A crystal structure of the bifunctional antibiotic simocyclinone D8, bound to DNA gyrase.

PubMed

Edwards, Marcus J; Flatman, Ruth H; Mitchenall, Lesley A; Stevenson, Clare E M; Le, Tung B K; Clarke, Thomas A; McKay, Adam R; Fiedler, Hans-Peter; Buttner, Mark J; Lawson, David M; Maxwell, Anthony

2009-12-04

Simocyclinones are bifunctional antibiotics that inhibit bacterial DNA gyrase by preventing DNA binding to the enzyme. We report the crystal structure of the complex formed between the N-terminal domain of the Escherichia coli gyrase A subunit and simocyclinone D8, revealing two binding pockets that separately accommodate the aminocoumarin and polyketide moieties of the antibiotic. These are close to, but distinct from, the quinolone-binding site, consistent with our observations that several mutations in this region confer resistance to both agents. Biochemical studies show that the individual moieties of simocyclinone D8 are comparatively weak inhibitors of gyrase relative to the parent compound, but their combination generates a more potent inhibitor. Our results should facilitate the design of drug molecules that target these unexploited binding pockets.

The effects of temperature and magnetic flux on electron transport through a four-channel DNA model

NASA Astrophysics Data System (ADS)

Lee, Sunhee; Hedin, Eric; Joe, Yong

2010-03-01

The temperature dependence of the conductivity of lambda phage DNA has been measured by Tran et al [1] experimentally, where the conductivity displayed strong (weak) temperature dependence above (below) a threshold temperature. In order to understand the temperature effects of electron transport theoretically, we study a two-dimensional and four-channel DNA model using a tight-binding (TB) Hamiltonian. The thermal effects within a TB model are incorporated into the hopping integral and the relative twist angle from its equilibrium value between base-pairs. Since these thermal structural fluctuations localize the electronic wave functions in DNA, we examine a temperature-dependent localization length, a temperature-driven transmission, and current-voltage characteristics in this system. In addition, we incorporate magnetic field effects into the analysis of the transmission through DNA in order to modulate the quantum interference between the electron paths that comprise the 4-channel structure. [1] P. Tran, B. Alavi, and G. Gruner, PRL 85, 1564 (2000).

Enhanced Stability of DNA Nanostructures by Incorporation of Unnatural Base Pairs.

PubMed

Liu, Qing; Liu, Guocheng; Wang, Ting; Fu, Jing; Li, Rujiao; Song, Linlin; Wang, Zhen-Gang; Ding, Baoquan; Chen, Fei

2017-11-03

Self-assembled DNA nanostructures hold great promise in the fields of nanofabrication, biosensing and nanomedicine. However, the inherent low stability of the DNA double helices, formed by weak interactions, largely hinders the assembly and functions of DNA nanostructures. In this study, we redesigned and constructed a six-arm DNA junction by incorporation of the unnatural base pairs 5-Me-isoC/isoG and A/2-thioT into the double helices. They not only retained the structural integrity of the DNA nanostructure, but also showed enhanced thermal stability and resistance to T7 Exonuclease digestion. This research may expand the applications of DNA nanostructures in nanofabrication and biomedical fields, and furthermore, the genetic alphabet expansion with unnatural base pairs may enable us to construct more complicated and diversified self-assembled DNA nanostructures. © 2017 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim.

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.

Membrane-Assisted Growth of DNA Origami Nanostructure Arrays

PubMed Central

2015-01-01

Biological membranes fulfill many important tasks within living organisms. In addition to separating cellular volumes, membranes confine the space available to membrane-associated proteins to two dimensions (2D), which greatly increases their probability to interact with each other and assemble into multiprotein complexes. We here employed two DNA origami structures functionalized with cholesterol moieties as membrane anchors—a three-layered rectangular block and a Y-shaped DNA structure—to mimic membrane-assisted assembly into hierarchical superstructures on supported lipid bilayers and small unilamellar vesicles. As designed, the DNA constructs adhered to the lipid bilayers mediated by the cholesterol anchors and diffused freely in 2D with diffusion coefficients depending on their size and number of cholesterol modifications. Different sets of multimerization oligonucleotides added to bilayer-bound origami block structures induced the growth of either linear polymers or two-dimensional lattices on the membrane. Y-shaped DNA origami structures associated into triskelion homotrimers and further assembled into weakly ordered arrays of hexagons and pentagons, which resembled the geometry of clathrin-coated pits. Our results demonstrate the potential to realize artificial self-assembling systems that mimic the hierarchical formation of polyhedral lattices on cytoplasmic membranes. PMID:25734977

