriers and endonuclease assays performed with varying ratios of endonuclease-deficient and endonuclease-proficient Mlh1-Mlh3. In addition, Mlh1-Mlh3 can generate religatable double-strand breaks and form an active nucleoprotein complex that can nick DNA substrates in trans. Together these observations argue that Mlh1-Mlh3 may not act like a canonical, RuvC-like Holliday junction resolvase and support a novel model in which Mlh1-Mlh3 is loaded onto DNA to form an activated polymer that cleaves DNA.

Induction of apoptosis by plumbagin through reactive oxygen species-mediated inhibition of topoisomerase II

DOE Office of Scientific and Technical Information (OSTI.GOV)

Kawiak, Anna; Piosik, Jacek; Stasilojc, Grzegorz

2007-09-15

Reactive oxygen species (ROS) have been recognized as key molecules, which can selectively modify proteins and therefore regulate cellular signalling including apoptosis. Plumbagin, a naphthoquinone exhibiting antitumor activity, is known to generate ROS and has been found to inhibit the activity of topoisomerase II (Topo II) through the stabilization of the Topo II-DNA cleavable complex. The objective of this research was to clarify the role of ROS and Topo II inhibition in the induction of apoptosis mediated by plumbagin. As determined by the comet assay, plumbagin induced DNA cleavage in HL-60 cells, whereas in a cell line with reduced Topomore » II activity-HL-60/MX2, the level of DNA damage was significantly decreased. The onset of DNA strand break formation in HL-60 cells was delayed in comparison with the generation of intracellular ROS. In HL-60/MX2 cells, ROS were generated at a similar rate, whereas a significant reduction in the level of DNA damage was detected. The pretreatment of cells with N-acetylcysteine (NAC) attenuated plumbagin-induced DNA damage, pointing out to the involvement of ROS generation in cleavable complex formation. These results suggest that plumbagin-induced ROS does not directly damage DNA but requires the involvement of Topo II. Furthermore, experiments carried out using light spectroscopy indicated no direct interactions between plumbagin and DNA. The induction of apoptosis was significantly delayed in HL-60/MX2 cells indicating the involvement of Topo II inhibition in plumbagin-mediated apoptosis. Thus, these findings strongly suggest ROS-mediated inhibition of Topo II as an important mechanism contributing to the apoptosis-inducing properties of plumbagin.« less

Electrostatic study of Alanine mutational effects on transcription: application to GATA-3:DNA interaction complex.

PubMed

El-Assaad, Atlal; Dawy, Zaher; Nemer, Georges

2015-01-01

Protein-DNA interaction is of fundamental importance in molecular biology, playing roles in functions as diverse as DNA transcription, DNA structure formation, and DNA repair. Protein-DNA association is also important in medicine; understanding Protein-DNA binding kinetics can assist in identifying disease root causes which can contribute to drug development. In this perspective, this work focuses on the transcription process by the GATA Transcription Factor (TF). GATA TF binds to DNA promoter region represented by `G,A,T,A' nucleotides sequence, and initiates transcription of target genes. When proper regulation fails due to some mutations on the GATA TF protein sequence or on the DNA promoter sequence (weak promoter), deregulation of the target genes might lead to various disorders. In this study, we aim to understand the electrostatic mechanism behind GATA TF and DNA promoter interactions, in order to predict Protein-DNA binding in the presence of mutations, while elaborating on non-covalent binding kinetics. To generate a family of mutants for the GATA:DNA complex, we replaced every charged amino acid, one at a time, with a neutral amino acid like Alanine (Ala). We then applied Poisson-Boltzmann electrostatic calculations feeding into free energy calculations, for each mutation. These calculations delineate the contribution to binding from each Ala-replaced amino acid in the GATA:DNA interaction. After analyzing the obtained data in view of a two-step model, we are able to identify potential key amino acids in binding. Finally, we applied the model to GATA-3:DNA (crystal structure with PDB-ID: 3DFV) binding complex and validated it against experimental results from the literature.

Role of indirect readout mechanism in TATA box binding protein-DNA interaction.

PubMed

Mondal, Manas; Choudhury, Devapriya; Chakrabarti, Jaydeb; Bhattacharyya, Dhananjay

2015-03-01

Gene expression generally initiates from recognition of TATA-box binding protein (TBP) to the minor groove of DNA of TATA box sequence where the DNA structure is significantly different from B-DNA. We have carried out molecular dynamics simulation studies of TBP-DNA system to understand how the DNA structure alters for efficient binding. We observed rigid nature of the protein while the DNA of TATA box sequence has an inherent flexibility in terms of bending and minor groove widening. The bending analysis of the free DNA and the TBP bound DNA systems indicate presence of some similar structures. Principal coordinate ordination analysis also indicates some structural features of the protein bound and free DNA are similar. Thus we suggest that the DNA of TATA box sequence regularly oscillates between several alternate structures and the one suitable for TBP binding is induced further by the protein for proper complex formation.

Human RAD50 makes a functional DNA-binding complex.

PubMed

Kinoshita, Eri; van Rossum-Fikkert, Sari; Sanchez, Humberto; Kertokalio, Aryandi; Wyman, Claire

2015-06-01

The MRE11-RAD50-NBS1 (MRN) complex has several distinct functions in DNA repair including important roles in both non-homologous end-joining (NHEJ) and homologous recombination (HR). The biochemical activities of MR(N) have been well characterized implying specific functional roles for the components. The arrangement of proteins in the complex implies interdependence of their biochemical activities making it difficult to separate specific functions. We obtained purified human RAD50 and observed that it binds ATP, undergoes ATP-dependent conformational changes as well as having ATPase activity. Scanning force microscopy analysis clearly showed that RAD50 binds DNA although not as oligomers. RAD50 alone was not functional in tethering DNA molecules. ATP increased formation of RAD50 multimers which were however globular lacking extended coiled coils, in contrast to the MR complex where ATP induced oligomers have obvious coiled coils protruding from a central domain. These results suggest that MRE11 is important in maintaining the structural arrangement of RAD50 in the protein complex and perhaps has a role in reinforcing proper alignment of the coiled coils in the ATP-bound state. Copyright © 2015 Elsevier B.V. and Société Française de Biochimie et Biologie Moléculaire (SFBBM). All rights reserved.

The structure of a ring-opened proliferating cell nuclear antigen-replication factor C complex revealed by fluorescence energy transfer.

PubMed

Zhuang, Zhihao; Yoder, Bonita L; Burgers, Peter M J; Benkovic, Stephen J

2006-02-21

Numerous proteins that function in DNA metabolic pathways are known to interact with the proliferating cell nuclear antigen (PCNA). The important function of PCNA in stimulating various cellular activities requires its topological linkage with DNA. Loading of the circular PCNA onto duplex DNA requires the activity of a clamp-loader [replication factor C (RFC)] complex and the energy derived from ATP hydrolysis. The mechanistic and structural details regarding PCNA loading by the RFC complex are still developing. In particular, the positive identification of a long-hypothesized structure of an open clamp-RFC complex as an intermediate in loading has remained elusive. In this study, we capture an open yeast PCNA clamp in a complex with RFC through fluorescence energy transfer experiments. We also follow the topological transitions of PCNA in the various steps of the clamp-loading pathway through both steady-state and stopped-flow fluorescence studies. We find that ATP effectively drives the clamp-loading process to completion with the formation of the closed PCNA bound to DNA, whereas ATPgammaS cannot. The information derived from this work complements that obtained from previous structural and mechanistic studies and provides a more complete picture of a eukaryotic clamp-loading pathway using yeast as a paradigm.

Interaction with Single-stranded DNA-binding Protein Stimulates Escherichia coli Ribonuclease HI Enzymatic Activity*

PubMed Central

Petzold, Christine; Marceau, Aimee H.; Miller, Katherine H.; Marqusee, Susan; Keck, James L.

2015-01-01

Single-stranded (ss) DNA-binding proteins (SSBs) bind and protect ssDNA intermediates formed during replication, recombination, and repair reactions. SSBs also directly interact with many different genome maintenance proteins to stimulate their enzymatic activities and/or mediate their proper cellular localization. We have identified an interaction formed between Escherichia coli SSB and ribonuclease HI (RNase HI), an enzyme that hydrolyzes RNA in RNA/DNA hybrids. The RNase HI·SSB complex forms by RNase HI binding the intrinsically disordered C terminus of SSB (SSB-Ct), a mode of interaction that is shared among all SSB interaction partners examined to date. Residues that comprise the SSB-Ct binding site are conserved among bacterial RNase HI enzymes, suggesting that RNase HI·SSB complexes are present in many bacterial species and that retaining the interaction is important for its cellular function. A steady-state kinetic analysis shows that interaction with SSB stimulates RNase HI activity by lowering the reaction Km. SSB or RNase HI protein variants that disrupt complex formation nullify this effect. Collectively our findings identify a direct RNase HI/SSB interaction that could play a role in targeting RNase HI activity to RNA/DNA hybrid substrates within the genome. PMID:25903123

Synthesis, Characterization, and Biological Activity Studies of Copper(II) Mixed Compound with Histamine and Nalidixic Acid

PubMed Central

Bivián-Castro, Egla Yareth; López, Mercedes G.; Pedraza-Reyes, Mario; Bernès, Sylvain; Mendoza-Díaz, Guillermo

2009-01-01

A mixed copper complex with deprotonated nalidixic acid (nal) and histamine (hsm) was synthesized and characterized by FTIR, UV-Vis, elemental analysis, and conductivity. The crystal structure of [Cu(hsm)(nal)H2O]Cl·3H2O (chn) showed a pentacoordinated cooper(II) in a square pyramidal geometry surrounded by two N atoms from hsm, two O atoms from the quinolone, and one apical water oxygen. Alteration of bacterial DNA structure and/or associated functions in vivo by [Cu(hsm)(nal)H2O]Cl·3H2O was demonstrated by the induction of a recA-lacZ fusion integrated at the amyE locus of a recombinant Bacillus subtilis strain. Results from circular dichroism and denaturation of calf thymus DNA (CT-DNA) suggested that increased amounts of copper complex were able to stabilize the double helix of DNA in vitro mainly by formation of hydrogen bonds between chn and the sugars of DNA minor groove. In vivo and in vitro biological activities of the chn complex were compared with the chemical nuclease [Cu(phen)(nal)H2O]NO3 · 3H2O (cpn) where phen is phenanthroline. PMID:19557138

An analysis of subunit exchange in the dimeric DNA-binding and DNA-bending protein, TF1.

PubMed

Andera, L; Schneider, G J; Geiduschek, E P

1994-01-01

TF1 is the Bacillus subtilis bacteriophage-encoded dimeric type II DNA-binding protein. This relative of the eubacterial HU proteins and of the Escherichia coli integration host factor binds preferentially to 5-(hydroxymethyluracil)-containing DNA. We have examined the dynamics of exchange of monomer subunits between molecules of dimeric TF1. The analysis takes advantage of the fact that replacement of phenylalanine with arginine at amino acid 61 in the beta-loop 'arm' of TF1 alters DNA-bending and -binding properties, generating DNA complexes with distinctively different mobilities in gel electrophoresis. New species of DNA-protein complexes were formed by mixtures of wild type and mutant TF1, reflecting the formation of heterodimeric TF1, and making the dynamics of monomer exchange between TF1 dimers accessible to a simple gel retardation analysis. Exchange was rapid at high protein concentrations, even at 0 degrees C, and is proposed to be capable of proceeding through an interaction of molecules of TF1 dimer rather than exclusively through dissociation into monomer subunits. Evidence suggesting that DNA-bound TF1 dimers do not exchange subunits readily is also presented.

Diversity in Requirement of Genetic and Epigenetic Factors for Centromere Function in Fungi ▿

PubMed Central

Roy, Babhrubahan; Sanyal, Kaustuv

2011-01-01

A centromere is a chromosomal region on which several proteins assemble to form the kinetochore. The centromere-kinetochore complex helps in the attachment of chromosomes to spindle microtubules to mediate segregation of chromosomes to daughter cells during mitosis and meiosis. In several budding yeast species, the centromere forms in a DNA sequence-dependent manner, whereas in most other fungi, factors other than the DNA sequence also determine the centromere location, as centromeres were able to form on nonnative sequences (neocentromeres) when native centromeres were deleted in engineered strains. Thus, in the absence of a common DNA sequence, the cues that have facilitated centromere formation on a specific DNA sequence for millions of years remain a mystery. Kinetochore formation is facilitated by binding of a centromere-specific histone protein member of the centromeric protein A (CENP-A) family that replaces a canonical histone H3 to form a specialized centromeric chromatin structure. However, the process of kinetochore formation on the rapidly evolving and seemingly diverse centromere DNAs in different fungal species is largely unknown. More interestingly, studies in various yeasts suggest that the factors required for de novo centromere formation (establishment) may be different from those required for maintenance (propagation) of an already established centromere. Apart from the DNA sequence and CENP-A, many other factors, such as posttranslational modification (PTM) of histones at centric and pericentric chromatin, RNA interference, and DNA methylation, are also involved in centromere formation, albeit in a species-specific manner. In this review, we discuss how several genetic and epigenetic factors influence the evolution of structure and function of centromeres in fungal species. PMID:21908596

Recognition of the Xenopus ribosomal core promoter by the transcription factor xUBF involves multiple HMG box domains and leads to an xUBF interdomain interaction.

PubMed

Leblanc, B; Read, C; Moss, T

1993-02-01

The interaction of the ribosomal transcription factor xUBF with the RNA polymerase I core promoter of Xenopus laevis has been studied both at the DNA and protein levels. It is shown that a single xUBF-DNA complex forms over the 40S initiation site (+1) and involves at least the DNA sequences between -20 and +60 bp. DNA sequences upstream of +10 and downstream of +18 are each sufficient to direct complex formation independently. HMG box 1 of xUBF independently recognizes the sequences -20 to -1 and +1 to +22 and the addition of the N-terminal dimerization domain to HMG box 1 stabilizes its interaction with these sequences approximately 10-fold. HMG boxes 2/3 interact with the DNA downstream of +22 and can independently position xUBF across the initiation site. The C-terminal segment of xUBF, HMG boxes 4, 5 or the acidic domain, directly or indirectly interact with HMG box 1, making the core promoter sequences between -11 and -15 hypersensitive to DNase. This interaction also requires the DNA sequences between +17 and +32, i.e. the HMG box 2/3 binding site. The data suggest extensive folding of the core promoter within the xUBF complex.

Comparing DNA damage-processing pathways by computer analysis of chromosome painting data.

PubMed

Levy, Dan; Vazquez, Mariel; Cornforth, Michael; Loucas, Bradford; Sachs, Rainer K; Arsuaga, Javier

2004-01-01

Chromosome aberrations are large-scale illegitimate rearrangements of the genome. They are indicative of DNA damage and informative about damage processing pathways. Despite extensive investigations over many years, the mechanisms underlying aberration formation remain controversial. New experimental assays such as multiplex fluorescent in situ hybridyzation (mFISH) allow combinatorial "painting" of chromosomes and are promising for elucidating aberration formation mechanisms. Recently observed mFISH aberration patterns are so complex that computer and graph-theoretical methods are needed for their full analysis. An important part of the analysis is decomposing a chromosome rearrangement process into "cycles." A cycle of order n, characterized formally by the cyclic graph with 2n vertices, indicates that n chromatin breaks take part in a single irreducible reaction. We here describe algorithms for computing cycle structures from experimentally observed or computer-simulated mFISH aberration patterns. We show that analyzing cycles quantitatively can distinguish between different aberration formation mechanisms. In particular, we show that homology-based mechanisms do not generate the large number of complex aberrations, involving higher-order cycles, observed in irradiated human lymphocytes.

Specific and non-specific interactions of ParB with DNA: implications for chromosome segregation

PubMed Central

Taylor, James A.; Pastrana, Cesar L.; Butterer, Annika; Pernstich, Christian; Gwynn, Emma J.; Sobott, Frank; Moreno-Herrero, Fernando; Dillingham, Mark S.

2015-01-01

The segregation of many bacterial chromosomes is dependent on the interactions of ParB proteins with centromere-like DNA sequences called parS that are located close to the origin of replication. In this work, we have investigated the binding of Bacillus subtilis ParB to DNA in vitro using a variety of biochemical and biophysical techniques. We observe tight and specific binding of a ParB homodimer to the parS sequence. Binding of ParB to non-specific DNA is more complex and displays apparent positive co-operativity that is associated with the formation of larger, poorly defined, nucleoprotein complexes. Experiments with magnetic tweezers demonstrate that non-specific binding leads to DNA condensation that is reversible by protein unbinding or force. The condensed DNA structure is not well ordered and we infer that it is formed by many looping interactions between neighbouring DNA segments. Consistent with this view, ParB is also able to stabilize writhe in single supercoiled DNA molecules and to bridge segments from two different DNA molecules in trans. The experiments provide no evidence for the promotion of non-specific DNA binding and/or condensation events by the presence of parS sequences. The implications of these observations for chromosome segregation are discussed. PMID:25572315

The role of Cas8 in type I CRISPR interference.

PubMed

Cass, Simon D B; Haas, Karina A; Stoll, Britta; Alkhnbashi, Omer S; Sharma, Kundan; Urlaub, Henning; Backofen, Rolf; Marchfelder, Anita; Bolt, Edward L

2015-05-05

CRISPR (clustered regularly interspaced short palindromic repeat) systems provide bacteria and archaea with adaptive immunity to repel invasive genetic elements. Type I systems use 'cascade' [CRISPR-associated (Cas) complex for antiviral defence] ribonucleoprotein complexes to target invader DNA, by base pairing CRISPR RNA (crRNA) to protospacers. Cascade identifies PAMs (protospacer adjacent motifs) on invader DNA, triggering R-loop formation and subsequent DNA degradation by Cas3. Cas8 is a candidate PAM recognition factor in some cascades. We analysed Cas8 homologues from type IB CRISPR systems in archaea Haloferax volcanii (Hvo) and Methanothermobacter thermautotrophicus (Mth). Cas8 was essential for CRISPR interference in Hvo and purified Mth Cas8 protein responded to PAM sequence when binding to nucleic acids. Cas8 interacted physically with Cas5-Cas7-crRNA complex, stimulating binding to PAM containing substrates. Mutation of conserved Cas8 amino acid residues abolished interference in vivo and altered catalytic activity of Cas8 protein in vitro. This is experimental evidence that Cas8 is important for targeting Cascade to invader DNA. © 2015 Authors.

Poly(ADP-ribose) polymerase 1 escorts XPC to UV-induced DNA lesions during nucleotide excision repair

PubMed Central

Robu, Mihaela; Shah, Rashmi G.; Purohit, Nupur K.; Zhou, Pengbo; Naegeli, Hanspeter

2017-01-01

Xeroderma pigmentosum C (XPC) protein initiates the global genomic subpathway of nucleotide excision repair (GG-NER) for removal of UV-induced direct photolesions from genomic DNA. The XPC has an inherent capacity to identify and stabilize at the DNA lesion sites, and this function is facilitated in the genomic context by UV-damaged DNA-binding protein 2 (DDB2), which is part of a multiprotein UV–DDB ubiquitin ligase complex. The nuclear enzyme poly(ADP-ribose) polymerase 1 (PARP1) has been shown to facilitate the lesion recognition step of GG-NER via its interaction with DDB2 at the lesion site. Here, we show that PARP1 plays an additional DDB2-independent direct role in recruitment and stabilization of XPC at the UV-induced DNA lesions to promote GG-NER. It forms a stable complex with XPC in the nucleoplasm under steady-state conditions before irradiation and rapidly escorts it to the damaged DNA after UV irradiation in a DDB2-independent manner. The catalytic activity of PARP1 is not required for the initial complex formation with XPC in the nucleoplasm but it enhances the recruitment of XPC to the DNA lesion site after irradiation. Using purified proteins, we also show that the PARP1–XPC complex facilitates the handover of XPC to the UV-lesion site in the presence of the UV–DDB ligase complex. Thus, the lesion search function of XPC in the genomic context is controlled by XPC itself, DDB2, and PARP1. Our results reveal a paradigm that the known interaction of many proteins with PARP1 under steady-state conditions could have functional significance for these proteins. PMID:28760956

Poly(ADP-ribose) polymerase 1 escorts XPC to UV-induced DNA lesions during nucleotide excision repair.

PubMed

Robu, Mihaela; Shah, Rashmi G; Purohit, Nupur K; Zhou, Pengbo; Naegeli, Hanspeter; Shah, Girish M

2017-08-15

Xeroderma pigmentosum C (XPC) protein initiates the global genomic subpathway of nucleotide excision repair (GG-NER) for removal of UV-induced direct photolesions from genomic DNA. The XPC has an inherent capacity to identify and stabilize at the DNA lesion sites, and this function is facilitated in the genomic context by UV-damaged DNA-binding protein 2 (DDB2), which is part of a multiprotein UV-DDB ubiquitin ligase complex. The nuclear enzyme poly(ADP-ribose) polymerase 1 (PARP1) has been shown to facilitate the lesion recognition step of GG-NER via its interaction with DDB2 at the lesion site. Here, we show that PARP1 plays an additional DDB2-independent direct role in recruitment and stabilization of XPC at the UV-induced DNA lesions to promote GG-NER. It forms a stable complex with XPC in the nucleoplasm under steady-state conditions before irradiation and rapidly escorts it to the damaged DNA after UV irradiation in a DDB2-independent manner. The catalytic activity of PARP1 is not required for the initial complex formation with XPC in the nucleoplasm but it enhances the recruitment of XPC to the DNA lesion site after irradiation. Using purified proteins, we also show that the PARP1-XPC complex facilitates the handover of XPC to the UV-lesion site in the presence of the UV-DDB ligase complex. Thus, the lesion search function of XPC in the genomic context is controlled by XPC itself, DDB2, and PARP1. Our results reveal a paradigm that the known interaction of many proteins with PARP1 under steady-state conditions could have functional significance for these proteins.

Influence of hydroxyl groups on the biological properties of cationic polymethacrylates as gene vectors.

PubMed

Ma, Ming; Li, Feng; Yuan, Zhe-fan; Zhuo, Ren-xi

2010-07-01

In this study poly(aminoethyl methacrylate) (PAEMA), poly(3-amino-2-hydroxypropyl methacrylate) (PAHPMA), poly(2-(2-aminoethylamino)ethyl methacrylate) (PAEAEMA) and poly(3-(2-aminoethylamino) 2-hydroxypropyl methacrylate) (PAEAHPMA) were synthesized using atom transfer radical polymerization to evaluate the effect of hydroxyl groups on the relative properties of cationic polymeric gene vectors. The results of heparin displacement assays showed that PAHPMA possessed a stronger binding capacity than PAEMA. PAHPMA/DNA complexes and PAEAHPMA/DNA complexes had lower zeta potentials than those of PAEMA and PAEAEMA. MTT assay results indicated that PAHPMA and PAEAHPMA exhibited obviously lower cytotoxicities than PAEMA and PAEAEMA. Subsequently, in vitro gene transfection studies in 293T cells without serum showed that PAHPMA exhibited a lower transfection efficiency than PAEMA and PAEAHPMA/DNA complexes possessed a similar transfection efficiency to PAEAEMA/DNA complexes. Moreover, PAHPMA and PAEAHPMA retained similar transfection efficiencies in DMEM with 10% serum, but PAEMA and PAEAEMA showed slightly lower transfection efficiencies than in the absence of serum. The reason for these phenomena might be attributed to the introduction of hydroxyl groups into PAHPMA and PAEAHPMA, i.e. the existence of hydroxyl groups might increase the binding capacity to DNA and at the same time decrease the surface charge of the polymer/DNA complexes due to the formation of hydrogen bonds between the polymers and DNA. Therefore, a lower zeta potential and stronger binding ability may result in a lower gene transfection efficiency. This effect of hydroxyl groups decreased with increasing amino group density on the polymer. Copyright 2010 Acta Materialia Inc. Published by Elsevier Ltd. All rights reserved.

Structure transition in lipids and nucleic acids of tumor cells under anticancer drugs applications

NASA Astrophysics Data System (ADS)

Dovbeshko, G. I.; Repnytska, O. P.; Tryndiak, V. P.; Todor, I. N.

2003-12-01

Interaction of DNA and phospholipids from Carcinoma Guerina resistant and sensitive cells of Wistar line rats with anti-cancer drugs - cis-platin and doxorubicin (DOX) have been studied in vivo and in vitro experiments. Surface enhanced infrared absorption (SEIRA) spectroscopy was applied for registration of conformational changes in DNA and lipids induced by anti-cancer drugs. It has been shown in vivo experiment that doxorubicin influences less structural disordering of the membrane than cis-platin. Cis-platin creates irreversible complex with memebrane phospholipids, strongly interacting with phosophates and carbohydrate chains. Doxorubicin influences the ordering of carbohydrate chains and does not strongly influence phosphate heads. This change seems to be partially reversible. In contrast, in vivo experiment the doxorubicin strongly influences the DNA structure, leading to DNA stabilization and formation of new H-bonds in DNA-doxorubicin complex. We have not registered the interaction of DNA with cis-platin in vivo experiment. Experiment in vitro for cis-platin incubation with phospholipids from cancer cells during 0.5 hour at 37°C has not shown those drastic structural peculiarities that it was observed in vivo experiments.

An electrochemical label-free and sensitive thrombin aptasensor based on graphene oxide modified pencil graphite electrode.

PubMed

Ahour, F; Ahsani, M K

2016-12-15

In this work, we tactfully constructed a novel label-free electrochemical aptasensor for rapid and facile detection of thrombin using graphene oxide (GO) and thrombin binding aptamer (TBA). The strategy relies on the preferential adsorption of single-stranded DNA (ssDNA) to GO over aptamer-target complexes. The TBA-thrombin complex formation was monitored by differential pulse voltammetry (DPV) using the guanine oxidation signal. In the absence of thrombin, the aptamers adsorbed onto the surface of GO leading to a strong background guanine oxidation signal. Conversely, in the presence of thrombin, the conformational transformation of TBA after incubating with the thrombin solution and formation of the aptamer-thrombin complexes which had weak binding ability to GO, leads to the desorption of TBA-thrombin complex from electrode surface and significant oxidation signal decrease. The selectivity of the biosensor was studied using other biological substances. The biosensor's signal was proportional to the thrombin concentration from 0.1 to 10nM with a detection limit of 0.07nM. Particularly, the proposed method could be widely applied to the aptamer-based determination of other target analytes. Copyright © 2016 Elsevier B.V. All rights reserved.

Binding of resveratrol to the minor groove of DNA sequences with AATT and TTAA segments induces differential stability.

PubMed

Nair, Maya S; D'Mello, Samar; Pant, Rashmi; Poluri, Krishna Mohan

2017-05-01

Interactions of a natural stilbene compound, resveratrol with two DNA sequences containing AATT/TTAA segments have been studied. Resveratrol is found to interact with both the sequences. The mode of interaction has been studied using absorption, steady state fluorescence and circular dichroism spectroscopic techniques. UV-visible absorption and fluorescence studies provided the information regarding the binding constants and the stoichiometry of binding, whereas circular dichroism studies depicted the structural changes in DNA upon resveratrol binding. Our results evidenced that, though resveratrol showed similar affinity to both the sequences, the mode of interactions was different. The binding constants of resveratrol to AATT/TTAA sequences were found to be 7.55×10 5 M -1 and 5.42×10 5 M -1 respectively. Spectroscopic data evidenced for a groove binding interaction. Melting studies showed that the binding of resveratrol induces differential stability to the DNA sequences d(CGTTAACG) 2 and d(CGAATTCG) 2 . Fluorescence data showed a stoichiometry of 1:1 for d(CGAATTCG) 2 -resveratrol complex and 1:4 for d(CGTTAACG) 2 -resveratrol complex. Molecular docking studies demonstrated that resveratrol binds to the minor groove region of both the sequences to form stable complexes with varied atomic contacts to the DNA bases or backbone. Both the complexes are stabilized by hydrogen bond formation. Our results evidenced that modulation of DNA sequence within the same bases can greatly alter the binding geometry and stability of the complex upon binding to small molecule inhibitor compounds like resveratrol. Copyright © 2017 Elsevier B.V. All rights reserved.

Complementary and partially complementary DNA duplexes tethered to a functionalized substrate: a molecular dynamics approach to biosensing.

PubMed

Monti, Susanna; Cacelli, Ivo; Ferretti, Alessandro; Prampolini, Giacomo; Barone, Vincenzo

2011-07-21

Molecular dynamics simulations (90 ns) of different DNA complexes attached to a functionalized substrate in solution were performed in order to clarify the behavior of mismatched DNA sequences captured by a tethered DNA probe (biochip). Examination of the trajectories revealed that the substrate influence and a series of cooperative events, including recognition, reorientation and reorganization of the bases, could induce the formation of stable duplexes having non-canonical arrangements. Major adjustment of the structures was observed when the mutated base was located in the end region of the chain close to the surface. This journal is © the Owner Societies 2011

Three-dimensional characterization of tethered microspheres by total internal reflection fluorescence microscopy

NASA Technical Reports Server (NTRS)

Blumberg, Seth; Gajraj, Arivalagan; Pennington, Matthew W.; Meiners, Jens-Christian

2005-01-01

Tethered particle microscopy is a powerful tool to study the dynamics of DNA molecules and DNA-protein complexes in single-molecule experiments. We demonstrate that stroboscopic total internal reflection microscopy can be used to characterize the three-dimensional spatiotemporal motion of DNA-tethered particles. By calculating characteristic measures such as symmetry and time constants of the motion, well-formed tethers can be distinguished from defective ones for which the motion is dominated by aberrant surface effects. This improves the reliability of measurements on tether dynamics. For instance, in observations of protein-mediated DNA looping, loop formation is distinguished from adsorption and other nonspecific events.

Inclusion complexes of β-cyclodextrin-dinitrocompounds as UV absorber for ballpoint pen ink.

PubMed

Srinivasan, Krishnan; Radhakrishnan, S; Stalin, Thambusamy

2014-08-14

2,4-Dinitrophenol (2,4-DNP), 2,4-dinitroaniline (2,4-DNA), 2,6-dinitroaniline (2,6-DNA) and 2,6-dinitrobenzoic acid (2,6-DNB) has appeared for the UV absorption bands in different wavelength region below 400 nm, a combination of these dinitro aromatic compounds gave the broad absorption spectra within the UV region. The absorption intensities have been increased by preparation of the inclusion complex of dinitro compounds with β-cyclodextrin (β-CD). Prepared inclusion complexes are used to improve the UV protection properties of the ball point pen ink against photo degradation. The formation of solid inclusion complexes was characterized by FT-IR, and (1)H NMR spectroscopy. The UV protecting properties of these inclusion complexes were calculated their sun protection factor (SPF) is also discussed. The stability of the ballpoint pen ink has been confirmed by UV-Visible spectroscopic method. Copyright © 2014 Elsevier B.V. All rights reserved.

Direct colorimetric detection of unamplified pathogen DNA by dextrin-capped gold nanoparticles.

PubMed

Baetsen-Young, Amy M; Vasher, Matthew; Matta, Leann L; Colgan, Phil; Alocilja, Evangelyn C; Day, Brad

2018-03-15

The interaction between gold nanoparticles (AuNPs) and nucleic acids has facilitated a variety of diagnostic applications, with further diversification of synthesis match bio-applications while reducing biotoxicity. However, DNA interactions with unique surface capping agents have not been fully defined. Using dextrin-capped AuNPs (d-AuNPs), we have developed a novel unamplified genomic DNA (gDNA) nanosensor, exploiting dispersion and aggregation characteristics of d-AuNPs, in the presence of gDNA, for sequence-specific detection. We demonstrate that d-AuNPs are stable in a five-fold greater salt concentration than citrate-capped AuNPs and the d-AuNPs were stabilized by single stranded DNA probe (ssDNAp). However, in the elevated salt concentrations of the DNA detection assay, the target reactions were surprisingly further stabilized by the formation of a ssDNAp-target gDNA complex. The results presented herein lead us to propose a mechanism whereby genomic ssDNA secondary structure formation during ssDNAp-to-target gDNA binding enables d-AuNP stabilization in elevated ionic environments. Using the assay described herein, we were successful in detecting as little as 2.94 fM of pathogen DNA, and using crude extractions of a pathogen matrix, as few as 18 spores/µL. Copyright © 2017 Elsevier B.V. All rights reserved.

New and old ways to control meiotic recombination.

PubMed

Phadnis, Naina; Hyppa, Randy W; Smith, Gerald R

2011-10-01

The unique segregation of homologs, rather than sister chromatids, at the first meiotic division requires the formation of crossovers (COs) between homologs by meiotic recombination in most species. Crossovers do not form at random along chromosomes. Rather, their formation is carefully controlled, both at the stage of formation of DNA double-strand breaks (DSBs) that can initiate COs and during the repair of these DSBs. Here, we review control of DSB formation and two recently recognized controls of DSB repair: CO homeostasis and CO invariance. Crossover homeostasis maintains a constant number of COs per cell when the total number of DSBs in a cell is experimentally or stochastically reduced. Crossover invariance maintains a constant CO density (COs per kb of DNA) across much of the genome despite strong DSB hotspots in some intervals. These recently uncovered phenomena show that CO control is even more complex than previously suspected. Copyright © 2011 Elsevier Ltd. All rights reserved.

ESI-MS Investigation of an Equilibrium between a Bimolecular Quadruplex DNA and a Duplex DNA/RNA Hybrid

NASA Astrophysics Data System (ADS)

Birrento, Monica L.; Bryan, Tracy M.; Samosorn, Siritron; Beck, Jennifer L.

2015-07-01

Electrospray ionization mass spectrometry (ESI-MS) conditions were optimized for simultaneous observation of a bimolecular qDNA and a Watson-Crick base-paired duplex DNA/RNA hybrid. The DNA sequence used was telomeric DNA, and the RNA contained the template for telomerase-mediated telomeric DNA synthesis. Addition of RNA to the quadruplex DNA (qDNA) resulted in formation of the duplex DNA/RNA hybrid. Melting profiles obtained using circular dichroism spectroscopy confirmed that the DNA/RNA hybrid exhibited greater thermal stability than the bimolecular qDNA in solution. Binding of a 13-substituted berberine ( 1) derivative to the bimolecular qDNA stabilized its structure as evidenced by an increase in its stability in the mass spectrometer, and an increase in its circular dichroism (CD) melting temperature of 10°C. The DNA/RNA hybrid did not bind the ligand extensively and its thermal stability was unchanged in the presence of ( 1). The qDNA-ligand complex resisted unfolding in the presence of excess RNA, limiting the formation of the DNA/RNA hybrid. Previously, it has been proposed that DNA secondary structures, such as qDNA, may be involved in the telomerase mechanism. DNA/RNA hybrid structures occur at the active site of telomerase. The results presented in the current work show that if telomeric DNA was folded into a qDNA structure, it is possible for a DNA/RNA hybrid to form as is required during template alignment. The discrimination of ligand ( 1) for binding to the bimolecular qDNA over the DNA/RNA hybrid positions it as a useful compound for probing the role(s), if any, of antiparallel qDNA in the telomerase mechanism.

HTLV-1 Tax Oncoprotein Subverts the Cellular DNA Damage Response via Binding to DNA-dependent Protein Kinase*S⃞

PubMed Central

Durkin, Sarah S.; Guo, Xin; Fryrear, Kimberly A.; Mihaylova, Valia T.; Gupta, Saurabh K.; Belgnaoui, S. Mehdi; Haoudi, Abdelali; Kupfer, Gary M.; Semmes, O. John

2008-01-01

Human T-cell leukemia virus type-1 is the causative agent for adult T-cell leukemia. Previous research has established that the viral oncoprotein Tax mediates the transformation process by impairing cell cycle control and cellular response to DNA damage. We showed previously that Tax sequesters huChk2 within chromatin and impairs the response to ionizing radiation. Here we demonstrate that DNA-dependent protein kinase (DNA-PK) is a member of the Tax·Chk2 nuclear complex. The catalytic subunit, DNA-PKcs, and the regulatory subunit, Ku70, were present. Tax-containing nuclear extracts showed increased DNA-PK activity, and specific inhibition of DNA-PK prevented Tax-induced activation of Chk2 kinase activity. Expression of Tax induced foci formation and phosphorylation of H2AX. However, Tax-induced constitutive signaling of the DNA-PK pathway impaired cellular response to new damage, as reflected in suppression of ionizing radiation-induced DNA-PK phosphorylation and γH2AX stabilization. Tax co-localized with phospho-DNA-PK into nuclear speckles and a nuclear excluded Tax mutant sequestered endogenous phospho-DNA-PK into the cytoplasm, suggesting that Tax interaction with DNA-PK is an initiating event. We also describe a novel interaction between DNA-PK and Chk2 that requires Tax. We propose that Tax binds to and stabilizes a protein complex with DNA-PK and Chk2, resulting in a saturation of DNA-PK-mediated damage repair response. PMID:18957425

The C-terminal region of Escherichia coli UvrC contributes to the flexibility of the UvrABC nucleotide excision repair system

PubMed Central

Verhoeven, Esther E. A.; van Kesteren, Marian; Turner, John J.; van der Marel, Gijs A.; van Boom, Jacques H.; Moolenaar, Geri F.; Goosen, Nora

2002-01-01

Nucleotide excision repair in Escherichia coli involves formation of the UvrB–DNA complex and subsequent DNA incisions on either site of the damage by UvrC. In this paper, we studied the incision of substrates with different damages in varying sequence contexts. We show that there is not always a correlation between the incision efficiency and the stability of the UvrB–DNA complex. Both stable and unstable UvrB–DNA complexes can be efficiently incised. However some lesions that give rise to stable UvrB–DNA complexes do result in a very low incision. We present evidence that this poor incision is due to sterical hindrance of the damage itself. In its C-terminal region UvrC contains two helix–hairpin–helix (HhH) motifs. Mutational analysis shows that these motifs constitute one functional unit, probably folded as one structural unit; the (HhH)2 domain. This (HhH)2 domain was previously shown to be important for the 5′ incision on a substrate containing a (cis-Pt)·GG adduct, but not for 3′ incision. Here we show that, mainly depending on the sequence context of the lesion, the (HhH)2 domain can be important for 3′ and/or 5′ incision. We propose that the (HhH)2 domain stabilises specific DNA structures required for the two incisions, thereby contributing to the flexibility of the UvrABC repair system. PMID:12034838

DNA in a Tunnel: A Comfy Spot for Recognition - or -The Structure of BsoBI Complexed with DNA. What can we Learn about Function via Structure Determination and how can this be Applied to Bone or Muscle Biology?