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

Egli, Martin; Pallan, Pradeep S.; Pattanayek, Rekha

An experimental rationalization of the structure type encountered in DNA and RNA by systematically investigating the chemical and physical properties of alternative nucleic acids has identified systems with a variety of sugar-phosphate backbones that are capable of Watson-Crick base pairing and in some cases cross-pairing with the natural nucleic acids. The earliest among the model systems tested to date, (4{prime} {yields} 6{prime})-linked oligo(2{prime},3{prime}-dideoxy-{beta}-d-glucopyranosyl)nucleotides or homo-DNA, shows stable self-pairing, but the pairing rules for the four natural bases are not the same as those in DNA. However, a complete interpretation and understanding of the properties of the hexapyranosyl (4{prime} {yields} 6{prime})more » family of nucleic acids has been impeded until now by the lack of detailed 3D-structural data. We have determined the crystal structure of a homo-DNA octamer. It reveals a weakly twisted right-handed duplex with a strong inclination between the hexose-phosphate backbones and base-pair axes, and highly irregular values for helical rise and twist at individual base steps. The structure allows a rationalization of the inability of allo-, altro-, and glucopyranosyl-based oligonucleotides to form stable pairing systems.« less

Dual color fluorescence quantitative detection of specific single-stranded DNA with molecular beacons and nucleic acid dye SYBR Green I.

PubMed

Xiang, Dong-Shan; Zhou, Guo-Hua; Luo, Ming; Ji, Xing-Hu; He, Zhi-Ke

2012-08-21

We have developed a dual color fluorescence quantitative detection method for specific single-stranded DNA with molecular beacons (MBs) and nucleic acid dye SYBR Green I by synchronous scanning fluorescence spectrometry. It is demonstrated by a reverse-transcription oligonucleotide sequence (target DNA, 33 bases) of RNA fragment of human immunodeficiency virus (HIV) as a model system. In the absence of target DNA, the MBs are in the stem-closed state, the fluorescence of 5-carboxy-X-rhodamine (ROX) is quenched by black hole quencher-2 (BHQ-2), and the interaction between SYBR Green I and the MBs is very weak. At this time the fluorescence signals of ROX and SYBR Green I are all very weak. In the presence of target DNA, MBs hybridize with target DNA and form a double-strand structure, the fluorophore ROX is separated from the quencher BHQ-2, and the fluorescence of ROX recovers. At the same time, SYBR Green I binds to hybridized dsDNA, whose fluorescence intensity is significantly enhanced. Thus, dual color fluorescence quantitative detection for the target DNA can be realized by synchronous scanning fluorescence spectrometry. In this strategy, the fluorescence signal of SYBR Green I is far larger than that of ROX, so the quantitative analysis of target DNA with the fluorescence intensity of SYBR Green I can significantly improve the detection sensitivity. In addition, the false-positive signals of MBs do not affect the fluorescence signals of nucleic acid dye SYBR Green I. Thereby, in the analysis of complex samples, quantitative analysis of target DNA with SYBR Green I can avoid the false-positive signals of MBs and improve the detection accuracy.

Conformational gating of DNA conductance

PubMed Central

Artés, Juan Manuel; Li, Yuanhui; Qi, Jianqing; Anantram, M. P.; Hihath, Joshua

2015-01-01

DNA is a promising molecule for applications in molecular electronics because of its unique electronic and self-assembly properties. Here we report that the conductance of DNA duplexes increases by approximately one order of magnitude when its conformation is changed from the B-form to the A-form. This large conductance increase is fully reversible, and by controlling the chemical environment, the conductance can be repeatedly switched between the two values. The conductance of the two conformations displays weak length dependencies, as is expected for guanine-rich sequences, and can be fit with a coherence-corrected hopping model. These results are supported by ab initio electronic structure calculations that indicate that the highest occupied molecular orbital is more disperse in the A-form DNA case. These results demonstrate that DNA can behave as a promising molecular switch for molecular electronics applications and also provide additional insights into the huge dispersion of DNA conductance values found in the literature. PMID:26648400

Conformational gating of DNA conductance.

PubMed

Artés, Juan Manuel; Li, Yuanhui; Qi, Jianqing; Anantram, M P; Hihath, Joshua

2015-12-09

DNA is a promising molecule for applications in molecular electronics because of its unique electronic and self-assembly properties. Here we report that the conductance of DNA duplexes increases by approximately one order of magnitude when its conformation is changed from the B-form to the A-form. This large conductance increase is fully reversible, and by controlling the chemical environment, the conductance can be repeatedly switched between the two values. The conductance of the two conformations displays weak length dependencies, as is expected for guanine-rich sequences, and can be fit with a coherence-corrected hopping model. These results are supported by ab initio electronic structure calculations that indicate that the highest occupied molecular orbital is more disperse in the A-form DNA case. These results demonstrate that DNA can behave as a promising molecular switch for molecular electronics applications and also provide additional insights into the huge dispersion of DNA conductance values found in the literature.

Theory and simulation of DNA-coated colloids: a guide for rational design.

PubMed

Angioletti-Uberti, Stefano; Mognetti, Bortolo M; Frenkel, Daan

2016-03-07

By exploiting the exquisite selectivity of DNA hybridization, DNA-coated colloids (DNACCs) can be made to self-assemble in a wide variety of structures. The beauty of this system stems largely from its exceptional versatility and from the fact that a proper choice of the grafted DNA sequences yields fine control over the colloidal interactions. Theory and simulations have an important role to play in the optimal design of self assembling DNACCs. At present, the powerful model-based design tools are not widely used, because the theoretical literature is fragmented and the connection between different theories is often not evident. In this Perspective, we aim to discuss the similarities and differences between the different models that have been described in the literature, their underlying assumptions, their strengths and their weaknesses. Using the tools described in the present Review, it should be possible to move towards a more rational design of novel self-assembling structures of DNACCs and, more generally, of systems where ligand-receptor are used to control interactions.

A mechanical mechanism for translocation of ring-shaped helicases on DNA and its demonstration in a macroscopic simulation system

NASA Astrophysics Data System (ADS)

Chou, Y. C.

2018-04-01

The asymmetry in the two-layered ring structure of helicases and the random thermal fluctuations of the helicase and DNA molecules are considered as the bases for the generation of the force required for translocation of the ring-shaped helicase on DNA. The helicase comprises a channel at its center with two unequal ends, through which strands of DNA can pass. The random collisions between the portion of the DNA strand in the central channel and the wall of the channel generate an impulsive force toward the small end. This impulsive force is the starting point for the helicase to translocate along the DNA with the small end in front. Such a physical mechanism may serve as a complementary for the chemomechanical mechanism of the translocation of helicase on DNA. When the helicase arrives at the junction of ssDNA and dsDNA (a fork), the collision between the helicase and the closest base pair may produce a sufficient impulsive force to break the weak hydrogen bond of the base pair. Thus, the helicase may advance and repeat the process of unwinding the dsDNA strand. This mechanism was tested in a macroscopic simulation system where the helicase was simulated using a truncated-cone structure and DNA was simulated with bead chains. Many features of translocation and unwinding such as translocation on ssDNA and dsDNA, unwinding of dsDNA, rewinding, strand switching, and Holliday junction resolution were reproduced.

Fabrication, characterization, and biological assessment of multilayer laminin γ2 DNA coatings on titanium surfaces.

PubMed

Yang, Guoli; Zhang, Jing; Dong, Wenjing; Liu, Li; Shi, Jue; Wang, Huiming

2016-03-21

The purpose of this work was to fabricate a multilayer laminin γ2 DNA coating on a titanium surface and evaluate its biological properties. A multilayer laminin γ2 DNA coating was fabricated on titanium using a layer-by-layer assembly technique. The rate of coating degradation was evaluated by detecting the amount of cDNA remaining. Surface analysis using X-ray photoelectron spectroscopy, atomic force microscopy, and surface contact angle measurements revealed the multilayer structure to consist of cationic lipid and confirmed that a laminin γ2 DNA layer could be fabricated on titanium via the layer-by-layer assembly process. The transfection efficiency was highest for five layers in the multilayer structure. HEK293 cells cultured on the multilayer films displayed significantly higher adhesion activity than the control group. The expression of laminin γ2 and the co-localization of integrin β4 and plectin were more obvious in HN4 cells cultured on the multilayer laminin γ2 DNA coating, while weak immunoreactivities were observed in the control group. We concluded that the DNA-loaded multilayer provided a surface with good biocompatibility and that the multilayer laminin γ2 DNA coating might be effective in improving cell adhesion and the formation of hemidesmosomes on titanium surfaces.