NASA Technical Reports Server (NTRS)

vanderWoerd, Mark

2004-01-01

The structure and function of a biologically active molecule are related. To understand its function, it is necessary (but not always sufficient) to know the structure of the molecule. There are many ways of relating the molecular function with the structure. Mutation analysis can identify pertinent amino acids of an enzyme, or alternatively structure comparison of the of two similar molecules with different function may lead to understanding which parts are responsible for a functional aspect, or a series of "structural cartoons" - enzyme structure, enzyme plus substrate, enzyme with transition state analog, and enzyme with product - may give insight in the function of a molecule. As an example we will discuss the structure and function of the restriction enzyme BsoBI from Bacillus stearothemzophilus in complex with its cognate DNA. The enzyme forms a unique complex with DNA in that it completely encircles the DNA. The structure reveals the enzyme-DNA contacts, how the DNA is distorted compared with the canonical forms, and elegantly shows how two distinct DNA sequences can be recognized with the same efficiency. Based on the structure we may also propose a hypothesis how the enzymatic mechanism works. The knowledge gained thru studies such as this one can be used to alter the function by changing the molecular structure. Usually this is done by design of inhibitors specifically active against and fitting into an active site of the enzyme of choice. In the case of BsoBI one of the objectives of the study was to alter the enzyme specificity. In bone biology there are many candidates available for molecular study in order to explain, alter, or (temporarily) suspend activity. For example, the understanding of a pathway that negatively regulates bone formation may be a good target for drug design to stimulate bone formation and have good potential as the basis for new countermeasures against bone loss. In principle the same approach may aid muscle atrophy, radiation damage, immune response changes and other risks identified for long-duration Space travel.

Simply amplified electrochemical aptasensor of ochratoxin A based on exonuclease-catalyzed target recycling.

PubMed

Tong, Ping; Zhang, Lan; Xu, Jing-Juan; Chen, Hong-Yuan

2011-11-15

A new "signal-on" aptasensor for ultrasensitive detection of Ochratoxin A (OTA) in wheat starch was developed based on exonuclease-catalyzed target recycling. To construct the aptasensor, a ferrocene (Fc) labeled probe DNA (S1) was immobilized on a gold electrode (GE) via Au-S bonding for the following hybridization with the complementary OTA aptamer, with the labeled Fc on S1 far from the GE surface. In the presence of analyte OTA, the formation of aptamer-OTA complex would result in not only the dissociation of aptamer from the double-strand DNA but also the transformation of the probe DNA into a hairpin structure. Subsequently, the OTA could be liberated from the aptamer-OTA complex for analyte recycling due to the employment of exonuclease, which is a single-stranded DNA specific exonuclease to selectively digest the appointed DNA (aptamer). Owing to the labeled Fc in close proximity to the electrode surface caused by the formation of the hairpin DNA and to the analyte recycling, differential pulse voltammetry (DPV) signal could be produced with enhanced signal amplification. Based on this strategy, an ultrasensitive aptasensor for the detection of OTA could be exhibited with a wide linear range of 0.005-10.0ngmL(-1) with a low detection limit (LOD) of 1.0pgmL(-1) OTA (at 3σ). The fabricated biosensor was then applied for the measurement of OTA in real wheat starch sample and validated by ELISA method. Copyright © 2011 Elsevier B.V. All rights reserved.

The formation of catalytically competent enzyme-substrate complex is not a bottleneck in lesion excision by human alkyladenine DNA glycosylase.

PubMed

Kuznetsov, N A; Kiryutin, A S; Kuznetsova, A A; Panov, M S; Barsukova, M O; Yurkovskaya, A V; Fedorova, O S

2017-04-01

Human alkyladenine DNA glycosylase (AAG) protects DNA from alkylated and deaminated purine lesions. AAG flips out the damaged nucleotide from the double helix of DNA and catalyzes the hydrolysis of the N-glycosidic bond to release the damaged base. To understand better, how the step of nucleotide eversion influences the overall catalytic process, we performed a pre-steady-state kinetic analysis of AAG interaction with specific DNA-substrates, 13-base pair duplexes containing in the 7th position 1-N6-ethenoadenine (εA), hypoxanthine (Hx), and the stable product analogue tetrahydrofuran (F). The combination of the fluorescence of tryptophan, 2-aminopurine, and 1-N6-ethenoadenine was used to record conformational changes of the enzyme and DNA during the processes of DNA lesion recognition, damaged base eversion, excision of the N-glycosidic bond, and product release. The thermal stability of the duplexes characterized by the temperature of melting, T m , and the rates of spontaneous opening of individual nucleotide base pairs were determined by NMR spectroscopy. The data show that the relative thermal stability of duplexes containing a particular base pair in position 7, (T m (F/T) < T m (εA/T) < T m (Hx/T) < T m (A/T)) correlates with the rate of reversible spontaneous opening of the base pair. However, in contrast to that, the catalytic lesion excision rate is two orders of magnitude higher for Hx-containing substrates than for substrates containing εA, proving that catalytic activity is not correlated with the stability of the damaged base pair. Our study reveals that the formation of the catalytically competent enzyme-substrate complex is not the bottleneck controlling the catalytic activity of AAG.

Local chromatin structure of heterochromatin regulates repeated DNA stability, nucleolus structure, and genome integrity

DOE Office of Scientific and Technical Information (OSTI.GOV)

Peng, Jamy C.

Heterochromatin constitutes a significant portion of the genome in higher eukaryotes; approximately 30% in Drosophila and human. Heterochromatin contains a high repeat DNA content and a low density of protein-encoding genes. In contrast, euchromatin is composed mostly of unique sequences and contains the majority of single-copy genes. Genetic and cytological studies demonstrated that heterochromatin exhibits regulatory roles in chromosome organization, centromere function and telomere protection. As an epigenetically regulated structure, heterochromatin formation is not defined by any DNA sequence consensus. Heterochromatin is characterized by its association with nucleosomes containing methylated-lysine 9 of histone H3 (H3K9me), heterochromatin protein 1 (HP1) thatmore » binds H3K9me, and Su(var)3-9, which methylates H3K9 and binds HP1. Heterochromatin formation and functions are influenced by HP1, Su(var)3-9, and the RNA interference (RNAi) pathway. My thesis project investigates how heterochromatin formation and function impact nuclear architecture, repeated DNA organization, and genome stability in Drosophila melanogaster. H3K9me-based chromatin reduces extrachromosomal DNA formation; most likely by restricting the access of repair machineries to repeated DNAs. Reducing extrachromosomal ribosomal DNA stabilizes rDNA repeats and the nucleolus structure. H3K9me-based chromatin also inhibits DNA damage in heterochromatin. Cells with compromised heterochromatin structure, due to Su(var)3-9 or dcr-2 (a component of the RNAi pathway) mutations, display severe DNA damage in heterochromatin compared to wild type. In these mutant cells, accumulated DNA damage leads to chromosomal defects such as translocations, defective DNA repair response, and activation of the G2-M DNA repair and mitotic checkpoints that ensure cellular and animal viability. My thesis research suggests that DNA replication, repair, and recombination mechanisms in heterochromatin differ from those in euchromatin. Remarkably, human euchromatin and fly heterochromatin share similar features; such as repeated DNA content, intron lengths and open reading frame sizes. Human cells likely stabilize their DNA content via mechanisms and factors similar to those in Drosophila heterochromatin. Furthermore, my thesis work raises implications for H3K9me and chromatin functions in complex-DNA genome stability, repeated DNA homogenization by molecular drive, and in genome reorganization through evolution.« less

Structural mechanisms of DNA binding and unwinding in bacterial RecQ helicases

DOE PAGES

Manthei, Kelly A.; Hill, Morgan C.; Burke, Jordan E.; ...

2015-03-23

RecQ helicases unwind remarkably diverse DNA structures as key components of many cellular processes. How RecQ enzymes accommodate different substrates in a unified mechanism that couples ATP hydrolysis to DNA unwinding is unknown. In this paper, the X-ray crystal structure of the Cronobacter sakazakii RecQ catalytic core domain bound to duplex DNA with a 3' single-stranded extension identifies two DNA-dependent conformational rearrangements: a winged-helix domain pivots ~90° to close onto duplex DNA, and a conserved aromatic-rich loop is remodeled to bind ssDNA. These changes coincide with a restructuring of the RecQ ATPase active site that positions catalytic residues for ATPmore » hydrolysis. Complex formation also induces a tight bend in the DNA and melts a portion of the duplex. Finally, this bending, coupled with translocation, could provide RecQ with a mechanism for unwinding duplex and other DNA structures.« less

Effects of TiO2 crystal structure on the luminescence quenching of [Ru(bpy)2(dppz)]2 +-intercalated into DNA

NASA Astrophysics Data System (ADS)

Chen, Linlin; Wang, Yi; Huang, Minggao; Li, Xiaodan; Zhu, Licai; Li, Hong

2017-06-01

The intercalation of [Ru(bpy)2(dppz)]2 + labeled as Ru(II) (bpy = 2,2‧-bipyridine and dppz = dipyrido[3,2,-a:2‧,3‧-c]phenazine) into herring sperm DNA leads to the formation of emissive Ru(II)-DNA dyads, which can be quenched by TiO2 nanoparticles (NPs) and sol-gel silica matrices at heterogeneous interfaces. The calcinations temperature exhibits a remarkable influence on the luminescence quenching of the Ru(II)-DNA dyads by TiO2 NPs. With increasing calcinations temperature in the range from 200 to 850 °C, the anatase-to-rutile TiO2 crystal structure transformation increases the average particle size and hydrodynamic diameter of TiO2 and DNA@TiO2. The anatase TiO2 has the stronger ability to unbind the Ru(II)-DNA dyads than the rutile TiO2 at room temperature. The TiO2 NPs and sol-gel silica matrices can quench the luminescence of the Ru(II) complex intercalated into DNA by selectively capturing the negatively DNA and positively charged Ru(II) complex to unbind the dyads, respectively. This present results provide new insights into the luminescence quenching and competitive binding of dye-labeled DNA dyads by inorganic NPs.

Nicotine drives neutrophil extracellular traps formation and accelerates collagen-induced arthritis.

PubMed

Lee, Jaejoon; Luria, Ayala; Rhodes, Christopher; Raghu, Harini; Lingampalli, Nithya; Sharpe, Orr; Rada, Balazs; Sohn, Dong Hyun; Robinson, William H; Sokolove, Jeremy

2017-04-01

The aim was to investigate the effects of nicotine on neutrophil extracellular traps (NETs) formation in current and non-smokers and on a murine model of RA. We compared spontaneous and phorbol 12-myristate 13-acetate-induced NETosis between current and non-smokers by DNA release binding. Nicotine-induced NETosis from non-smokers was assessed by DNA release binding, NET-specific (myeloperoxidase (MPO)-DNA complex) ELISA and real-time fluorescence microscopy. We also used immunofluorescent staining to detect nicotinic acetylcholine receptors (nAChRs) on neutrophils and performed a functional analysis to assess the role of nAChRs in nicotine-induced NETosis. Finally, we investigated the effects of systemic nicotine exposure on arthritis severity and NETosis in the CIA mouse model. Neutrophils derived from current smokers displayed elevated levels of spontaneous and phorbol 12-myristate 13-acetate-induced NETosis. Nicotine induced dose-dependent NETosis in ex vivo neutrophils from healthy non-smokers, and co-incubation with ACPA-immune complexes or TNF-α facilitated a synergistic effect on NETosis. Real-time fluorescence microscopy revealed robust formation of NET-like structures in nicotine-exposed neutrophils. Immunofluorescent staining demonstrated the presence of the α7 subunit of the nAChR on neutrophils. Stimulation of neutrophils with an α7-specific nAChR agonist induced NETosis, whereas pretreatment with an nAChR antagonist attenuated nicotine-induced NETosis. Nicotine administration to mice with CIA exacerbated inflammatory arthritis, with higher plasma levels of NET-associated MPO-DNA complex. We demonstrate that nicotine is a potent inducer of NETosis, which may play an important role in accelerating arthritis in the CIA model. This study generates awareness of and the mechanisms by which nicotine-containing products, including e-cigarettes, may have deleterious effects on patients with RA. Published by Oxford University Press 2016. This work is written by US Government employees and is in the public domain in the United States.

Interactions of Kid-Kis toxin-antitoxin complexes with the parD operator-promoter region of plasmid R1 are piloted by the Kis antitoxin and tuned by the stoichiometry of Kid-Kis oligomers.

PubMed

Monti, Maria C; Hernández-Arriaga, Ana M; Kamphuis, Monique B; López-Villarejo, Juan; Heck, Albert J R; Boelens, Rolf; Díaz-Orejas, Ramón; van den Heuvel, Robert H H

2007-01-01

The parD operon of Escherichia coli plasmid R1 encodes a toxin-antitoxin system, which is involved in plasmid stabilization. The toxin Kid inhibits cell growth by RNA degradation and its action is neutralized by the formation of a tight complex with the antitoxin Kis. A fascinating but poorly understood aspect of the kid-kis system is its autoregulation at the transcriptional level. Using macromolecular (tandem) mass spectrometry and DNA binding assays, we here demonstrate that Kis pilots the interaction of the Kid-Kis complex in the parD regulatory region and that two discrete Kis-binding regions are present on parD. The data clearly show that only when the Kis concentration equals or exceeds the Kid concentration a strong cooperative effect exists between strong DNA binding and Kid2-Kis2-Kid2-Kis2 complex formation. We propose a model in which transcriptional repression of the parD operon is tuned by the relative molar ratio of the antitoxin and toxin proteins in solution. When the concentration of the toxin exceeds that of the antitoxin tight Kid2-Kis2-Kid2 complexes are formed, which only neutralize the lethal activity of Kid. Upon increasing the Kis concentration, (Kid2-Kis2)n complexes repress the kid-kis operon.

DNA Shape Dominates Sequence Affinity in Nucleosome Formation

NASA Astrophysics Data System (ADS)

Freeman, Gordon S.; Lequieu, Joshua P.; Hinckley, Daniel M.; Whitmer, Jonathan K.; de Pablo, Juan J.

2014-10-01

Nucleosomes provide the basic unit of compaction in eukaryotic genomes, and the mechanisms that dictate their position at specific locations along a DNA sequence are of central importance to genetics. In this Letter, we employ molecular models of DNA and proteins to elucidate various aspects of nucleosome positioning. In particular, we show how DNA's histone affinity is encoded in its sequence-dependent shape, including subtle deviations from the ideal straight B-DNA form and local variations of minor groove width. By relying on high-precision simulations of the free energy of nucleosome complexes, we also demonstrate that, depending on DNA's intrinsic curvature, histone binding can be dominated by bending interactions or electrostatic interactions. More generally, the results presented here explain how sequence, manifested as the shape of the DNA molecule, dominates molecular recognition in the problem of nucleosome positioning.

Structural Variation of Type I-F CRISPR RNA Guided DNA Surveillance.

PubMed

Pausch, Patrick; Müller-Esparza, Hanna; Gleditzsch, Daniel; Altegoer, Florian; Randau, Lennart; Bange, Gert

2017-08-17

CRISPR-Cas systems are prokaryotic immune systems against invading nucleic acids. Type I CRISPR-Cas systems employ highly diverse, multi-subunit surveillance Cascade complexes that facilitate duplex formation between crRNA and complementary target DNA for R-loop formation, retention, and DNA degradation by the subsequently recruited nuclease Cas3. Typically, the large subunit recognizes bona fide targets through the PAM (protospacer adjacent motif), and the small subunit guides the non-target DNA strand. Here, we present the Apo- and target-DNA-bound structures of the I-Fv (type I-F variant) Cascade lacking the small and large subunits. Large and small subunits are functionally replaced by the 5' terminal crRNA cap Cas5fv and the backbone protein Cas7fv, respectively. Cas5fv facilitates PAM recognition from the DNA major groove site, in contrast to all other described type I systems. Comparison of the type I-Fv Cascade with an anti-CRISPR protein-bound I-F Cascade reveals that the type I-Fv structure differs substantially at known anti-CRISPR protein target sites and might therefore be resistant to viral Cascade interception. Copyright © 2017 Elsevier Inc. All rights reserved.

In vitro DNA binding studies of therapeutic and prophylactic drug citral.

PubMed

Alam, Md Fazle; Varshney, Supriya; Khan, Masood Alam; Laskar, Amaj Ahmed; Younus, Hina

2018-07-01

The study of drug-DNA interactions is of great importance, as it paves the way towards the design of better therapeutic agents. Here, the interaction of DNA with a therapeutic and prophylactic drug citral has been studied. We have attempted to ascertain the mode of binding of citral with calf thymus DNA (Ct-DNA) through various biophysical techniques. Analysis of the UV-visible absorbance spectra and fluorescence spectra indicated the formation of a complex between citral and Ct-DNA. Competitive binding assays with ethidium bromide (EB), acridine orange (AO) and Hoechst 33258 reflected that citral possibly intercalates within the Ct-DNA. These observations were further confirmed by circular dichroism (CD) spectral analysis, viscosity measurements, DNA melting and molecular docking studies. This study is expected to contribute to a better understanding of molecular mechanisms of citral, and design of new drugs in the future. Copyright © 2018 Elsevier B.V. All rights reserved.

RPA binds histone H3-H4 and functions in DNA replication-coupled nucleosome assembly.

PubMed

Liu, Shaofeng; Xu, Zhiyun; Leng, He; Zheng, Pu; Yang, Jiayi; Chen, Kaifu; Feng, Jianxun; Li, Qing

2017-01-27

DNA replication-coupled nucleosome assembly is essential to maintain genome integrity and retain epigenetic information. Multiple involved histone chaperones have been identified, but how nucleosome assembly is coupled to DNA replication remains elusive. Here we show that replication protein A (RPA), an essential replisome component that binds single-stranded DNA, has a role in replication-coupled nucleosome assembly. RPA directly binds free H3-H4. Assays using a synthetic sequence that mimics freshly unwound single-stranded DNA at replication fork showed that RPA promotes DNA-(H3-H4) complex formation immediately adjacent to double-stranded DNA. Further, an RPA mutant defective in H3-H4 binding exhibited attenuated nucleosome assembly on nascent chromatin. Thus, we propose that RPA functions as a platform for targeting histone deposition to replication fork, through which RPA couples nucleosome assembly with ongoing DNA replication. Copyright © 2017, American Association for the Advancement of Science.

Nuclear Proximity of Mtr4 with RNA exosome restricts DNA mutational asymmetry

PubMed Central

Lim, Junghyun; Giri, Pankaj Kumar; Kazadi, David; Laffleur, Brice; Zhang, Wanwei; Grinstein, Veronika; Pefanis, Evangelos; Brown, Lewis M.; Ladewig, Erik; Martin, Ophélie; Chen, Yuling; Rabadan, Raul; Boyer, François; Rothschild, Gerson; Cogné, Michel; Pinaud, Eric; Deng, Haiteng; Basu, Uttiya

2017-01-01

SUMMARY The distribution of sense and antisense strand DNA mutations on transcribed duplex DNA contributes to the development of immune and neural systems along with the progression of cancer. Because developmentally matured B cells undergo biologically programmed strand-specific DNA mutagenesis at focal DNA/RNA hybrid structures, they make a convenient system to investigate strand-specific mutagenesis mechanisms. We demonstrate that the sense and antisense strand DNA mutagenesis at the immunoglobulin heavy chain locus and some other regions of the B cell genome depends upon localized RNA processing protein complex formation in the nucleus. Both the physical proximity and coupled activities of RNA helicase Mtr4 (and Senataxin) with the noncoding RNA processing function of RNA exosome determine the strand specific distribution of DNA mutations. Our study suggests that strand-specific DNA mutagenesis-associated mechanisms will play major roles in other undiscovered aspects of organismic development. PMID:28431250

High-frequency promoter firing links THO complex function to heavy chromatin formation.

PubMed

Mouaikel, John; Causse, Sébastien Z; Rougemaille, Mathieu; Daubenton-Carafa, Yves; Blugeon, Corinne; Lemoine, Sophie; Devaux, Frédéric; Darzacq, Xavier; Libri, Domenico

2013-11-27

The THO complex is involved in transcription, genome stability, and messenger ribonucleoprotein (mRNP) formation, but its precise molecular function remains enigmatic. Under heat shock conditions, THO mutants accumulate large protein-DNA complexes that alter the chromatin density of target genes (heavy chromatin), defining a specific biochemical facet of THO function and a powerful tool of analysis. Here, we show that heavy chromatin distribution is dictated by gene boundaries and that the gene promoter is necessary and sufficient to convey THO sensitivity in these conditions. Single-molecule fluorescence in situ hybridization measurements show that heavy chromatin formation correlates with an unusually high firing pace of the promoter with more than 20 transcription events per minute. Heavy chromatin formation closely follows the modulation of promoter firing and strongly correlates with polymerase occupancy genome wide. We propose that the THO complex is required for tuning the dynamic of gene-nuclear pore association and mRNP release to the same high pace of transcription initiation. Copyright © 2013 The Authors. Published by Elsevier Inc. All rights reserved.

Single-stranded nucleic acids promote SAMHD1 complex formation.

PubMed

Tüngler, Victoria; Staroske, Wolfgang; Kind, Barbara; Dobrick, Manuela; Kretschmer, Stefanie; Schmidt, Franziska; Krug, Claudia; Lorenz, Mike; Chara, Osvaldo; Schwille, Petra; Lee-Kirsch, Min Ae

2013-06-01

SAM domain and HD domain-containing protein 1 (SAMHD1) is a dGTP-dependent triphosphohydrolase that degrades deoxyribonucleoside triphosphates (dNTPs) thereby limiting the intracellular dNTP pool. Mutations in SAMHD1 cause Aicardi-Goutières syndrome (AGS), an inflammatory encephalopathy that mimics congenital viral infection and that phenotypically overlaps with the autoimmune disease systemic lupus erythematosus. Both disorders are characterized by activation of the antiviral cytokine interferon-α initiated by immune recognition of self nucleic acids. Here we provide first direct evidence that SAMHD1 associates with endogenous nucleic acids in situ. Using fluorescence cross-correlation spectroscopy, we demonstrate that SAMHD1 specifically interacts with ssRNA and ssDNA and establish that nucleic acid-binding and formation of SAMHD1 complexes are mutually dependent. Interaction with nucleic acids and complex formation do not require the SAM domain, but are dependent on the HD domain and the C-terminal region of SAMHD1. We finally demonstrate that mutations associated with AGS exhibit both impaired nucleic acid-binding and complex formation implicating that interaction with nucleic acids is an integral aspect of SAMHD1 function.

MCM-BP regulates unloading of the MCM2–7 helicase in late S phase

PubMed Central

Nishiyama, Atsuya; Frappier, Lori; Méchali, Marcel

2011-01-01

Origins of DNA replication are licensed by recruiting MCM2–7 to assemble the prereplicative complex (pre-RC). How MCM2–7 is inactivated or removed from chromatin at the end of S phase is still unclear. Here, we show that MCM-BP can disassemble the MCM2–7 complex and might function as an unloader of MCM2–7 from chromatin. In Xenopus egg extracts, MCM-BP exists in a stable complex with MCM7, but is not associated with the MCM2–7 hexameric complex. MCM-BP accumulates in nuclei in late S phase, well after the loading of MCM2–7 onto chromatin. MCM-BP immunodepletion in Xenopus egg extracts inhibits replication-dependent MCM dissociation without affecting pre-RC formation and DNA replication. When excess MCM-BP is incubated with Xenopus egg extracts or immunopurified MCM2–7, it binds to MCM proteins and promotes disassembly of the MCM2–7 complex. Recombinant MCM-BP also releases MCM2–7 from isolated late-S-phase chromatin, but this activity is abolished when DNA replication is blocked. MCM-BP silencing in human cells also delays MCM dissociation in late S phase. We propose that MCM-BP plays a key role in the mechanism by which pre-RC is cleared from replicated DNA in vertebrate cells. PMID:21196493

The octamer-binding proteins form multi-protein--DNA complexes with the HSV alpha TIF regulatory protein.

PubMed Central

Kristie, T M; LeBowitz, J H; Sharp, P A

1989-01-01

The herpes simplex virus transactivator, alpha TIF, stimulates transcription of the alpha/immediate early genes via a cis-acting site containing an octamer element and a conserved flanking sequence. The alpha TIF protein, produced in a baculovirus expression system, nucleates the formation of at least two DNA--protein complexes on this regulatory element. Both of these complexes contain the ubiquitous Oct-1 protein, whose POU domain alone is sufficient to allow assembly of the alpha TIF-dependent complexes. A second member of the POU domain family, the lymphoid specific Oct-2 protein, can also be assembled into similar complexes at high concentrations of alpha TIF protein. These complexes contain at least two cellular proteins in addition to Oct-1. One of these proteins is present in both insect and HeLa cells and probably recognizes sequences in the cis element. The second cellular protein, only present in HeLa cells, probably binds by protein-protein interactions. Images PMID:2556266

The octamer-binding proteins form multi-protein--DNA complexes with the HSV alpha TIF regulatory protein.

PubMed

Kristie, T M; LeBowitz, J H; Sharp, P A

1989-12-20

The herpes simplex virus transactivator, alpha TIF, stimulates transcription of the alpha/immediate early genes via a cis-acting site containing an octamer element and a conserved flanking sequence. The alpha TIF protein, produced in a baculovirus expression system, nucleates the formation of at least two DNA--protein complexes on this regulatory element. Both of these complexes contain the ubiquitous Oct-1 protein, whose POU domain alone is sufficient to allow assembly of the alpha TIF-dependent complexes. A second member of the POU domain family, the lymphoid specific Oct-2 protein, can also be assembled into similar complexes at high concentrations of alpha TIF protein. These complexes contain at least two cellular proteins in addition to Oct-1. One of these proteins is present in both insect and HeLa cells and probably recognizes sequences in the cis element. The second cellular protein, only present in HeLa cells, probably binds by protein-protein interactions.

Synthesis, crystal structure, antimicrobial activity and DNA-binding of hydrogen-bonded proton-transfer complex of 2,6-diaminopyridine with picric acid.

PubMed

Khan, Ishaat M; Ahmad, Afaq; Ullah, M F

2011-04-04

A proton-transfer (charge transfer) complex formed on the reaction between 2,6-diaminopyridine (donor) and picric acid (acceptor) was synthesized and characterized by FTIR, (1)H NMR, thermal and elemental analysis. The crystal structure determined by single-crystal X-ray diffraction indicates that cation and anion are joined together by strong N(+)-H- -O(-) type hydrogen bonds. The hydrogen-bonded charge transfer (HBCT) complex was screened for its pharmacology such as antimicrobial activity against various fungal and bacterial strains and Calf thymus DNA-binding. The results showed that HBCT complex (100μg/ml) exhibited good antibacterial antifungal activity as that of standard antibiotics Tetracycline and Nystatin. A molecular frame work through H-bonding interactions between neighboring moieties is found to be responsible for high melting point of resulting complex. This has been attributed to the formation of 1:1 HBCT complex. Copyright © 2011 Elsevier B.V. All rights reserved.

Thermodynamic Investigation and Mixed Ligand Complex Formation of 1,4-Bis-(3-aminopropyl)-piperazine and Biorelevant Ligands.

PubMed

El-Sherif, Ahmed A; Shehata, Mohamed R; Shoukry, Mohamed M; Barakat, Mohammad H

2012-01-01

Thermodynamic parameters for protonation of 1,4-bis(3-aminopropyl)-piperazine (BAPP) and its metal complexation with some divalent metal ions were determined in aqueous solution at constant ionic strength (0.1 M NaNO(3)) using a potentiometric technique. The order of -ΔG(0) and -ΔH(0) was found to obey Co(2+) < Ni(2+) < Cu(2+) > Zn(2+), in accordance with the Irving-Williams order. The formation equilibria of zinc (II) complexes and the ternary complexes Zn(BAPP)L, where L = amino acid, amides, or DNA constituents), have been investigated. Ternary complexes are formed by a simultaneous mechanism. The concentration distribution of the complexes in solution was evaluated as a function of pH. Stoichiometry and stability constants for the complexes formed are reported and discussed. The stability of ternary complexes was quantitatively compared with their corresponding binary complexes in terms of the parameter Δlog K.

Thermodynamic Investigation and Mixed Ligand Complex Formation of 1,4-Bis-(3-aminopropyl)-piperazine and Biorelevant Ligands

PubMed Central

El-Sherif, Ahmed A.; Shehata, Mohamed R.; Shoukry, Mohamed M.; Barakat, Mohammad H.

2012-01-01

Thermodynamic parameters for protonation of 1,4-bis(3-aminopropyl)-piperazine (BAPP) and its metal complexation with some divalent metal ions were determined in aqueous solution at constant ionic strength (0.1 M NaNO3) using a potentiometric technique. The order of –ΔG0 and –ΔH0 was found to obey Co2+ < Ni2+ < Cu2+ > Zn2+, in accordance with the Irving-Williams order. The formation equilibria of zinc (II) complexes and the ternary complexes Zn(BAPP)L, where L = amino acid, amides, or DNA constituents), have been investigated. Ternary complexes are formed by a simultaneous mechanism. The concentration distribution of the complexes in solution was evaluated as a function of pH. Stoichiometry and stability constants for the complexes formed are reported and discussed. The stability of ternary complexes was quantitatively compared with their corresponding binary complexes in terms of the parameter Δlog K. PMID:23226992

Quenching Enhancement of the Singlet Excited State of Pheophorbide-a by DNA in the Presence of the Quinone Carboquone

PubMed Central

Díaz-Espinosa, Yisaira; Crespo-Hernández, Carlos E.; Alegría, Antonio E.; García, Carmelo; Arce, Rafael

2011-01-01

Changes in the emission fluorescence intensity of pheophorbide-a (PHEO) in the presence of carboquone (CARBOQ) were used to obtain the association constant, the number of CARBOQ molecules interacting with PHEO, and the fluorescence quantum yield of the complex. Excitation spectra of mixtures of PHEO and CARBOQ in ethanol (EtOH) show an unresolved doublet in the red-most excitation band of PHEO, indicating the formation of a loose ground-state complex. The 1:1 CARBOQ–PHEO complex shows a higher fluorescence quantum yield in EtOH (0.41 ± 0.02) than in buffer solution (0.089 ± 0.002), which is also higher than that of the PHEO monomer (0.28). Quenching of the PHEO fluorescence by DNA nucleosides and double-stranded oligonucleotides was also observed and the bimolecular quenching rate constants were determined. The quenching rate constant increase as the oxidation potential of the DNA nucleoside increases. Larger quenching constants were obtained in the presence of CARBOQ suggesting that CARBOQ enhances DNA photo-oxidation, presumably by inhibiting the back–electron-transfer reaction from the photoreduced PHEO to the oxidized base. Thus, the enhanced DNA-base photosensitized oxidation by PHEO in the presence of CARBOQ may be related to the large extent by which this quinone covalently binds to DNA, as previously reported. PMID:21138440

DNA (Cytosine-C5) methyltransferase inhibition by oligodeoxyribonucleotides containing 2-(1H)-pyrimidinone (zebularine aglycon) at the enzymatic target site.

PubMed

van Bemmel, Dana M; Brank, Adam S; Eritja, Ramon; Marquez, Victor E; Christman, Judith K

2009-09-15

Aberrant cytosine methylation in promoter regions leads to gene silencing associated with cancer progression. A number of DNA methyltransferase inhibitors are known to reactivate silenced genes; including 5-azacytidine and 2-(1H)-pyrimidinone riboside (zebularine). Zebularine is a more stable, less cytotoxic inhibitor compared to 5-azacytidine. To determine the mechanistic basis for this difference, we carried out a detailed comparisons of the interaction between purified DNA methyltransferases and oligodeoxyribonucleotides (ODNs) containing either 5-azacytosine or 2-(1H)-pyrimidinone in place of the cytosine targeted for methylation. When incorporated into small ODNs, the rate of C5 DNA methyltransferase inhibition by both nucleosides is essentially identical. However, the stability and reversibility of the enzyme complex in the absence and presence of cofactor differs. 5-Azacytosine ODNs form complexes with C5 DNA methyltransferases that are irreversible when the 5-azacytosine ring is intact. ODNs containing 2-(1H)-pyrimidinone at the enzymatic target site are competitive inhibitors of both prokaryotic and mammalian DNA C5 methyltransferases. We determined that the ternary complexes between the enzymes, 2-(1H)-pyrimidinone inhibitor, and the cofactor S-adenosyl methionine are maintained through the formation of a reversible covalent interaction. The differing stability and reversibility of the covalent bonds may partially account for the observed differences in cytotoxicity between zebularine and 5-azacytidine inhibitors.

DNA (Cytosine-C5) Methyltransferase Inhibition by Oligodeoxyribonucleotides Containing 2-(1H)-Pyrimidinone (Zebularine Aglycon) at the Enzymatic Target Site

PubMed Central

van Bemmel, Dana M.; Brank, Adam S.; Eritja, Ramon; Marquez, Victor E.; Christman, Judith K.

2009-01-01

Aberrant cytosine methylation in promoter regions leads to gene silencing associated with cancer progression. A number of DNA methyltransferase inhibitors are known to reactivate silenced genes; including 5-azacytidine and 2-(1H)-pyrimidinone riboside (zebularine). Zebularine is a more stable, less cytotoxic inhibitor compared to 5-azacytidine. To determine the mechanistic basis for this difference, we carried out a detailed comparisons of the interaction between purified DNA methyltransferases and oligodeoxyribonucleotides (ODNs) containing either 5-azacytosine or 2-(1H)-pyrimidinone in place of the cytosine targeted for methylation. When incorporated into small ODNs, the rate of C5 DNA methyltransferase inhibition by both nucleosides is essentially identical. However, the stability and reversibility of the enzyme complex in the absence and presence of cofactor differs. 5-Azacytosine ODNs form complexes with C5 DNA methyltransferases that are irreversible when the 5-azacytosine ring is intact. ODNs containing 2-(1H)-pyrimidinone at the enzymatic target site are competitive inhibitors of both prokaryotic and mammalian DNA C5 methyltransferases. We determined that the ternary complexes between the enzymes, 2-(1H)-pyrimidinone inhibitor, and the cofactor S-adenosyl methionine are maintained through the formation of a reversible covalent interaction. The differing stability and reversibility of the covalent bonds may partially account for the observed differences in cytotoxicity between zebularine and 5-azacytidine inhibitors. PMID:19467223

A Novel RNA Polymerase I Transcription Initiation Factor, TIF-IE, Commits rRNA Genes by Interaction with TIF-IB, Not by DNA Binding

PubMed Central

Al-Khouri, Anna Maria; Paule, Marvin R.

2002-01-01

In the small, free-living amoeba Acanthamoeba castellanii, rRNA transcription requires, in addition to RNA polymerase I, a single DNA-binding factor, transcription initiation factor IB (TIF-IB). TIF-IB is a multimeric protein that contains TATA-binding protein (TBP) and four TBP-associated factors that are specific for polymerase I transcription. TIF-IB is required for accurate and promoter-specific initiation of rRNA transcription, recruiting and positioning the polymerase on the start site by protein-protein interaction. In A. castellanii, partially purified TIF-IB can form a persistent complex with the ribosomal DNA (rDNA) promoter while homogeneous TIF-IB cannot. An additional factor, TIF-IE, is required along with homogeneous TIF-IB for the formation of a stable complex on the rDNA core promoter. We show that TIF-IE by itself, however, does not bind to the rDNA promoter and thus differs in its mechanism from the upstream binding factor and upstream activating factor, which carry out similar complex-stabilizing functions in vertebrates and yeast, respectively. In addition to its presence in impure TIF-IB, TIF-IE is found in highly purified fractions of polymerase I, with which it associates. Renaturation of polypeptides excised from sodium dodecyl sulfate-polyacrylamide gels showed that a 141-kDa polypeptide possesses all the known activities of TIF-IE. PMID:11784852

A novel RNA polymerase I transcription initiation factor, TIF-IE, commits rRNA genes by interaction with TIF-IB, not by DNA binding.

PubMed

Al-Khouri, Anna Maria; Paule, Marvin R

2002-02-01

In the small, free-living amoeba Acanthamoeba castellanii, rRNA transcription requires, in addition to RNA polymerase I, a single DNA-binding factor, transcription initiation factor IB (TIF-IB). TIF-IB is a multimeric protein that contains TATA-binding protein (TBP) and four TBP-associated factors that are specific for polymerase I transcription. TIF-IB is required for accurate and promoter-specific initiation of rRNA transcription, recruiting and positioning the polymerase on the start site by protein-protein interaction. In A. castellanii, partially purified TIF-IB can form a persistent complex with the ribosomal DNA (rDNA) promoter while homogeneous TIF-IB cannot. An additional factor, TIF-IE, is required along with homogeneous TIF-IB for the formation of a stable complex on the rDNA core promoter. We show that TIF-IE by itself, however, does not bind to the rDNA promoter and thus differs in its mechanism from the upstream binding factor and upstream activating factor, which carry out similar complex-stabilizing functions in vertebrates and yeast, respectively. In addition to its presence in impure TIF-IB, TIF-IE is found in highly purified fractions of polymerase I, with which it associates. Renaturation of polypeptides excised from sodium dodecyl sulfate-polyacrylamide gels showed that a 141-kDa polypeptide possesses all the known activities of TIF-IE.

DNA damage mediated transcription arrest: Step back to go forward.

PubMed

Mullenders, Leon

2015-12-01

The disturbance of DNA helix conformation by bulky DNA damage poses hindrance to transcription elongating due to stalling of RNA polymerase at transcription blocking lesions. Stalling of RNA polymerase provokes the formation of R-loops, i.e. the formation of a DNA-RNA hybrid and a displaced single stranded DNA strand as well as displacement of spliceosomes. R-loops are processed into DNA single and double strand breaks by NER factors depending on TC-NER factors leading to genome instability. Moreover, stalling of RNA polymerase induces a strong signal for cell cycle arrest and apoptosis. These toxic and mutagenic effects are counteracted by a rapid recruitment of DNA repair proteins to perform transcription coupled nucleotide excision repair (TC-NER) to remove the blocking DNA lesions and to restore transcription. Recent studies have highlighted the role of backtracking of RNA polymerase to facilitate TC-NER and identified novel factors that play key roles in TC-NER and in restoration of transcription. On the molecular level these factors facilitate stability of the repair complex by promotion and regulation of various post-translational modifications of NER factors and chromatin substrate. In addition, the continuous flow of new factors that emerge from screening assays hints to several regulatory levels to safeguard the integrity of transcription elongation after disturbance by DNA damage that have yet to be explored. Copyright © 2015 Elsevier B.V. All rights reserved.

Mutation of a Conserved Active Site Residue Converts Tyrosyl-DNA Phosphodiesterase l Into a DNA Topoisomerase l-Dependent Poison

DOE Office of Scientific and Technical Information (OSTI.GOV)

He,X.; van Waardenburg, R.; Babaoglu, K.

2007-01-01

Tyrosyl-DNA phosphodiesterase 1 (Tdp1) catalyzes the resolution of 3' and 5' phospho-DNA adducts. A defective mutant, associated with the recessive neurodegenerative disease SCAN1, accumulates Tdp1-DNA complexes in vitro. To assess the conservation of enzyme architecture, a 2.0 {angstrom} crystal structure of yeast Tdp1 was determined that is very similar to human Tdp1. Poorly conserved regions of primary structure are peripheral to an essentially identical catalytic core. Enzyme mechanism was also conserved, because the yeast SCAN1 mutant (H{sub 432}R) enhanced cell sensitivity to the DNA topoisomerase I (Top1) poison camptothecin. A more severe Top1-dependent lethality of Tdp1H{sub 432}N was drug-independent, coincidingmore » with increased covalent Top1-DNA and Tdp1-DNA complex formation in vivo. However, both H432 mutants were recessive to wild-type Tdp1. Thus, yeast H{sub 432} acts in the general acid/base catalytic mechanism of Tdp1 to resolve 3' phosphotyrosyl and 3' phosphoamide linkages. However, the distinct pattern of mutant Tdp1 activity evident in yeast cells, suggests a more severe defect in Tdp1H{sub 432}N-catalyzed resolution of 3' phospho-adducts.« less

Preparation and testing of quaternized chitosan nanoparticles as gene delivery vehicles.