Fabrication, characterization, and biological assessment of multilayer laminin γ2 DNA coatings on titanium surfaces

PubMed Central

Yang, Guoli; Zhang, Jing; Dong, Wenjing; Liu, Li; Shi, Jue; Wang, Huiming

2016-01-01

The purpose of this work was to fabricate a multilayer laminin γ2 DNA coating on a titanium surface and evaluate its biological properties. A multilayer laminin γ2 DNA coating was fabricated on titanium using a layer-by-layer assembly technique. The rate of coating degradation was evaluated by detecting the amount of cDNA remaining. Surface analysis using X-ray photoelectron spectroscopy, atomic force microscopy, and surface contact angle measurements revealed the multilayer structure to consist of cationic lipid and confirmed that a laminin γ2 DNA layer could be fabricated on titanium via the layer-by-layer assembly process. The transfection efficiency was highest for five layers in the multilayer structure. HEK293 cells cultured on the multilayer films displayed significantly higher adhesion activity than the control group. The expression of laminin γ2 and the co-localization of integrin β4 and plectin were more obvious in HN4 cells cultured on the multilayer laminin γ2 DNA coating, while weak immunoreactivities were observed in the control group. We concluded that the DNA-loaded multilayer provided a surface with good biocompatibility and that the multilayer laminin γ2 DNA coating might be effective in improving cell adhesion and the formation of hemidesmosomes on titanium surfaces. PMID:26996815

Fabrication, characterization, and biological assessment of multilayer laminin γ2 DNA coatings on titanium surfaces

NASA Astrophysics Data System (ADS)

Yang, Guoli; Zhang, Jing; Dong, Wenjing; Liu, Li; Shi, Jue; Wang, Huiming

2016-03-01

The purpose of this work was to fabricate a multilayer laminin γ2 DNA coating on a titanium surface and evaluate its biological properties. A multilayer laminin γ2 DNA coating was fabricated on titanium using a layer-by-layer assembly technique. The rate of coating degradation was evaluated by detecting the amount of cDNA remaining. Surface analysis using X-ray photoelectron spectroscopy, atomic force microscopy, and surface contact angle measurements revealed the multilayer structure to consist of cationic lipid and confirmed that a laminin γ2 DNA layer could be fabricated on titanium via the layer-by-layer assembly process. The transfection efficiency was highest for five layers in the multilayer structure. HEK293 cells cultured on the multilayer films displayed significantly higher adhesion activity than the control group. The expression of laminin γ2 and the co-localization of integrin β4 and plectin were more obvious in HN4 cells cultured on the multilayer laminin γ2 DNA coating, while weak immunoreactivities were observed in the control group. We concluded that the DNA-loaded multilayer provided a surface with good biocompatibility and that the multilayer laminin γ2 DNA coating might be effective in improving cell adhesion and the formation of hemidesmosomes on titanium surfaces.

Molecular modelling study of changes induced by netropsin binding to nucleosome core particles.

PubMed Central

Pérez, J J; Portugal, J

1990-01-01

It is well known that certain sequence-dependent modulators in structure appear to determine the rotational positioning of DNA on the nucleosome core particle. That preference is rather weak and could be modified by some ligands as netropsin, a minor-groove binding antibiotic. We have undertaken a molecular modelling approach to calculate the relative energy of interaction between a DNA molecule and the protein core particle. The histones particle is considered as a distribution of positive charges on the protein surface that interacts with the DNA molecule. The molecular electrostatic potentials for the DNA, simulated as a discontinuous cylinder, were calculated using the values for all the base pairs. Computing these parameters, we calculated the relative energy of interaction and the more stable rotational setting of DNA. The binding of four molecules of netropsin to this model showed that a new minimum of energy is obtained when the DNA turns toward the protein surface by about 180 degrees, so a new energetically favoured structure appears where netropsin binding sites are located facing toward the histones surface. The effect of netropsin could be explained in terms of an induced change in the phasing of DNA on the core particle. The induced rotation is considered to optimize non-bonded contacts between the netropsin molecules and the DNA backbone. PMID:2165249

Characterization of biochemical properties of Bacillus subtilis RecQ helicase.

PubMed

Qin, Wei; Liu, Na-Nv; Wang, Lijun; Zhou, Min; Ren, Hua; Bugnard, Elisabeth; Liu, Jie-Lin; Zhang, Lin-Hu; Vendôme, Jeremie; Hu, Jin-Shan; Xi, Xu Guang

2014-12-01

RecQ family helicases function as safeguards of the genome. Unlike Escherichia coli, the Gram-positive Bacillus subtilis bacterium possesses two RecQ-like homologues, RecQ[Bs] and RecS, which are required for the repair of DNA double-strand breaks. RecQ[Bs] also binds to the forked DNA to ensure a smooth progression of the cell cycle. Here we present the first biochemical analysis of recombinant RecQ[Bs]. RecQ[Bs] binds weakly to single-stranded DNA (ssDNA) and blunt-ended double-stranded DNA (dsDNA) but strongly to forked dsDNA. The protein exhibits a DNA-stimulated ATPase activity and ATP- and Mg(2+)-dependent DNA helicase activity with a 3' → 5' polarity. Molecular modeling shows that RecQ[Bs] shares high sequence and structure similarity with E. coli RecQ. Surprisingly, RecQ[Bs] resembles the truncated Saccharomyces cerevisiae Sgs1 and human RecQ helicases more than RecQ[Ec] with regard to its enzymatic activities. Specifically, RecQ[Bs] unwinds forked dsDNA and DNA duplexes with a 3'-overhang but is inactive on blunt-ended dsDNA and 5'-overhung duplexes. Interestingly, RecQ[Bs] unwinds blunt-ended DNA with structural features, including nicks, gaps, 5'-flaps, Kappa joints, synthetic replication forks, and Holliday junctions. We discuss these findings in the context of RecQ[Bs]'s possible functions in preserving genomic stability. Copyright © 2014, American Society for Microbiology. All Rights Reserved.

Conformation-dependent DNA attraction

NASA Astrophysics Data System (ADS)

Li, Weifeng; Nordenskiöld, Lars; Zhou, Ruhong; Mu, Yuguang

2014-05-01

Understanding how DNA molecules interact with other biomolecules is related to how they utilize their functions and is therefore critical for understanding their structure-function relationships. For a long time, the existence of Z-form DNA (a left-handed double helical version of DNA, instead of the common right-handed B-form) has puzzled the scientists, and the definitive biological significance of Z-DNA has not yet been clarified. In this study, the effects of DNA conformation in DNA-DNA interactions are explored by molecular dynamics simulations. Using umbrella sampling, we find that for both B- and Z-form DNA, surrounding Mg2+ ions always exert themselves to screen the Coulomb repulsion between DNA phosphates, resulting in very weak attractive force. On the contrary, a tight and stable bound state is discovered for Z-DNA in the presence of Mg2+ or Na+, benefiting from their hydrophobic nature. Based on the contact surface and a dewetting process analysis, a two-stage binding process of Z-DNA is outlined: two Z-DNA first attract each other through charge screening and Mg2+ bridges to phosphate groups in the same way as that of B-DNA, after which hydrophobic contacts of the deoxyribose groups are formed via a dewetting effect, resulting in stable attraction between two Z-DNA molecules. The highlighted hydrophobic nature of Z-DNA interaction from the current study may help to understand the biological functions of Z-DNA in gene transcription.Understanding how DNA molecules interact with other biomolecules is related to how they utilize their functions and is therefore critical for understanding their structure-function relationships. For a long time, the existence of Z-form DNA (a left-handed double helical version of DNA, instead of the common right-handed B-form) has puzzled the scientists, and the definitive biological significance of Z-DNA has not yet been clarified. In this study, the effects of DNA conformation in DNA-DNA interactions are explored by molecular dynamics simulations. Using umbrella sampling, we find that for both B- and Z-form DNA, surrounding Mg2+ ions always exert themselves to screen the Coulomb repulsion between DNA phosphates, resulting in very weak attractive force. On the contrary, a tight and stable bound state is discovered for Z-DNA in the presence of Mg2+ or Na+, benefiting from their hydrophobic nature. Based on the contact surface and a dewetting process analysis, a two-stage binding process of Z-DNA is outlined: two Z-DNA first attract each other through charge screening and Mg2+ bridges to phosphate groups in the same way as that of B-DNA, after which hydrophobic contacts of the deoxyribose groups are formed via a dewetting effect, resulting in stable attraction between two Z-DNA molecules. The highlighted hydrophobic nature of Z-DNA interaction from the current study may help to understand the biological functions of Z-DNA in gene transcription. Electronic supplementary information (ESI) available. See DOI: 10.1039/c3nr03235c

Improved DNA hybridization parameters by Twisted Intercalating Nucleic Acid (TINA).