PubMed

Li, Guang-Feng; Wang, Jing-Cheng; Feng, Xin-Min; Liu, Zhen-Dong; Jiang, Chao-Yong; Yang, Jian-Dong

2015-04-01

The aim of this study was to synthesize a chitosan (CS) derivative, a quaternary ammonium salt crystal called N-2-hydroxypropyl trimethyl ammonium chloride chitosan (HACC), and test a series of HACC and pEGFP-DNA complexes at different weight ratios for their efficiency of gene delivery into human cells. CS was modified with cationic etherifying agent to obtain the CS derivative. Fourier transform infrared spectra were recorded on KBr pellets with a spectrometer. (1)H nuclear magnetic resonance (NMR) spectra of HACC were obtained using a spectrometer. HACC was subsequently used to prepare HACC/DNA complexes at different weight ratios by coacervation method. The resulting particle size and surface charge were assessed by laser light scattering using a zeta potential analyzer. The HACC/DNA complex formation and DNA protection in the nanoparticle complex was investigated by gel mobility shift assay and DNase I protection assay, respectively. The cytotoxicity of HACC and HACC/DNA nanoparticles was evaluated by MTT assay using (mesenchymal stem cell) MSC lines. The nanoscale structure of the particles was obtained by transmission electron microscope (TEM). The FTIR spectrum of HACC showed the characteristic quaternary ammonium group absorption band at 1475 cm(-1), which indicated the presence of quaternary ammonium group. The successful synthesis of HACC was also confirmed by (1)H NMR spectrum. HACC showed good solubility in water and was electropositive. HACC efficiently packed and protected pEGFP-DNA at a weight ratio of 10. With increased weight ratios, the surface charge of the composite particle increased from negative to positive, the average particle size increased, and HACC nanoparticle had a higher carrying efficiency. The nanoparticles released DNA in two distinct phases, and 55 % was released within the first 20 h of solubilization. The nanoparticles under TEM showed circular or oval shapes. The particles exhibited no cytotoxicity against human cells. No significant difference in gene delivery efficiency was detected between HACC/pEGFP-GDNF and liposome/pEGFP-GDNF complexes (33.8 vs. 34 %, P = 0.363). In this study, HACC was successfully synthesized, and HACC/DNA complex assembled efficiently. HACC showed strong DNA binding affinity and high protection of DNA and was non-cytotoxic to human cells. The particles had appropriate nanostructure, mean diameter, and DNA release time. The results suggest that HACC nanoparticles are a novel tool for efficient and safe gene delivery.

Interaction with Single-stranded DNA-binding Protein Stimulates Escherichia coli Ribonuclease HI Enzymatic Activity

DOE Office of Scientific and Technical Information (OSTI.GOV)

Petzold, Christine; Marceau, Aimee H.; Miller, Katherine H.

Single-stranded (ss) DNA-binding proteins (SSBs) bind and protect ssDNA intermediates formed during replication, recombination, and repair reactions. SSBs also directly interact with many different genome maintenance proteins to stimulate their enzymatic activities and/or mediate their proper cellular localization. We have identified an interaction formed between Escherichia coli SSB and ribonuclease HI (RNase HI), an enzyme that hydrolyzes RNA in RNA/DNA hybrids. The RNase HI·SSB complex forms by RNase HI binding the intrinsically disordered C terminus of SSB (SSB-Ct), a mode of interaction that is shared among all SSB interaction partners examined to date. Residues that comprise the SSB-Ct binding sitemore » are conserved among bacterial RNase HI enzymes, suggesting that RNase HI·SSB complexes are present in many bacterial species and that retaining the interaction is important for its cellular function. A steady-state kinetic analysis shows that interaction with SSB stimulates RNase HI activity by lowering the reaction Km. SSB or RNase HI protein variants that disrupt complex formation nullify this effect. Collectively our findings identify a direct RNase HI/SSB interaction that could play a role in targeting RNase HI activity to RNA/DNA hybrid substrates within the genome.« less

[Evaluation of the immunological activity and safety of group B meningococcal vaccine prepared from a natural complex of specific polysaccharide and outer membrane proteins].

PubMed

Kuvakina, V I; Golovina, L I; Mishina, A I; Skirda, T A; Bobyleva, G V; Mikheeva, N G; Chernyshova, T F; Temper, R M; Ermolenko, Z N

2002-01-01

Immunological activity and safety of group B meningococcal vaccine prepared from a natural complex of specific polysaccharide and outer membrane proteins were under study. The immunological safety of the vaccine was evaluated by the absence of antibodies to denaturated and native DNA (d-DNA and n-DNA). As shown with the use of the enzyme immunoassay (EIA), the administration of the vaccine did not induce antibody formation to d-DNA and n-DNA during the observation period. The titer of bactericidal antibodies in the immune bacteriolysis assay (IBA) to the vaccine strain B:2b:P1.2 after immunization increased four-fold and greater in 80% of the vaccinated persons. The significant increase of bactericidal antibodies to heterologous strains B:2a:P1.2 and B:15:P1.7 was registered in 20-30% of the vaccinees, respectively. A month after the repeated vaccination an increase in specific IgG antibodies to the complex antigen was found to occur according to EIA results. The use of RIB made it possible to evaluate the preventive activity of group B meningococcal vaccine as a whole and to suppose that the vaccine induced mainly type-specific response.

Interaction with Single-stranded DNA-binding Protein Stimulates Escherichia coli Ribonuclease HI Enzymatic Activity.

PubMed

Petzold, Christine; Marceau, Aimee H; Miller, Katherine H; Marqusee, Susan; Keck, James L

2015-06-05

Single-stranded (ss) DNA-binding proteins (SSBs) bind and protect ssDNA intermediates formed during replication, recombination, and repair reactions. SSBs also directly interact with many different genome maintenance proteins to stimulate their enzymatic activities and/or mediate their proper cellular localization. We have identified an interaction formed between Escherichia coli SSB and ribonuclease HI (RNase HI), an enzyme that hydrolyzes RNA in RNA/DNA hybrids. The RNase HI·SSB complex forms by RNase HI binding the intrinsically disordered C terminus of SSB (SSB-Ct), a mode of interaction that is shared among all SSB interaction partners examined to date. Residues that comprise the SSB-Ct binding site are conserved among bacterial RNase HI enzymes, suggesting that RNase HI·SSB complexes are present in many bacterial species and that retaining the interaction is important for its cellular function. A steady-state kinetic analysis shows that interaction with SSB stimulates RNase HI activity by lowering the reaction Km. SSB or RNase HI protein variants that disrupt complex formation nullify this effect. Collectively our findings identify a direct RNase HI/SSB interaction that could play a role in targeting RNase HI activity to RNA/DNA hybrid substrates within the genome. © 2015 by The American Society for Biochemistry and Molecular Biology, Inc.

Mechanism of synergistic DNA damage induced by the hydroquinone metabolite of brominated phenolic environmental pollutants and Cu(II): Formation of DNA-Cu complex and site-specific production of hydroxyl radicals.

PubMed

Shao, Bo; Mao, Li; Qu, Na; Wang, Ya-Fen; Gao, Hui-Ying; Li, Feng; Qin, Li; Shao, Jie; Huang, Chun-Hua; Xu, Dan; Xie, Lin-Na; Shen, Chen; Zhou, Xiang; Zhu, Ben-Zhan

2017-03-01

2,6-Dibromohydroquinone (2,6-DBrHQ) has been identified as an reactive metabolite of many brominated phenolic environmental pollutants such as tetrabromobisphenol-A (TBBPA), bromoxynil and 2,4,6-tribromophenol, and was also found as one of disinfection byproducts in drinking water. In this study, we found that the combination of 2,6-DBrHQ and Cu(II) together could induce synergistic DNA damage as measured by double strand breakage in plasmid DNA and 8-oxo-7,8-dihydro-2'-deoxyguanosine (8-oxodG) formation, while either of them alone has no effect. 2,6-DBrHQ/Cu(II)-induced DNA damage could be inhibited by the Cu(I)-specific chelating agent bathocuproine disulfonate and catalase, but not by superoxide dismutase, nor by the typical hydroxyl radical (•OH) scavengers such as DMSO and mannitol. Interestingly, we found that Cu(II)/Cu(I) could be combined with DNA to form DNA-Cu(II)/Cu(I) complex by complementary application of low temperature direct ESR, circular dichroism, cyclic voltammetry and oxygen consumption methods; and the highly reactive •OH were produced synergistically by DNA-bound-Cu(I) with H 2 O 2 produced by the redox reactions between 2,6-DBrHQ and Cu(II), which then immediately attack DNA in a site-specific manner as demonstrated by both fluorescent method and by ESR spin-trapping studies. Further DNA sequencing investigations provided more direct evidence that 2,6-DBrHQ/Cu(II) caused preferential cleavage at guanine, thymine and cytosine residues. Based on these data, we proposed that the synergistic DNA damage induced by 2,6-DBrHQ/Cu(II) might be due to the synergistic and site-specific production of •OH near the binding site of copper and DNA. Our findings may have broad biological and environmental implications for future research on the carcinogenic polyhalogenated phenolic compounds. Copyright © 2017 Elsevier Inc. All rights reserved.

A chemistry/physics pathway with nanofibrous scaffolds for gene delivery.

PubMed

Wan, Fen; Tang, Zhaohui; He, Weidong; Chu, Benjamin

2010-10-21

This perspective is to introduce a new pathway for non-viral gene delivery by taking advantage of nanofibrous scaffolds as gene storage devices, gene carriers and homing devices. During gene delivery to the target, the DNA has to be protected in order to pass through a set of barriers before reaching the nucleus. The DNA can form a complex with polycations, and numerous publications exist on how to stabilize the DNA fragments by natural and synthetic materials. Electrospun nanofibrous scaffolds can be used to store the DNA, especially in the form of a more stabilized polyplex, and then to deliver the DNA (polyplex) to cells that are attached to the scaffold. While each essential step has been tested experimentally, the overall yet untested process, especially for in vivo experiments, may lead to a promising specific approach for gene/drug storage and delivery. The pathway described herein is based mainly on our understanding of the physics and chemistry of gene storage and delivery processes, in contrast to using pure biological concepts. Novel biodegradable, biocompatible nanofibrous materials with imbedded DNA (e.g., in the polyplex form) can then be designed to fabricate an intelligent scaffold for gene delivery. To achieve the above goal, the first step is to stabilize the DNA so that it can be incorporated into nanofibrous scaffolds. In this respect, we shall discuss the different methods of DNA/gene condensation and complex formation, and then explain the strategy used to incorporate DNA into electrospun nanofibers. Solvent-induced DNA condensation and then encapsulation were achieved. However, the released naked DNA was not sufficiently protected for gene transfection in cells. The objective of the current perspective is to suggest that, instead of the solvent-induced DNA condensation, one can combine the recently developed polyplex formation by using branched polyethyleneimine (bPEI). More importantly, free bPEI can be incorporated into the nanofibers separately so that during the gene delivery step, the presence of a predesigned amount of free bPEI can greatly increase the gene transfection efficiency, as has been reported recently by Chi Wu and his coworkers. Thus, a physics/chemistry-based pathway that utilizes nanofibrous scaffolds for gene delivery is within reach.

Multivalent DNA-binding properties of the HMG-1 proteins.

PubMed Central

Maher, J F; Nathans, D

1996-01-01

HMG-I proteins are DNA-binding proteins thought to affect the formation and function of transcription complexes. Each protein contains three DNA-binding motifs, known as AT-hooks, that bind in the minor groove of AT tracts in DNA. Multiple AT-hooks within a polypeptide chain should contact multiple AT tracts, but the rules governing these interactions have not been defined. In this study, we demonstrate that high-affinity binding uses two or three appropriately spaced AT tracts as a single multivalent binding site. These principles have implications for binding to regulatory elements such as the interferon beta enhancer, TATA boxes, and serum response elements. Images Fig. 1 Fig. 2 Fig. 3 Fig. 4 Fig. 5 PMID:8692884

Positive and negative ion mode comparison for the determination of DNA/peptide noncovalent binding sites through the formation of "three-body" noncovalent fragment ions.

PubMed

Brahim, Bessem; Tabet, Jean-Claude; Alves, Sandra

2018-02-01

Gas-phase fragmentation of single strand DNA-peptide noncovalent complexes is investigated in positive and negative electrospray ionization modes.Collision-induced dissociation experiments, performed on the positively charged noncovalent complex precursor ions, have confirmed the trend previously observed in negative ion mode, i.e. a high stability of noncovalent complexes containing very basic peptidic residues (i.e. R > K) and acidic nucleotide units (i.e. Thy units), certainly incoming from the existence of salt bridge interactions. Independent of the ion polarity, stable noncovalent complex precursor ions were found to dissociate preferentially through covalent bond cleavages of the partners without disrupting noncovalent interactions. The resulting DNA fragment ions were found to be still noncovalently linked to the peptides. Additionally, the losses of an internal nucleic fragment producing "three-body" noncovalent fragment ions were also observed in both ion polarities, demonstrating the spectacular salt bridge interaction stability. The identical fragmentation patterns (regardless of the relative fragment ion abundances) observed in both polarities have shown a common location of salt bridge interaction certainly preserved from solution. Nonetheless, most abundant noncovalent fragment ions (and particularly three-body ones) are observed from positively charged noncovalent complexes. Therefore, we assume that, independent of the preexisting salt bridge interaction and zwitterion structures, multiple covalent bond cleavages from single-stranded DNA/peptide complexes rely on an excess of positive charges in both electrospray ionization ion polarities.

Association of herpes simplex virus regulatory protein ICP22 with transcriptional complexes containing EAP, ICP4, RNA polymerase II, and viral DNA requires posttranslational modification by the U(L)13 proteinkinase.

PubMed Central

Leopardi, R; Ward, P L; Ogle, W O; Roizman, B

1997-01-01

The expression of herpes simplex virus 1 gamma (late) genes requires functional alpha proteins (gamma1 genes) and the onset of viral DNA synthesis (gamma2 genes). We report that late in infection after the onset of viral DNA synthesis, cell nuclei exhibit defined structures which contain two viral regulatory proteins (infected cell proteins 4 and 22) required for gamma gene expression, RNA polymerase II, a host nucleolar protein (EAP or L22) known to be associated with ribosomes and to bind small RNAs, including the Epstein-Barr virus small nuclear RNAs, and newly synthesized progeny DNA. The formation of these complexes required the onset of viral DNA synthesis. The association of infected cell protein 22, a highly posttranslationally processed protein, with these structures did not occur in cells infected with a viral mutant deleted in the genes U(L)13 and U(S)3, each of which specifies a protein kinase known to phosphorylate the protein. PMID:8995634

Replication Protein A (RPA) deficiency activates the Fanconi anemia DNA repair pathway.

PubMed

Jang, Seok-Won; Jung, Jin Ki; Kim, Jung Min

2016-09-01

The Fanconi anemia (FA) pathway regulates DNA inter-strand crosslink (ICL) repair. Despite our greater understanding of the role of FA in ICL repair, its function in the preventing spontaneous genome instability is not well understood. Here, we show that depletion of replication protein A (RPA) activates the FA pathway. RPA1 deficiency increases chromatin recruitment of FA core complex, leading to FANCD2 monoubiquitination (FANCD2-Ub) and foci formation in the absence of DNA damaging agents. Importantly, ATR depletion, but not ATM, abolished RPA1 depletion-induced FANCD2-Ub, suggesting that ATR activation mediated FANCD2-Ub. Interestingly, we found that depletion of hSSB1/2-INTS3, a single-stranded DNA-binding protein complex, induces FANCD2-Ub, like RPA1 depletion. More interestingly, depletion of either RPA1 or INTS3 caused increased accumulation of DNA damage in FA pathway deficient cell lines. Taken together, these results indicate that RPA deficiency induces activation of the FA pathway in an ATR-dependent manner, which may play a role in the genome maintenance.

Condensin suppresses recombination and regulates double-strand break processing at the repetitive ribosomal DNA array to ensure proper chromosome segregation during meiosis in budding yeast.

PubMed

Li, Ping; Jin, Hui; Yu, Hong-Guo

2014-10-01

During meiosis, homologues are linked by crossover, which is required for bipolar chromosome orientation before chromosome segregation at anaphase I. The repetitive ribosomal DNA (rDNA) array, however, undergoes little or no meiotic recombination. Hyperrecombination can cause chromosome missegregation and rDNA copy number instability. We report here that condensin, a conserved protein complex required for chromosome organization, regulates double-strand break (DSB) formation and repair at the rDNA gene cluster during meiosis in budding yeast. Condensin is highly enriched at the rDNA region during prophase I, released at the prophase I/metaphase I transition, and reassociates with rDNA before anaphase I onset. We show that condensin plays a dual role in maintaining rDNA stability: it suppresses the formation of Spo11-mediated rDNA breaks, and it promotes DSB processing to ensure proper chromosome segregation. Condensin is unnecessary for the export of rDNA breaks outside the nucleolus but required for timely repair of meiotic DSBs. Our work reveals that condensin coordinates meiotic recombination with chromosome segregation at the repetitive rDNA sequence, thereby maintaining genome integrity. © 2014 Li et al. This article is distributed by The American Society for Cell Biology under license from the author(s). Two months after publication it is available to the public under an Attribution–Noncommercial–Share Alike 3.0 Unported Creative Commons License (http://creativecommons.org/licenses/by-nc-sa/3.0).

Structure and Mechanism of Action of the BRCA2 Breast Cancer Tumor Suppressor

PubMed Central

Malivert, Laurent; McIlwraith, Michael J.; Pape, Tillman; West, Stephen C.; Zhang, Xiaodong

2014-01-01

Mutations in BRCA2 increase susceptibility to breast, ovarian and prostate cancers. The product of human BRCA2, BRCA2 protein, plays a key role in the repair of DNA double strand breaks and interstrand crosslinks by RAD51-mediated homologous recombination. Here, we present a biochemical and structural characterization of full length (3,418 amino acid) BRCA2, alone and in complex with RAD51. We show that BRCA2 facilitates nucleation of RAD51 filaments at multiple sites on single-stranded DNA. Three-dimensional electron microscopy reconstructions revealed that BRCA2 exists as a dimer and that two oppositely-oriented sets of RAD51 molecules bind the dimer. Single stranded DNA binds along the long axis of BRCA2, such that only one set of RAD51 monomers can form a productive complex with DNA and establish filament formation. Our data define the molecular mechanism by which this tumor suppressor facilitates RAD51-mediated homologous recombinational repair. PMID:25282148

DNA-Methylation: Master or Slave of Neural Fate Decisions?

PubMed Central

Stricker, Stefan H.; Götz, Magdalena

2018-01-01

The pristine formation of complex organs depends on sharp temporal and spatial control of gene expression. Therefore, epigenetic mechanisms have been frequently attributed a central role in controlling cell fate determination. A prime example for this is the first discovered and still most studied epigenetic mark, DNA methylation, and the development of the most complex mammalian organ, the brain. Recently, the field of epigenetics has advanced significantly: new DNA modifications were discovered, epigenomic profiling became widely accessible, and methods for targeted epigenomic manipulation have been developed. Thus, it is time to challenge established models of epigenetic gene regulation. Here, we review the current state of knowledge about DNA modifications, their epigenomic distribution, and their regulatory role. We will summarize the evidence suggesting they possess crucial roles in neurogenesis and discuss whether this likely includes lineage choice regulation or rather effects on differentiation. Finally, we will attempt an outlook on how questions, which remain unresolved, could be answered soon. PMID:29449798

Modulation of the Pyrococcus abyssi NucS Endonuclease Activity by Replication Clamp at Functional and Structural Levels*

PubMed Central

Creze, Christophe; Ligabue, Alessio; Laurent, Sébastien; Lestini, Roxane; Laptenok, Sergey P.; Khun, Joelle; Vos, Marten H.; Czjzek, Mirjam; Myllykallio, Hannu; Flament, Didier

2012-01-01

Pyrococcus abyssi NucS is the founding member of a new family of structure-specific DNA endonucleases that interact with the replication clamp proliferating cell nuclear antigen (PCNA). Using a combination of small angle x-ray scattering and surface plasmon resonance analyses, we demonstrate the formation of a stable complex in solution, in which one molecule of the PabNucS homodimer binds to the outside surface of the PabPCNA homotrimer. Using fluorescent labels, PCNA is shown to increase the binding affinity of NucS toward single-strand/double-strand junctions on 5′ and 3′ flaps, as well as to modulate the cleavage specificity on the branched DNA structures. Our results indicate that the presence of a single major contact between the PabNucS and PabPCNA proteins, together with the complex-induced DNA bending, facilitate conformational flexibility required for specific cleavage at the single-strand/double-strand DNA junction. PMID:22431731

Modulation of the Pyrococcus abyssi NucS endonuclease activity by replication clamp at functional and structural levels.

PubMed

Creze, Christophe; Ligabue, Alessio; Laurent, Sébastien; Lestini, Roxane; Laptenok, Sergey P; Khun, Joelle; Vos, Marten H; Czjzek, Mirjam; Myllykallio, Hannu; Flament, Didier

2012-05-04

Pyrococcus abyssi NucS is the founding member of a new family of structure-specific DNA endonucleases that interact with the replication clamp proliferating cell nuclear antigen (PCNA). Using a combination of small angle x-ray scattering and surface plasmon resonance analyses, we demonstrate the formation of a stable complex in solution, in which one molecule of the PabNucS homodimer binds to the outside surface of the PabPCNA homotrimer. Using fluorescent labels, PCNA is shown to increase the binding affinity of NucS toward single-strand/double-strand junctions on 5' and 3' flaps, as well as to modulate the cleavage specificity on the branched DNA structures. Our results indicate that the presence of a single major contact between the PabNucS and PabPCNA proteins, together with the complex-induced DNA bending, facilitate conformational flexibility required for specific cleavage at the single-strand/double-strand DNA junction.

BIOPHYSICAL PROPERTIES OF NUCLEIC ACIDS AT SURFACES RELEVANT TO MICROARRAY PERFORMANCE.

PubMed

Rao, Archana N; Grainger, David W

2014-04-01

Both clinical and analytical metrics produced by microarray-based assay technology have recognized problems in reproducibility, reliability and analytical sensitivity. These issues are often attributed to poor understanding and control of nucleic acid behaviors and properties at solid-liquid interfaces. Nucleic acid hybridization, central to DNA and RNA microarray formats, depends on the properties and behaviors of single strand (ss) nucleic acids (e.g., probe oligomeric DNA) bound to surfaces. ssDNA's persistence length, radius of gyration, electrostatics, conformations on different surfaces and under various assay conditions, its chain flexibility and curvature, charging effects in ionic solutions, and fluorescent labeling all influence its physical chemistry and hybridization under assay conditions. Nucleic acid (e.g., both RNA and DNA) target interactions with immobilized ssDNA strands are highly impacted by these biophysical states. Furthermore, the kinetics, thermodynamics, and enthalpic and entropic contributions to DNA hybridization reflect global probe/target structures and interaction dynamics. Here we review several biophysical issues relevant to oligomeric nucleic acid molecular behaviors at surfaces and their influences on duplex formation that influence microarray assay performance. Correlation of biophysical aspects of single and double-stranded nucleic acids with their complexes in bulk solution is common. Such analysis at surfaces is not commonly reported, despite its importance to microarray assays. We seek to provide further insight into nucleic acid-surface challenges facing microarray diagnostic formats that have hindered their clinical adoption and compromise their research quality and value as genomics tools.

Formation of stable and functional HIV-1 nucleoprotein complexes in vitro.

PubMed

Tanchou, V; Gabus, C; Rogemond, V; Darlix, J L

1995-10-06

HIV genomic RNA resides within the nucleocapsid, in the interior of the virus, which serves to protect the RNA against nuclease degradation and to promote its reverse transcription. To investigate the role of nucleocapsid protein (NCp7) in the stability and replication of genomic RNA within the nucleocapsid, we used NCp7, reverse transcriptase (RT) and RNAs representing the 5' and 3' regions of the genome to reconstitute functional HIV-1 nucleocapsids. The nucleoprotein complexes generated in vitro were found to be stable, which, according to biochemical and genetic data, probably results from the tight binding of NCp7 molecules to the RNA and strong NCp7/NCp7 interactions. The nucleoprotein complexes efficiently protected viral RNA against RNase degradation and, at the same time, promoted viral DNA synthesis by RT. DNA strand transfer from the 5' to the 3' RNA template was very efficient in nucleoprotein complexes formed in the presence of both RNAs, but not when the RNAs were in separate complexes. These results indicate that the in vitro reconstituted HIV-1 nucleoprotein complexes function like virion nucleocapsids and thus provide a way to study at the molecular level this viral substructure and the synthesis of proviral DNA, and to search for new anti-HIV agents.

Translocation and gross deletion breakpoints in human inherited disease and cancer II: Potential involvement of repetitive sequence elements in secondary structure formation between DNA ends.

PubMed

Chuzhanova, Nadia; Abeysinghe, Shaun S; Krawczak, Michael; Cooper, David N

2003-09-01

Translocations and gross deletions are responsible for a significant proportion of both cancer and inherited disease. Although such gene rearrangements are nonuniformly distributed in the human genome, the underlying mutational mechanisms remain unclear. We have studied the potential involvement of various types of repetitive sequence elements in the formation of secondary structure intermediates between the single-stranded DNA ends that recombine during rearrangements. Complexity analysis was used to assess the potential of these ends to form secondary structures, the maximum decrease in complexity consequent to a gross rearrangement being used as an indicator of the type of repeat and the specific DNA ends involved. A total of 175 pairs of deletion/translocation breakpoint junction sequences available from the Gross Rearrangement Breakpoint Database [GRaBD; www.uwcm.ac.uk/uwcm/mg/grabd/grabd.html] were analyzed. Potential secondary structure was noted between the 5' flanking sequence of the first breakpoint and the 3' flanking sequence of the second breakpoint in 49% of rearrangements and between the 5' flanking sequence of the second breakpoint and the 3' flanking sequence of the first breakpoint in 36% of rearrangements. Inverted repeats, inversions of inverted repeats, and symmetric elements were found in association with gross rearrangements at approximately the same frequency. However, inverted repeats and inversions of inverted repeats accounted for the vast majority (83%) of deletions plus small insertions, symmetric elements for one-half of all antigen receptor-mediated translocations, while direct repeats appear only to be involved in mediating simple deletions. These findings extend our understanding of illegitimate recombination by highlighting the importance of secondary structure formation between single-stranded DNA ends at breakpoint junctions. Copyright 2003 Wiley-Liss, Inc.

Relative stabilities of triple helices composed of combinations of DNA, RNA and 2'-O-methyl-RNA backbones: chimeric circular oligonucleotides as probes.

PubMed

Wang, S; Kool, E T

1995-04-11

Described is a systematic study of the effects of varied backbone structure on the stabilities of pyr.pur.pyr triple helices. The effects were measured using six circular 34 base oligonucleotides containing DNA (D), RNA (R) and/or 2'-O-methyl-RNA (M) residues designed to bind a complementary single-stranded purine target strand by triple helix formation. Eighteen different backbone combinations were studied at pH 5.5 and 7.0 by optical melting experiments and the results compared with the stabilities of the corresponding Watson-Crick duplexes. When the target purine strand is DNA, all circles form pH-dependent triple helical complexes which are considerably stronger than the duplexes alone. When RNA is the target, five of the nine complexes studied are of the pH-dependent triplex type and the other four complexes are not significantly stronger than the corresponding duplexes. The results are useful in the design of the highest affinity ligands for single- and double-stranded DNAs and RNAs and also point out novel ways to engender DNA- or RNA-selective binding.

The TubR-centromere complex adopts a double-ring segrosome structure in Type III partition systems.

PubMed

Martín-García, Bárbara; Martín-González, Alejandro; Carrasco, Carolina; Hernández-Arriaga, Ana M; Ruíz-Quero, Rubén; Díaz-Orejas, Ramón; Aicart-Ramos, Clara; Moreno-Herrero, Fernando; Oliva, María A

2018-05-14

In prokaryotes, the centromere is a specialized segment of DNA that promotes the assembly of the segrosome upon binding of the Centromere Binding Protein (CBP). The segrosome structure exposes a specific surface for the interaction of the CBP with the motor protein that mediates DNA movement during cell division. Additionally, the CBP usually controls the transcriptional regulation of the segregation system as a cell cycle checkpoint. Correct segrosome functioning is therefore indispensable for accurate DNA segregation. Here, we combine biochemical reconstruction and structural and biophysical analysis to bring light to the architecture of the segrosome complex in Type III partition systems. We present the particular features of the centromere site, tubC, of the model system encoded in Clostridium botulinum prophage c-st. We find that the split centromere site contains two different iterons involved in the binding and spreading of the CBP, TubR. The resulting nucleoprotein complex consists of a novel double-ring structure that covers part of the predicted promoter. Single molecule data provides a mechanism for the formation of the segrosome structure based on DNA bending and unwinding upon TubR binding.

Dlx5 Homeodomain: DNA Complex: Structure, Binding and Effect of Mutations Related to Split Hand and Foot Malformation Syndrome

DOE Office of Scientific and Technical Information (OSTI.GOV)

Proudfoot, Andrew; Axelrod, Herbert L.; Geralt, Michael

The Dlx5 homeodomain is a transcription factor related to the Drosophila Distal-less gene that is associated with breast and lung cancer, lymphoma, Rett syndrome and osteoporosis in humans. Mutations in the DLX5 gene have been linked to deficiencies in craniofacial and limb development in higher eukaryotes, including Split Hand and Foot Malformation-1 (SHFM-1) in humans. Our characterization of a Dlx5 homeodomain–(CGACTAATTAGTCG) 2 complex by NMR spectroscopy paved the way for determination of its crystal structure at 1.85 Å resolution that enabled rationalization of the effects of disease-related mutations on the protein function. A remarkably subtle mutation, Q186H, is linked tomore » SHFM-1; this change likely affects affinity of DNA binding by disrupting water-mediated interactions with the DNA major groove. A more subtle effect is implicated for the Q178P mutation, which is not in direct contact with the DNA. Our data indicate that these mutations diminish the ability of the Dlx5 homeodomain to recognize and bind target DNAs, and likely destabilize the formation of functional complexes.« less

Dlx5 Homeodomain: DNA Complex: Structure, Binding and Effect of Mutations Related to Split Hand and Foot Malformation Syndrome

DOE PAGES

Proudfoot, Andrew; Axelrod, Herbert L.; Geralt, Michael; ...

2016-01-29

The Dlx5 homeodomain is a transcription factor related to the Drosophila Distal-less gene that is associated with breast and lung cancer, lymphoma, Rett syndrome and osteoporosis in humans. Mutations in the DLX5 gene have been linked to deficiencies in craniofacial and limb development in higher eukaryotes, including Split Hand and Foot Malformation-1 (SHFM-1) in humans. Our characterization of a Dlx5 homeodomain–(CGACTAATTAGTCG) 2 complex by NMR spectroscopy paved the way for determination of its crystal structure at 1.85 Å resolution that enabled rationalization of the effects of disease-related mutations on the protein function. A remarkably subtle mutation, Q186H, is linked tomore » SHFM-1; this change likely affects affinity of DNA binding by disrupting water-mediated interactions with the DNA major groove. A more subtle effect is implicated for the Q178P mutation, which is not in direct contact with the DNA. Our data indicate that these mutations diminish the ability of the Dlx5 homeodomain to recognize and bind target DNAs, and likely destabilize the formation of functional complexes.« less

Interaction of the replication terminator protein of Bacillus subtilis with DNA probed by NMR spectroscopy

DOE Office of Scientific and Technical Information (OSTI.GOV)

Hastings, Adam F.; Otting, Gottfried; Folmer, Rutger H.A.

2005-09-23

Termination of DNA replication in Bacillus subtilis involves the polar arrest of replication forks by a specific complex formed between the dimeric 29 kDa replication terminator protein (RTP) and DNA terminator sites. We have used NMR spectroscopy to probe the changes in {sup 1}H-{sup 15}N correlation spectra of a {sup 15}N-labelled RTP.C110S mutant upon the addition of a 21 base pair symmetrical DNA binding site. Assignment of the {sup 1}H-{sup 15}N correlations was achieved using a suite of triple resonance NMR experiments with {sup 15}N,{sup 13}C,70% {sup 2}H enriched protein recorded at 800 MHz and using TROSY pulse sequences. Perturbationsmore » to {sup 1}H-{sup 15}N spectra revealed that the N-termini, {alpha}3-helices and several loops are affected by the binding interaction. An analysis of this data in light of the crystallographically determined apo- and DNA-bound forms of RTP.C110S revealed that the NMR spectral perturbations correlate more closely to protein structural changes upon complex formation rather than to interactions at the protein-DNA interface.« less

Optimizing DNA nanotechnology through coarse-grained modeling: a two-footed DNA walker.

PubMed

Ouldridge, Thomas E; Hoare, Rollo L; Louis, Ard A; Doye, Jonathan P K; Bath, Jonathan; Turberfield, Andrew J

2013-03-26

DNA has enormous potential as a programmable material for creating artificial nanoscale structures and devices. For more complex systems, however, rational design and optimization can become difficult. We have recently proposed a coarse-grained model of DNA that captures the basic thermodynamic, structural, and mechanical changes associated with the fundamental process in much of DNA nanotechnology, the formation of duplexes from single strands. In this article, we demonstrate that the model can provide powerful insight into the operation of complex nanotechnological systems through a detailed investigation of a two-footed DNA walker that is designed to step along a reusable track, thereby offering the possibility of optimizing the design of such systems. We find that applying moderate tension to the track can have a large influence on the operation of the walker, providing a bias for stepping forward and helping the walker to recover from undesirable overstepped states. Further, we show that the process by which spent fuel detaches from the walker can have a significant impact on the rebinding of the walker to the track, strongly influencing walker efficiency and speed. Finally, using the results of the simulations, we propose a number of modifications to the walker to improve its operation.

The interaction of HMGB1 and linker histones occurs through their acidic and basic tails.

PubMed

Cato, Laura; Stott, Katherine; Watson, Matthew; Thomas, Jean O

2008-12-31

H1 and HMGB1 bind to linker DNA in chromatin, in the vicinity of the nucleosome dyad. They appear to have opposing effects on the nucleosome, H1 stabilising it by "sealing" two turns of DNA around the octamer, and HMGB1 destabilising it, probably by bending the adjacent DNA. Their presence in chromatin might be mutually exclusive. Displacement/replacement of one by the other as a result of their highly dynamic binding in vivo might, in principle, involve interactions between them. Chemical cross-linking and gel-filtration show that a 1:1 linker histone/HMGB1 complex is formed, which persists at physiological ionic strength, and that complex formation requires the acidic tail of HMGB1. NMR spectroscopy shows that the linker histone binds, predominantly through its basic C-terminal domain, to the acidic tail of HMGB1, thereby disrupting the interaction of the tail with the DNA-binding faces of the HMG boxes. A potential consequence of this interaction is enhanced DNA binding by HMGB1, and concomitantly lowered affinity of H1 for DNA. In a chromatin context, this might facilitate displacement of H1 by HMGB1.

SUMO regulates p21Cip1 intracellular distribution and with p21Cip1 facilitates multiprotein complex formation in the nucleolus upon DNA damage.

PubMed

Brun, Sonia; Abella, Neus; Berciano, Maria T; Tapia, Olga; Jaumot, Montserrat; Freire, Raimundo; Lafarga, Miguel; Agell, Neus

2017-01-01

We previously showed that p21Cip1 transits through the nucleolus on its way from the nucleus to the cytoplasm and that DNA damage inhibits this transit and induces the formation of p21Cip1-containing intranucleolar bodies (INoBs). Here, we demonstrate that these INoBs also contain SUMO-1 and UBC9, the E2 SUMO-conjugating enzyme. Furthermore, whereas wild type SUMO-1 localized in INoBs, a SUMO-1 mutant, which is unable to conjugate with proteins, does not, suggesting the presence of SUMOylated proteins at INoBs. Moreover, depletion of the SUMO-conjugating enzyme UBC9 or the sumo hydrolase SENP2 changed p21Cip1 intracellular distribution. In addition to SUMO-1 and p21Cip1, cell cycle regulators and DNA damage checkpoint proteins, including Cdk2, Cyclin E, PCNA, p53 and Mdm2, and PML were also detected in INoBs. Importantly, depletion of UBC9 or p21Cip1 impacted INoB biogenesis and the nucleolar accumulation of the cell cycle regulators and DNA damage checkpoint proteins following DNA damage. The impact of p21Cip1 and SUMO-1 on the accumulation of proteins in INoBs extends also to CRM1, a nuclear exportin that is also important for protein translocation from the cytoplasm to the nucleolus. Thus, SUMO and p21Cip1 regulate the transit of proteins through the nucleolus, and that disruption of nucleolar export by DNA damage induces SUMO and p21Cip1 to act as hub proteins to form a multiprotein complex in the nucleolus.

SUMO regulates p21Cip1 intracellular distribution and with p21Cip1 facilitates multiprotein complex formation in the nucleolus upon DNA damage

PubMed Central

Brun, Sonia; Abella, Neus; Berciano, Maria T.; Tapia, Olga; Jaumot, Montserrat; Freire, Raimundo; Lafarga, Miguel

2017-01-01

We previously showed that p21Cip1 transits through the nucleolus on its way from the nucleus to the cytoplasm and that DNA damage inhibits this transit and induces the formation of p21Cip1-containing intranucleolar bodies (INoBs). Here, we demonstrate that these INoBs also contain SUMO-1 and UBC9, the E2 SUMO-conjugating enzyme. Furthermore, whereas wild type SUMO-1 localized in INoBs, a SUMO-1 mutant, which is unable to conjugate with proteins, does not, suggesting the presence of SUMOylated proteins at INoBs. Moreover, depletion of the SUMO-conjugating enzyme UBC9 or the sumo hydrolase SENP2 changed p21Cip1 intracellular distribution. In addition to SUMO-1 and p21Cip1, cell cycle regulators and DNA damage checkpoint proteins, including Cdk2, Cyclin E, PCNA, p53 and Mdm2, and PML were also detected in INoBs. Importantly, depletion of UBC9 or p21Cip1 impacted INoB biogenesis and the nucleolar accumulation of the cell cycle regulators and DNA damage checkpoint proteins following DNA damage. The impact of p21Cip1 and SUMO-1 on the accumulation of proteins in INoBs extends also to CRM1, a nuclear exportin that is also important for protein translocation from the cytoplasm to the nucleolus. Thus, SUMO and p21Cip1 regulate the transit of proteins through the nucleolus, and that disruption of nucleolar export by DNA damage induces SUMO and p21Cip1 to act as hub proteins to form a multiprotein complex in the nucleolus. PMID:28582471

Dimerization site 2 of the bacterial DNA-binding protein H-NS is required for gene silencing and stiffened nucleoprotein filament formation.