PubMed

Schneider, Uffe Vest

2012-01-01

This thesis establishes oligonucleotide design rules and applications of a novel group of DNA stabilizing molecules collectively called Twisted Intercalating Nucleic Acid - TINA. Three peer-reviewed publications form the basis for the thesis. One publication describes an improved and rapid method for determination of DNA melting points and two publications describe the effects of positioning TINA molecules in parallel triplex helix and antiparallel duplex helix forming DNA structures. The third publication establishes that TINA molecules containing oligonucleotides improve an antiparallel duplex hybridization based capture assay's analytical sensitivity compared to conventionel DNA oligonucleotides. Clinical microbiology is traditionally based on pathogenic microorganisms' culture and serological tests. The introduction of DNA target amplification methods like PCR has improved the analytical sensitivity and total turn around time involved in clinical diagnostics of infections. Due to the relatively weak hybridization between the two strands of double stranded DNA, a number of nucleic acid stabilizing molecules have been developed to improve the sensitivity of DNA based diagnostics through superior binding properties. A short introduction is given to Watson-Crick and Hoogsteen based DNA binding and the derived DNA structures. A number of other nucleic acid stabilizing molecules are described. The stabilizing effect of TINA molecules on different DNA structures is discussed and considered in relation to other nucleic acid stabilizing molecules and in relation to future use of TINA containing oligonucleotides in clinical diagnostics and therapy. In conclusion, design of TINA modified oligonucleotides for antiparallel duplex helixes and parallel triplex helixes follows simple purpose dependent rules. TINA molecules are well suited for improving multiplex PCR assays and can be used as part of novel technologies. Future research should test whether combinations of TINA molecules and other nucleic acid stabilizing molecules can increase analytical sensitivity whilst maintaining nucleobase mismatch discrimination in triplex helix based diagnostic assays.

Genetic population structure in the yellow mongoose, Cynictis penicillata.

PubMed

Van Vuuren, B J; Robinson, T J

1997-12-01

Phylogeographic structure was determined for the yellow mongoose, Cynictis penicillata, using mtDNA RFLPs and control region sequences. The RFLP analysis revealed 13 haplotypes which showed weak geographical patterning consistent with a recent range expansion from a refugial population(s). An analysis of molecular variance (AMOVA) revealed no correspondence between mtDNA phylogeography and subspecies delimitation, nor between matrilines and areas characterized by a high incidence of the viverrid-type rabies, of which the yellow mongoose is the principal vector. The lack of structure was also shown by control region sequences although four of the maternal lineages shared a near-perfect 81 bp repeat. We speculate that regional hot spots of the viverrid rabies biotype reflect population density differences in the yellow mongoose that are not underscored by genetic partitioning, at least at the level of resolution provided by our analyses.

Chicken Fetal Liver DNA Damage and Adduct Formation by Activation-Dependent DNA-Reactive Carcinogens and Related Compounds of Several Structural Classes

PubMed Central

Williams, Gary M.; Duan, Jian-Dong; Brunnemann, Klaus D.; Iatropoulos, Michael J.; Vock, Esther; Deschl, Ulrich

2014-01-01

The chicken egg genotoxicity assay (CEGA), which utilizes the liver of an intact and aseptic embryo-fetal test organism, was evaluated using four activation-dependent DNA-reactive carcinogens and four structurally related less potent carcinogens or non-carcinogens. In the assay, three daily doses of test substances were administered to eggs containing 9–11-day-old fetuses and the fetal livers were assessed for two endpoints, DNA breaks using the alkaline single cell gel electrophoresis (comet) assay and DNA adducts using the 32P-nucleotide postlabeling (NPL) assay. The effects of four carcinogens of different structures requiring distinct pathways of bioactivation, i.e., 2-acetylaminofluorene (AAF), aflatoxin B1 (AFB1), benzo[a]pyrene (B[a]P), and diethylnitrosamine (DEN), were compared with structurally related non-carcinogens fluorene (FLU) and benzo[e]pyrene (B[e]P) or weak carcinogens, aflatoxin B2 (AFB2) and N-nitrosodiethanolamine (NDELA). The four carcinogens all produced DNA breaks at microgram or low milligram total doses, whereas less potent carcinogens and non-carcinogens yielded borderline or negative results, respectively, at higher doses. AAF and B[a]P produced DNA adducts, whereas none was found with the related comparators FLU or B[e]P, consistent with comet results. DEN and NDELA were also negative for adducts, as expected in the case of DEN for an alkylating agent in the standard NPL assay. Also, AFB1 and AFB2 were negative in NPL, as expected, due to the nature of ring opened aflatoxin adducts, which are resistant to enzymatic digestion. Thus, the CEGA, using comet and NPL, is capable of detection of the genotoxicity of diverse DNA-reactive carcinogens, while not yielding false positives for non-carcinogens. PMID:24973097

Quenching the chemiluminescence of acridinium ester by graphene oxide for label-free and homogeneous DNA detection.

PubMed

He, Yi; Huang, Guangming; Cui, Hua

2013-11-13

It was found that graphene oxide (GO) could effectively quench the chemiluminescence (CL) emission from a acridinium ester (AE)-hydrogen peroxide system. By taking advantage of this quenching effect, as a proof of concept, a label-free and homogeneous DNA assay was developed for the detection of Mycobacterium tuberculosis DNA. In the absence of target DNA, both probe DNA and AE were absorbed on the surface of GO, producing a weak CL emission owing to the CL quenching effect of GO. However, in the presence of target DNA, a double-stranded structure of DNA was generated, leading to the release of the oligonucleotide from the GO surface. AE favors binding with double-stranded DNA, which will be released from the GO surface; thus, the quenching effect of GO will be no longer effective and a strong CL signal can be observed. This assay can detect M. tuberculosis DNA with a detection limit of 0.65 nM. This sensitivity is lower than that of previously reported electrochemical detection.

Low-temperature 1H NMR spectroscopic study of hydration properties of a hybrid system based on nanosilica, DNA and doxorubicin in the presence of C60 fullerene

NASA Astrophysics Data System (ADS)

Turov, V. V.; Prylutskyy, Yu. I.; Ugnivenko, A. P.; Barvinchenko, V. N.; Krupskaya, T. V.; Tsierkezos, N. G.; Ritter, U.

2014-03-01

The structure of hydrate cover layers of SiO2-DNA-Dox (where Dox: doxorubicin) and SiO2-DNA-Dox-C60 fullerene hybrids was studied by means of low-temperature 1H NMR spectroscopy in tetrachloromethane. The hydration properties of SiO2-DNA-Dox nanomaterials combined with fullerenes and their derivatives are extremely important for their further use as therapeutics in cancer treatment and for safety reasons. The findings reveal that the hydration properties of the hybrids differ from those of the solid DNA particulates or SiO2-DNA systems due to the existence of different types of water clusters, namely the weakly (WAW) and strongly associated water (SAW) clusters. For SAW clusters the radial distributions as well as the distributions of change in Gibbs free energy due to adsorptive interactions at the surfaces of the investigated systems were obtained.

Mitochondrial myopathies.

PubMed

DiMauro, Salvatore

2006-11-01

Our understanding of mitochondrial diseases (defined restrictively as defects of the mitochondrial respiratory chain) is expanding rapidly. In this review, I will give the latest information on disorders affecting predominantly or exclusively skeletal muscle. The most recently described mitochondrial myopathies are due to defects in nuclear DNA, including coenzyme Q10 deficiency and mutations in genes controlling mitochondrial DNA abundance and structure, such as POLG, TK2, and MPV17. Barth syndrome, an X-linked recessive mitochondrial myopathy/cardiopathy, is associated with decreased amount and altered structure of cardiolipin, the main phospholipid of the inner mitochondrial membrane, but a secondary impairment of respiratory chain function is plausible. The role of mutations in protein-coding genes of mitochondrial DNA in causing isolated myopathies has been confirmed. Mutations in tRNA genes of mitochondrial DNA can also cause predominantly myopathic syndromes and--contrary to conventional wisdom--these mutations can be homoplasmic. Defects in the mitochondrial respiratory chain impair energy production and almost invariably involve skeletal muscle, causing exercise intolerance, cramps, recurrent myoglobinuria, or fixed weakness, which often affects extraocular muscles and results in droopy eyelids (ptosis) and progressive external ophthalmoplegia.

A fluorescent aptasensor based on a DNA pyramid nanostructure for ultrasensitive detection of ochratoxin A.

PubMed

Nameghi, Morteza Alinezhad; Danesh, Noor Mohammad; Ramezani, Mohammad; Hassani, Faezeh Vahdati; Abnous, Khalil; Taghdisi, Seyed Mohammad

2016-08-01

Analytical techniques for detection of ochratoxin A (OTA) in food products and blood serum are of great significance. In this study, a fluorescent aptasensor was developed for sensitive and specific detection of OTA, based on a DNA pyramid nanostructure (DPN) and PicoGreen (PG) dye. The designed aptasensor inherits characteristics of DPN, such as high stability and capacity for PG loading. PG, as a fluorescent dye, could bind to double-stranded DNA (dsDNA). In the absence of OTA, the pyramid structure of DPN remains intact, leading to a very strong fluorescence emission. Because of higher affinity of aptamer for its target relative to its complementary strand, upon addition of target, the pyramid structure of DPN is disassembled, leading to a weak fluorescence emission. The presented aptasensor showed high specificity toward OTA with a limit of detection (LOD) as low as 0.135 nM. Besides, the designed sensing strategy was successfully utilized to recognize OTA in serum and grape juice with LODs of 0.184 and 0.149 nM, respectively.