PubMed

Yamanaka, Yuki; Winardhi, Ricksen S; Yamauchi, Erika; Nishiyama, So-Ichiro; Sowa, Yoshiyuki; Yan, Jie; Kawagishi, Ikuro; Ishihama, Akira; Yamamoto, Kaneyoshi

2018-06-15

The bacterial nucleoid-associated protein H-NS is a DNA-binding protein, playing a major role in gene regulation. To regulate transcription, H-NS silences genes, including horizontally acquired foreign genes. Escherichia coli H-NS is 137 residues long and consists of two discrete and independent structural domains: an N-terminal oligomerization domain and a C-terminal DNA-binding domain, joined by a flexible linker. The N-terminal oligomerization domain is composed of two dimerization sites, dimerization sites 1 and 2, which are both required for H-NS oligomerization, but the exact role of dimerization site 2 in gene silencing is unclear. To this end, we constructed a whole set of single amino acid substitution variants spanning residues 2 to 137. Using a well-characterized H-NS target, the slp promoter of the glutamic acid-dependent acid resistance (GAD) cluster promoters, we screened for any variants defective in gene silencing. Focusing on the function of dimerization site 2, we analyzed four variants, I70C/I70A and L75C/L75A, which all could actively bind DNA but are defective in gene silencing. Atomic force microscopy analysis of DNA-H-NS complexes revealed that all of these four variants formed condensed complexes on DNA, whereas WT H-NS formed rigid and extended nucleoprotein filaments, a conformation required for gene silencing. Single-molecule stretching experiments confirmed that the four variants had lost the ability to form stiffened filaments. We conclude that dimerization site 2 of H-NS plays a key role in the formation of rigid H-NS nucleoprotein filament structures required for gene silencing. © 2018 by The American Society for Biochemistry and Molecular Biology, Inc.

Effect of Escherichia coli DNA binding protein on the transcription of single-stranded phage M13 DNA by Escherichia coli RNA polymerase

DOE Office of Scientific and Technical Information (OSTI.GOV)

Niyogi, S.K.; Ratrie, H. III; Datta, A.K.

E. coli DNA binding protein strongly inhibits the transcription of single-stranded rather than double-stranded phage M13 DNA by E. coli RNA polymerase. This inhibition cannot be significantly overcome by increasing the concentration of RNA polymerase. Nor does the order of addition of binding protein affect its inhibitory property: inhibition is evident whether binding protein is added before or after the formation of the RNA polymerase--DNA complex. Inhibition is also observed if binding protein is added at various times after initiation of RNA synthesis. Maximal inhibition occurs at a binding protein-to-DNA ratio (w/w) of about 8:1. This corresponds to one bindingmore » protein molecule covering about 30 nucleotides, in good agreement with values obtained by physical measurements.« less

MicroCantilever (MC) based nanomechanical sensor for detection of molecular interactions

DOE Office of Scientific and Technical Information (OSTI.GOV)

Kang, Kyung

Specific aims of this study are to investigate the mechanism governing surface stress generation associated with chemical or molecular binding on functionalized microcantilevers. Formation of affinity complexes on cantilever surfaces leads to charge redistribution, configurational change and steric hindrance between neighboring molecules resulting in surface stress change and measureable cantilever deformation. A novel interferometry technique employing two adjacent micromachined cantilevers (a sensing/reference pair) was utilized to measure the cantilever deformation. The sensing principle is that binding/reaction of specific chemical or biological species on the sensing cantilever transduces to mechanical deformation. The differential bending of the sensing cantilever respect to themore » reference cantilever ensures that measured response is insensitive to environmental disturbances. As a proof of principle for the measurement technique, surface stress changes associated with: self-assembly of alkanethiol, hybridization of ssDNA, and the formation of cocaine-aptamer complexes were measured. Dissociation constant (K d) for each molecular reaction was utilized to estimate the surface coverage of affinity complexes. In the cases of DNA hybridization and cocaine-aptamer binding, measured surface stress was found to be dependent on the surface coverage of the affinity complexes. In order to achieve a better sensitivity for DNA hybridization, immobilization of receptor molecules was modified to enhance the deformation of underlying surface. Single-stranded DNA (ssDNA) strands with thiol-modification on both 3-foot and 5-foot ends were immobilized on the gold surface such that both ends are attached to the gold surface. Immobilization condition was controlled to obtain similar receptor density as single-thiolated DNA strands. Hybridization of double-thiolated DNA strands leads to an almost two orders of magnitude increase in cantilever deformation. In both DNA hybridization and the conventional mode for cocaine detection, the lowest detectable concentration was determined by binding activity between the ligand and receptor molecules. In order to overcome this limitation for cocaine detection, a novel competition sensing mode that relies on rate of aptamers unbinding from the cantilever due to either diffusion or reaction with cocaine as target ligands in solution was investigated. The rate of unbinding is found to be dependent on the concentration of cocaine molecules. A model based on diffusion-reaction equation was developed to explain the experimental observation. Experimental results indicate that the competition mode reduces the lowest detectable threshold to 200 nM which is comparable to that achieved analytical techniques such as mass spectrometry.« less

Uncovering the self-assembly of DNA nanostructures by thermodynamics and kinetics.

PubMed

Wei, Xixi; Nangreave, Jeanette; Liu, Yan

2014-06-17

CONSPECTUS: DNA nanotechnology is one of the most flourishing interdisciplinary research fields. DNA nanostructures can be designed to self-assemble into a variety of periodic or aperiodic patterns of different shapes and length scales. They can be used as scaffolds for organizing other nanoparticles, proteins, and chemical groups, leveraging their functions for creating complex bioinspired materials that may serve as smart drug delivery systems, in vitro or in vivo biomolecular computing platforms, and diagnostic devices. Achieving optimal structural features, efficient assembly protocols, and precise functional group positioning and modification requires a thorough understanding of the thermodynamics and kinetics of the DNA nanostructure self-assembly process. The most common real-time measurement strategies include monitoring changes in UV absorbance based on the hyperchromic effect of DNA, and the emission signal changes of DNA intercalating dyes or covalently conjugated fluorescent dyes/pairs that accompany temperature dependent structural changes. Thermodynamic studies of a variety of DNA nanostructures have been performed, from simple double stranded DNA formation to more complex origami assembly. The key parameters that have been evaluated in terms of stability and cooperativity include the overall dimensions, the folding path of the scaffold, crossover and nick point arrangement, length and sequence of single strands, and salt and ion concentrations. DNA tile-tile interactions through sticky end hybridization have also been analyzed, and the steric inhibition and rigidity of tiles turn out to be important factors. Many kinetic studies have also been reported, and most are based on double stranded DNA formation. A two-state assumption and the hypothesis of several intermediate states have been applied to determine the rate constant and activation energy of the DNA hybridization process. A few simulated models were proposed to represent the structural, mechanical, and kinetic properties of DNA hybridization. The kinetics of strand displacement reactions has also been studied as a special case of DNA hybridization. The thermodynamic and kinetic characteristics of DNA nanostructures have been exploited to develop rapid and isothermal annealing protocols. It is conceivable that a more thorough understanding of the DNA assembly process could be used to guide the structural design process and optimize the conditions for assembly, manipulation, and functionalization, thus benefiting both upstream design and downstream applications.

Structural and Functional Assessment of APOBEC3G Macromolecular Complexes

PubMed Central

Polevoda, Bogdan; McDougall, William M.; Bennett, Ryan P.; Salter, Jason D.; Smith, Harold C.

2016-01-01

There are eleven members in the human APOBEC family of proteins that are evolutionarily related through their zinc-dependent cytidine deaminase domains. The human APOBEC gene clusters arose on chromosome 6 and 22 through gene duplication and divergence to where current day APOBEC proteins are functionally diverse and broadly expressed in tissues. APOBEC serve enzymatic and non enzymatic functions in cells. In both cases, formation of higher-order structures driven by APOBEC protein-protein interactions and binding to RNA and/or single stranded DNA are integral to their function. In some circumstances, these interactions are regulatory and modulate APOBEC activities. We are just beginning to understand how macromolecular interactions drive processes such as APOBEC subcellular compartmentalization, formation of holoenzyme complexes, gene targeting, foreign DNA restriction, anti-retroviral activity, formation of ribonucleoprotein particles and APOBEC degradation. Protein-protein and protein-nucleic acid cross-linking methods coupled with mass spectrometry, electrophoretic mobility shift assays, glycerol gradient sedimentation, fluorescence anisotropy and APOBEC deaminase assays are enabling mapping of interacting surfaces that are essential for these functions. The goal of this methods review is through example of our research on APOBEC3G, describe the application of cross-linking methods to characterize and quantify macromolecular interactions and their functional implications. Given the homology in structure and function, it is proposed that these methods will be generally applicable to the discovery process for other APOBEC and RNA and DNA editing and modifying proteins. PMID:26988126

Cooperative motion of a key positively charged residue and metal ions for DNA replication catalyzed by human DNA Polymerase-η.

PubMed

Genna, Vito; Gaspari, Roberto; Dal Peraro, Matteo; De Vivo, Marco

2016-04-07

Trans-lesion synthesis polymerases, like DNA Polymerase-η (Pol-η), are essential for cell survival. Pol-η bypasses ultraviolet-induced DNA damages via a two-metal-ion mechanism that assures DNA strand elongation, with formation of the leaving group pyrophosphate (PPi). Recent structural and kinetics studies have shown that Pol-η function depends on the highly flexible and conserved Arg61 and, intriguingly, on a transient third ion resolved at the catalytic site, as lately observed in other nucleic acid-processing metalloenzymes. How these conserved structural features facilitate DNA replication, however, is still poorly understood. Through extended molecular dynamics and free energy simulations, we unravel a highly cooperative and dynamic mechanism for DNA elongation and repair, which is here described by an equilibrium ensemble of structures that connect the reactants to the products in Pol-η catalysis. We reveal that specific conformations of Arg61 help facilitate the recruitment of the incoming base and favor the proper formation of a pre-reactive complex in Pol-η for efficient DNA editing. Also, we show that a third transient metal ion, which acts concertedly with Arg61, serves as an exit shuttle for the leaving PPi. Finally, we discuss how this effective and cooperative mechanism for DNA repair may be shared by other DNA-repairing polymerases. © The Author(s) 2016. Published by Oxford University Press on behalf of Nucleic Acids Research.

Platinum anti-cancer drugs: Free radical mechanism of Pt-DNA adduct formation and anti-neoplastic effect.

PubMed

Fong, Clifford W

2016-06-01

The literature on the anti-neoplastic effects of Pt drugs provides substantial evidence that free radical may be involved in the formation of Pt-DNA adducts and other cytotoxic effects. The conditions specific to cancerous tumours are more conducive to free radical mechanisms than the commonly accepted hydrolysis nucleophilic-electrophilic mechanism of Pt-DNA adduct formation. Molecular orbital studies of the adiabatic attachment of hydrated electrons to Pt drugs reveal that there is a significant lengthening of the Pt-X bond (where X is Cl, O in cisplatin, carboplatin and some pyrophosphate-Pt drugs but not oxaliplatin) in the anion radical species. This observation is consistent with a dissociative electron transfer (DET) mechanism for the formation of Pt-DNA adducts. A DET reaction mechanism is proposed for the reaction of Pt drugs with guanine which involves a quasi-inner sphere 2 electron transfer process involving a transient intermediate 5 co-ordinated activated anion radical species {R2Pt---Cl(G)(Cl)•}*(-) (where R is an ammine group, and G is guanine) and the complex has an elongated Pt---Cl (or Pt---O) bond. A DET mechanism is also proposed when Pt drugs are activated by reaction with free radicals such as HO•, CO3•(-), O2•(-) but do not react with DNA bases to form adducts, but form Pt-protein adducts with proteins such ezrin, FAS, DR5, TNFR1 etc. The DET mechanism may not occur with oxaliplatin. Copyright © 2016 Elsevier Inc. All rights reserved.

Dynamic epigenetic states of maize centromeres

PubMed Central

Liu, Yalin; Su, Handong; Zhang, Jing; Liu, Yang; Han, Fangpu; Birchler, James A.

2015-01-01

The centromere is a specialized chromosomal region identified as the major constriction, upon which the kinetochore complex is formed, ensuring accurate chromosome orientation and segregation during cell division. The rapid evolution of centromere DNA sequence and the conserved centromere function are two contradictory aspects of centromere biology. Indeed, the sole presence of genetic sequence is not sufficient for centromere formation. Various dicentric chromosomes with one inactive centromere have been recognized. It has also been found that de novo centromere formation is common on fragments in which centromeric DNA sequences are lost. Epigenetic factors play important roles in centromeric chromatin assembly and maintenance. Non-disjunction of the supernumerary B chromosome centromere is independent of centromere function, but centromere pairing during early prophase of meiosis I requires an active centromere. This review discusses recent studies in maize about genetic and epigenetic elements regulating formation and maintenance of centromere chromatin, as well as centromere behavior in meiosis. PMID:26579154

Dynamic epigenetic states of maize centromeres.

PubMed

Liu, Yalin; Su, Handong; Zhang, Jing; Liu, Yang; Han, Fangpu; Birchler, James A

2015-01-01

The centromere is a specialized chromosomal region identified as the major constriction, upon which the kinetochore complex is formed, ensuring accurate chromosome orientation and segregation during cell division. The rapid evolution of centromere DNA sequence and the conserved centromere function are two contradictory aspects of centromere biology. Indeed, the sole presence of genetic sequence is not sufficient for centromere formation. Various dicentric chromosomes with one inactive centromere have been recognized. It has also been found that de novo centromere formation is common on fragments in which centromeric DNA sequences are lost. Epigenetic factors play important roles in centromeric chromatin assembly and maintenance. Non-disjunction of the supernumerary B chromosome centromere is independent of centromere function, but centromere pairing during early prophase of meiosis I requires an active centromere. This review discusses recent studies in maize about genetic and epigenetic elements regulating formation and maintenance of centromere chromatin, as well as centromere behavior in meiosis.

Interactions of Kid–Kis toxin–antitoxin complexes with the parD operator-promoter region of plasmid R1 are piloted by the Kis antitoxin and tuned by the stoichiometry of Kid–Kis oligomers

PubMed Central

Monti, Maria C.; Hernández-Arriaga, Ana M.; Kamphuis, Monique B.; López-Villarejo, Juan; Heck, Albert J. R.; Boelens, Rolf; Díaz-Orejas, Ramón; van den Heuvel, Robert H. H.

2007-01-01

The parD operon of Escherichia coli plasmid R1 encodes a toxin–antitoxin system, which is involved in plasmid stabilization. The toxin Kid inhibits cell growth by RNA degradation and its action is neutralized by the formation of a tight complex with the antitoxin Kis. A fascinating but poorly understood aspect of the kid–kis system is its autoregulation at the transcriptional level. Using macromolecular (tandem) mass spectrometry and DNA binding assays, we here demonstrate that Kis pilots the interaction of the Kid–Kis complex in the parD regulatory region and that two discrete Kis-binding regions are present on parD. The data clearly show that only when the Kis concentration equals or exceeds the Kid concentration a strong cooperative effect exists between strong DNA binding and Kid2–Kis2–Kid2–Kis2 complex formation. We propose a model in which transcriptional repression of the parD operon is tuned by the relative molar ratio of the antitoxin and toxin proteins in solution. When the concentration of the toxin exceeds that of the antitoxin tight Kid2–Kis2–Kid2 complexes are formed, which only neutralize the lethal activity of Kid. Upon increasing the Kis concentration, (Kid2–Kis2)n complexes repress the kid–kis operon. PMID:17317682

Structural Basis for DNA Recognition by the Two-Component Response Regulator RcsB.

PubMed

Filippova, Ekaterina V; Zemaitaitis, Bozena; Aung, Theint; Wolfe, Alan J; Anderson, Wayne F

2018-02-27

RcsB is a highly conserved transcription regulator of the Rcs phosphorelay system, a complex two-component signal transduction system (N. Majdalani and S. Gottesman, Annu Rev Microbiol 59:379-405, 2005; A. J. Wolfe, Curr Opin Microbiol 13:204-209, 2010, https://doi.org/10.1016/j.mib.2010.01.002; D. J. Clarke, Future Microbiol 5:1173-1184, 2010, https://doi.org/10.2217/fmb.10.83). RcsB plays an important role in virulence and pathogenicity in human hosts by regulating biofilm formation. RcsB can regulate transcription alone or together with its auxiliary transcription regulators by forming heterodimers. This complexity allows RcsB to regulate transcription of more than 600 bacterial genes in response to different stresses (D. Wang et al., Mol Plant Microbe Interact 25:6-17, 2012, https://doi.org/10.1094/MPMI-08-11-0207). Despite increasing knowledge of RcsB importance, molecular mechanisms that drive the ability of RcsB to control transcription of a large number of genes remain unclear. Here, we present crystal structures of unphosphorylated RcsB in complex with the consensus DNA-binding sequence of 22-mer (DNA22) and 18-mer (DNA18) of the flhDC operon from Escherichia coli determined at 3.15- and 3.37-Å resolution, respectively. The results of our structural analysis combined with the results of in vitro binding assays provide valuable insights to the protein regulatory mechanism, demonstrate how RcsB recognizes target DNA sequences, and reveal a unique oligomeric state that allows RcsB to form homo- and heterodimers. This information will help us understand the complex mechanisms of transcriptional regulation by RcsB in bacteria. IMPORTANCE RcsB is a well-studied two-component response regulator of the Rcs phosphorelay system, conserved within the family Enterobacteriaceae , which includes many pathogens. It is a global regulator, controlling more than 5% of bacterial genes associated with capsule biosynthesis, flagellar biogenesis, cell wall biosynthesis, antibiotic resistance, biofilm formation, and virulence in pathogens. Knowledge of RcsB structure represents a unique opportunity to explore mechanisms that regulate the Rcs phosphorelay system and its role in the family Enterobacteriaceae . Copyright © 2018 Filippova et al.

Aberrant topoisomerase-1 DNA lesions are pathogenic in neurodegenerative genome instability syndromes.

PubMed

Katyal, Sachin; Lee, Youngsoo; Nitiss, Karin C; Downing, Susanna M; Li, Yang; Shimada, Mikio; Zhao, Jingfeng; Russell, Helen R; Petrini, John H J; Nitiss, John L; McKinnon, Peter J

2014-06-01

DNA damage is considered to be a prime factor in several spinocerebellar neurodegenerative diseases; however, the DNA lesions underpinning disease etiology are unknown. We observed the endogenous accumulation of pathogenic topoisomerase-1 (Top1)-DNA cleavage complexes (Top1ccs) in murine models of ataxia telangiectasia and spinocerebellar ataxia with axonal neuropathy 1. We found that the defective DNA damage response factors in these two diseases cooperatively modulated Top1cc turnover in a non-epistatic and ATM kinase-independent manner. Furthermore, coincident neural inactivation of ATM and DNA single-strand break repair factors, including tyrosyl-DNA phosphodiesterase-1 or XRCC1, resulted in increased Top1cc formation and excessive DNA damage and neurodevelopmental defects. Notably, direct Top1 poisoning to elevate Top1cc levels phenocopied the neuropathology of the mouse models described above. Our results identify a critical endogenous pathogenic lesion associated with neurodegenerative syndromes arising from DNA repair deficiency, indicating that genome integrity is important for preventing disease in the nervous system.

Formation and Biological Targets of Quinones: Cytotoxic versus Cytoprotective Effects

PubMed Central

2016-01-01

Quinones represent a class of toxicological intermediates, which can create a variety of hazardous effects in vivo including, acute cytotoxicity, immunotoxicity, and carcinogenesis. In contrast, quinones can induce cytoprotection through the induction of detoxification enzymes, anti-inflammatory activities, and modification of redox status. The mechanisms by which quinones cause these effects can be quite complex. The various biological targets of quinones depend on their rate and site of formation and their reactivity. Quinones are formed through a variety of mechanisms from simple oxidation of catechols/hydroquinones catalyzed by a variety of oxidative enzymes and metal ions to more complex mechanisms involving initial P450-catalyzed hydroxylation reactions followed by two-electron oxidation. Quinones are Michael acceptors, and modification of cellular processes could occur through alkylation of crucial cellular proteins and/or DNA. Alternatively, quinones are highly redox active molecules which can redox cycle with their semiquinone radical anions leading to the formation of reactive oxygen species (ROS) including superoxide, hydrogen peroxide, and ultimately the hydroxyl radical. Production of ROS can alter redox balance within cells through the formation of oxidized cellular macromolecules including lipids, proteins, and DNA. This perspective explores the varied biological targets of quinones including GSH, NADPH, protein sulfhydryls [heat shock proteins, P450s, cyclooxygenase-2 (COX-2), glutathione S-transferase (GST), NAD(P)H:quinone oxidoreductase 1, (NQO1), kelch-like ECH-associated protein 1 (Keap1), IκB kinase (IKK), and arylhydrocarbon receptor (AhR)], and DNA. The evidence strongly suggests that the numerous mechanisms of quinone modulations (i.e., alkylation versus oxidative stress) can be correlated with the known pathology/cytoprotection of the parent compound(s) that is best described by an inverse U-shaped dose–response curve. PMID:27617882

Distinct DNA-binding surfaces in the ATPase and linker domains of MutLγ determine its substrate specificities and exert separable functions in meiotic recombination and mismatch repair

PubMed Central

2017-01-01

Mlh1-Mlh3 (MutLγ) is a mismatch repair factor with a central role in formation of meiotic crossovers, presumably through resolution of double Holliday junctions. MutLγ has DNA-binding, nuclease, and ATPase activities, but how these relate to one another and to in vivo functions are unclear. Here, we combine biochemical and genetic analyses to characterize Saccharomyces cerevisiae MutLγ. Limited proteolysis and atomic force microscopy showed that purified recombinant MutLγ undergoes ATP-driven conformational changes. In vitro, MutLγ displayed separable DNA-binding activities toward Holliday junctions (HJ) and, surprisingly, single-stranded DNA (ssDNA), which was not predicted from current models. MutLγ bound DNA cooperatively, could bind multiple substrates simultaneously, and formed higher-order complexes. FeBABE hydroxyl radical footprinting indicated that the DNA-binding interfaces of MutLγ for ssDNA and HJ substrates only partially overlap. Most contacts with HJ substrates were located in the linker regions of MutLγ, whereas ssDNA contacts mapped within linker regions as well as the N-terminal ATPase domains. Using yeast genetic assays for mismatch repair and meiotic recombination, we found that mutations within different DNA-binding surfaces exert separable effects in vivo. For example, mutations within the Mlh1 linker conferred little or no meiotic phenotype but led to mismatch repair deficiency. Interestingly, mutations in the N-terminal domain of Mlh1 caused a stronger meiotic defect than mlh1Δ, suggesting that the mutant proteins retain an activity that interferes with alternative recombination pathways. Furthermore, mlh3Δ caused more chromosome missegregation than mlh1Δ, whereas mlh1Δ but not mlh3Δ partially alleviated meiotic defects of msh5Δ mutants. These findings illustrate functional differences between Mlh1 and Mlh3 during meiosis and suggest that their absence impinges on chromosome segregation not only via reduced formation of crossovers. Taken together, our results offer insights into the structure-function relationships of the MutLγ complex and reveal unanticipated genetic relationships between components of the meiotic recombination machinery. PMID:28505149

Molecular mechanism of transcription inhibition by phage T7 gp2 protein.

PubMed

Mekler, Vladimir; Minakhin, Leonid; Sheppard, Carol; Wigneshweraraj, Sivaramesh; Severinov, Konstantin

2011-11-11

Escherichia coli T7 bacteriophage gp2 protein is a potent inhibitor of host RNA polymerase (RNAP). gp2 inhibits formation of open promoter complex by binding to the β' jaw, an RNAP domain that interacts with downstream promoter DNA. Here, we used an engineered promoter with an optimized sequence to obtain and characterize a specific promoter complex containing RNAP and gp2. In this complex, localized melting of promoter DNA is initiated but does not propagate to include the point of the transcription start. As a result, the complex is transcriptionally inactive. Using a highly sensitive RNAP beacon assay, we performed quantitative real-time measurements of specific binding of the RNAP-gp2 complex to promoter DNA and various promoter fragments. In this way, the effect of gp2 on RNAP interaction with promoters was dissected. As expected, gp2 greatly decreased RNAP affinity to downstream promoter duplex. However, gp2 also inhibited RNAP binding to promoter fragments that lacked downstream promoter DNA that interacts with the β' jaw. The inhibition was caused by gp2-mediated decrease of the RNAP binding affinity to template and non-template strand segments of the transcription bubble downstream of the -10 promoter element. The inhibition of RNAP interactions with single-stranded segments of the transcription bubble by gp2 is a novel effect, which may occur via allosteric mechanism that is set in motion by the gp2 binding to the β' jaw. Copyright © 2011 Elsevier Ltd. All rights reserved.

Modified nucleotides reveal the indirect role of the central base pairs in stabilizing the lac repressor-operator complex.

PubMed Central

Zhang, X; Gottlieb, P A

1995-01-01

Guanine residues in the lac operator were replaced by 2-aminopurine or purine analogues, pairing the modified nucleotides with C. The observed equilibrium dissociation constants for lac repressor binding to substituted operators were measured in 10 mM Tris, 150 mM KCl, 0.1 mM EDTA, 0.1 mM DTE, pH 7.6 at 25 degrees C. These measurements revealed five positions that destabilized the complex when substituted with either analogue. Two positions, which are related by a 2-fold symmetry, are in the major groove of the operator thought to directly interact with the protein. Three sites were in the central region of the operator. A purine analogue at a sixth site perturbed the local DNA structure and destabilized the complex. Alkylation interference experiments of the 2-aminopurine substituted operators demonstrated that, of the five affected, two substitutions displayed altered phosphate interference patterns at the phosphate adjacent to the substituted base. For these operators, complex formation was measured in different concentrations of KCl to assess the contribution of counterion release to the bimolecular process. The results indicated that both complexes were similar to wild-type, although minor changes were observed. The Kobs of the complex was then measured when 2-aminopurine or purine analogues were paired with uracil nucleotide, a base pair that serves to stabilize the DNA. The introduction of the new base pairs revealed two effects on the bimolecular interaction. For those operator sites that are thought to perturb the interaction directly, the affinity of the complex was weakened to levels observed for the singly-substituted operators. In contrast, the nucleotides of 2-aminopurine paired with uracil positioned in the central region of the operator served to enhance the stability of the complex. The purine-uracil base pair substitution on the other hand had a significant destabilizing effect on the interaction. We propose that the central base pairs modulate binding of the complex by altering the intrinsic properties of the DNA. Two specific attributes are required to achieve the lowest free energy of interaction. The DNA must have two interstrand hydrogen bonds to stabilize the duplex and it must have properties associated with directional bending or unwinding. This analysis does not rule out contributions by direct interactions between the protein and the central region of the operator but underscores how indirect effects play a major role in complex formation in this system. Images PMID:7784203

Triplex-forming oligonucleotides: a third strand for DNA nanotechnology

PubMed Central

2018-01-01

Abstract DNA self-assembly has proved to be a useful bottom-up strategy for the construction of user-defined nanoscale objects, lattices and devices. The design of these structures has largely relied on exploiting simple base pairing rules and the formation of double-helical domains as secondary structural elements. However, other helical forms involving specific non-canonical base-base interactions have introduced a novel paradigm into the process of engineering with DNA. The most notable of these is a three-stranded complex generated by the binding of a third strand within the duplex major groove, generating a triple-helical (‘triplex’) structure. The sequence, structural and assembly requirements that differentiate triplexes from their duplex counterparts has allowed the design of nanostructures for both dynamic and/or structural purposes, as well as a means to target non-nucleic acid components to precise locations within a nanostructure scaffold. Here, we review the properties of triplexes that have proved useful in the engineering of DNA nanostructures, with an emphasis on applications that hitherto have not been possible by duplex formation alone. PMID:29228337

Novel readout method for molecular diagnostic assays based on optical measurements of magnetic nanobead dynamics.

PubMed

Donolato, Marco; Antunes, Paula; Bejhed, Rebecca S; Zardán Gómez de la Torre, Teresa; Østerberg, Frederik W; Strömberg, Mattias; Nilsson, Mats; Strømme, Maria; Svedlindh, Peter; Hansen, Mikkel F; Vavassori, Paolo

2015-02-03

We demonstrate detection of DNA coils formed from a Vibrio cholerae DNA target at picomolar concentrations using a novel optomagnetic approach exploiting the dynamic behavior and optical anisotropy of magnetic nanobead (MNB) assemblies. We establish that the complex second harmonic optical transmission spectra of MNB suspensions measured upon application of a weak uniaxial AC magnetic field correlate well with the rotation dynamics of the individual MNBs. Adding a target analyte to the solution leads to the formation of permanent MNB clusters, namely, to the suppression of the dynamic MNB behavior. We prove that the optical transmission spectra are highly sensitive to the formation of permanent MNB clusters and, thereby to the target analyte concentration. As a specific clinically relevant diagnostic case, we detect DNA coils formed via padlock probe recognition and isothermal rolling circle amplification and benchmark against a commercial equipment. The results demonstrate the fast optomagnetic readout of rolling circle products from bacterial DNA utilizing the dynamic properties of MNBs in a miniaturized and low-cost platform requiring only a transparent window in the chip.

Lamellar cationic lipid-DNA complexes from lipids with a strong preference for planar geometry: A Minimal Electrostatic Model.

PubMed

Perico, Angelo; Manning, Gerald S

2014-11-01

We formulate and analyze a minimal model, based on condensation theory, of the lamellar cationic lipid (CL)-DNA complex of alternately charged lipid bilayers and DNA monolayers in a salt solution. Each lipid bilayer, composed by a random mixture of cationic and neutral lipids, is assumed to be a rigid uniformly charged plane. Each DNA monolayer, located between two lipid bilayers, is formed by the same number of parallel DNAs with a uniform separation distance. For the electrostatic calculation, the model lipoplex is collapsed to a single plane with charge density equal to the net lipid and DNA charge. The free energy difference between the lamellar lipoplex and a reference state of the same number of free lipid bilayers and free DNAs, is calculated as a function of the fraction of CLs, of the ratio of the number of CL charges to the number of negative charges of the DNA phosphates, and of the total number of planes. At the isoelectric point the free energy difference is minimal. The complex formation, already favoured by the decrease of the electrostatic charging free energy, is driven further by the free energy gain due to the release of counterions from the DNAs and from the lipid bilayers, if strongly charged. This minimal model compares well with experiment for lipids having a strong preference for planar geometry and with major features of more detailed models of the lipoplex. © 2014 Wiley Periodicals, Inc.

Kinetics and Thermodynamics of DNA Processing by Wild Type DNA-Glycosylase Endo III and Its Catalytically Inactive Mutant Forms.

PubMed

Kladova, Olga A; Krasnoperov, Lev N; Kuznetsov, Nikita A; Fedorova, Olga S

2018-03-30

Endonuclease III (Endo III or Nth) is one of the key enzymes responsible for initiating the base excision repair of oxidized or reduced pyrimidine bases in DNA. In this study, a thermodynamic analysis of structural rearrangements of the specific and nonspecific DNA-duplexes during their interaction with Endo III is performed based on stopped-flow kinetic data. 1,3-diaza-2-oxophenoxazine (tC O ), a fluorescent analog of the natural nucleobase cytosine, is used to record multistep DNA binding and lesion recognition within a temperature range (5-37 °C). Standard Gibbs energy, enthalpy, and entropy of the specific steps are derived from kinetic data using Van't Hoff plots. The data suggest that enthalpy-driven exothermic 5,6-dihydrouracil (DHU) recognition and desolvation-accompanied entropy-driven adjustment of the enzyme-substrate complex into a catalytically active state play equally important parts in the overall process. The roles of catalytically significant amino acids Lys120 and Asp138 in the DNA lesion recognition and catalysis are identified. Lys120 participates not only in the catalytic steps but also in the processes of local duplex distortion, whereas substitution Asp138Ala leads to a complete loss of the ability of Endo III to distort a DNA double chain during enzyme-DNA complex formation.

Carbon nanotubes as gene carriers: Focus on internalization pathways related to functionalization and properties.

PubMed

Caoduro, Cécile; Hervouet, Eric; Girard-Thernier, Corine; Gharbi, Tijani; Boulahdour, Hatem; Delage-Mourroux, Régis; Pudlo, Marc

2017-02-01

Carbon nanotubes represent promising transporters for delivery of DNA and other biomolecules into living cells. Various methods of CNTs surface functionalization have been developed. These are essential to improve CNTs dispersibility and permit their interactions with biological structures that broaden their use in advanced biomedical applications. The present review discusses the different single walled carbon nanotubes and multiwalled carbon nanotubes functionalization methods, leading to the formation of optimized and functionalized-CNT complexes with DNA. F-CNTs are recognized as efficient and promising gene carriers. Emphasis is then placed on the processes used by f-CNTs/DNA complexes to cross cell membranes. Energy independent pathways and uptake mechanisms dependent on energy, such as endocytosis or phagocytosis, are reported by many studies, and if these mechanisms seem contradictory at first sight, a detailed review of the literature illustrates that they are rather complementary. Preferential use of one or the other depends on the DNA and CNTs chemical nature and physical parameters, experimental procedures and cell types. Efficient non-viral gene delivery is desirable, yet challenging. CNTs appear as a promising solution to penetrate into cells and successfully deliver DNA. Moreover, the field of use of CNTs as gene carrier is large and is currently growing. This critical review summarizes the development and evaluation of CNTs as intracellular gene delivery system and provides an overview of functionalized CNTs/DNA cellular uptake mechanisms, depending on several parameters of CNTs/DNA complexes. Copyright © 2016 Acta Materialia Inc. Published by Elsevier Ltd. All rights reserved.

Ternary polyplex micelles with PEG shells and intermediate barrier to complexed DNA cores for efficient systemic gene delivery.

PubMed

Li, Junjie; Chen, Qixian; Zha, Zengshi; Li, Hui; Toh, Kazuko; Dirisala, Anjaneyulu; Matsumoto, Yu; Osada, Kensuke; Kataoka, Kazunori; Ge, Zhishen

2015-07-10

Simultaneous achievement of prolonged retention in blood circulation and efficient gene transfection activity in target tissues has always been a major challenge hindering in vivo applications of nonviral gene vectors via systemic administration. Herein, we constructed novel rod-shaped ternary polyplex micelles (TPMs) via complexation between the mixed block copolymers of poly(ethylene glycol)-b-poly{N'-[N-(2-aminoethyl)-2-aminoethyl]aspartamide} (PEG-b-PAsp(DET)) and poly(N-isopropylacrylamide)-b-PAsp(DET) (PNIPAM-b-PAsp(DET)) and plasmid DNA (pDNA) at room temperature, exhibiting distinct temperature-responsive formation of a hydrophobic intermediate layer between PEG shells and pDNA cores through facile temperature increase from room temperature to body temperature (~37 °C). As compared with binary polyplex micelles of PEG-b-PAsp(DET) (BPMs), TPMs were confirmed to condense pDNA into a more compact structure, which achieved enhanced tolerability to nuclease digestion and strong counter polyanion exchange. In vitro gene transfection results demonstrated TPMs exhibiting enhanced gene transfection efficiency due to efficient cellular uptake and endosomal escape. Moreover, in vivo performance evaluation after intravenous injection confirmed that TPMs achieved significantly prolonged blood circulation, high tumor accumulation, and promoted gene expression in tumor tissue. Moreover, TPMs loading therapeutic pDNA encoding an anti-angiogenic protein remarkably suppressed tumor growth following intravenous injection into H22 tumor-bearing mice. These results suggest TPMs with PEG shells and facilely engineered intermediate barrier to inner complexed pDNA have great potentials as systemic nonviral gene vectors for cancer gene therapy. Copyright © 2015 Elsevier B.V. All rights reserved.

The RNA Splicing Response to DNA Damage.

PubMed

Shkreta, Lulzim; Chabot, Benoit

2015-10-29

The number of factors known to participate in the DNA damage response (DDR) has expanded considerably in recent years to include splicing and alternative splicing factors. While the binding of splicing proteins and ribonucleoprotein complexes to nascent transcripts prevents genomic instability by deterring the formation of RNA/DNA duplexes, splicing factors are also recruited to, or removed from, sites of DNA damage. The first steps of the DDR promote the post-translational modification of splicing factors to affect their localization and activity, while more downstream DDR events alter their expression. Although descriptions of molecular mechanisms remain limited, an emerging trend is that DNA damage disrupts the coupling of constitutive and alternative splicing with the transcription of genes involved in DNA repair, cell-cycle control and apoptosis. A better understanding of how changes in splice site selection are integrated into the DDR may provide new avenues to combat cancer and delay aging.

The RNA Splicing Response to DNA Damage

PubMed Central

Shkreta, Lulzim; Chabot, Benoit

2015-01-01

The number of factors known to participate in the DNA damage response (DDR) has expanded considerably in recent years to include splicing and alternative splicing factors. While the binding of splicing proteins and ribonucleoprotein complexes to nascent transcripts prevents genomic instability by deterring the formation of RNA/DNA duplexes, splicing factors are also recruited to, or removed from, sites of DNA damage. The first steps of the DDR promote the post-translational modification of splicing factors to affect their localization and activity, while more downstream DDR events alter their expression. Although descriptions of molecular mechanisms remain limited, an emerging trend is that DNA damage disrupts the coupling of constitutive and alternative splicing with the transcription of genes involved in DNA repair, cell-cycle control and apoptosis. A better understanding of how changes in splice site selection are integrated into the DDR may provide new avenues to combat cancer and delay aging. PMID:26529031

DOE Office of Scientific and Technical Information (OSTI.GOV)

Rajan, Rakhi; Taneja, Bhupesh; Mondragón, Alfonso

Topoisomerase V is an archaeal type I topoisomerase that is unique among topoisomerases due to presence of both topoisomerase and DNA repair activities in the same protein. It is organized as an N-terminal topoisomerase domain followed by 24 tandem helix-hairpin-helix (HhH) motifs. Structural studies have shown that the active site is buried by the (HhH) motifs. Here we show that the N-terminal domain can relax DNA in the absence of any HhH motifs and that the HhH motifs are required for stable protein-DNA complex formation. Crystal structures of various topoisomerase V fragments show changes in the relative orientation of themore » domains mediated by a long bent linker helix, and these movements are essential for the DNA to enter the active site. Phosphate ions bound to the protein near the active site helped model DNA in the topoisomerase domain and show how topoisomerase V may interact with DNA.« less

MMSET is dynamically regulated during cell-cycle progression and promotes normal DNA replication.

PubMed

Evans, Debra L; Zhang, Haoxing; Ham, Hyoungjun; Pei, Huadong; Lee, SeungBaek; Kim, JungJin; Billadeau, Daniel D; Lou, Zhenkun

2016-01-01

The timely and precise duplication of cellular DNA is essential for maintaining genome integrity and is thus tightly-regulated. During mitosis and G1, the Origin Recognition Complex (ORC) binds to future replication origins, coordinating with multiple factors to load the minichromosome maintenance (MCM) complex onto future replication origins as part of the pre-replication complex (pre-RC). The pre-RC machinery, in turn, remains inactive until the subsequent S phase when it is required for replication fork formation, thereby initiating DNA replication. Multiple myeloma SET domain-containing protein (MMSET, a.k.a. WHSC1, NSD2) is a histone methyltransferase that is frequently overexpressed in aggressive cancers and is essential for normal human development. Several studies have suggested a role for MMSET in cell-cycle regulation; however, whether MMSET is itself regulated during cell-cycle progression has not been examined. In this study, we report that MMSET is degraded during S phase in a cullin-ring ligase 4-Cdt2 (CRL4(Cdt2)) and proteasome-dependent manner. Notably, we also report defects in DNA replication and a decreased association of pre-RC factors with chromatin in MMSET-depleted cells. Taken together, our results suggest a dynamic regulation of MMSET levels throughout the cell cycle, and further characterize the role of MMSET in DNA replication and cell-cycle progression.

A cyclometalated iridium(III) complex used as a conductor for the electrochemical sensing of IFN-γ

NASA Astrophysics Data System (ADS)

Miao, Xiangmin; Ko, Chung-Nga; Vellaisamy, Kasipandi; Li, Zongbing; Yang, Guanjun; Leung, Chung-Hang; Ma, Dik-Lung

2017-02-01

A novel iridium(III) complex was prepared and used as a conductor for sensitive and enzyme-free electrochemical detection of interferon gamma (IFN-γ). This assay is based on a dual signal amplification mechanism involving positively charged gold nanoparticles ((+)AuNPs) and hybridization chain reaction (HCR). To construct the sensor, nafion (Nf) and (+)AuNPs composite membrane was first immobilized onto the electrode surface. Subsequently, a loop-stem structured capture probe (CP) containing a special IFN-γ interact strand was modified onto the (+)AuNP surface via the formation of Au-S bonds. Upon addition of IFN-γ, the loop-stem structure of CP was opened, and the newly exposed “sticky” region of CP then hybridized with DNA hairpin-1 (H1), which in turn opened its hairpin structure for hybridizing with DNA hairpin-2 (H2). Happen of HCR between H1 and H2 thus generated a polymeric duplex DNA (dsDNA) chain. Meanwhile, the iridium(III) complex could interact with the grooves of the dsDNA polymer, producing a strong current signal that was proportional to IFN-γ concentration. Thus, sensitive detection of IFN-γ could be realized with a detection limit down to 16.3 fM. Moreover, satisfied results were achieved by using this method for the detection of IFN-γ in human serum samples.

Spectroscopic Study of the Binding of Netropsin and Hoechst 33258 to Nucleic Acids

NASA Astrophysics Data System (ADS)

Vardevanyan, P. O.; Parsadanyan, M. A.; Antonyan, A. P.; Sahakyan, V. G.

2018-05-01

The interaction of groove binding compounds — peptide antibiotic (polyamide) netropsin and fluorescent dye (bisbenzimidazole) Hoechst 33258 — with the double-stranded DNA and synthetic double-stranded polynucleotide poly(rA)-poly(rU) has been studied by spectrophotometry. Absorption spectra of these ligand complexes with nucleic acids have been obtained. Spectral changes at the complexation of individual ligands with the mentioned nucleic acids reveal the similarity of binding of each of these ligands with both DNA and RNA. Based on the spectroscopic measurements, the binding parameters of netropsin and Hoechst 33258 binding to DNA and poly(rA)-poly(rU) - K and n, as well as the thermodynamic parameters ΔS, ΔG, and ΔH have been determined. It was found that the binding of Hoechst 33258 to both nucleic acids is accompanied by a positive change in enthalpy, while in the case of netropsin the change in enthalpy is negative. Moreover, the contribution of entropy to the formation of the complexes is more pronounced in the case of Hoechst 33258.

Bio-Organic Optoelectronic Devices Using DNA

NASA Astrophysics Data System (ADS)

Singh, Thokchom Birendra; Sariciftci, Niyazi Serdar; Grote, James G.

Biomolecular DNA, as a marine waste product from salmon processing, has been exploited as biodegradable polymeric material for photonics and electronics. For preparing high optical quality thin films of DNA, a method using DNA with cationic surfactants such as DNA-cetyltrimethylammonium, CTMA has been applied. This process enhances solubility and processing for thin film fabrication. These DNA-CTMA complexes resulted in the formation of self-assembled supramolecular films. Additionally, the molecular weight can be tailored to suit the application through sonication. It revealed that DNA-CTMA complexes were thermostable up to 230 ∘ C. UV-VIS absorption shows that these thin films have high transparency from 350 to about 1,700 nm. Due to its nature of large band gap and large dielectric constant, thin films of DNA-CTMA has been successfully used in multiple applications such as organic light emitting diodes (OLED), a cladding and host material in nonlinear optical devices, and organic field-effect transistors (OFET). Using this DNA based biopolymers as a gate dielectric layer, OFET devices were fabricated that exhibits current-voltage characteristics with low voltages as compared with using other polymer-based dielectrics. Using a thin film of DNA-CTMA based biopolymer as the gate insulator and pentacene as the organic semiconductor, we have demonstrated a bio-organic FET or BioFET in which the current was modulated over three orders of magnitude using gate voltages less than 10 V. Given the possibility to functionalise the DNA film customised for specific purposes viz. biosensing, DNA-CTMA with its unique structural, optical and electronic properties results in many applications that are extremely interesting.

Deciphering the mechanism of interaction of edifenphos with calf thymus DNA

NASA Astrophysics Data System (ADS)

Ahmad, Ajaz; Ahmad, Masood

2018-01-01

Edifenphos is an important organophosphate pesticide with many antifungal and anti-insecticidal properties but it may cause potential hazards to human health. In this work, we have tried to explore the binding mode of action and mechanism of edifenphos to calf thymus DNA (CT-DNA). Several experiments such as ultraviolet-visible absorption spectra and emission spectroscopy showed complex formation between edifenphos and CT-DNA and low binding constant values supporting groove binding mode. These results were further confirmed by circular dichroism (CD), CT-DNA melting studies, viscosity measurements, density functional theory and molecular docking. CD study suggests that edifenphos does not alter native structure of CT-DNA. Isothermal calorimetry reveals that binding of edifenphos with CT-DNA is enthalpy driven process. Competitive binding assay and effect of ionic strength showed that edifenphos binds to CT-DNA via groove binding manner. Hence, edifenphos is a minor groove binder preferably interacting with A-T regions with docking score - 6.84 kJ/mol.

Overcoming a nucleosomal barrier to replication

PubMed Central

Chang, Han-Wen; Pandey, Manjula; Kulaeva, Olga I.; Patel, Smita S.; Studitsky, Vasily M.

2016-01-01

Efficient overcoming and accurate maintenance of chromatin structure and associated histone marks during DNA replication are essential for normal functioning of the daughter cells. However, the molecular mechanisms of replication through chromatin are unknown. We have studied traversal of uniquely positioned mononucleosomes by T7 replisome in vitro. Nucleosomes present a strong, sequence-dependent barrier for replication, with particularly strong pausing of DNA polymerase at the +(31–40) and +(41–65) regions of the nucleosomal DNA. The exonuclease activity of T7 DNA polymerase increases the overall rate of progression of the replisome through a nucleosome, likely by resolving nonproductive complexes. The presence of nucleosome-free DNA upstream of the replication fork facilitates the progression of DNA polymerase through the nucleosome. After replication, at least 50% of the nucleosomes assume an alternative conformation, maintaining their original positions on the DNA. Our data suggest a previously unpublished mechanism for nucleosome maintenance during replication, likely involving transient formation of an intranucleosomal DNA loop. PMID:27847876

Unusually Strong Binding to the DNA Minor Groove by a Highly Twisted Benzimidazole-Diphenylether: Induced Fit and Bound Water†

PubMed Central

Tanious, Farial A.; Laine, William; Peixoto, Paul; Bailly, Christian; Goodwin, Kristie D.; Lewis, Mark A.; Long, Eric C.; Georgiadis, Millie M.; Tidwell, Richard R.; Wilson, W. David

2008-01-01

RT29 is a dicationic diamidine derivative that does not obey the classical “rules” for shape and functional group placement that are expected to result in strong binding and specific recognition of the DNA minor groove. The compound contains a benzimidazole-diphenyl ether core that is flanked by the amidine cations. The diphenyl ether is highly twisted and gives the entire compound too much curvature to fit well to the shape of the minor groove. DNaseI footprinting, fluorescence intercalator displacement studies and circular dichroism spectra, however, indicate that the compound is an AT specific minor groove binding agent. Even more surprisingly, quantitative biosensor-surface plasmon resonance and isothermal titration calorimetric results indicate that the compound binds with exceptional strength to certain AT sequences in DNA with a large negative enthalpy of binding. Crystallographic results for the DNA complex of RT29 compared to calculated results for the free compound show that the compound undergoes significant conformational changes to enhance its minor groove interactions. In addition, a water molecule is incorporated directly into the complex to complete the compound-DNA interface and it forms an essential link between the compound and base pair edges at the floor of the minor groove. The calculated ΔCp value for complex formation is substantially less than the experimentally observed value in support of water being an intrinsic part of the complex with a major contribution to the ΔCp value. Both the induced fit conformational changes of the compound and the bound water are essential for strong binding to DNA by RT29. PMID:17506529

Targeting the Blind Spot of Polycationic Nanocarrier-Based siRNA Delivery

PubMed Central

Zheng, Mengyao; Pavan, Giovanni M.; Neeb, Manuel; Schaper, Andreas K.; Danani, Andrea; Klebe, Gerhard; Merkel, Olivia M.; Kissel, Thomas

2013-01-01

Polycationic nanocarriers attract increasing attention to the field of siRNA delivery. We investigated the self-assembly of siRNA vs pDNA with polycations, which are broadly used for nonviral gene and siRNA delivery. Although polyethyleneimine (PEI) was routinely adopted as siRNA carrier based on its efficacy in delivering pDNA, it has not been investigated yet why PEI efficiently delivers pDNA to cells but is controversially discussed in terms of efficacy for siRNA delivery. We are the first to investigate the self-assembly of PEI/siRNA vs PEI/pDNA and the steps of complexation and aggregation through different levels of hierarchy on the atomic and molecular scale with the novel synergistic use of molecular modeling, molecular dynamics simulation, isothermal titration calorimetry, and other characterization techniques. We are also the fist to elucidate atomic interactions, size, shape, stoichiometry, and association dynamics for polyplexes containing siRNA vs pDNA. Our investigation highlights differences in the hierarchical mechanism of formation of related polycation–siRNA and polycation–pDNA complexes. The results of fluorescence quenching assays indicated a biphasic behavior of siRNA binding with polycations where molecular reorganization of the siRNA within the polycations occurred at lower N/P ratios (nitrogen/phosphorus). Our results, for the first time, emphasize a biphasic behavior in siRNA complexation and the importance of low N/P ratios, which allow for excellent siRNA delivery efficiency. Our investigation highlights the formulation of siRNA complexes from a thermodynamic point of view and opens new perspectives to advance the rational design of new siRNA delivery systems. PMID:23036046

Dual chromatin recognition by the histone deacetylase complex HCHC is required for proper DNA methylation in Neurospora crassa

PubMed Central

Honda, Shinji; Bicocca, Vincent T.; Gessaman, Jordan D.; Rountree, Michael R.; Yokoyama, Ayumi; Yu, Eun Y.; Selker, Jeanne M. L.; Selker, Eric U.

2016-01-01

DNA methylation, heterochromatin protein 1 (HP1), histone H3 lysine 9 (H3K9) methylation, histone deacetylation, and highly repeated sequences are prototypical heterochromatic features, but their interrelationships are not fully understood. Prior work showed that H3K9 methylation directs DNA methylation and histone deacetylation via HP1 in Neurospora crassa and that the histone deacetylase complex HCHC is required for proper DNA methylation. The complex consists of the chromodomain proteins HP1 and chromodomain protein 2 (CDP-2), the histone deacetylase HDA-1, and the AT-hook motif protein CDP-2/HDA-1–associated protein (CHAP). We show that the complex is required for proper chromosome segregation, dissect its function, and characterize interactions among its components. Our analyses revealed the existence of an HP1-based DNA methylation pathway independent of its chromodomain. The pathway partially depends on CHAP but not on the CDP-2 chromodomain. CDP-2 serves as a bridge between the recognition of H3K9 trimethylation (H3K9me3) by HP1 and the histone deacetylase activity of HDA-1. CHAP is also critical for HDA-1 localization to heterochromatin. Specifically, the CHAP zinc finger interacts directly with the HDA-1 argonaute-binding protein 2 (Arb2) domain, and the CHAP AT-hook motifs recognize heterochromatic regions by binding to AT-rich DNA. Our data shed light on the interrelationships among the prototypical heterochromatic features and support a model in which dual recognition by the HP1 chromodomain and the CHAP AT-hooks are required for proper heterochromatin formation. PMID:27681634

Spectro Analytical, Computational and In Vitro Biological Studies of Novel Substituted Quinolone Hydrazone and it's Metal Complexes.

PubMed

Nagula, Narsimha; Kunche, Sudeepa; Jaheer, Mohmed; Mudavath, Ravi; Sivan, Sreekanth; Ch, Sarala Devi

2018-01-01

Some novel transition metal [Cu (II), Ni (II) and Co (II)] complexes of nalidixic acid hydrazone have been prepared and characterized by employing spectro-analytical techniques viz: elemental analysis, 1 H-NMR, Mass, UV-Vis, IR, TGA-DTA, SEM-EDX, ESR and Spectrophotometry studies. The HyperChem 7.5 software was used for geometry optimization of title compound in its molecular and ionic forms. Quantum mechanical parameters, contour maps of highest occupied molecular orbitals (HOMO) and lowest unoccupied molecular orbitals (LUMO) and corresponding binding energy values were computed using semi empirical single point PM3 method. The stoichiometric equilibrium studies of metal complexes carried out spectrophotometrically using Job's continuous variation and mole ratio methods inferred formation of 1:2 (ML 2 ) metal complexes in respective systems. The title compound and its metal complexes screened for antibacterial and antifungal properties, exemplified improved activity in metal complexes. The studies of nuclease activity for the cleavage of CT- DNA and MTT assay for in vitro cytotoxic properties involving metal complexes exhibited high activity. In addition, the DNA binding properties of Cu (II), Ni (II) and Co (II) complexes investigated by electronic absorption and fluorescence measurements revealed their good binding ability and commended agreement of K b values obtained from both the techniques. Molecular docking studies were also performed to find the binding affinity of synthesized compounds with DNA (PDB ID: 1N37) and "Thymidine phosphorylase from E.coli" (PDB ID: 4EAF) protein targets.

Cohesin organizes chromatin loops at DNA replication factories

PubMed Central

Guillou, Emmanuelle; Ibarra, Arkaitz; Coulon, Vincent; Casado-Vela, Juan; Rico, Daniel; Casal, Ignacio; Schwob, Etienne; Losada, Ana; Méndez, Juan

2010-01-01

Genomic DNA is packed in chromatin fibers organized in higher-order structures within the interphase nucleus. One level of organization involves the formation of chromatin loops that may provide a favorable environment to processes such as DNA replication, transcription, and repair. However, little is known about the mechanistic basis of this structuration. Here we demonstrate that cohesin participates in the spatial organization of DNA replication factories in human cells. Cohesin is enriched at replication origins and interacts with prereplication complex proteins. Down-regulation of cohesin slows down S-phase progression by limiting the number of active origins and increasing the length of chromatin loops that correspond with replicon units. These results give a new dimension to the role of cohesin in the architectural organization of interphase chromatin, by showing its participation in DNA replication. PMID:21159821

Recognition of the DNA sequence by an inorganic crystal surface

PubMed Central

Sampaolese, Beatrice; Bergia, Anna; Scipioni, Anita; Zuccheri, Giampaolo; Savino, Maria; Samorì, Bruno; De Santis, Pasquale

2002-01-01

The sequence-dependent curvature is generally recognized as an important and biologically relevant property of DNA because it is involved in the formation and stability of association complexes with proteins. When a DNA tract, intrinsically curved for the periodical recurrence on the same strand of A-tracts phased with the B-DNA periodicity, is deposited on a flat surface, it exposes to that surface either a T- or an A-rich face. The surface of a freshly cleaved mica crystal recognizes those two faces and preferentially interacts with the former one. Statistical analysis of scanning force microscopy (SFM) images provides evidence of this recognition between an inorganic crystal surface and nanoscale structures of double-stranded DNA. This finding could open the way toward the use of the sequence-dependent adhesion to specific crystal faces for nanotechnological purposes. PMID:12361979

Alternative Lengthening of Telomeres Mediated by Mitotic DNA Synthesis Engages Break-Induced Replication Processes

PubMed Central

Min, Jaewon; Wright, Woodring E.

2017-01-01

ABSTRACT Alternative lengthening of telomeres (ALT) is a telomerase-independent telomere maintenance mechanism that occurs in a subset of cancers. By analyzing telomerase-positive cells and their human TERC knockout-derived ALT human cell lines, we show that ALT cells harbor more fragile telomeres representing telomere replication problems. ALT-associated replication defects trigger mitotic DNA synthesis (MiDAS) at telomeres in a RAD52-dependent, but RAD51-independent, manner. Telomeric MiDAS is a conservative DNA synthesis process, potentially mediated by break-induced replication, similar to type II ALT survivors in Saccharomyces cerevisiae. Replication stresses induced by ectopic oncogenic expression of cyclin E, G-quadruplexes, or R-loop formation facilitate the ALT pathway and lead to telomere clustering, a hallmark of ALT cancers. The TIMELESS/TIPIN complex suppresses telomere clustering and telomeric MiDAS, whereas the SMC5/6 complex promotes them. In summary, ALT cells exhibit more telomere replication defects that result in persistent DNA damage responses at telomeres, leading to the engagement of telomeric MiDAS (spontaneous mitotic telomere synthesis) that is triggered by DNA replication stress, a potential driver of genomic duplications in cancer. PMID:28760773

BIOPHYSICAL PROPERTIES OF NUCLEIC ACIDS AT SURFACES RELEVANT TO MICROARRAY PERFORMANCE

PubMed Central

Rao, Archana N.; Grainger, David W.

2014-01-01

Both clinical and analytical metrics produced by microarray-based assay technology have recognized problems in reproducibility, reliability and analytical sensitivity. These issues are often attributed to poor understanding and control of nucleic acid behaviors and properties at solid-liquid interfaces. Nucleic acid hybridization, central to DNA and RNA microarray formats, depends on the properties and behaviors of single strand (ss) nucleic acids (e.g., probe oligomeric DNA) bound to surfaces. ssDNA’s persistence length, radius of gyration, electrostatics, conformations on different surfaces and under various assay conditions, its chain flexibility and curvature, charging effects in ionic solutions, and fluorescent labeling all influence its physical chemistry and hybridization under assay conditions. Nucleic acid (e.g., both RNA and DNA) target interactions with immobilized ssDNA strands are highly impacted by these biophysical states. Furthermore, the kinetics, thermodynamics, and enthalpic and entropic contributions to DNA hybridization reflect global probe/target structures and interaction dynamics. Here we review several biophysical issues relevant to oligomeric nucleic acid molecular behaviors at surfaces and their influences on duplex formation that influence microarray assay performance. Correlation of biophysical aspects of single and double-stranded nucleic acids with their complexes in bulk solution is common. Such analysis at surfaces is not commonly reported, despite its importance to microarray assays. We seek to provide further insight into nucleic acid-surface challenges facing microarray diagnostic formats that have hindered their clinical adoption and compromise their research quality and value as genomics tools. PMID:24765522

Photoactivation of Diiodido-Pt(IV) Complexes Coupled to Upconverting Nanoparticles.

PubMed

Perfahl, Stefanie; Natile, Marta M; Mohamad, Heba S; Helm, Christiane A; Schulzke, Carola; Natile, Giovanni; Bednarski, Patrick J

2016-07-05

The preparation, characterization, and surface modification of upconverting lanthanide-doped hexagonal NaGdF4 nanocrystals attached to light sensitive diiodido-Pt(IV) complexes is presented. The evaluation for photoactivation and cytotoxicity of the novel carboxylated diiodido-Pt(IV) cytotoxic prodrugs by near-infrared (NIR) light (λ = 980 nm) is also reported. We attempted two different strategies for attachment of light-sensitive diiodido-Pt(IV) complexes to Yb,Er- and Yb,Tm-doped β-NaGdF4 upconverting nanoparticles (UCNPs) in order to provide nanohybrids, which offer unique opportunities for selective drug activation within the tumor cells and subsequent spatiotemporal controlled drug release by NIR-to-visible light-upconversion: (A) covalent attachment of the Pt(IV) complex via amide bond formation and (B) carboxylate exchange of oleate on the surface of the UCNPs with diiodido-Pt(IV) carboxylato complexes. Initial feasibility studies showed that NIR applied by a 980 nm laser had only a slight effect on the stability of the various diiodido-Pt(IV) complexes, but when UCNPs were present more rapid loss of the ligand-metal-charge transfer (LMCT) bands of the diiodido-Pt(IV) complexes was observed. Furthermore, Pt released from the Pt(IV) complexes platinated calf-thymus DNA (ct-DNA) more rapidly when NIR was applied compared to dark controls. Of the two attachment strategies, method A with the covalently attached diiodido-Pt(IV) carboxylates via amide bond formation proved to be the most effective method for generating UCNPs that release Pt when irradiated with NIR; the released Pt was also able to bind irreversibly to calf thymus DNA. Nonetheless, only ca. 20% of the Pt on the surface of the UCNPs was in the Pt(IV) oxidation state, the rest was Pt(II), indicating chemical reduction of the diiodido-Pt(IV) prodrug by the UCNPs. Cytotoxicity studies with the various UCNP-Pt conjugates and constructs, tested on human leukemia HL60 cells in culture, indicated a substantial increase in cytotoxicity when modified UCNPs were combined with five rounds of 30 min irradiation with NIR compared to dark controls, but NIR alone also had a significant cytotoxic effect at this duration.

Structural and functional properties of the HIV-1 RNA-tRNA(Lys)3 primer complex annealed by the nucleocapsid protein: comparison with the heat-annealed complex.

PubMed Central

Brulé, Fabienne; Marquet, Roland; Rong, Liwei; Wainberg, Mark A; Roques, Bernard P; Le Grice, Stuart F J; Ehresmann, Bernard; Ehresmann, Chantal

2002-01-01

The conversion of the single-stranded RNA genome into double-stranded DNA by virus-coded reverse transcriptase (RT) is an essential step of the retrovirus life cycle. In human immunodeficiency virus type 1 (HIV-1), RT uses the cellular tRNA(Lys)3 to initiate the (-) strand DNA synthesis. Placement of the primer tRNA(Lys)3 involves binding of its 3'-terminal 18 nt to a complementary region of genomic RNA termed PBS. However, the PBS sequence is not the unique determinant of primer usage and additional contacts are important. This placement is believed to be achieved in vivo by the nucleocapsid domain of Gag or by the mature protein NCp. Up to now, structural information essentially arose from heat-annealed primer-template complexes (Isel et al., J Mol Biol, 1995, 247:236-250; Isel et al., EMBO J, 1999, 18:1038-1048). Here, we investigated the formation of the primer-template complex mediated by NCp and compared structural and functional properties of heat- and NCp-annealed complexes. We showed that both heat- and NCp-mediated procedures allow comparable high yields of annealing. Then, we investigated structural features of both kinds of complexes by enzymatic probing, and we compared their relative efficiency in (-) strong stop DNA synthesis. We did not find any significant differences between these complexes, suggesting that information derived from the heat-annealed complex can be transposed to the NCp-mediated complex and most likely to complexes formed in vivo. PMID:11873759

Spontaneous translocation of antitumor oxaliplatin, its enantiomeric analogue, and Cisplatin from one strand to another in double-helical DNA.

PubMed

Malina, Jaroslav; Natile, Giovanni; Brabec, Viktor

2013-09-02

Oxaliplatin and cisplatin belong to the class of platinum-based anticancer agents. Formation of DNA adducts by these complexes and the consequences for its structure and function, is the mechanistic paradigm by which these drugs exert their antitumor activity. We show that employing short oligonucleotide duplexes containing single, site-specific 1,3-intrastrand cross-links of oxaliplatin, its enantiomeric analogue, or cisplatin and by using gel electrophoresis that under physiological conditions the coordination bonds between platinum and the N7 position of guanine residues involved in the cross-links of the Pt(II) complexes can be cleaved. This cleavage may lead to linkage isomerization reactions between these metallodrugs and double-helical DNA. For instance, approximately 25 % 1,3-intrastrand cross-links of the platinum complexes isomerized after 192 h (at 310 K in 200 mM NaClO4). Differential scanning calorimetry of duplexes containing single, site-specific cross-links of oxaliplatin, its enantiomeric analogue, and cisplatin reveals that one of the driving forces that leads to the lability of DNA cross-links of these metallodrugs is a difference between the thermodynamic destabilization induced by the cross-link and by the adduct into which it could isomerize. The rearrangements may proceed in the way that cross-links originally formed in one strand of the DNA can spontaneously translocate from one DNA strand to its complementary counterpart, which may evoke walking of the platinum complex on DNA molecule. In addition, the differences in the kinetics of the rearrangement reactions and the thermodynamic destabilization of DNA observed for adducts of oxaliplatin and its enantiomeric analogue confirm that the chirality at the carrier 1,2-diaminocyclohexane ligand can considerably affect structural and other physical properties of DNA adducts and consequently their biological effects. In aggregate, interesting generalization of the results described in this work might be that the migration of oxaliplatin, its enantiomeric analogue, or cisplatin from one strand to another in double-helical DNA controlled by energetic signatures of these agents might contribute to a better understanding of their cytotoxic and mutagenic potential. Copyright © 2013 WILEY‐VCH Verlag GmbH & Co. KGaA, Weinheim.

Evidence for the role of DNA strand passage in the mechanism of action of microcin B17 on DNA gyrase.

PubMed

Pierrat, Olivier A; Maxwell, Anthony

2005-03-22

Microcin B17 (MccB17) is a DNA gyrase poison; in previous work, this bacterial toxin was found to slowly and incompletely inhibit the reactions of supercoiling and relaxation of DNA by gyrase and to stabilize the cleavage complex, depending on the presence of ATP and the DNA topology. We now show that the action of MccB17 on the gyrase ATPase reaction and cleavage complex formation requires a linear DNA fragment of more than 150 base pairs. MccB17 is unable to stimulate the ATPase reaction by stabilizing the weak interactions between short linear DNA fragments (70 base pairs or less) and gyrase, in contrast with the quinolone ciprofloxacin. However, MccB17 can affect the ATP-dependent relaxation of DNA by gyrase lacking its DNA-wrapping or ATPase domains. From these findings, we propose a mode of action of MccB17 requiring a DNA molecule long enough to allow the transport of a segment through the DNA gate of the enzyme. Furthermore, we suggest that MccB17 may trap a transient intermediate state of the gyrase reaction present only during DNA strand passage and enzyme turnover. The proteolytic signature of MccB17 from trypsin treatment of the full enzyme requires DNA and ATP and shows a protection of the C-terminal 47-kDa domain of gyrase, indicating the involvement of this domain in the toxin mode of action and consistent with its proposed role in the mechanism of DNA strand passage. We suggest that the binding site of MccB17 is in the C-terminal domain of GyrB.

Hsp90 is required for the activity of a hepatitis B virus reverse transcriptase.

PubMed Central

Hu, J; Seeger, C

1996-01-01

The heat shock protein Hsp90 is known as an essential component of several signal transduction pathways and has now been identified as an essential host factor for hepatitis B virus replication. Hsp90 interacts with the viral reverse transcriptase to facilitate the formation of a ribonucleoprotein (RNP) complex between the polymerase and an RNA ligand. This RNP complex is required early in replication for viral assembly and initiation of DNA synthesis through a protein-priming mechanism. These results thus invoke a role for the Hsp90 pathway in the formation of an RNP. Images Fig. 1 Fig. 2 Fig. 3 Fig. 4 Fig. 5 Fig. 6 PMID:8577714

Interactions of Ku70/80 with Double-Strand DNA: Energetic, Dynamics, and Functional Implications

NASA Technical Reports Server (NTRS)

Hu, Shaowen; Cucinotta, Francis A.

2010-01-01

Space radiation is a proficient inducer of DNA damage leading to mutation, aberrant cell signaling, and cancer formation. Ku is among the first responding proteins in nucleus to recognize and bind the DNA double strand breaks (DSBs) whenever they are introduced. Once loaded Ku works as a scaffold to recruit other repair factors of non-homologous end joining and facilitates the following repair processes. The crystallographic study of the Ku70/80 heterodimer indicate the core structure of this protein shows virtually no conformational change after binding with DNA. To investigate the dynamical features as well as the energetic characteristics of Ku-DNA binding, we conduct multi-nanosecond molecular dynamics simulations of a modeled Ku70/80 structure and several complexes with two 24-bp DNA duplexes. Free energy calculations show significant energy differences between the complexes with Ku bound at DSBs and those with Ku associated at an internal site of a chromosome. The results also reveal detailed interactions between different nucleotides and the amino acids along the DNA-binding cradle of Ku, indicating subtle binding preference of Ku at specific DNA sequences. The covariance matrix analyses along the trajectories demonstrate the protein is stimulated to undergo correlated motions of different domains once bound to DNA ends. Additionally, principle component analyses identify these low frequency collective motions suitable for binding with and translocation along duplex DNA. It is proposed that the modification of dynamical properties of Ku upon binding with DSBs may provide a signal for the further recruitment of other repair factors such as DNA-PKcs, XLF, and XRCC4.

The kinetochore prevents centromere-proximal crossover recombination during meiosis

PubMed Central

Vincenten, Nadine; Kuhl, Lisa-Marie; Lam, Isabel; Oke, Ashwini; Kerr, Alastair RW; Hochwagen, Andreas; Fung, Jennifer; Keeney, Scott; Vader, Gerben; Marston, Adèle L

2015-01-01

During meiosis, crossover recombination is essential to link homologous chromosomes and drive faithful chromosome segregation. Crossover recombination is non-random across the genome, and centromere-proximal crossovers are associated with an increased risk of aneuploidy, including Trisomy 21 in humans. Here, we identify the conserved Ctf19/CCAN kinetochore sub-complex as a major factor that minimizes potentially deleterious centromere-proximal crossovers in budding yeast. We uncover multi-layered suppression of pericentromeric recombination by the Ctf19 complex, operating across distinct chromosomal distances. The Ctf19 complex prevents meiotic DNA break formation, the initiating event of recombination, proximal to the centromere. The Ctf19 complex independently drives the enrichment of cohesin throughout the broader pericentromere to suppress crossovers, but not DNA breaks. This non-canonical role of the kinetochore in defining a chromosome domain that is refractory to crossovers adds a new layer of functionality by which the kinetochore prevents the incidence of chromosome segregation errors that generate aneuploid gametes. DOI: http://dx.doi.org/10.7554/eLife.10850.001 PMID:26653857

DNA interrogation by the CRISPR RNA-guided endonuclease Cas9.

PubMed

Sternberg, Samuel H; Redding, Sy; Jinek, Martin; Greene, Eric C; Doudna, Jennifer A

2014-03-06

The clustered regularly interspaced short palindromic repeats (CRISPR)-associated enzyme Cas9 is an RNA-guided endonuclease that uses RNA-DNA base-pairing to target foreign DNA in bacteria. Cas9-guide RNA complexes are also effective genome engineering agents in animals and plants. Here we use single-molecule and bulk biochemical experiments to determine how Cas9-RNA interrogates DNA to find specific cleavage sites. We show that both binding and cleavage of DNA by Cas9-RNA require recognition of a short trinucleotide protospacer adjacent motif (PAM). Non-target DNA binding affinity scales with PAM density, and sequences fully complementary to the guide RNA but lacking a nearby PAM are ignored by Cas9-RNA. Competition assays provide evidence that DNA strand separation and RNA-DNA heteroduplex formation initiate at the PAM and proceed directionally towards the distal end of the target sequence. Furthermore, PAM interactions trigger Cas9 catalytic activity. These results reveal how Cas9 uses PAM recognition to quickly identify potential target sites while scanning large DNA molecules, and to regulate scission of double-stranded DNA.

DNA interrogation by the CRISPR RNA-guided endonuclease Cas9

NASA Astrophysics Data System (ADS)

Sternberg, Samuel H.; Redding, Sy; Jinek, Martin; Greene, Eric C.; Doudna, Jennifer A.

2014-03-01

The clustered regularly interspaced short palindromic repeats (CRISPR)-associated enzyme Cas9 is an RNA-guided endonuclease that uses RNA-DNA base-pairing to target foreign DNA in bacteria. Cas9-guide RNA complexes are also effective genome engineering agents in animals and plants. Here we use single-molecule and bulk biochemical experiments to determine how Cas9-RNA interrogates DNA to find specific cleavage sites. We show that both binding and cleavage of DNA by Cas9-RNA require recognition of a short trinucleotide protospacer adjacent motif (PAM). Non-target DNA binding affinity scales with PAM density, and sequences fully complementary to the guide RNA but lacking a nearby PAM are ignored by Cas9-RNA. Competition assays provide evidence that DNA strand separation and RNA-DNA heteroduplex formation initiate at the PAM and proceed directionally towards the distal end of the target sequence. Furthermore, PAM interactions trigger Cas9 catalytic activity. These results reveal how Cas9 uses PAM recognition to quickly identify potential target sites while scanning large DNA molecules, and to regulate scission of double-stranded DNA.

Localization Microscopy Analyses of MRE11 Clusters in 3D-Conserved Cell Nuclei of Different Cell Lines.

PubMed

Eryilmaz, Marion; Schmitt, Eberhard; Krufczik, Matthias; Theda, Franziska; Lee, Jin-Ho; Cremer, Christoph; Bestvater, Felix; Schaufler, Wladimir; Hausmann, Michael; Hildenbrand, Georg

2018-01-22

In radiation biophysics, it is a subject of nowadays research to investigate DNA strand break repair in detail after damage induction by ionizing radiation. It is a subject of debate as to what makes up the cell's decision to use a certain repair pathway and how the repair machinery recruited in repair foci is spatially and temporarily organized. Single-molecule localization microscopy (SMLM) allows super-resolution analysis by precise localization of single fluorescent molecule tags, resulting in nuclear structure analysis with a spatial resolution in the 10 nm regime. Here, we used SMLM to study MRE11 foci. MRE11 is one of three proteins involved in the MRN-complex (MRE11-RAD50-NBS1 complex), a prominent DNA strand resection and broken end bridging component involved in homologous recombination repair (HRR) and alternative non-homologous end joining (a-NHEJ). We analyzed the spatial arrangements of antibody-labelled MRE11 proteins in the nuclei of a breast cancer and a skin fibroblast cell line along a time-course of repair (up to 48 h) after irradiation with a dose of 2 Gy. Different kinetics for cluster formation and relaxation were determined. Changes in the internal nano-scaled structure of the clusters were quantified and compared between the two cell types. The results indicate a cell type-dependent DNA damage response concerning MRE11 recruitment and cluster formation. The MRE11 data were compared to H2AX phosphorylation detected by γH2AX molecule distribution. These data suggested modulations of MRE11 signal frequencies that were not directly correlated to DNA damage induction. The application of SMLM in radiation biophysics offers new possibilities to investigate spatial foci organization after DNA damaging and during subsequent repair.

Localization Microscopy Analyses of MRE11 Clusters in 3D-Conserved Cell Nuclei of Different Cell Lines

PubMed Central

Eryilmaz, Marion; Schmitt, Eberhard; Krufczik, Matthias; Theda, Franziska; Lee, Jin-Ho; Cremer, Christoph; Bestvater, Felix; Schaufler, Wladimir; Hildenbrand, Georg

2018-01-01

In radiation biophysics, it is a subject of nowadays research to investigate DNA strand break repair in detail after damage induction by ionizing radiation. It is a subject of debate as to what makes up the cell’s decision to use a certain repair pathway and how the repair machinery recruited in repair foci is spatially and temporarily organized. Single-molecule localization microscopy (SMLM) allows super-resolution analysis by precise localization of single fluorescent molecule tags, resulting in nuclear structure analysis with a spatial resolution in the 10 nm regime. Here, we used SMLM to study MRE11 foci. MRE11 is one of three proteins involved in the MRN-complex (MRE11-RAD50-NBS1 complex), a prominent DNA strand resection and broken end bridging component involved in homologous recombination repair (HRR) and alternative non-homologous end joining (a-NHEJ). We analyzed the spatial arrangements of antibody-labelled MRE11 proteins in the nuclei of a breast cancer and a skin fibroblast cell line along a time-course of repair (up to 48 h) after irradiation with a dose of 2 Gy. Different kinetics for cluster formation and relaxation were determined. Changes in the internal nano-scaled structure of the clusters were quantified and compared between the two cell types. The results indicate a cell type-dependent DNA damage response concerning MRE11 recruitment and cluster formation. The MRE11 data were compared to H2AX phosphorylation detected by γH2AX molecule distribution. These data suggested modulations of MRE11 signal frequencies that were not directly correlated to DNA damage induction. The application of SMLM in radiation biophysics offers new possibilities to investigate spatial foci organization after DNA damaging and during subsequent repair. PMID:29361783

Structural, biocomplexation and gene delivery properties of hydroxyethylated gemini surfactants with varied spacer length.

PubMed

Zakharova, Lucia Ya; Gabdrakhmanov, Dinar R; Ibragimova, Alsu R; Vasilieva, Elmira A; Nizameev, Irek R; Kadirov, Marsil K; Ermakova, Elena A; Gogoleva, Natalia E; Faizullin, Dzhigangir A; Pokrovsky, Andrey G; Korobeynikov, Vladislav A; Cheresiz, Sergey V; Zuev, Yuriy F

2016-04-01

Gemini surfactants with hexadecyl tails and hydroxyethylated head groups bridged with tetramethylene (G4), hexamethylene (G6) and dodecamethylene (G12) spacers were shown to self-assemble at the lower critical micelle concentration compared to their conventional m-s-m analogs. The lipoplex formation and the plasmid DNA transfer into different kinds of host cells were studied. In the case of eukaryotic cells, high transfection efficacy has been demonstrated for DNA-gemini complexes, which increased as follows: G6G4>G12 has been obtained in the case of transformation of bacterial cells with plasmid DNA-gemini complexes, mediated by electroporation technique. Solely G6 shows transformation efficacy exceeding the control result (uncomplexed DNA), while the inhibitory effect occurs for G4 and G12. Analysis of physico-chemical features of single surfactants and lipoplexes shows that compaction and condensation effects change as follows: G6

Cyclic Peptidic Mimetics of Apollo Peptides Targeting Telomeric Repeat Binding Factor 2 (TRF2) and Apollo Interaction.

PubMed

Chen, Xia; Liu, Liu; Chen, Yong; Yang, Yuting; Yang, Chao-Yie; Guo, Tianyue; Lei, Ming; Sun, Haiying; Wang, Shaomeng

2018-05-10

Telomeric repeat binding factor 2 (TRF2) is a telomere-associated protein that plays an important role in the formation of the 3' single strand DNA overhang and the "T loop", two structures critical for the stability of the telomeres. Apollo is a 5'-exonuclease recruited by TRF2 to the telomere and contributes to the formation of the 3' single strand DNA overhang. Knocking down of Apollo can induce DNA damage response similar to that caused by the knocking down of TRF2. In this Letter, we report the design and synthesis of a class of cyclic peptidic mimetics of the TRFH binding motif of Apollo (Apollo TBM ). We found conformational control of the C terminal residues of Apollo TBM can effectively improve the binding affinity. We have obtained a crystal structure of a cyclic peptidic Apollo peptide mimetic ( 34 ) complexed with TRF2, which provides valuable guidance to the future design of TRF2 inhibitors.

Casein Kinase 1 and Phosphorylation of Cohesin Subunit Rec11 (SA3) Promote Meiotic Recombination through Linear Element Formation.

PubMed

Phadnis, Naina; Cipak, Lubos; Polakova, Silvia; Hyppa, Randy W; Cipakova, Ingrid; Anrather, Dorothea; Karvaiova, Lucia; Mechtler, Karl; Smith, Gerald R; Gregan, Juraj

2015-05-01

Proper meiotic chromosome segregation, essential for sexual reproduction, requires timely formation and removal of sister chromatid cohesion and crossing-over between homologs. Early in meiosis cohesins hold sisters together and also promote formation of DNA double-strand breaks, obligate precursors to crossovers. Later, cohesin cleavage allows chromosome segregation. We show that in fission yeast redundant casein kinase 1 homologs, Hhp1 and Hhp2, previously shown to regulate segregation via phosphorylation of the Rec8 cohesin subunit, are also required for high-level meiotic DNA breakage and recombination. Unexpectedly, these kinases also mediate phosphorylation of a different meiosis-specific cohesin subunit Rec11. This phosphorylation in turn leads to loading of linear element proteins Rec10 and Rec27, related to synaptonemal complex proteins of other species, and thereby promotes DNA breakage and recombination. Our results provide novel insights into the regulation of chromosomal features required for crossing-over and successful reproduction. The mammalian functional homolog of Rec11 (STAG3) is also phosphorylated during meiosis and appears to be required for fertility, indicating wide conservation of the meiotic events reported here.

DOE Office of Scientific and Technical Information (OSTI.GOV)

Damian, Luminita, E-mail: luminitadamian@microcal.eu.com; Universite de Toulouse, UPS, IPBS, F-31077 Toulouse; IUB, School of Engineering and Science, D-28727 Bremen

Is single-strand DNA translatable? Since the 60s, the question still remains whether or not DNA could be directly translated into protein. Some discrepancies in the results were reported about functional translation of single-strand DNA but all results converged on a similar behavior of RNA and ssDNA in the initiation step. Isothermal Titration Calorimetry method was used to determine thermodynamic constants of interaction between single-strand DNA and S30 extract of Escherichia coli. Our results showed that the binding was not affected by the nature of the template tested and the dissociation constants were in the same range when ssDNA (K{sub d}more » = 3.62 {+-} 2.1 x 10{sup -8} M) or the RNA corresponding sequence (K{sub d} = 2.7 {+-} 0.82 x 10{sup -8} M) bearing SD/ATG sequences were used. The binding specificity was confirmed by antibiotic interferences which block the initiation complex formation. These results suggest that the limiting step in translation of ssDNA is the elongation process.« less

Structures of minimal catalytic fragments of topoisomerase V reveals conformational changes relevant for DNA binding

PubMed Central

Rajan, Rakhi; Taneja, Bhupesh; Mondragón, Alfonso

2010-01-01

Summary Topoisomerase V is an archaeal type I topoisomerase that is unique among topoisomerases due to presence of both topoisomerase and DNA repair activities in the same protein. It is organized as an N-terminal topoisomerase domain followed by 24 tandem helix hairpin helix (HhH) motifs. Structural studies have shown that the active site is buried by the (HhH) motifs. Here we show that the N-terminal domain can relax DNA in the absence of any HhH motifs and that the HhH motifs are required for stable protein-DNA complex formation. Crystal structures of various topoisomerase V fragments show changes in the relative orientation of the domains mediated by a long bent linker helix, and these movements are essential for the DNA to enter the active site. Phosphate ions bound to the protein near the active site helped model DNA in the topoisomerase domain and shows how topoisomerase V may interact with DNA. PMID:20637419

Structural dynamics and interactions of Xeroderma pigmentosum complementation group A (XPA98-210) with damaged DNA.

PubMed

Pradhan, Sushmita; Mattaparthi, Venkata Satish Kumar

2017-10-25

Nucleotide excision repair (NER) in higher organisms repair massive DNA abrasions caused by ultraviolet rays, and various mutagens, where Xeroderma pigmentosum group A (XPA) protein is known to be involved in damage recognition step. Any mutations in XPA cause classical Xeroderma pigmentosum disease. The extent to which XPA is required in the NER is still unclear. Here, we present the comparative study on the structural and conformational changes in globular DNA binding domain of XPA 98-210 in DNA bound and DNA free state. Atomistic molecular dynamics simulation was carried out for both XPA 98-210 systems using AMBER force fields. We observed that XPA 98-210 in presence of damaged DNA exhibited more structural changes compared to XPA 98-210 in its free form. When XPA is in contact with DNA, we found marked stability of the complex due to the formation of characteristic longer antiparallel β-sheets consisting mainly lysine residues.

Computational investigation of fullerene-DNA interactions: Implications of fullerene's size and functionalization on DNA structure and binding energetics.

PubMed

Papavasileiou, Konstantinos D; Avramopoulos, Aggelos; Leonis, Georgios; Papadopoulos, Manthos G

2017-06-01

DNA is the building block of life, as it carries the biological information controlling development, function and reproduction of all organisms. However, its central role in storing and transferring genetic information can be severely hindered by molecules with structure altering abilities. Fullerenes are nanoparticles that find a broad spectrum of uses, but their toxicological effects on living organisms upon exposure remain unclear. The present study examines the interactions of a diverse array of fullerenes with DNA, by means of Molecular Dynamics and MM-PBSA methodologies, with special focus on structural deformations that may hint toxicity implications. Our results show that pristine and hydroxylated fullerenes have no unwinding effects upon DNA structure, with the latter displaying binding preference to the DNA major groove, achieved by both direct formation of hydrogen bonds and water molecule mediation. Fluorinated derivatives are capable of penetrating DNA structure, forming intercalative complexes with high binding affinities. Copyright © 2017 Elsevier Inc. All rights reserved.

Clathrate colloidal crystals

NASA Astrophysics Data System (ADS)

Lin, Haixin; Lee, Sangmin; Sun, Lin; Spellings, Matthew; Engel, Michael; Glotzer, Sharon C.; Mirkin, Chad A.

2017-03-01

DNA-programmable assembly has been used to deliberately synthesize hundreds of different colloidal crystals spanning dozens of symmetries, but the complexity of the achieved structures has so far been limited to small unit cells. We assembled DNA-modified triangular bipyramids (~250-nanometer long edge, 177-nanometer short edge) into clathrate architectures. Electron microscopy images revealed that at least three different structures form as large single-domain architectures or as multidomain materials. Ordered assemblies, isostructural to clathrates, were identified with the help of molecular simulations and geometric analysis. These structures are the most sophisticated architectures made via programmable assembly, and their formation can be understood based on the shape of the nanoparticle building blocks and mode of DNA functionalization.

Self-assembled catalytic DNA nanostructures for synthesis of para-directed polyaniline.

PubMed

Wang, Zhen-Gang; Zhan, Pengfei; Ding, Baoquan

2013-02-26

Templated synthesis has been considered as an efficient approach to produce polyaniline (PANI) nanostructures. The features of DNA molecules enable a DNA template to be an intriguing template for fabrication of emeraldine PANI. In this work, we assembled HRP-mimicking DNAzyme with different artificial DNA nanostructures, aiming to manipulate the molecular structures and morphologies of PANI nanostructures through the controlled DNA self-assembly. UV-vis absorption spectra were used to investigate the molecular structures of PANI and monitor kinetic growth of PANI. It was found that PANI was well-doped at neutral pH and the redox behaviors of the resultant PANI were dependent on the charge density of the template, which was controlled by the template configurations. CD spectra indicated that the PANI threaded tightly around the helical DNA backbone, resulting in the right handedness of PANI. These reveal the formation of the emeraldine form of PANI that was doped by the DNA. The morphologies of the resultant PANI were studied by AFM and SEM. It was concluded from the imaging and spectroscopic kinetic results that PANI grew preferably from the DNAzyme sites and then expanded over the template to form 1D PANI nanostructures. The strategy of the DNAzyme-DNA template assembly brings several advantages in the synthesis of para-coupling PANI, including the region-selective growth of PANI, facilitating the formation of a para-coupling structure and facile regulation. We believe this study contributes significantly to the fabrication of doped PANI nanopatterns with controlled complexity, and the development of DNA nanotechnology.

Purification of ribonucleoproteins by a novel approach: isolation of the SSB1 ribonucleoprotein from yeast and demonstration that it has no role in mRNA splicing.

PubMed

Cusick, M E

1992-12-29

A novel approach is described to purify potential ribonucleoproteins (RNP) of yeast. The method assays a yeast RNP complex, assembled in vitro on actin pre-mRNA, by low-ionic strength acrylamide gel electrophoresis. The minimal protein components of this RNP complex were three proteins, one of 30 kDa and two at 42-44 kDa, defined by formation of the complex on biotinylated-RNA, binding of this complex to avidin-agarose, and salt elution of the protein in the biotinylated-RNP complex. Using the assay for RNP complex formation, an RNP protein was purified to homogeneity on the basis of its affinity towards single-stranded DNA and RNA. This RNP protein turned out to be identical to a known RNP protein, the single-stranded binding protein 1 (ssb1) of yeast, on the basis of identical gel electrophoretic migration, antibody cross-reactivity, and identical properties on the gel complex formation assay. In vitro mRNA splicing was normal in extracts made from a yeast strain missing ssb1 (ssb1- strain). Addition of anti-ssb1 antibody to splicing extracts made from a wild type strain did not inhibit or diminish splicing. Instead, mRNA splicing was reproducibly stimulated several fold, indicating competition between ssb1 and splicing factors for binding to single-stranded RNA in the extracts. RNP complexes still formed in the ssb1- strain, demonstrating that it would be possible to purify other RNP proteins from this strain using the gel complex formation assay.

[Identification of C(2)M interacting proteins by yeast two-hybrid screening].

PubMed

Yue, Shan-shan; Xia, Lai-xin

2015-11-01

The synaptonemal complex (SC) is a huge structure which assembles between the homologous chromosomes during meiotic prophase I. Drosophila germ cell-specific nucleoprotein C(2)M clustering at chromosomes can induce SC formation. To further study the molecular function and mechanism of C(2)M in meiosis, we constructed a bait vector for C(2)M and used the yeast two-hybrid system to identify C(2)M interacting proteins. Forty interacting proteins were obtained, including many DNA and histone binding proteins, ATP synthases and transcription factors. Gene silencing assays in Drosophila showed that two genes, wech and Psf1, may delay the disappearance of SC. These results indicate that Wech and Psf1 may form a complex with C(2)M to participate in the formation or stabilization of the SC complex.

Hypomorphism in human NSMCE2 linked to primordial dwarfism and insulin resistance

PubMed Central

Payne, Felicity; Colnaghi, Rita; Rocha, Nuno; Seth, Asha; Harris, Julie; Carpenter, Gillian; Bottomley, William E.; Wheeler, Eleanor; Wong, Stephen; Saudek, Vladimir; Savage, David; O’Rahilly, Stephen; Carel, Jean-Claude; Barroso, Inês; O’Driscoll, Mark; Semple, Robert

2014-01-01

Structural maintenance of chromosomes (SMC) complexes are essential for maintaining chromatin structure and regulating gene expression. Two the three known SMC complexes, cohesin and condensin, are important for sister chromatid cohesion and condensation, respectively; however, the function of the third complex, SMC5–6, which includes the E3 SUMO-ligase NSMCE2 (also widely known as MMS21) is less clear. Here, we characterized 2 patients with primordial dwarfism, extreme insulin resistance, and gonadal failure and identified compound heterozygous frameshift mutations in NSMCE2. Both mutations reduced NSMCE2 expression in patient cells. Primary cells from one patient showed increased micronucleus and nucleoplasmic bridge formation, delayed recovery of DNA synthesis, and reduced formation of foci containing Bloom syndrome helicase (BLM) after hydroxyurea-induced replication fork stalling. These nuclear abnormalities in patient dermal fibroblast were restored by expression of WT NSMCE2, but not a mutant form lacking SUMO-ligase activity. Furthermore, in zebrafish, knockdown of the NSMCE2 ortholog produced dwarfism, which was ameliorated by reexpression of WT, but not SUMO-ligase–deficient NSMCE. Collectively, these findings support a role for NSMCE2 in recovery from DNA damage and raise the possibility that loss of its function produces dwarfism through reduced tolerance of replicative stress. PMID:25105364

Hypomorphism in human NSMCE2 linked to primordial dwarfism and insulin resistance.

PubMed

Payne, Felicity; Colnaghi, Rita; Rocha, Nuno; Seth, Asha; Harris, Julie; Carpenter, Gillian; Bottomley, William E; Wheeler, Eleanor; Wong, Stephen; Saudek, Vladimir; Savage, David; O'Rahilly, Stephen; Carel, Jean-Claude; Barroso, Inês; O'Driscoll, Mark; Semple, Robert

2014-09-01

Structural maintenance of chromosomes (SMC) complexes are essential for maintaining chromatin structure and regulating gene expression. Two the three known SMC complexes, cohesin and condensin, are important for sister chromatid cohesion and condensation, respectively; however, the function of the third complex, SMC5-6, which includes the E3 SUMO-ligase NSMCE2 (also widely known as MMS21) is less clear. Here, we characterized 2 patients with primordial dwarfism, extreme insulin resistance, and gonadal failure and identified compound heterozygous frameshift mutations in NSMCE2. Both mutations reduced NSMCE2 expression in patient cells. Primary cells from one patient showed increased micronucleus and nucleoplasmic bridge formation, delayed recovery of DNA synthesis, and reduced formation of foci containing Bloom syndrome helicase (BLM) after hydroxyurea-induced replication fork stalling. These nuclear abnormalities in patient dermal fibroblast were restored by expression of WT NSMCE2, but not a mutant form lacking SUMO-ligase activity. Furthermore, in zebrafish, knockdown of the NSMCE2 ortholog produced dwarfism, which was ameliorated by reexpression of WT, but not SUMO-ligase-deficient NSMCE. Collectively, these findings support a role for NSMCE2 in recovery from DNA damage and raise the possibility that loss of its function produces dwarfism through reduced tolerance of replicative stress.

DNA-Protein Cross-Links: Formation, Structural Identities, and Biological Outcomes.

PubMed

Tretyakova, Natalia Y; Groehler, Arnold; Ji, Shaofei

2015-06-16

Noncovalent DNA-protein interactions are at the heart of normal cell function. In eukaryotic cells, genomic DNA is wrapped around histone octamers to allow for chromosomal packaging in the nucleus. Binding of regulatory protein factors to DNA directs replication, controls transcription, and mediates cellular responses to DNA damage. Because of their fundamental significance in all cellular processes involving DNA, dynamic DNA-protein interactions are required for cell survival, and their disruption is likely to have serious biological consequences. DNA-protein cross-links (DPCs) form when cellular proteins become covalently trapped on DNA strands upon exposure to various endogenous, environmental and chemotherapeutic agents. DPCs progressively accumulate in the brain and heart tissues as a result of endogenous exposure to reactive oxygen species and lipid peroxidation products, as well as normal cellular metabolism. A range of structurally diverse DPCs are found following treatment with chemotherapeutic drugs, transition metal ions, and metabolically activated carcinogens. Because of their considerable size and their helix-distorting nature, DPCs interfere with the progression of replication and transcription machineries and hence hamper the faithful expression of genetic information, potentially contributing to mutagenesis and carcinogenesis. Mass spectrometry-based studies have identified hundreds of proteins that can become cross-linked to nuclear DNA in the presence of reactive oxygen species, carcinogen metabolites, and antitumor drugs. While many of these proteins including histones, transcription factors, and repair proteins are known DNA binding partners, other gene products with no documented affinity for DNA also participate in DPC formation. Furthermore, multiple sites within DNA can be targeted for cross-linking including the N7 of guanine, the C-5 methyl group of thymine, and the exocyclic amino groups of guanine, cytosine, and adenine. This structural complexity complicates structural and biological studies of DPC lesions. Two general strategies have been developed for creating DNA strands containing structurally defined, site-specific DPCs. Enzymatic methodologies that trap DNA modifying proteins on their DNA substrate are site specific and efficient, but do not allow for systematic studies of DPC lesion structure on their biological outcomes. Synthetic methodologies for DPC formation are based on solid phase synthesis of oligonucleotide strands containing protein-reactive unnatural DNA bases. The latter approach allows for a wider range of protein substrates to be conjugated to DNA and affords a greater flexibility for the attachment sites within DNA. In this Account, we outline the chemistry of DPC formation in cells, describe our recent efforts to identify the cross-linked proteins by mass spectrometry, and discuss various methodologies for preparing DNA strands containing structurally defined, site specific DPC lesions. Polymerase bypass experiments conducted with model DPCs indicate that the biological outcomes of these bulky lesions are strongly dependent on the peptide/protein size and the exact cross-linking site within DNA. Future studies are needed to elucidate the mechanisms of DPC repair and their biological outcomes in living cells.

DNA-Protein Cross-links: Formation, Structural Identities, and Biological Outcomes

PubMed Central

Tretyakova, Natalia Y.; Groehler, Arnold; Ji, Shaofei

2015-01-01

CONSPECTUS Non-covalent DNA-protein interactions are at the heart of normal cell function. In eukaryotic cells, genomic DNA is wrapped around histone octamers to allow for chromosomal packaging in the nucleus. Binding of regulatory protein factors to DNA directs replication, controls transcription, and mediates cellular responses to DNA damage. Because of their fundamental significance in all cellular processes involving DNA, dynamic DNA-protein interactions are required for cell survival, and their disruption is likely to have serious biological consequences. DNA-protein cross-links (DPCs) form when cellular proteins become covalently trapped on DNA strands upon exposure to various endogenous, environmental and chemotherapeutic agents. DPCs progressively accumulate in the brain and heart tissues as a result of endogenous exposure to reactive oxygen species and lipid peroxidation products, as well as normal cellular metabolism. A range of structurally diverse DPCs are found following treatment with chemotherapeutic drugs, transition metal ions, and metabolically activated carcinogens. Because of their considerable size and their helix-distorting nature, DPCs interfere with the progression of replication and transcription machineries and hence hamper the faithful expression of genetic information, potentially contributing to mutagenesis and carcinogenesis. Mass spectrometry-based studies have identified hundreds of proteins that can become cross-linked to nuclear DNA in the presence of reactive oxygen species, carcinogen metabolites, and antitumor drugs. While many of these proteins including histones, transcription factors, and repair proteins are known DNA binding partners, other gene products with no documented affinity for DNA also participate in DPC formation. Furthermore, multiple sites within DNA can be targeted for cross-linking including the N7 of guanine, the C-5 methyl group of thymine, and the exocyclic amino groups of guanine, cytosine, and adenine. This structural complexity complicates structural and biological studies of DPC lesions. Two general strategies have been developed for creating DNA strands containing structurally defined, site-specific DPCs. Enzymatic methodologies that trap DNA modifying proteins on their DNA substrate are site specific and efficient, but do not allow for systematic studies of DPC lesion structure on their biological outcomes. Synthetic methodologies for DPC formation are based on solid phase synthesis of oligonucleotide strands containing protein-reactive unnatural DNA bases. The latter approach allows for a wider range of protein substrates to be conjugated to DNA and affords a greater flexibility for the attachment sites within DNA. In this Account, we outline the chemistry of DPC formation in cells, describe our recent efforts to identify the cross-linked proteins by mass spectrometry, and discuss various methodologies for preparing DNA strands containing structurally defined, site specific DPC lesions. Polymerase bypass experiments conducted with model DPCs indicate that the biological outcomes of these bulky lesions are strongly dependent on the peptide/protein size and the exact cross-linking site within DNA. Future studies are needed to elucidate the mechanisms of DPC repair and their biological outcomes in living cells. PMID:26032357

Synthesis, spectroscopic characterization and structural investigations of a new charge transfer complex of 2,6-diaminopyridine with 3,5-dinitrobenzoic acid: DNA binding and antimicrobial studies

NASA Astrophysics Data System (ADS)

Khan, Ishaat M.; Ahmad, Afaq; Kumar, Sarvendra

2013-03-01

A new charge transfer (CT) complex [(DAPH)+(DNB)-] consisting of 2,6-diaminopyridine (DAP) as donor and 3,5-dinitrobenzoic acid (DNB-H) as acceptor, was synthesized and characterized by FTIR, 1H and 13C NMR, ESI mass spectroscopic and X-ray crystallographic techniques. The hydrogen bonding (N+-H⋯O-) plays an important role to consolidate the cation and anion together. CT complex shows a considerable interaction with Calf thymus DNA. The CT complex was also tested for its antibacterial activity against two Gram-positive bacteria Staphylococcus aureus and Bacillus subtilis and two Gram-negative bacteria Escherichia coli and Pseudomonas aeruginosa strains by using Tetracycline as standard, and antifungal property against Aspergillus niger, Candida albicans, and Penicillium sp. by using Nystatin as standard. The results were compared with standard drugs and significant conclusions were obtained. A polymeric net work through H-bonding interactions between neighboring moieties was observed. This has been attributed to the formation of 1:1 type CT complex.

Association of a Platinum Complex to a G-Quadruplex Ligand Enhances Telomere Disruption.

PubMed

Charif, Razan; Granotier-Beckers, Christine; Bertrand, Hélène Charlotte; Poupon, Joël; Ségal-Bendirdjian, Evelyne; Teulade-Fichou, Marie-Paule; Boussin, François D; Bombard, Sophie

2017-08-21

Telomeres protect the ends of chromosomes against illegitimate recombination and repair. They can be targets for G-quadruplex ligands and platinum complexes due to their repeated G-rich sequences. Protection of telomeres is ensured by a complex of six proteins, including TRF2, which inhibits the DNA damage response pathway. We analyzed telomere modifications induced in cancer cells by the experimental hybrid platinum complex, Pt-MPQ, comprising both an ethylene diamine monofunctional platinum complex and a G-quadruplex recognition moiety (MPQ). Pt-MPQ promotes the displacement of two telomeric proteins (TRF2 and TRF1) from telomeres, as well as the formation of telomere damage and telomere sister losses, whereas the control compound MPQ does not. This suggests that the platinum moiety potentiates the targeting of the G-quadruplex ligand to telomeres, opening a new perspective for telomere biology and anticancer therapy. Interestingly, the chemotherapy drug cisplatin, which has no specific affinity for G-quadruplex structures, partially induces the TRF2 delocalization from telomeres but produces less telomeric DNA damage, suggesting that this TRF2 displacement could be independent of G-quadruplex recognition.

The subunits of the S-phase checkpoint complex Mrc1/Tof1/Csm3: dynamics and interdependence

PubMed Central

2014-01-01

Background The S-phase checkpoint aims to prevent cells from generation of extensive single-stranded DNA that predisposes to genome instability. The S. cerevisiae complex Tof1/Csm3/Mrc1 acts to restrain the replicative MCM helicase when DNA synthesis is prohibited. Keeping the replication machinery intact allows restart of the replication fork when the block is relieved. Although the subunits of the Tof1/Csm3/Mrc1 complex are well studied, the impact of every single subunit on the triple complex formation and function needs to be established. Findings This work studies the cellular localization and the chromatin binding of GFP-tagged subunits when the complex is intact and when a subunit is missing. We demonstrate that the complex is formed in cell nucleus, not the cytoplasm, as Tof1, Csm3 and Mrc1 enter the nucleus independently from one another. Via in situ chromatin binding assay we show that a Tof1-Csm3 dimer formation and chromatin binding is required to ensure the attachment of Mrc1 to chromatin. Our study indicates that the translocation into the nucleus is not the process to regulate the timing of chromatin association of Mrc1. We also studied the nuclear behavior of Mrc1 subunit in the process of adaptation to the presence hydroxyurea. Our results indicate that after prolonged HU incubation, cells bypass the S-phase checkpoint and proceed throughout the cell cycle. This process is accompanied by Mrc1 chromatin detachment and Rad53 dephosphorylation. Conclusions In S. cerevisiae the subunits of the S-phase checkpoint complex Mrc1/Tof1/Csm3 independently enter the cell nucleus, where a Tof1-Csm3 dimer is formed to ensure the chromatin binding of Mrc1 and favor DNA replication and S-phase checkpoint fork arrest. In the process of adaptation to the presence of hydroxyurea Mrc1 is detached from chromatin and Rad53 checkpoint activity is diminished in order to allow S-phase checkpoint escape and completion of the cell cycle. PMID:25379053

The subunits of the S-phase checkpoint complex Mrc1/Tof1/Csm3: dynamics and interdependence.

PubMed

Uzunova, Sonya Dimitrova; Zarkov, Alexander Stefanov; Ivanova, Anna Marianova; Stoynov, Stoyno Stefanov; Nedelcheva-Veleva, Marina Nedelcheva

2014-01-01

The S-phase checkpoint aims to prevent cells from generation of extensive single-stranded DNA that predisposes to genome instability. The S. cerevisiae complex Tof1/Csm3/Mrc1 acts to restrain the replicative MCM helicase when DNA synthesis is prohibited. Keeping the replication machinery intact allows restart of the replication fork when the block is relieved. Although the subunits of the Tof1/Csm3/Mrc1 complex are well studied, the impact of every single subunit on the triple complex formation and function needs to be established. This work studies the cellular localization and the chromatin binding of GFP-tagged subunits when the complex is intact and when a subunit is missing. We demonstrate that the complex is formed in cell nucleus, not the cytoplasm, as Tof1, Csm3 and Mrc1 enter the nucleus independently from one another. Via in situ chromatin binding assay we show that a Tof1-Csm3 dimer formation and chromatin binding is required to ensure the attachment of Mrc1 to chromatin. Our study indicates that the translocation into the nucleus is not the process to regulate the timing of chromatin association of Mrc1. We also studied the nuclear behavior of Mrc1 subunit in the process of adaptation to the presence hydroxyurea. Our results indicate that after prolonged HU incubation, cells bypass the S-phase checkpoint and proceed throughout the cell cycle. This process is accompanied by Mrc1 chromatin detachment and Rad53 dephosphorylation. In S. cerevisiae the subunits of the S-phase checkpoint complex Mrc1/Tof1/Csm3 independently enter the cell nucleus, where a Tof1-Csm3 dimer is formed to ensure the chromatin binding of Mrc1 and favor DNA replication and S-phase checkpoint fork arrest. In the process of adaptation to the presence of hydroxyurea Mrc1 is detached from chromatin and Rad53 checkpoint activity is diminished in order to allow S-phase checkpoint escape and completion of the cell cycle.

Study of nucleic acid-gold nanorod interactions and detecting nucleic acid hybridization using gold nanorod solutions in the presence of sodium citrate.

PubMed

Kanjanawarut, Roejarek; Su, Xiaodi

2010-09-01

In this study, the authors report that sodium citrate can aggregate hexadecyl-trimethyl-ammonium ion(+)-coated gold nanorods (AuNRs), and nucleic acids of different charge and structure properties, i.e., single-stranded DNA (ssDNA), double-stranded DNA (dsDNA), single-stranded peptide nucleic acid (PNA), and PNA-DNA complex, can bind to the AuNRs and therefore retard the sodium citrate-induced aggregation to different extents. The discovery that hybridized dsDNA (and the PNA-DNA complex) has a more pronounced protection effect than ssDNA (and PNA) allows the authors to develop a homogeneous phase AuNRs-based UV-visible (UV-vis) spectral assay for detecting specific sequences of oligonucleotides (20 mer) with a single-base-mismatch selectivity and a limit of detection of 5 nM. This assay involves no tedious bioconjugation and on-particle hybridization. The simple "set and test" format allows for a highly efficient hybridization in a homogeneous phase and a rapid display of the results in less than a minute. By measuring the degree of reduction in AuNR aggregation in the presence of different nucleic acid samples, one can assess how different nucleic acids interact with the AuNRs to complement the knowledge of spherical gold nanoparticles. Besides UV-vis characterization, transmission electron microscopy and zeta potential measurements were conduced to provide visual evidence of the particle aggregation and to support the discussion of the assay principle.

High Fractional Occupancy of a Tandem Maf Recognition Element and Its Role in Long-Range β-Globin Gene Regulation

PubMed Central

Stees, Jared R.; Hossain, Mir A.; Sunose, Tomoki; Kudo, Yasushi; Pardo, Carolina E.; Nabilsi, Nancy H.; Darst, Russell P.; Poudyal, Rosha; Igarashi, Kazuhiko; Kladde, Michael P.

2015-01-01

Enhancers and promoters assemble protein complexes that ultimately regulate the recruitment and activity of RNA polymerases. Previous work has shown that at least some enhancers form stable protein complexes, leading to the formation of enhanceosomes. We analyzed protein-DNA interactions in the murine β-globin gene locus using the methyltransferase accessibility protocol for individual templates (MAPit). The data show that a tandem Maf recognition element (MARE) in locus control region (LCR) hypersensitive site 2 (HS2) reveals a remarkably high degree of occupancy during differentiation of mouse erythroleukemia cells. Most of the other transcription factor binding sites in LCR HS2 or in the adult β-globin gene promoter regions exhibit low fractional occupancy, suggesting highly dynamic protein-DNA interactions. Targeting of an artificial zinc finger DNA-binding domain (ZF-DBD) to the HS2 tandem MARE caused a reduction in the association of MARE-binding proteins and transcription complexes at LCR HS2 and the adult βmajor-globin gene promoter but did not affect expression of the βminor-globin gene. The data demonstrate that a stable MARE-associated footprint in LCR HS2 is important for the recruitment of transcription complexes to the adult βmajor-globin gene promoter during erythroid cell differentiation. PMID:26503787

Determination of the DNA-binding kinetics of three related but heteroimmune bacteriophage repressors using EMSA and SPR analysis

PubMed Central

Henriksson-Peltola, Petri; Sehlén, Wilhelmina; Haggård-Ljungquist, Elisabeth

2007-01-01

Bacteriophages P2, P2 Hy dis and WΦ are very similar but heteroimmune Escherichia coli phages. The structural genes show over 96% identity, but the repressors show between 43 and 63% identities. Furthermore, the operators, which contain two directly repeated sequences, vary in sequence, length, location relative to the promoter and spacing between the direct repeats. We have compared the in vivo effects of the wild type and mutated operators on gene expression with the complexes formed between the repressors and their wild type or mutated operators using electrophoretic mobility shift assay (EMSA), and real-time kinetics of the protein–DNA interactions using surface plasmon resonance (SPR) analysis. Using EMSA, the repressors formed different protein–DNA complexes, and only WΦ was significantly affected by point mutations. However, SPR analysis showed a reduced association rate constant and an increased dissociation rate constant for P2 and WΦ operator mutants. The association rate constants of P2 Hy dis was too fast to be determined. The P2 Hy dis dissociation response curves were shown to be triphasic, while both P2 and WΦ C were biphasic. Thus, the kinetics of complex formation and the nature of the complexes formed differ extensively between these very closely related phages. PMID:17412705

How can macromolecular crowding inhibit biological reactions? The enhanced formation of DNA nanoparticles

PubMed Central

Hou, Sen; Trochimczyk, Piotr; Sun, Lili; Wisniewska, Agnieszka; Kalwarczyk, Tomasz; Zhang, Xuzhu; Wielgus-Kutrowska, Beata; Bzowska, Agnieszka; Holyst, Robert

2016-01-01

In contrast to the already known effect that macromolecular crowding usually promotes biological reactions, solutions of PEG 6k at high concentrations stop the cleavage of DNA by HindIII enzyme, due to the formation of DNA nanoparticles. We characterized the DNA nanoparticles and probed the prerequisites for their formation using multiple techniques such as fluorescence correlation spectroscopy, dynamic light scattering, fluorescence analytical ultracentrifugation etc. In >25% PEG 6k solution, macromolecular crowding promotes the formation of DNA nanoparticles with dimensions of several hundreds of nanometers. The formation of DNA nanoparticles is a fast and reversible process. Both plasmid DNA (2686 bp) and double-stranded/single-stranded DNA fragment (66bp/nt) can form nanoparticles. We attribute the enhanced nanoparticle formation to the depletion effect of macromolecular crowding. This study presents our idea to enhance the formation of DNA nanoparticles by macromolecular crowding, providing the first step towards a final solution to efficient gene therapy. PMID:26903405

How can macromolecular crowding inhibit biological reactions? The enhanced formation of DNA nanoparticles.

PubMed

Hou, Sen; Trochimczyk, Piotr; Sun, Lili; Wisniewska, Agnieszka; Kalwarczyk, Tomasz; Zhang, Xuzhu; Wielgus-Kutrowska, Beata; Bzowska, Agnieszka; Holyst, Robert

2016-02-23

In contrast to the already known effect that macromolecular crowding usually promotes biological reactions, solutions of PEG 6k at high concentrations stop the cleavage of DNA by HindIII enzyme, due to the formation of DNA nanoparticles. We characterized the DNA nanoparticles and probed the prerequisites for their formation using multiple techniques such as fluorescence correlation spectroscopy, dynamic light scattering, fluorescence analytical ultracentrifugation etc. In >25% PEG 6k solution, macromolecular crowding promotes the formation of DNA nanoparticles with dimensions of several hundreds of nanometers. The formation of DNA nanoparticles is a fast and reversible process. Both plasmid DNA (2686 bp) and double-stranded/single-stranded DNA fragment (66 bp/nt) can form nanoparticles. We attribute the enhanced nanoparticle formation to the depletion effect of macromolecular crowding. This study presents our idea to enhance the formation of DNA nanoparticles by macromolecular crowding, providing the first step towards a final solution to efficient gene therapy.

UV Damage-Induced Phosphorylation of HBO1 Triggers CRL4DDB2-Mediated Degradation To Regulate Cell Proliferation

PubMed Central

Matsunuma, Ryoichi; Ohhata, Tatsuya; Kitagawa, Kyoko; Sakai, Satoshi; Uchida, Chiharu; Shiotani, Bunsyo; Matsumoto, Masaki; Nakayama, Keiichi I.; Ogura, Hiroyuki; Shiiya, Norihiko; Kitagawa, Masatoshi

2015-01-01

Histone acetyltransferase binding to ORC-1 (HBO1) is a critically important histone acetyltransferase for forming the prereplicative complex (pre-RC) at the replication origin. Pre-RC formation is completed by loading of the MCM2-7 heterohexameric complex, which functions as a helicase in DNA replication. HBO1 recruited to the replication origin by CDT1 acetylates histone H4 to relax the chromatin conformation and facilitates loading of the MCM complex onto replication origins. However, the acetylation status and mechanism of regulation of histone H3 at replication origins remain elusive. HBO1 positively regulates cell proliferation under normal cell growth conditions. Whether HBO1 regulates proliferation in response to DNA damage is poorly understood. In this study, we demonstrated that HBO1 was degraded after DNA damage to suppress cell proliferation. Ser50 and Ser53 of HBO1 were phosphorylated in an ATM/ATR DNA damage sensor-dependent manner after UV treatment. ATM/ATR-dependently phosphorylated HBO1 preferentially interacted with DDB2 and was ubiquitylated by CRL4DDB2. Replacement of endogenous HBO1 in Ser50/53Ala mutants maintained acetylation of histone H3K14 and impaired cell cycle regulation in response to UV irradiation. Our findings demonstrate that HBO1 is one of the targets in the DNA damage checkpoint. These results show that ubiquitin-dependent control of the HBO1 protein contributes to cell survival during UV irradiation. PMID:26572825

The DNA damage checkpoint pathway promotes extensive resection and nucleotide synthesis to facilitate homologous recombination repair and genome stability in fission yeast.

PubMed

Blaikley, Elizabeth J; Tinline-Purvis, Helen; Kasparek, Torben R; Marguerat, Samuel; Sarkar, Sovan; Hulme, Lydia; Hussey, Sharon; Wee, Boon-Yu; Deegan, Rachel S; Walker, Carol A; Pai, Chen-Chun; Bähler, Jürg; Nakagawa, Takuro; Humphrey, Timothy C

2014-05-01

DNA double-strand breaks (DSBs) can cause chromosomal rearrangements and extensive loss of heterozygosity (LOH), hallmarks of cancer cells. Yet, how such events are normally suppressed is unclear. Here we identify roles for the DNA damage checkpoint pathway in facilitating homologous recombination (HR) repair and suppressing extensive LOH and chromosomal rearrangements in response to a DSB. Accordingly, deletion of Rad3(ATR), Rad26ATRIP, Crb2(53BP1) or Cdc25 overexpression leads to reduced HR and increased break-induced chromosome loss and rearrangements. We find the DNA damage checkpoint pathway facilitates HR, in part, by promoting break-induced Cdt2-dependent nucleotide synthesis. We also identify additional roles for Rad17, the 9-1-1 complex and Chk1 activation in facilitating break-induced extensive resection and chromosome loss, thereby suppressing extensive LOH. Loss of Rad17 or the 9-1-1 complex results in a striking increase in break-induced isochromosome formation and very low levels of chromosome loss, suggesting the 9-1-1 complex acts as a nuclease processivity factor to facilitate extensive resection. Further, our data suggest redundant roles for Rad3ATR and Exo1 in facilitating extensive resection. We propose that the DNA damage checkpoint pathway coordinates resection and nucleotide synthesis, thereby promoting efficient HR repair and genome stability. © The Author(s) 2014. Published by Oxford University Press.

Pivotal Impacts of Retrotransposon Based Invasive RNAs on Evolution.

PubMed

Habibi, Laleh; Salmani, Hamzeh

2017-01-01

RNAs have long been described as the mediators of gene expression; they play a vital role in the structure and function of cellular complexes. Although the role of RNAs in the prokaryotes is mainly confined to these basic functions, the effects of these molecules in regulating the gene expression and enzymatic activities have been discovered in eukaryotes. Recently, a high-resolution analysis of the DNA obtained from different organisms has revealed a fundamental impact of the RNAs in shaping the genomes, heterochromatin formation, and gene creation. Deep sequencing of the human genome revealed that about half of our DNA is comprised of repetitive sequences (remnants of transposable element movements) expanded mostly through RNA-mediated processes. ORF2 encoded by L1 retrotransposons is a cellular reverse transcriptase which is mainly responsible for RNA invasion of various transposable elements (L1s, Alus, and SVAs) and cellular mRNAs in to the genomic DNA. In addition to increasing retroelements copy number; genomic expansion in association with centromere, telomere, and heterochromatin formation as well as pseudogene creation are the evolutionary consequences of this RNA-based activity. Threatening DNA integrity by disrupting the genes and forming excessive double strand breaks is another effect of this invasion. Therefore, repressive mechanisms have been evolved to control the activities of these invasive intracellular RNAs. All these mechanisms now have essential roles in the complex cellular functions. Therefore, it can be concluded that without direct action of RNA networks in shaping the genome and in the development of different cellular mechanisms, the evolution of higher eukaryotes would not be possible.

Pivotal Impacts of Retrotransposon Based Invasive RNAs on Evolution

PubMed Central

Habibi, Laleh; Salmani, Hamzeh

2017-01-01

RNAs have long been described as the mediators of gene expression; they play a vital role in the structure and function of cellular complexes. Although the role of RNAs in the prokaryotes is mainly confined to these basic functions, the effects of these molecules in regulating the gene expression and enzymatic activities have been discovered in eukaryotes. Recently, a high-resolution analysis of the DNA obtained from different organisms has revealed a fundamental impact of the RNAs in shaping the genomes, heterochromatin formation, and gene creation. Deep sequencing of the human genome revealed that about half of our DNA is comprised of repetitive sequences (remnants of transposable element movements) expanded mostly through RNA-mediated processes. ORF2 encoded by L1 retrotransposons is a cellular reverse transcriptase which is mainly responsible for RNA invasion of various transposable elements (L1s, Alus, and SVAs) and cellular mRNAs in to the genomic DNA. In addition to increasing retroelements copy number; genomic expansion in association with centromere, telomere, and heterochromatin formation as well as pseudogene creation are the evolutionary consequences of this RNA-based activity. Threatening DNA integrity by disrupting the genes and forming excessive double strand breaks is another effect of this invasion. Therefore, repressive mechanisms have been evolved to control the activities of these invasive intracellular RNAs. All these mechanisms now have essential roles in the complex cellular functions. Therefore, it can be concluded that without direct action of RNA networks in shaping the genome and in the development of different cellular mechanisms, the evolution of higher eukaryotes would not be possible. PMID:29067016

DNA Binding and Phosphorylation Regulate the Core Structure of the NF-κB p50 Transcription Factor

NASA Astrophysics Data System (ADS)

Vonderach, Matthias; Byrne, Dominic P.; Barran, Perdita E.; Eyers, Patrick A.; Eyers, Claire E.

2018-06-01

The NF-κB transcription factors are known to be extensively phosphorylated, with dynamic site-specific modification regulating their ability to dimerize and interact with DNA. p50, the proteolytic product of p105 (NF-κB1), forms homodimers that bind DNA but lack intrinsic transactivation function, functioning as repressors of transcription from κB promoters. Here, we examine the roles of specific phosphorylation events catalysed by either protein kinase A (PKAc) or Chk1, in regulating the functions of p50 homodimers. LC-MS/MS analysis of proteolysed p50 following in vitro phosphorylation allows us to define Ser328 and Ser337 as PKAc- and Chk1-mediated modifications, and pinpoint an additional four Chk1 phosphosites: Ser65, Thr152, Ser242 and Ser248. Native mass spectrometry (MS) reveals Chk1- and PKAc-regulated disruption of p50 homodimer formation through Ser337. Additionally, we characterise the Chk1-mediated phosphosite, Ser242, as a regulator of DNA binding, with a S242D p50 phosphomimetic exhibiting a > 10-fold reduction in DNA binding affinity. Conformational dynamics of phosphomimetic p50 variants, including S242D, are further explored using ion-mobility MS (IM-MS). Finally, comparative theoretical modelling with experimentally observed p50 conformers, in the absence and presence of DNA, reveals that the p50 homodimer undergoes conformational contraction during electrospray ionisation that is stabilised by complex formation with κB DNA.

Expression of the leukemia-associated CBF{beta}/SMMHC chimeric gene causes transformation of 3T3 cells

DOE Office of Scientific and Technical Information (OSTI.GOV)

Hajra, A.; Liu, P.; Collins, E.S.

1994-09-01

A pericentric inversion of chromosome 16 (inv(16)(p13;q22)) is consistently seen in acute myeloid leukemia of the M4Eo subtype. This inversion fuses almost the entire coding region of the gene encoding of the {beta} subunit of the heterodimeric transcription factor CBF/PEBP2 to the region of the MYH11 gene encoding the rod domain for the smooth muscle myosin heavy chain (SMMHC). To investigate the biological properties of the CBF{beta}/SMMHC fusion protein, we have generated 3T3 cell lines that stably express the CBF{beta}/SMMHC chimeric cDNA or the normal, nonchimeric CBF{beta} and SMMHC cDNAs. 3T3 cells expressing CBF{beta}/SMMHC acquire a transformed phenotype, as indicatedmore » by altered cell morphology, formation of foci, and growth in soft agar. Cells constitutively overexpressing the normal CBF{beta} cDNA or the rod region of SMMHC remain nontransformed. Western blot analysis using antibodies to CBF{beta} and the SMMHC rod demonstrates that stably transfected cells express the appropriate chimeric or normal protein. Electrophoretic mobility shift assays reveal that cells transformed by the chimeric cDNA do not have a CBF-DNA complex of the expected mobility, but instead contain a large complex with CBF DNA-binding activity that fails to migrate out of the gel wells. In order to define the regions of CBF{beta}/SMMHC necessary for 3T3 transformation, we have stably transfected cells with mutant CBF{beta}/SMMHC cDNAs containing various deletions of the coding region. Analysis of these cell lines indicates that the transformation property of CBF{beta}/SMMHC requires regions of CBF{beta} known to be necessary for association with the DNA-binding CBF{alpha} subunit, and also requires an intact SMMHC carboxyl terminus, which is necessary for formation of the coiled coil domain of the myosin rod.« less

Dynamic binding of replication protein a is required for DNA repair

PubMed Central

Chen, Ran; Subramanyam, Shyamal; Elcock, Adrian H.; Spies, Maria; Wold, Marc S.

2016-01-01

Replication protein A (RPA), the major eukaryotic single-stranded DNA (ssDNA) binding protein, is essential for replication, repair and recombination. High-affinity ssDNA-binding by RPA depends on two DNA binding domains in the large subunit of RPA. Mutation of the evolutionarily conserved aromatic residues in these two domains results in a separation-of-function phenotype: aromatic residue mutants support DNA replication but are defective in DNA repair. We used biochemical and single-molecule analyses, and Brownian Dynamics simulations to determine the molecular basis of this phenotype. Our studies demonstrated that RPA binds to ssDNA in at least two modes characterized by different dissociation kinetics. We also showed that the aromatic residues contribute to the formation of the longer-lived state, are required for stable binding to short ssDNA regions and are needed for RPA melting of partially duplex DNA structures. We conclude that stable binding and/or the melting of secondary DNA structures by RPA is required for DNA repair, including RAD51 mediated DNA strand exchange, but is dispensable for DNA replication. It is likely that the binding modes are in equilibrium and reflect dynamics in the RPA–DNA complex. This suggests that dynamic binding of RPA to DNA is necessary for different cellular functions. PMID:27131385

A complex of RAG-1 and RAG-2 proteins persists on DNA after single-strand cleavage at V(D)J recombination signal sequences.

PubMed Central

Grawunder, U; Lieber, M R

1997-01-01

The recombination activating gene (RAG) 1 and 2 proteins are required for initiation of V(D)J recombination in vivo and have been shown to be sufficient to introduce DNA double-strand breaks at recombination signal sequences (RSSs) in a cell-free assay in vitro. RSSs consist of a highly conserved palindromic heptamer that is separated from a slightly less conserved A/T-rich nonamer by either a 12 or 23 bp spacer of random sequence. Despite the high sequence specificity of RAG-mediated cleavage at RSSs, direct binding of the RAG proteins to these sequences has been difficult to demonstrate by standard methods. Even when this can be demonstrated, questions about the order of events for an individual RAG-RSS complex will require methods that monitor aspects of the complex during transitions from one step of the reaction to the next. Here we have used template-independent DNA polymerase terminal deoxynucleotidyl transferase (TdT) in order to assess occupancy of the reaction intermediates by the RAG complex during the reaction. In addition, this approach allows analysis of the accessibility of end products of a RAG-catalyzed cleavage reaction for N nucleotide addition. The results indicate that RAG proteins form a long-lived complex with the RSS once the initial nick is generated, because the 3'-OH group at the nick remains obstructed for TdT-catalyzed N nucleotide addition. In contrast, the 3'-OH group generated at the signal end after completion of the cleavage reaction can be efficiently tailed by TdT, suggesting that the RAG proteins disassemble from the signal end after DNA double-strand cleavage has been completed. Therefore, a single RAG complex maintains occupancy from the first step (nick formation) to the second step (cleavage). In addition, the results suggest that N region diversity at V(D)J junctions within rearranged immunoglobulin and T cell receptor gene loci can only be introduced after the generation of RAG-catalyzed DNA double-strand breaks, i.e. during the DNA end joining phase of the V(D)J recombination reaction. PMID:9060432

Enhancement of photoisomerization of polymethine dyes in complexes with biomacromolecules

NASA Astrophysics Data System (ADS)

Tatikolov, Alexander S.; Akimkin, Timofei M.; Pronkin, Pavel G.; Yarmoluk, Sergiy M.

2013-01-01

Photochemical processes (photoisomerization and generation of the triplet state) of the thiacarbocyanine dyes 3,3',9-trimethylthiacarbocyanine iodide (Cyan 2), 3,3'-diethyl-9-methylthiacarbocyanine iodide (DMTC), and 3,3',9-triethylthiacarbocyanine iodide (TETC) in complexes with biomacromolecules—DNA and chondroitin-4-sulfate—were studied by flash photolysis. It has been shown that, along with generation of the triplet state, enhancement of the photoisomer formation is observed for Cyan 2 and DMTC complexed with the biomolecules. This effect can be explained by the influence of the biopolymer matrix on the potential energy curves of the photoisomerization process.

RPA and XPA interaction with DNA structures mimicking intermediates of the late stages in nucleotide excision repair

PubMed Central

Maltseva, Ekaterina A.

2018-01-01

Replication protein A (RPA) and the xeroderma pigmentosum group A (XPA) protein are indispensable for both pathways of nucleotide excision repair (NER). Here we analyze the interaction of RPA and XPA with DNA containing a flap and different size gaps that imitate intermediates of the late NER stages. Using gel mobility shift assays, we found that RPA affinity for DNA decreased when DNA contained both extended gap and similar sized flap in comparison with gapped-DNA structure. Moreover, crosslinking experiments with the flap-gap DNA revealed that RPA interacts mainly with the ssDNA platform within the long gap and contacts flap in DNA with a short gap. XPA exhibits higher affinity for bubble-DNA structures than to flap-gap-containing DNA. Protein titration analysis showed that formation of the RPA-XPA-DNA ternary complex depends on the protein concentration ratio and these proteins can function as independent players or in tandem. Using fluorescently-labelled RPA, direct interaction of this protein with XPA was detected and characterized quantitatively. The data obtained allow us to suggest that XPA can be involved in the post-incision NER stages via its interaction with RPA. PMID:29320546

RPA and XPA interaction with DNA structures mimicking intermediates of the late stages in nucleotide excision repair.

PubMed

Krasikova, Yuliya S; Rechkunova, Nadejda I; Maltseva, Ekaterina A; Lavrik, Olga I

2018-01-01

Replication protein A (RPA) and the xeroderma pigmentosum group A (XPA) protein are indispensable for both pathways of nucleotide excision repair (NER). Here we analyze the interaction of RPA and XPA with DNA containing a flap and different size gaps that imitate intermediates of the late NER stages. Using gel mobility shift assays, we found that RPA affinity for DNA decreased when DNA contained both extended gap and similar sized flap in comparison with gapped-DNA structure. Moreover, crosslinking experiments with the flap-gap DNA revealed that RPA interacts mainly with the ssDNA platform within the long gap and contacts flap in DNA with a short gap. XPA exhibits higher affinity for bubble-DNA structures than to flap-gap-containing DNA. Protein titration analysis showed that formation of the RPA-XPA-DNA ternary complex depends on the protein concentration ratio and these proteins can function as independent players or in tandem. Using fluorescently-labelled RPA, direct interaction of this protein with XPA was detected and characterized quantitatively. The data obtained allow us to suggest that XPA can be involved in the post-incision NER stages via its interaction with RPA.

RNA binding to APOBEC3G induces the disassembly of functional deaminase complexes by displacing single-stranded DNA substrates

PubMed Central

Polevoda, Bogdan; McDougall, William M.; Tun, Bradley N.; Cheung, Michael; Salter, Jason D.; Friedman, Alan E.; Smith, Harold C.

2015-01-01

APOBEC3G (A3G) DNA deaminase activity requires a holoenzyme complex whose assembly on nascent viral reverse transcripts initiates with A3G dimers binding to ssDNA followed by formation of higher-order A3G homo oligomers. Catalytic activity is inhibited when A3G binds to RNA. Our prior studies suggested that RNA inhibited A3G binding to ssDNA. In this report, near equilibrium binding and gel shift analyses showed that A3G assembly and disassembly on ssDNA was an ordered process involving A3G dimers and multimers thereof. Although, fluorescence anisotropy showed that A3G had similar nanomolar affinity for RNA and ssDNA, RNA stochastically dissociated A3G dimers and higher-order oligomers from ssDNA, suggesting a different modality for RNA binding. Mass spectrometry mapping of A3G peptides cross-linked to nucleic acid suggested ssDNA only bound to three peptides, amino acids (aa) 181–194 in the N-terminus and aa 314–320 and 345–374 in the C-terminus that were part of a continuous exposed surface. RNA bound to these peptides and uniquely associated with three additional peptides in the N- terminus, aa 15–29, 41–52 and 83–99, that formed a continuous surface area adjacent to the ssDNA binding surface. The data predict a mechanistic model of RNA inhibition of ssDNA binding to A3G in which competitive and allosteric interactions determine RNA-bound versus ssDNA-bound conformational states. PMID:26424853

Hda Monomerization by ADP Binding Promotes Replicase Clamp-mediated DnaA-ATP Hydrolysis*S⃞

PubMed Central

Su'etsugu, Masayuki; Nakamura, Kenta; Keyamura, Kenji; Kudo, Yuka; Katayama, Tsutomu

2008-01-01

ATP-DnaA is the initiator of chromosomal replication in Escherichia coli, and the activity of DnaA is regulated by the regulatory inactivation of the DnaA (RIDA) system. In this system, the Hda protein promotes DnaA-ATP hydrolysis to produce inactive ADP-DnaA in a mechanism that is mediated by the DNA-loaded form of the replicase sliding clamp. In this study, we first revealed that hda translation uses an unusual initiation codon, CUG, located downstream of the annotated initiation codon. The CUG initiation codon could be used for restricting the Hda level, as this initiation codon has a low translation efficiency, and the cellular Hda level is only ∼100 molecules per cell. Hda translated using the correct reading frame was purified and found to have a high RIDA activity in vitro. Moreover, we found that Hda has a high affinity for ADP but not for other nucleotides, including ATP. ADP-Hda was active in the RIDA system in vitro and stable in a monomeric state, whereas apo-Hda formed inactive homomultimers. Both ADP-Hda and apo-Hda could form complexes with the DNA-loaded clamp; however, only ADP-Hda-DNA-clamp complexes were highly functional in the following interaction with DnaA. Formation of ADP-Hda was also observed in vivo, and mutant analysis suggested that ADP binding is crucial for cellular Hda activity. Thus, we propose that ADP is a crucial Hda ligand that promotes the activated conformation of the protein. ADP-dependent monomerization might enable the arginine finger of the Hda AAA+ domain to be accessible to ATP bound to the DnaA AAA+ domain. PMID:18977760

Hda monomerization by ADP binding promotes replicase clamp-mediated DnaA-ATP hydrolysis.

PubMed

Su'etsugu, Masayuki; Nakamura, Kenta; Keyamura, Kenji; Kudo, Yuka; Katayama, Tsutomu

2008-12-26

ATP-DnaA is the initiator of chromosomal replication in Escherichia coli, and the activity of DnaA is regulated by the regulatory inactivation of the DnaA (RIDA) system. In this system, the Hda protein promotes DnaA-ATP hydrolysis to produce inactive ADP-DnaA in a mechanism that is mediated by the DNA-loaded form of the replicase sliding clamp. In this study, we first revealed that hda translation uses an unusual initiation codon, CUG, located downstream of the annotated initiation codon. The CUG initiation codon could be used for restricting the Hda level, as this initiation codon has a low translation efficiency, and the cellular Hda level is only approximately 100 molecules per cell. Hda translated using the correct reading frame was purified and found to have a high RIDA activity in vitro. Moreover, we found that Hda has a high affinity for ADP but not for other nucleotides, including ATP. ADP-Hda was active in the RIDA system in vitro and stable in a monomeric state, whereas apo-Hda formed inactive homomultimers. Both ADP-Hda and apo-Hda could form complexes with the DNA-loaded clamp; however, only ADP-Hda-DNA-clamp complexes were highly functional in the following interaction with DnaA. Formation of ADP-Hda was also observed in vivo, and mutant analysis suggested that ADP binding is crucial for cellular Hda activity. Thus, we propose that ADP is a crucial Hda ligand that promotes the activated conformation of the protein. ADP-dependent monomerization might enable the arginine finger of the Hda AAA+ domain to be accessible to ATP bound to the DnaA AAA+ domain.

Mitochondrial Respiration Is Reduced in Atherosclerosis, Promoting Necrotic Core Formation and Reducing Relative Fibrous Cap Thickness.

PubMed

Yu, Emma P K; Reinhold, Johannes; Yu, Haixiang; Starks, Lakshi; Uryga, Anna K; Foote, Kirsty; Finigan, Alison; Figg, Nichola; Pung, Yuh-Fen; Logan, Angela; Murphy, Michael P; Bennett, Martin

2017-12-01

Mitochondrial DNA (mtDNA) damage is present in murine and human atherosclerotic plaques. However, whether endogenous levels of mtDNA damage are sufficient to cause mitochondrial dysfunction and whether decreasing mtDNA damage and improving mitochondrial respiration affects plaque burden or composition are unclear. We examined mitochondrial respiration in human atherosclerotic plaques and whether augmenting mitochondrial respiration affects atherogenesis. Human atherosclerotic plaques showed marked mitochondrial dysfunction, manifested as reduced mtDNA copy number and oxygen consumption rate in fibrous cap and core regions. Vascular smooth muscle cells derived from plaques showed impaired mitochondrial respiration, reduced complex I expression, and increased mitophagy, which was induced by oxidized low-density lipoprotein. Apolipoprotein E-deficient (ApoE -/- ) mice showed decreased mtDNA integrity and mitochondrial respiration, associated with increased mitochondrial reactive oxygen species. To determine whether alleviating mtDNA damage and increasing mitochondrial respiration affects atherogenesis, we studied ApoE -/- mice overexpressing the mitochondrial helicase Twinkle (Tw + /ApoE -/- ). Tw + /ApoE -/- mice showed increased mtDNA integrity, copy number, respiratory complex abundance, and respiration. Tw + /ApoE -/- mice had decreased necrotic core and increased fibrous cap areas, and Tw + /ApoE -/- bone marrow transplantation also reduced core areas. Twinkle increased vascular smooth muscle cell mtDNA integrity and respiration. Twinkle also promoted vascular smooth muscle cell proliferation and protected both vascular smooth muscle cells and macrophages from oxidative stress-induced apoptosis. Endogenous mtDNA damage in mouse and human atherosclerosis is associated with significantly reduced mitochondrial respiration. Reducing mtDNA damage and increasing mitochondrial respiration decrease necrotic core and increase fibrous cap areas independently of changes in reactive oxygen species and may be a promising therapeutic strategy in atherosclerosis. © 2017 The Authors.

Formation of Nucleobases from the UV Irradiation of Pyrimidine in Interstellar Ice Analogs

NASA Technical Reports Server (NTRS)

Milam, Stefanie N.; Nuevo, Michel; Sandford, Scott A.; Elsila, Jamie E.; Dworkin, Jason P.

2010-01-01

Previous laboratory simulations showed that complex molecules, including prebiotic compounds/can be formed under interstellar conditions from the vacuum UV irradiation of interstellar ice analogs containing H2O, CO, NH3 etc. Although some complex prebiotic species have not been confirmed In the interstellar medium, they are known to be present in meteorites. Nucleobases, the building blocks of DNA and RNA, have also been detected in meteorites. Here, we present a study of the formation of pyrimidine-based compounds from the UV irradiation of pyrimidine in H2O- and/or NH3-ices at 20-30 K, Our results show that various derivatives, induding the nucleobases uracil and cytosine, are formed under these conditions.

Specific interaction of mutant p53 with regions of matrix attachment region DNA elements (MARs) with a high potential for base-unpairing

PubMed Central

Will, Katrin; Warnecke, Gabriele; Wiesmüller, Lisa; Deppert, Wolfgang

1998-01-01

Mutant, but not wild-type p53 binds with high affinity to a variety of MAR-DNA elements (MARs), suggesting that MAR-binding of mutant p53 relates to the dominant-oncogenic activities proposed for mutant p53. MARs recognized by mutant p53 share AT richness and contain variations of an AATATATTT “DNA-unwinding motif,” which enhances the structural dynamics of chromatin and promotes regional DNA base-unpairing. Mutant p53 specifically interacted with MAR-derived oligonucleotides carrying such unwinding motifs, catalyzing DNA strand separation when this motif was located within a structurally labile sequence environment. Addition of GC-clamps to the respective MAR-oligonucleotides or introducing mutations into the unwinding motif strongly reduced DNA strand separation, but supported the formation of tight complexes between mutant p53 and such oligonucleotides. We conclude that the specific interaction of mutant p53 with regions of MAR-DNA with a high potential for base-unpairing provides the basis for the high-affinity binding of mutant p53 to MAR-DNA. PMID:9811860

The role of host DNA ligases in hepadnavirus covalently closed circular DNA formation

PubMed Central

Long, Quanxin; Yan, Ran; Hu, Jieli; Cai, Dawei; Kim, Elena S.; Zhang, Hu; Liu, Yuanjie

2017-01-01

Hepadnavirus covalently closed circular (ccc) DNA is the bona fide viral transcription template, which plays a pivotal role in viral infection and persistence. Upon infection, the non-replicative cccDNA is converted from the incoming and de novo synthesized viral genomic relaxed circular (rc) DNA, presumably through employment of the host cell’s DNA repair mechanisms in the nucleus. The conversion of rcDNA into cccDNA requires preparation of the extremities at the nick/gap regions of rcDNA for strand ligation. After screening 107 cellular DNA repair genes, we herein report that the cellular DNA ligase (LIG) 1 and 3 play a critical role in cccDNA formation. Ligase inhibitors or functional knock down/out of LIG1/3 significantly reduced cccDNA production in an in vitro cccDNA formation assay, and in cccDNA-producing cells without direct effect on viral core DNA replication. In addition, transcomplementation of LIG1/3 in the corresponding knock-out or knock-down cells was able to restore cccDNA formation. Furthermore, LIG4, a component in non-homologous end joining DNA repair apparatus, was found to be responsible for cccDNA formation from the viral double stranded linear (dsl) DNA, but not rcDNA. In conclusion, we demonstrate that hepadnaviruses utilize the whole spectrum of host DNA ligases for cccDNA formation, which sheds light on a coherent molecular pathway of cccDNA biosynthesis, as well as the development of novel antiviral strategies for treatment of hepatitis B. PMID:29287110

Design, characterization, teratogenicity testing, antibacterial, antifungal and DNA interaction of few high spin Fe(II) Schiff base amino acid complexes

NASA Astrophysics Data System (ADS)

Abdel-Rahman, Laila H.; El-Khatib, Rafat M.; Nassr, Lobna A. E.; Abu-Dief, Ahmed M.; Lashin, Fakhr El-Din

2013-07-01

In this study, new Fe(II) Schiff base amino acid chelates derived from the condensation of o-hydroxynaphthaldehyde with L-alanine, L-phenylalanine, L-aspartic acid, L-histidine and L-arginine were synthesized and characterized via elemental, thermogravimetric analysis, molar conductance, IR, electronic, mass spectra and magnetic moment measurements. The stoichiometry and the stability constants of the complexes were determined spectrophotometrically. Correlation of all spectroscopic data suggested that Schiff bases ligands exhibited tridentate with ONO sites coordinating to the metal ions via protonated phenolic-OH, azomethine-N and carboxylate-O with the general formulae [Fe(HL)2]·nH2O. But in case of L-histidine, the ligand acts as tetradentate via deprotonated phenolic-OH, azomethine-N, carboxylate-O and N-imidazole ring ([FeL(H2O)2]·2H2O), where HL = mono anion and L = dianion of the ligand. The structure of the prepared complexes is suggested to be octahedral. The prepared complexes were tested for their teratogenicity on chick embryos and found to be safe until a concentration of 100 μg/egg with full embryos formation. Moreover, the interaction between CT-DNA and the investigated complexes were followed by spectrophotometric and viscosity measurements. It was found that, the prepared complexes bind to DNA via classical intercalative mode and showed a different DNA activity with the sequence: nhi > nari > nali > nasi > nphali. Furthermore, the free ligands and their complexes are screened for their in vitro antibacterial and antifungal activity against three types of bacteria, Escherichia coli, Pseudomonas aeruginosa and Bacillus cereus and three types of anti fungal cultures, Penicillium purpurogenium, Aspergillus flavus and Trichotheium rosium in order to assess their antimicrobial potential. The results show that the metal complexes are more reactive with respect to their corresponding Schiff base amino acid ligands.

Tailored protein encapsulation into a DNA host using geometrically organized supramolecular interactions

NASA Astrophysics Data System (ADS)

Sprengel, Andreas; Lill, Pascal; Stegemann, Pierre; Bravo-Rodriguez, Kenny; Schöneweiß, Elisa-C.; Merdanovic, Melisa; Gudnason, Daniel; Aznauryan, Mikayel; Gamrad, Lisa; Barcikowski, Stephan; Sanchez-Garcia, Elsa; Birkedal, Victoria; Gatsogiannis, Christos; Ehrmann, Michael; Saccà, Barbara

2017-02-01

The self-organizational properties of DNA have been used to realize synthetic hosts for protein encapsulation. However, current strategies of DNA-protein conjugation still limit true emulation of natural host-guest systems, whose formation relies on non-covalent bonds between geometrically matching interfaces. Here we report one of the largest DNA-protein complexes of semisynthetic origin held in place exclusively by spatially defined supramolecular interactions. Our approach is based on the decoration of the inner surface of a DNA origami hollow structure with multiple ligands converging to their corresponding binding sites on the protein surface with programmable symmetry and range-of-action. Our results demonstrate specific host-guest recognition in a 1:1 stoichiometry and selectivity for the guest whose size guarantees sufficient molecular diffusion preserving short intermolecular distances. DNA nanocontainers can be thus rationally designed to trap single guest molecules in their native form, mimicking natural strategies of molecular recognition and anticipating a new method of protein caging.

Single molecule techniques in DNA repair: A primer

PubMed Central

Hughes, Craig D.; Simons, Michelle; Mackenzie, Cassidy E.; Van Houten, Bennett; Kad, Neil M.

2016-01-01

A powerful new approach has become much more widespread and offers insights into aspects of DNA repair unattainable with billions of molecules. Single molecule techniques can be used to image, manipulate or characterize the action of a single repair protein on a single strand of DNA. This allows search mechanisms to be probed, and the effects of force to be understood. These physical aspects can dominate a biochemical reaction, where at the ensemble level their nuances are obscured. In this paper we discuss some of the many technical advances that permit study at the single molecule level. We focus on DNA repair to which these techniques are actively being applied. DNA repair is also a process that encompasses so much of what single molecule studies benefit – searching for targets, complex formation, sequential biochemical reactions and substrate hand-off to name just a few. We discuss how single molecule biophysics is poised to transform our understanding of biological systems, in particular DNA repair. PMID:24819596

Atomistic Molecular Dynamics Simulations of Mitochondrial DNA Polymerase γ: Novel Mechanisms of Function and Pathogenesis.

PubMed

Euro, Liliya; Haapanen, Outi; Róg, Tomasz; Vattulainen, Ilpo; Suomalainen, Anu; Sharma, Vivek

2017-03-07

DNA polymerase γ (Pol γ) is a key component of the mitochondrial DNA replisome and an important cause of neurological diseases. Despite the availability of its crystal structures, the molecular mechanism of DNA replication, the switch between polymerase and exonuclease activities, the site of replisomal interactions, and functional effects of patient mutations that do not affect direct catalysis have remained elusive. Here we report the first atomistic classical molecular dynamics simulations of the human Pol γ replicative complex. Our simulation data show that DNA binding triggers remarkable changes in the enzyme structure, including (1) completion of the DNA-binding channel via a dynamic subdomain, which in the apo form blocks the catalytic site, (2) stabilization of the structure through the distal accessory β-subunit, and (3) formation of a putative transient replisome-binding platform in the "intrinsic processivity" subdomain of the enzyme. Our data indicate that noncatalytic mutations may disrupt replisomal interactions, thereby causing Pol γ-associated neurodegenerative disorders.

Structural and Functional Basis for an EBNA1 Hexameric Ring in Epstein-Barr Virus Episome Maintenance.

PubMed

Deakyne, Julianna S; Malecka, Kimberly A; Messick, Troy E; Lieberman, Paul M

2017-10-01

Epstein-Barr virus (EBV) establishes a stable latent infection that can persist for the life of the host. EBNA1 is required for the replication, maintenance, and segregation of the latent episome, but the structural features of EBNA1 that confer each of these functions are not completely understood. Here, we have solved the X-ray crystal structure of an EBNA1 DNA-binding domain (DBD) and discovered a novel hexameric ring oligomeric form. The oligomeric interface pivoted around residue T585 as a joint that links and stabilizes higher-order EBNA1 complexes. Substitution mutations around the interface destabilized higher-order complex formation and altered the cooperative DNA-binding properties of EBNA1. Mutations had both positive and negative effects on EBNA1-dependent DNA replication and episome maintenance with OriP. We found that one naturally occurring polymorphism in the oligomer interface (T585P) had greater cooperative DNA binding in vitro , minor defects in DNA replication, and pronounced defects in episome maintenance. The T585P mutant was compromised for binding to OriP in vivo as well as for assembling the origin recognition complex subunit 2 (ORC2) and trimethylated histone 3 lysine 4 (H3K4me3) at OriP. The T585P mutant was also compromised for forming stable subnuclear foci in living cells. These findings reveal a novel oligomeric structure of EBNA1 with an interface subject to naturally occurring polymorphisms that modulate EBNA1 functional properties. We propose that EBNA1 dimers can assemble into higher-order oligomeric structures important for diverse functions of EBNA1. IMPORTANCE Epstein-Barr virus is a human gammaherpesvirus that is causally associated with various cancers. Carcinogenic properties are linked to the ability of the virus to persist in the latent form for the lifetime of the host. EBNA1 is a sequence-specific DNA-binding protein that is consistently expressed in EBV tumors and is the only viral protein required to maintain the viral episome during latency. The structural and biochemical mechanisms by which EBNA1 allows the long-term persistence of the EBV genome are currently unclear. Here, we have solved the crystal structure of an EBNA1 hexameric ring and characterized key residues in the interface required for higher-order complex formation and long-term plasmid maintenance. Copyright © 2017 American Society for Microbiology.

Pseudomonas aeruginosa AmrZ Binds to Four Sites in the algD Promoter, Inducing DNA-AmrZ Complex Formation and Transcriptional Activation.

PubMed

Xu, Binjie; Soukup, Randal J; Jones, Christopher J; Fishel, Richard; Wozniak, Daniel J

2016-10-01

During late stages of cystic fibrosis pulmonary infections, Pseudomonas aeruginosa often overproduces the exopolysaccharide alginate, protecting the bacterial community from host immunity and antimicrobials. The transcription of the alginate biosynthesis operon is under tight control by a number of factors, including AmrZ, the focus of this study. Interestingly, multiple transcription factors interact with the far-upstream region of this promoter (PalgD), in which one AmrZ binding site has been identified previously. The mechanisms of AmrZ binding and subsequent activation remain unclear and require more-detailed investigation. In this study, in-depth examinations elucidated four AmrZ binding sites, and their disruption eliminated AmrZ binding and promoter activation. Furthermore, our in vitro fluorescence resonance energy transfer experiments suggest that AmrZ holds together multiple binding sites in PalgD and thereafter induces the formation of higher-order DNA-AmrZ complexes. To determine the importance of interactions between those AmrZ oligomers in the cell, a DNA phasing experiment was performed. PalgD transcription was significantly impaired when the relative phase between AmrZ binding sites was reversed (5 bp), while a full-DNA-turn insertion (10 bp) restored promoter activity. Taken together, the investigations presented here provide a deeper mechanistic understanding of AmrZ-mediated binding to PalgD IMPORTANCE: Overproduction of the exopolysaccharide alginate provides protection to Pseudomonas aeruginosa against antimicrobial treatments and is associated with chronic P. aeruginosa infections in the lungs of cystic fibrosis patients. In this study, we combined a variety of microbiological, genetic, biochemical, and biophysical approaches to investigate the activation of the alginate biosynthesis operon promoter by a key transcription factor named AmrZ. This study has provided important new information on the mechanism of activation of this extremely complex promoter. Copyright © 2016, American Society for Microbiology. All Rights Reserved.

Association, intrinsic shape, and molecular recognition: Elucidating DNA biophysics through coarse-grained simulation

NASA Astrophysics Data System (ADS)

Freeman, Gordon Samuel

DNA is of central importance in biology as it is responsible for carrying, copying, and translating the genetic code into the building blocks that comprise life. In order to accomplish these tasks, the DNA molecule must be versatile and robust. Indeed, the underlying molecular interactions that allow DNA to execute these tasks are complex and their origins are only beginning to be understood. While experiments are able to elucidate many key biophysical phenomena, there remain many unanswered questions. Molecular simulation is able to shed light on phenomena at the molecular scale and provide information that is missing from experimental views of DNA behavior. In this dissertation I use state-of-the-art coarse-grained DNA models to address two key problems. In the first, metadynamics calculations are employed to uncover the free energy surface of two complimentary DNA strands. This free energy surface takes on the appearance of a hybridization funnel and reveals candidates for intermediate states in the hybridization of short DNA oligomers. Such short oligomers are important building blocks for DNA-driven self-assembly and the mechanism of hybridization in this regime is not well understood. The second problem is that of nucleosome formation. Nucleosomes are the fundamental subunit of genome compaction in the nucleus of a cell. As such, nucleosomes are a key epigenetic factor and affect gene expression and the ability of DNA-binding proteins to locate and bind to the appropriate position in the genome. However, the factors that drive nucleosome positioning are not well understood. While DNA sequence is known to affect nucleosome formation, the mechanism by which it does so has not been established and a number of hypotheses explaining this sequence-dependence exist in the literature. I demonstrate that DNA shape dominates this process with contributions arising from both intrinsic DNA curvature as well as DNA-protein interactions driven by sequence-dependent variations in minor groove dimensions.

In Vitro Interactions between 17β-Estradiol and DNA Result in Formation of the Hormone-DNA Complexes

PubMed Central

Heger, Zbynek; Guran, Roman; Zitka, Ondrej; Beklova, Miroslava; Adam, Vojtech; Kizek, Rene

2014-01-01

Beyond the role of 17β-estradiol (E2) in reproduction and during the menstrual cycle, it has been shown to modulate numerous physiological processes such as cell proliferation, apoptosis, inflammation and ion transport in many tissues. The pathways in which estrogens affect an organism have been partially described, although many questions still exist regarding estrogens’ interaction with biomacromolecules. Hence, the present study showed the interaction of four oligonucleotides (17, 20, 24 and/or 38-mer) with E2. The strength of these interactions was evaluated using optical methods, showing that the interaction is influenced by three major factors, namely: oligonucleotide length, E2 concentration and interaction time. In addition, the denaturation phenomenon of DNA revealed that the binding of E2 leads to destabilization of hydrogen bonds between the nitrogenous bases of DNA strands resulting in a decrease of their melting temperatures (Tm). To obtain a more detailed insight into these interactions, MALDI-TOF mass spectrometry was employed. This study revealed that E2 with DNA forms non-covalent physical complexes, observed as the mass shifts for app. 270 Da (Mr of E2) to higher molecular masses. Taken together, our results indicate that E2 can affect biomacromolecules, as circulating oligonucleotides, which can trigger mutations, leading to various unwanted effects. PMID:25089777

Interaction of the Retinoblastoma Protein with Orc1 and Its Recruitment to Human Origins of DNA Replication

PubMed Central

Mendoza-Maldonado, Ramiro; Paolinelli, Roberta; Galbiati, Laura; Giadrossi, Sara; Giacca, Mauro

2010-01-01

Background The retinoblastoma protein (Rb) is a crucial regulator of cell cycle progression by binding with E2F transcription factor and repressing the expression of a variety of genes required for the G1-S phase transition. Methodology/Principal Findings Here we show that Rb and E2F1 directly participate in the control of initiation of DNA replication in human HeLa, U2OS and T98G cells by specifically binding to origins of DNA replication in a cell cycle regulated manner. We show that, both in vitro and inside the cells, the largest subunit of the origin recognition complex (Orc1) specifically binds hypo-phosphorylated Rb and that this interaction is competitive with the binding of Rb to E2F1. The displacement of Rb-bound Orc1 by E2F1 at origins of DNA replication marks the progression of the G1 phase of the cell cycle toward the G1-S border. Conclusions/Significance The participation of Rb and E2F1 in the formation of the multiprotein complex that binds origins of DNA replication in mammalian cells appears to represent an effective mechanism to couple the expression of genes required for cell cycle progression to the activation of DNA replication. PMID:21085491

Inactive DNMT3B Splice Variants Modulate De Novo DNA Methylation

PubMed Central

Gordon, Catherine A.; Hartono, Stella R.; Chédin, Frédéric

2013-01-01

Inactive DNA methyltransferase (DNMT) 3B splice isoforms are associated with changes in DNA methylation, yet the mechanisms by which they act remain largely unknown. Using biochemical and cell culture assays, we show here that the inactive DNMT3B3 and DNMT3B4 isoforms bind to and regulate the activity of catalytically competent DNMT3A or DNMT3B molecules. DNMT3B3 modestly stimulated the de novo methylation activity of DNMT3A and also counteracted the stimulatory effects of DNMT3L, therefore leading to subtle and contrasting effects on activity. DNMT3B4, by contrast, significantly inhibited de novo DNA methylation by active DNMT3 molecules, most likely due to its ability to reduce the DNA binding affinity of co-complexes, thereby sequestering them away from their substrate. Immunocytochemistry experiments revealed that in addition to their effects on the intrinsic catalytic function of active DNMT3 enzymes, DNMT3B3 and DNMT34 drive distinct types of chromatin compaction and patterns of histone 3 lysine 9 tri-methylation (H3K9me3) deposition. Our findings suggest that regulation of active DNMT3 members through the formation of co-complexes with inactive DNMT3 variants is a general mechanism by which DNMT3 variants function. This may account for some of the changes in DNA methylation patterns observed during development and disease. PMID:23894490

Arabidopsis SEPALLATA proteins differ in cooperative DNA-binding during the formation of floral quartet-like complexes

PubMed Central

Jetha, Khushboo; Theißen, Günter; Melzer, Rainer

2014-01-01

The SEPALLATA (SEP) genes of Arabidopsis thaliana encode MADS-domain transcription factors that specify the identity of all floral organs. The four Arabidopsis SEP genes function in a largely yet not completely redundant manner. Here, we analysed interactions of the SEP proteins with DNA. All of the proteins were capable of forming tetrameric quartet-like complexes on DNA fragments carrying two sequence elements termed CArG-boxes. Distances between the CArG-boxes for strong cooperative DNA-binding were in the range of 4–6 helical turns. However, SEP1 also bound strongly to CArG-box pairs separated by smaller or larger distances, whereas SEP2 preferred large and SEP4 preferred small inter-site distances for binding. Cooperative binding of SEP3 was comparatively weak for most of the inter-site distances tested. All SEP proteins constituted floral quartet-like complexes together with the floral homeotic proteins APETALA3 (AP3) and PISTILLATA (PI) on the target genes AP3 and SEP3. Our results suggest an important part of an explanation for why the different SEP proteins have largely, but not completely redundant functions in determining floral organ identity: they may bind to largely overlapping, but not identical sets of target genes that differ in the arrangement and spacing of the CArG-boxes in their cis-regulatory regions. PMID:25183521

Neurospora Importin α Is Required for Normal Heterochromatic Formation and DNA Methylation

PubMed Central

Klocko, Andrew D.; Rountree, Michael R.; Grisafi, Paula L.; Hays, Shan M.; Adhvaryu, Keyur K.; Selker, Eric U.

2015-01-01

Heterochromatin and associated gene silencing processes play roles in development, genome defense, and chromosome function. In many species, constitutive heterochromatin is decorated with histone H3 tri-methylated at lysine 9 (H3K9me3) and cytosine methylation. In Neurospora crassa, a five-protein complex, DCDC, catalyzes H3K9 methylation, which then directs DNA methylation. Here, we identify and characterize a gene important for DCDC function, dim-3 (defective in methylation-3), which encodes the nuclear import chaperone NUP-6 (Importin α). The critical mutation in dim-3 results in a substitution in an ARM repeat of NUP-6 and causes a substantial loss of H3K9me3 and DNA methylation. Surprisingly, nuclear transport of all known proteins involved in histone and DNA methylation, as well as a canonical transport substrate, appear normal in dim-3 strains. Interactions between DCDC members also appear normal, but the nup-6dim-3 allele causes the DCDC members DIM-5 and DIM-7 to mislocalize from heterochromatin and NUP-6dim-3 itself is mislocalized from the nuclear envelope, at least in conidia. GCN-5, a member of the SAGA histone acetyltransferase complex, also shows altered localization in dim-3, raising the possibility that NUP-6 is necessary to localize multiple chromatin complexes following nucleocytoplasmic transport. PMID:25793375

UV-induced replication arrest in the xeroderma pigmentosum variant leads to DNA double-strand breaks, γ-H2AX formation, and Mre11 relocalization

PubMed Central

Limoli, Charles L.; Giedzinski, Erich; Bonner, William M.; Cleaver, James E.

2002-01-01

UV-induced replication arrest in the xeroderma pigmentosum variant (XPV) but not in normal cells leads to an accumulation of the Mre11/Rad50/Nbs1 complex and phosphorylated histone H2AX (γ-H2AX) in large nuclear foci at sites of stalled replication forks. These complexes have been shown to signal the presence of DNA damage, in particular, double-strand breaks (DSBs). This finding suggests that UV damage leads to the formation of DSBs during the course of replication arrest. After UV irradiation, XPV cells showed a fluence-dependent increase in the yield of γ-H2AX foci that paralleled the production of Mre11 foci. The percentage of foci-positive cells increased rapidly (10–15%) up to fluences of 10 J⋅m−2 before saturating at higher fluences. Frequencies of γ-H2AX and Mre11 foci both reached maxima at 4 h after UV irradiation. This pattern contrasts sharply to the situation observed after x-irradiation, where peak levels of γ-H2AX foci were found to precede the formation of Mre11 foci by several hours. The nuclear distributions of γ-H2AX and Mre11 were found to colocalize spatially after UV- but not x-irradiation. UV-irradiated XPV cells showed a one-to-one correspondence between Mre11 and γ-H2AX foci-positive cells. These results show that XPV cells develop DNA DSBs during the course of UV-induced replication arrest. These UV-induced foci occur in cells that are unable to carry out efficient bypass replication of UV damage and may contribute to further genetic variation. PMID:11756691

Electrostatic occlusion and quaternary structural ion pairing are key determinants of Cu(I)-mediated allostery in the copper-sensing operon repressor (CsoR).

PubMed

Chang, Feng-Ming James; Martin, Julia E; Giedroc, David P

2015-04-21

The copper-sensing operon repressor (CsoR) is an all-α-helical disc-shaped D2-symmetric homotetramer that forms a 2:1 tetramer/DNA operator complex and represses the expression of copper-resistance genes in a number of bacteria. A previous bioinformatics analysis of CsoR-family repressors distributes Cu(I)-sensing CsoRs in four of seven distinct clades on the basis of global sequence similarity. In this work, we define energetically important determinants of DNA binding in the apo-state (ΔΔGbind), and for allosteric negative coupling of Cu(I) binding to DNA binding (ΔΔGc) in a model clade IV CsoR from Geobacillus thermodenitrificans (Gt) of known structure, by selectively targeting for mutagenesis those charged residues uniquely conserved in clade IV CsoRs. These include a folded N-terminal "tail" and a number of Cu(I)-sensor and clade-specific residues that when mapped onto a model of Cu(I)-bound Gt CsoR define a path across one face of the tetramer. We find that Cu(I)-binding prevents formation of the 2:1 "sandwich" complex rather than DNA binding altogether. Folding of the N-terminal tail (residues R18, E22, R74) upon Cu-binding to the periphery of the tetramer inhibits assembly of the 2:1 apoprotein-DNA complex. In contrast, Ala substitution of residues that surround the central "hole" (R65, K101) in the tetramer, as well R48, impact DNA binding. We also identify a quaternary structural ion-pair, E73-K101″, that crosses the tetramer interface, charge-reversal of which restores DNA binding activity, allosteric regulation by Cu(I), and transcriptional derepression by Cu(I) in cells. These findings suggest an "electrostatic occlusion" model, in which basic residues important for DNA binding and/or allostery become sequestered via ion-pairing specifically in the Cu(I)-bound state, and this aids in copper-dependent disassembly of a repression complex.

Crystal Structure of the Vaccinia Virus DNA Polymerase Holoenzyme Subunit D4 in Complex with the A20 N-Terminal Domain

PubMed Central

Contesto-Richefeu, Céline; Tarbouriech, Nicolas; Brazzolotto, Xavier; Betzi, Stéphane; Morelli, Xavier; Burmeister, Wim P.; Iseni, Frédéric

2014-01-01

Vaccinia virus polymerase holoenzyme is composed of the DNA polymerase E9, the uracil-DNA glycosylase D4 and A20, a protein with no known enzymatic activity. The D4/A20 heterodimer is the DNA polymerase co-factor whose function is essential for processive DNA synthesis. Genetic and biochemical data have established that residues located in the N-terminus of A20 are critical for binding to D4. However, no information regarding the residues of D4 involved in A20 binding is yet available. We expressed and purified the complex formed by D4 and the first 50 amino acids of A20 (D4/A201–50). We showed that whereas D4 forms homodimers in solution when expressed alone, D4/A201–50 clearly behaves as a heterodimer. The crystal structure of D4/A201–50 solved at 1.85 Å resolution reveals that the D4/A20 interface (including residues 167 to 180 and 191 to 206 of D4) partially overlaps the previously described D4/D4 dimer interface. A201–50 binding to D4 is mediated by an α-helical domain with important leucine residues located at the very N-terminal end of A20 and a second stretch of residues containing Trp43 involved in stacking interactions with Arg167 and Pro173 of D4. Point mutations of the latter residues disturb D4/A201–50 formation and reduce significantly thermal stability of the complex. Interestingly, small molecule docking with anti-poxvirus inhibitors selected to interfere with D4/A20 binding could reproduce several key features of the D4/A201–50 interaction. Finally, we propose a model of D4/A201–50 in complex with DNA and discuss a number of mutants described in the literature, which affect DNA synthesis. Overall, our data give new insights into the assembly of the poxvirus DNA polymerase cofactor and may be useful for the design and rational improvement of antivirals targeting the D4/A20 interface. PMID:24603707

A novel host factor for integration of mycobacteriophage L5

PubMed Central

Pedulla, Marisa L.; Lee, Mong Hong; Lever, Dawn C.; Hatfull, Graham F.

1996-01-01

Bacterial integration host factors (IHFs) play central roles in the cellular processes of recombination, DNA replication, transcription, and bacterial pathogenesis. We describe here a novel mycobacterial IHF (mIHF) of Mycobacterium smegmatis and Mycobacterium tuberculosis that stimulates integration of mycobacteriophage L5. mIHF is the product of a single gene and is unrelated at the sequence level to other integration host factors. By itself, mIHF does not bind preferentially to attP DNA, although it significantly alters the pattern of integrase (Int) binding, promoting the formation of specific integrase–mIHF–attP intasome complexes. PMID:8986825

Escherichia coli promoter sequences predict in vitro RNA polymerase selectivity.

PubMed

Mulligan, M E; Hawley, D K; Entriken, R; McClure, W R

1984-01-11

We describe a simple algorithm for computing a homology score for Escherichia coli promoters based on DNA sequence alone. The homology score was related to 31 values, measured in vitro, of RNA polymerase selectivity, which we define as the product KBk2, the apparent second order rate constant for open complex formation. We found that promoter strength could be predicted to within a factor of +/-4.1 in KBk2 over a range of 10(4) in the same parameter. The quantitative evaluation was linked to an automated (Apple II) procedure for searching and evaluating possible promoters in DNA sequence files.

Complexes of oligo(poly)nucleotides with structural anomalies

NASA Astrophysics Data System (ADS)

Dolinnaya, N. G.; Gryaznova, O. I.

1989-08-01

The results of studies on the structure and properties of DNA-RNA hybrids and complexes of oligo(poly)nucleotides containing non-canonical base pairs or unpaired bases both within and at the ends of the double helix are surveyed. The methods used in the study of such systems are briefly characterised: X-ray diffraction analysis, NMR and UV spectroscopy, circular dichroism, scanning microcalorimetry, etc. A comparative analysis of the influence of the non-canonical pairs on the structure and the energetic and kinetic parameters of the formation and dissociation of the oligonucleotide complexes has been carried out. The question of the stability of the non-canonical pairs as a function of their nature and position in the double helix is considered. The mechanisms of the formation of the hydrogen bonds between the bases of non-complementary pairs are discussed. The bibliography includes 171 references.

Mutations in genes encoding condensin complex proteins cause microcephaly through decatenation failure at mitosis

PubMed Central

Martin, Carol-Anne; Murray, Jennie E.; Carroll, Paula; Leitch, Andrea; Mackenzie, Karen J.; Halachev, Mihail; Fetit, Ahmed E.; Keith, Charlotte; Bicknell, Louise S.; Fluteau, Adeline; Gautier, Philippe; Hall, Emma A.; Joss, Shelagh; Soares, Gabriela; Silva, João; Bober, Michael B.; Duker, Angela; Wise, Carol A.; Quigley, Alan J.; Phadke, Shubha R.; Wood, Andrew J.; Vagnarelli, Paola; Jackson, Andrew P.

2016-01-01

Compaction of chromosomes is essential for accurate segregation of the genome during mitosis. In vertebrates, two condensin complexes ensure timely chromosome condensation, sister chromatid disentanglement, and maintenance of mitotic chromosome structure. Here, we report that biallelic mutations in NCAPD2, NCAPH, or NCAPD3, encoding subunits of these complexes, cause microcephaly. In addition, hypomorphic Ncaph2 mice have significantly reduced brain size, with frequent anaphase chromatin bridge formation observed in apical neural progenitors during neurogenesis. Such DNA bridges also arise in condensin-deficient patient cells, where they are the consequence of failed sister chromatid disentanglement during chromosome compaction. This results in chromosome segregation errors, leading to micronucleus formation and increased aneuploidy in daughter cells. These findings establish “condensinopathies” as microcephalic disorders, with decatenation failure as an additional disease mechanism for microcephaly, implicating mitotic chromosome condensation as a key process ensuring mammalian cerebral cortex size. PMID:27737959

Annonalide and derivatives: Semisynthesis, cytotoxic activities and studies on interaction of annonalide with DNA.

PubMed

Marques, Ricardo A; Gomes, Akenaton O C V; de Brito, Maria V; Dos Santos, Ana L P; da Silva, Gladyane S; de Lima, Leandro B; Nunes, Fátima M; de Mattos, Marcos C; de Oliveira, Fátima C E; do Ó Pessoa, Cláudia; de Moraes, Manoel O; de Fátima, Ângelo; Franco, Lucas L; Silva, Marina de M; Dantas, Maria Dayanne de A; Santos, Josué C C; Figueiredo, Isis M; da Silva-Júnior, Edeíldo F; de Aquino, Thiago M; de Araújo-Júnior, João X; de Oliveira, Maria C F; Leslie Gunatilaka, A A

2018-02-01

The cytotoxic activity of the pimarane diterpene annonalide (1) and nine of its semisynthetic derivatives (2-10) was investigated against the human tumor cell lines HL-60 (leukemia), PC-3 (prostate adenocarcinoma), HepG2 (hepatocellular carcinoma), SF-295 (glioblastoma) and HCT-116 (colon cancer), and normal mouse fibroblast (L929) cells. The preparation of 2-10 involved derivatization of the side chain of 1 at C-13. Except for 2, all derivatives are being reported for the first time. Most of the tested compounds presented IC 50 s below 4.0 μM, being considered potential antitumor agents. The structures of all new compounds were elucidated by spectroscopic analyses including 2D NMR and HRMS. Additionally, the interaction of annonalide (1) with ctDNA was evaluated using spectroscopic techniques, and the formation of a supramolecular complex with the macromolecule was confirmed. Competition assays with fluorescent probes (Hoechst and ethidium bromide) and theoretical studies confirmed that 1 interacts preferentially via DNA intercalation with stoichiometric ratio of 1:1 (1:ctDNA). The ΔG value was calculated as -28.24 kJ mol -1 , and indicated that the interaction process occurs spontaneously. Docking studies revealed that van der Walls is the most important interaction in 1-DNA and EB-DNA complexes, and that both ligands (1 and EB) interact with the same DNA residues (DA6, DA17 and DT19). Copyright © 2018. Published by Elsevier B.V.

Molecular spectroscopic and thermodynamic studies on the interaction of anti-platelet drug ticlopidine with calf thymus DNA

NASA Astrophysics Data System (ADS)

Afrin, Shumaila; Rahman, Yusra; Sarwar, Tarique; Husain, Mohammed Amir; Ali, Abad; Shamsuzzaman; Tabish, Mohammad

2017-11-01

Ticlopidine is an anti-platelet drug which belongs to the thienopyridine structural family and exerts its effect by functioning as an ADP receptor inhibitor. Ticlopidine inhibits the expression of TarO gene in S. aureus and may provide protection against MRSA. Groove binding agents are known to disrupt the transcription factor DNA complex and consequently inhibit gene expression. Understanding the mechanism of interaction of ticlopidine with DNA can prove useful in the development of a rational drug designing system. At present, there is no such study on the interaction of anti-platelet drugs with nucleic acids. A series of biophysical experiments were performed to ascertain the binding mode between ticlopidine and calf thymus DNA. UV-visible and fluorescence spectroscopic experiments confirmed the formation of a complex between ticlopidine and calf thymus DNA. Moreover, the values of binding constant were found to be in the range of 103 M- 1, which is indicative of groove binding between ticlopidine and calf thymus DNA. These results were further confirmed by studying the effect of denaturation on double stranded DNA, iodide quenching, viscometric studies, thermal melting profile as well as CD spectral analysis. The thermodynamic profile of the interaction was also determined using isothermal titration calorimetric studies. The reaction was found to be endothermic and the parameters obtained were found to be consistent with those of known groove binders. In silico molecular docking studies further corroborated well with the experimental results.

Secure and effective gene delivery system of plasmid DNA coated by polynucleotide.

PubMed

Kodama, Yukinobu; Ohkubo, Chikako; Kurosaki, Tomoaki; Egashira, Kanoko; Sato, Kayoko; Fumoto, Shintaro; Nishida, Koyo; Higuchi, Norihide; Kitahara, Takashi; Nakamura, Tadahiro; Sasaki, Hitoshi

2015-01-01

Polynucleotides are anionic macromolecules which are expected to transfer into the targeted cells through specific uptake mechanisms. So, we developed polynucleotides coating complexes of plasmid DNA (pDNA) and polyethylenimine (PEI) for a secure and efficient gene delivery system and evaluated their usefulness. Polyadenylic acid (polyA), polyuridylic acid (polyU), polycytidylic acid (polyC), and polyguanylic acid (polyG) were examined as the coating materials. pDNA/PEI/polyA, pDNA/PEI/polyU, and pDNA/PEI/polyC complexes formed nanoparticles with a negative surface charge although pDNA/PEI/polyG was aggregated. The pDNA/PEI/polyC complex showed high transgene efficiency in B16-F10 cells although there was little efficiency in pDNA/PEI/polyA and pDNA/PEI/polyU complexes. An inhibition study strongly indicated the specific uptake mechanism of pDNA/PEI/polyC complex. Polynucleotide coating complexes had lower cytotoxicity than pDNA/PEI complex. The pDNA/PEI/polyC complex showed high gene expression selectively in the spleen after intravenous injection into mice. The pDNA/PEI/polyC complex showed no agglutination with erythrocytes and no acute toxicity although these were observed in pDNA/PEI complex. Thus, we developed polynucleotide coating complexes as novel vectors for clinical gene therapy, and the pDNA/PEI/polyC complex as a useful candidate for a gene delivery system.

DNA binding specificity of the basic-helix-loop-helix protein MASH-1.

PubMed

Meierhan, D; el-Ariss, C; Neuenschwander, M; Sieber, M; Stackhouse, J F; Allemann, R K

1995-09-05

Despite the high degree of sequence similarity in their basic-helix-loop-helix (BHLH) domains, MASH-1 and MyoD are involved in different biological processes. In order to define possible differences between the DNA binding specificities of these two proteins, we investigated the DNA binding properties of MASH-1 by circular dichroism spectroscopy and by electrophoretic mobility shift assays (EMSA). Upon binding to DNA, the BHLH domain of MASH-1 underwent a conformational change from a mainly unfolded to a largely alpha-helical form, and surprisingly, this change was independent of the specific DNA sequence. The same conformational transition could be induced by the addition of 20% 2,2,2-trifluoroethanol. The apparent dissociation constants (KD) of the complexes of full-length MASH-1 with various oligonucleotides were determined from half-saturation points in EMSAs. MASH-1 bound as a dimer to DNA sequences containing an E-box with high affinity KD = 1.4-4.1 x 10(-14) M2). However, the specificity of DNA binding was low. The dissociation constant for the complex between MASH-1 and the highest affinity E-box sequence (KD = 1.4 x 10(-14) M2) was only a factor of 10 smaller than for completely unrelated DNA sequences (KD = approximately 1 x 10(-13) M2). The DNA binding specificity of MASH-1 was not significantly increased by the formation of an heterodimer with the ubiquitous E12 protein. MASH-1 and MyoD displayed similar binding site preferences, suggesting that their different target gene specificities cannot be explained solely by differential DNA binding. An explanation for these findings is provided on the basis of the known crystal structure of the BHLH domain of MyoD.

A SWI/SNF Chromatin Remodelling Protein Controls Cytokinin Production through the Regulation of Chromatin Architecture

PubMed Central

Jégu, Teddy; Domenichini, Séverine; Blein, Thomas; Ariel, Federico; Christ, Aurélie; Kim, Soon-Kap; Crespi, Martin; Boutet-Mercey, Stéphanie; Mouille, Grégory; Bourge, Mickaël; Hirt, Heribert; Bergounioux, Catherine; Raynaud, Cécile; Benhamed, Moussa

2015-01-01

Chromatin architecture determines transcriptional accessibility to DNA and consequently gene expression levels in response to developmental and environmental stimuli. Recently, chromatin remodelers such as SWI/SNF complexes have been recognized as key regulators of chromatin architecture. To gain insight into the function of these complexes during root development, we have analyzed Arabidopsis knock-down lines for one sub-unit of SWI/SNF complexes: BAF60. Here, we show that BAF60 is a positive regulator of root development and cell cycle progression in the root meristem via its ability to down-regulate cytokinin production. By opposing both the deposition of active histone marks and the formation of a chromatin regulatory loop, BAF60 negatively regulates two crucial target genes for cytokinin biosynthesis (IPT3 and IPT7) and one cell cycle inhibitor (KRP7). Our results demonstrate that SWI/SNF complexes containing BAF60 are key factors governing the equilibrium between formation and dissociation of a chromatin loop controlling phytohormone production and cell cycle progression. PMID:26457678

Direct Spectroscopic Study of Reconstituted Transcription Complexes Reveals That Intrinsic Termination Is Driven Primarily by Thermodynamic Destabilization of the Nucleic Acid Framework*S

PubMed Central

Datta, Kausiki; von Hippel, Peter H.

2008-01-01

Changes in near UV circular dichroism (CD) and fluorescence spectra of site-specifically placed pairs of 2-aminopurine residues have been used to probe the roles of the RNA hairpin and the RNA-DNA hybrid in controlling intrinsic termination of transcription. Functional transcription complexes were assembled directly by mixing preformed nucleic acid scaffolds of defined sequence with T7 RNA polymerase (RNAP). Scaffolds containing RNA hairpins immediately upstream of a GC-rich hybrid formed complexes of reduced stability, whereas the same hairpins adjacent to a hybrid of rU-dA base pairs triggered complex dissociation and transcript release. 2-Aminopurine probes at the upstream ends of the hairpin stems show that the hairpins open on RNAP binding and that stem re-formation begins after one or two RNA bases on the downstream side of the stem have emerged from the RNAP exit tunnel. Hairpins directly adjacent to the RNA-DNA hybrid weaken RNAP binding, decrease elongation efficiency, and disrupt the upstream end of the hybrid as well as interfere with the movement of the template base at the RNAP active site. Probing the edges of the DNA transcription bubble demonstrates that termination hairpins prevent translocation of the RNAP, suggesting that they transiently “lock” the polymerase to the nucleic acid scaffold and, thus, hold the RNA-DNA hybrid “in frame.” At intrinsic terminators the weak rU-dA hybrid and the adjacent termination hairpin combine to destabilize the elongation complex sufficiently to permit significant transcript release, whereas hairpin-dependent pausing provides time for the process to go to completion. PMID:18070878

Uracil DNA glycosylase BKRF3 contributes to Epstein-Barr virus DNA replication through physical interactions with proteins in viral DNA replication complex.

PubMed

Su, Mei-Tzu; Liu, I-Hua; Wu, Chia-Wei; Chang, Shu-Ming; Tsai, Ching-Hwa; Yang, Pei-Wen; Chuang, Yu-Chia; Lee, Chung-Pei; Chen, Mei-Ru

2014-08-01

Epstein-Barr virus (EBV) BKRF3 shares sequence homology with members of the uracil-N-glycosylase (UNG) protein family and has DNA glycosylase activity. Here, we explored how BKRF3 participates in the DNA replication complex and contributes to viral DNA replication. Exogenously expressed Flag-BKRF3 was distributed mostly in the cytoplasm, whereas BKRF3 was translocated into the nucleus and colocalized with the EBV DNA polymerase BALF5 in the replication compartment during EBV lytic replication. The expression level of BKRF3 increased gradually during viral replication, coupled with a decrease of cellular UNG2, suggesting BKRF3 enzyme activity compensates for UNG2 and ensures the fidelity of viral DNA replication. In immunoprecipitation-Western blotting, BKRF3 was coimmuno-precipitated with BALF5, the polymerase processivity factor BMRF1, and the immediate-early transactivator Rta. Coexpression of BMRF1 appeared to facilitate the nuclear targeting of BKRF3 in immunofluorescence staining. Residues 164 to 255 of BKRF3 were required for interaction with Rta and BALF5, whereas residues 81 to 166 of BKRF3 were critical for BMRF1 interaction in glutathione S-transferase (GST) pulldown experiments. Viral DNA replication was defective in cells harboring BKRF3 knockout EBV bacmids. In complementation assays, the catalytic mutant BKRF3(Q90L,D91N) restored viral DNA replication, whereas the leucine loop mutant BKRF3(H213L) only partially rescued viral DNA replication, coupled with a reduced ability to interact with the viral DNA polymerase and Rta. Our data suggest that BKRF3 plays a critical role in viral DNA synthesis predominantly through its interactions with viral proteins in the DNA replication compartment, while its enzymatic activity may be supplementary for uracil DNA glycosylase (UDG) function during virus replication. Catalytic activities of both cellular UDG UNG2 and viral UDGs contribute to herpesviral DNA replication. To ensure that the enzyme activity executes at the right time and the right place in DNA replication forks, complex formation with other components in the DNA replication machinery provides an important regulation for UDG function. In this study, we provide the mechanism for EBV UDG BKRF3 nuclear targeting and the interacting domains of BKRF3 with viral DNA replication proteins. Through knockout and complementation approaches, we further demonstrate that in addition to UDG activity, the interaction of BKRF3 with viral proteins in the replication compartment is crucial for efficient viral DNA replication. Copyright © 2014, American Society for Microbiology. All Rights Reserved.

Individual retrotransposon integrants are differentially controlled by KZFP/KAP1-dependent histone methylation, DNA methylation and TET-mediated hydroxymethylation in naïve embryonic stem cells.

PubMed

Coluccio, Andrea; Ecco, Gabriela; Duc, Julien; Offner, Sandra; Turelli, Priscilla; Trono, Didier

2018-02-26

The KZFP/KAP1 (KRAB zinc finger proteins/KRAB-associated protein 1) system plays a central role in repressing transposable elements (TEs) and maintaining parent-of-origin DNA methylation at imprinting control regions (ICRs) during the wave of genome-wide reprogramming that precedes implantation. In naïve murine embryonic stem cells (mESCs), the genome is maintained highly hypomethylated by a combination of TET-mediated active demethylation and lack of de novo methylation, yet KAP1 is tethered by sequence-specific KZFPs to ICRs and TEs where it recruits histone and DNA methyltransferases to impose heterochromatin formation and DNA methylation. Here, upon removing either KAP1 or the cognate KZFP, we observed rapid TET2-dependent accumulation of 5hmC at both ICRs and TEs. In the absence of the KZFP/KAP1 complex, ICRs lost heterochromatic histone marks and underwent both active and passive DNA demethylation. For KAP1-bound TEs, 5mC hydroxylation correlated with transcriptional reactivation. Using RNA-seq, we further compared the expression profiles of TEs upon Kap1 removal in wild-type, Dnmt and Tet triple knockout mESCs. While we found that KAP1 represents the main effector of TEs repression in all three settings, we could additionally identify specific groups of TEs further controlled by DNA methylation. Furthermore, we observed that in the absence of TET proteins, activation upon Kap1 depletion was blunted for some TE integrants and increased for others. Our results indicate that the KZFP/KAP1 complex maintains heterochromatin and DNA methylation at ICRs and TEs in naïve embryonic stem cells partly by protecting these loci from TET-mediated demethylation. Our study further unveils an unsuspected level of complexity in the transcriptional control of the endovirome by demonstrating often integrant-specific differential influences of histone-based heterochromatin modifications, DNA methylation and 5mC oxidation in regulating TEs expression.

Mispair-specific Recruitment of the Mlh1-Pms1 Complex Identifies Repair Substrates of the Saccharomyces cerevisiae Msh2-Msh3 Complex*

PubMed Central

Srivatsan, Anjana; Bowen, Nikki; Kolodner, Richard D.

2014-01-01

DNA mismatch repair is initiated by either the Msh2-Msh6 or the Msh2-Msh3 mispair recognition heterodimer. Here we optimized the expression and purification of Saccharomyces cerevisiae Msh2-Msh3 and performed a comparative study of Msh2-Msh3 and Msh2-Msh6 for mispair binding, sliding clamp formation, and Mlh1-Pms1 recruitment. Msh2-Msh3 formed sliding clamps and recruited Mlh1-Pms1 on +1, +2, +3, and +4 insertion/deletions and CC, AA, and possibly GG mispairs, whereas Msh2-Msh6 formed mispair-dependent sliding clamps and recruited Mlh1-Pms1 on 7 of the 8 possible base:base mispairs, the +1 insertion/deletion mispair, and to a low level on the +2 but not the +3 or +4 insertion/deletion mispairs and not on the CC mispair. The mispair specificity of sliding clamp formation and Mlh1-Pms1 recruitment but not mispair binding alone correlated best with genetic data on the mispair specificity of Msh2-Msh3- and Msh2-Msh6-dependent mismatch repair in vivo. Analysis of an Msh2-Msh6/Msh3 chimeric protein and mutant Msh2-Msh3 complexes showed that the nucleotide binding domain and communicating regions but not the mispair binding domain of Msh2-Msh3 are responsible for the extremely rapid dissociation of Msh2-Msh3 sliding clamps from DNA relative to that seen for Msh2-Msh6, and that amino acid residues predicted to stabilize Msh2-Msh3 interactions with bent, strand-separated mispair-containing DNA are more critical for the recognition of small +1 insertion/deletions than larger +4 insertion/deletions. PMID:24550389

Mispair-specific recruitment of the Mlh1-Pms1 complex identifies repair substrates of the Saccharomyces cerevisiae Msh2-Msh3 complex.

PubMed

Srivatsan, Anjana; Bowen, Nikki; Kolodner, Richard D

2014-03-28

DNA mismatch repair is initiated by either the Msh2-Msh6 or the Msh2-Msh3 mispair recognition heterodimer. Here we optimized the expression and purification of Saccharomyces cerevisiae Msh2-Msh3 and performed a comparative study of Msh2-Msh3 and Msh2-Msh6 for mispair binding, sliding clamp formation, and Mlh1-Pms1 recruitment. Msh2-Msh3 formed sliding clamps and recruited Mlh1-Pms1 on +1, +2, +3, and +4 insertion/deletions and CC, AA, and possibly GG mispairs, whereas Msh2-Msh6 formed mispair-dependent sliding clamps and recruited Mlh1-Pms1 on 7 of the 8 possible base:base mispairs, the +1 insertion/deletion mispair, and to a low level on the +2 but not the +3 or +4 insertion/deletion mispairs and not on the CC mispair. The mispair specificity of sliding clamp formation and Mlh1-Pms1 recruitment but not mispair binding alone correlated best with genetic data on the mispair specificity of Msh2-Msh3- and Msh2-Msh6-dependent mismatch repair in vivo. Analysis of an Msh2-Msh6/Msh3 chimeric protein and mutant Msh2-Msh3 complexes showed that the nucleotide binding domain and communicating regions but not the mispair binding domain of Msh2-Msh3 are responsible for the extremely rapid dissociation of Msh2-Msh3 sliding clamps from DNA relative to that seen for Msh2-Msh6, and that amino acid residues predicted to stabilize Msh2-Msh3 interactions with bent, strand-separated mispair-containing DNA are more critical for the recognition of small +1 insertion/deletions than larger +4 insertion/deletions.

Asilomar moments: formative framings in recombinant DNA and solar climate engineering research.

PubMed

Schäfer, Stefan; Low, Sean

2014-12-28

We examine the claim that in governance for solar climate engineering research, and especially field tests, there is no need for external governance beyond existing mechanisms such as peer review and environmental impact assessments that aim to assess technically defined risks to the physical environment. By drawing on the historical debate on recombinant DNA research, we show that defining risks is not a technical question but a complex process of narrative formation. Governance emerges from within, and as a response to, narratives of what is at stake in a debate. In applying this finding to the case of climate engineering, we find that the emerging narrative differs starkly from the narrative that gave meaning to rDNA technology during its formative period, with important implications for governance. While the narrative of rDNA technology was closed down to narrowly focus on technical risks, that of climate engineering continues to open up and includes social, political and ethical issues. This suggests that, in order to be legitimate, governance must take into account this broad perception of what constitutes the relevant issues and risks of climate engineering, requiring governance that goes beyond existing mechanisms that focus on technical risks. Even small-scale field tests with negligible impacts on the physical environment warrant additional governance as they raise broader concerns that go beyond the immediate impacts of individual experiments. © 2014 The Author(s) Published by the Royal Society. All rights reserved.

Diorganotin(IV) complexes of biologically potent 4(3H)-quinazolinone derived Schiff bases: synthesis, spectroscopic characterization, DNA interaction studies and antimicrobial activity.

PubMed

Prasad, Kollur Shiva; Kumar, Linganna Shiva; Chandan, Shivamallu; Jayalakshmi, Basvegowda; Revanasiddappa, Hosakere D

2011-10-15

Four Schiff base ligands and their corresponding organotin(IV) complexes have been synthesized and characterized by elemental analyses, IR, (1)H NMR, MS and thermal studies. The Schiff bases are obtained by the condensation of 3-amino-2-methyl-4(3H)-quinazolinone with different substituted aldehydes. The elemental analysis data suggest the stoichiometry to be 1:1 ratio formation. Infrared spectral data agreed with the coordination to the central metal ion through imine nitrogen, lactam oxygen and deprotonated phenolic oxygen atoms. All the synthesized compounds have been evaluated for antimicrobial activity against selected species of microorganisms. In addition, DNA binding/cleavage capacity of the compounds was analyzed by absorption spectroscopy, viscosity measurements and gel electrophoresis methods. Copyright © 2011 Elsevier B.V. All rights reserved.

Sub-diffusion and trapped dynamics of neutral and charged probes in DNA-protein coacervates

NASA Astrophysics Data System (ADS)

Arfin, Najmul; Yadav, Avinash Chand; Bohidar, H. B.

2013-11-01

The physical mechanism leading to the formation of large intermolecular DNA-protein complexes has been studied. Our study aims to explain the occurrence of fast coacervation dynamics at the charge neutralization point, followed by the appearance of smaller complexes and slower coacervation dynamics as the complex experiences overcharging. Furthermore, the electrostatic potential and probe mobility was investigated to mimic the transport of DNA / DNA-protein complex in a DNA-protein complex coacervate medium [N. Arfin and H. B. Bohidar, J. Phys. Chem. B 116, 13192 (2012)] by assigning neutral, negative, or positive charge to the probe particle. The mobility of the neutral probe was maximal at low matrix concentrations and showed random walk behavior, while its mobility ceased at the jamming concentration of c = 0.6, showing sub-diffusion and trapped dynamics. The positively charged probe showed sub-diffusive random walk followed by trapped dynamics, while the negatively charged probe showed trapping with occasional hopping dynamics at much lower concentrations. Sub-diffusion of the probe was observed in all cases under consideration, where the electrostatic interaction was used exclusively as the dominant force involved in the dynamics. For neutral and positive probes, the mean square displacement ⟨R2⟩ exhibits a scaling with time as ⟨R2⟩ ˜ tα, distinguishing random walk and trapped dynamics at α = 0.64 ± 0.04 at c = 0.12 and c = 0.6, respectively. In addition, the same scaling factors with the exponent β = 0.64 ± 0.04 can be used to distinguish random walk and trapped dynamics for the neutral and positive probes using the relation between the number of distinct sites visited by the probe, S(t), which follows the scaling, S(t) ˜ tβ/ln (t). Our results established the occurrence of a hierarchy of diffusion dynamics experienced by a probe in a dense medium that is either charged or neutral.

Reaction of protein chloramines with DNA and nucleosides: evidence for the formation of radicals, protein-DNA cross-links and DNA fragmentation.

PubMed Central

Hawkins, Clare L; Pattison, David I; Davies, Michael J

2002-01-01

Stimulated phagocyte cells produce the oxidant HOCl, via the release of the enzyme myeloperoxidase and hydrogen peroxide. HOCl is important in bacterial cell killing, but excessive or misplaced generation can damage the host tissue and may lead to the development of certain diseases such as cancer. The role of HOCl in the oxidation of isolated proteins, DNA and their components has been investigated extensively, but little work has been performed on the protein-DNA (nucleosome) complexes present in eukaryotic cell nuclei. Neither the selectivity of damage in such complexes nor the possibility of transfer of damage from the protein to DNA or vice versa, has been studied. In the present study, kinetic modelling has been employed to predict that reaction occurs predominantly with the protein and not with the DNA in the nucleosome, using molar HOCl excesses of up to 200-fold. With 50-200-fold excesses, 50-80% of the HOCl is predicted to react with histone lysine and histidine residues to yield chloramines. The yield and stability of such chloramines predicted by these modelling studies agrees well with experimental data. Decomposition of these species gives protein-derived, nitrogen-centred radicals, probably on the lysine side chains, as characterized by the EPR and spin-trapping experiments. It is shown that isolated lysine, histidine, peptide and protein chloramines can react with plasmid DNA to cause strand breaks. The protection against such damage afforded by the radical scavengers Trolox (a water-soluble alpha-tocopherol derivative) and 5,5-dimethyl-1-pyrroline-N-oxide suggests a radical-mediated process. The EPR experiments and product analyses have also provided evidence for the rapid addition of protein radicals, formed on chloramine decomposition, to pyrimidine nucleosides to give nucleobase radicals. Further evidence for the formation of such covalent cross-links has been obtained from experiments performed using (3)H-lysine and (14)C-histidine chloramines. These results are consistent with the predictions of the kinetic model and suggest that histones are major targets for HOCl in the nucleosome. Furthermore, the resulting protein chloramines and the radicals derived from them may act as contributing agents in HOCl-mediated DNA oxidation. PMID:12010123

DNA/RNA hybrid substrates modulate the catalytic activity of purified AID.

PubMed

Abdouni, Hala S; King, Justin J; Ghorbani, Atefeh; Fifield, Heather; Berghuis, Lesley; Larijani, Mani

2018-01-01

Activation-induced cytidine deaminase (AID) converts cytidine to uridine at Immunoglobulin (Ig) loci, initiating somatic hypermutation and class switching of antibodies. In vitro, AID acts on single stranded DNA (ssDNA), but neither double-stranded DNA (dsDNA) oligonucleotides nor RNA, and it is believed that transcription is the in vivo generator of ssDNA targeted by AID. It is also known that the Ig loci, particularly the switch (S) regions targeted by AID are rich in transcription-generated DNA/RNA hybrids. Here, we examined the binding and catalytic behavior of purified AID on DNA/RNA hybrid substrates bearing either random sequences or GC-rich sequences simulating Ig S regions. If substrates were made up of a random sequence, AID preferred substrates composed entirely of DNA over DNA/RNA hybrids. In contrast, if substrates were composed of S region sequences, AID preferred to mutate DNA/RNA hybrids over substrates composed entirely of DNA. Accordingly, AID exhibited a significantly higher affinity for binding DNA/RNA hybrid substrates composed specifically of S region sequences, than any other substrates composed of DNA. Thus, in the absence of any other cellular processes or factors, AID itself favors binding and mutating DNA/RNA hybrids composed of S region sequences. AID:DNA/RNA complex formation and supporting mutational analyses suggest that recognition of DNA/RNA hybrids is an inherent structural property of AID. Copyright © 2017 Elsevier Ltd. All rights reserved.