Cdc13 N-Terminal Dimerization DNA Binding and Telomere Length Regulation

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

M Mitchell; J Smith; M Mason

The essential yeast protein Cdc13 facilitates chromosome end replication by recruiting telomerase to telomeres, and together with its interacting partners Stn1 and Ten1, it protects chromosome ends from nucleolytic attack, thus contributing to genome integrity. Although Cdc13 has been studied extensively, the precise role of its N-terminal domain (Cdc13N) in telomere length regulation remains unclear. Here we present a structural, biochemical, and functional characterization of Cdc13N. The structure reveals that this domain comprises an oligonucleotide/oligosaccharide binding (OB) fold and is involved in Cdc13 dimerization. Biochemical data show that Cdc13N weakly binds long, single-stranded, telomeric DNA in a fashion that ismore » directly dependent on domain oligomerization. When introduced into full-length Cdc13 in vivo, point mutations that prevented Cdc13N dimerization or DNA binding caused telomere shortening or lengthening, respectively. The multiple DNA binding domains and dimeric nature of Cdc13 offer unique insights into how it coordinates the recruitment and regulation of telomerase access to the telomeres.« less

Standard atomic volumes in double-stranded DNA and packing in protein–DNA interfaces

PubMed Central

Nadassy, Katalin; Tomás-Oliveira, Isabel; Alberts, Ian; Janin, Joël; Wodak, Shoshana J.

2001-01-01

Standard volumes for atoms in double-stranded B-DNA are derived using high resolution crystal structures from the Nucleic Acid Database (NDB) and compared with corresponding values derived from crystal structures of small organic compounds in the Cambridge Structural Database (CSD). Two different methods are used to compute these volumes: the classical Voronoi method, which does not depend on the size of atoms, and the related Radical Planes method which does. Results show that atomic groups buried in the interior of double-stranded DNA are, on average, more tightly packed than in related small molecules in the CSD. The packing efficiency of DNA atoms at the interfaces of 25 high resolution protein–DNA complexes is determined by computing the ratios between the volumes of interfacial DNA atoms and the corresponding standard volumes. These ratios are found to be close to unity, indicating that the DNA atoms at protein–DNA interfaces are as closely packed as in crystals of B-DNA. Analogous volume ratios, computed for buried protein atoms, are also near unity, confirming our earlier conclusions that the packing efficiency of these atoms is similar to that in the protein interior. In addition, we examine the number, volume and solvent occupation of cavities located at the protein–DNA interfaces and compared them with those in the protein interior. Cavities are found to be ubiquitous in the interfaces as well as inside the protein moieties. The frequency of solvent occupation of cavities is however higher in the interfaces, indicating that those are more hydrated than protein interiors. Lastly, we compare our results with those obtained using two different measures of shape complementarity of the analysed interfaces, and find that the correlation between our volume ratios and these measures, as well as between the measures themselves, is weak. Our results indicate that a tightly packed environment made up of DNA, protein and solvent atoms plays a significant role in protein–DNA recognition. PMID:11504874

Structural basis for bifunctional zinc(II) macrocyclic complex recognition of thymine bulges in DNA.

PubMed

del Mundo, Imee Marie A; Siters, Kevin E; Fountain, Matthew A; Morrow, Janet R

2012-05-07

The zinc(II) complex of 1-(4-quinoylyl)methyl-1,4,7,10-tetraazacyclododecane (cy4q) binds selectively to thymine bulges in DNA and to a uracil bulge in RNA. Binding constants are in the low-micromolar range for thymine bulges in the stems of hairpins, for a thymine bulge in a DNA duplex, and for a uracil bulge in an RNA hairpin. Binding studies of Zn(cy4q) to a series of hairpins containing thymine bulges with different flanking bases showed that the complex had a moderate selectivity for thymine bulges with neighboring purines. The dissociation constants of the most strongly bound Zn(cy4q)-DNA thymine bulge adducts were 100-fold tighter than similar sequences with fully complementary stems or than bulges containing cytosine, guanine, or adenine. In order to probe the role of the pendent group, three additional zinc(II) complexes containing 1,4,7,10-tetraazacyclododecane (cyclen) with aromatic pendent groups were studied for binding to DNA including 1-(2-quinolyl)methyl-1,4,7,10-tetraazacyclododecane (cy2q), 1-(4-biphenyl)methyl-1,4,7,10-tetraazacyclododecane (cybp), and 5-(1,4,7,10-tetraazacyclododecan-1-ylsulfonyl)-N,N-dimethylnaphthalen-1-amine (dsc). The Zn(cybp) complex binds with moderate affinity but little selectivity to DNA hairpins with thymine bulges and to DNA lacking bulges. Similarly, Zn(dsc) binds weakly both to thymine bulges and hairpins with fully complementary stems. The zinc(II) complex of cy2q has the 2-quinolyl moiety bound to the Zn(II) center, as shown by (1)H NMR spectroscopy and pH-potentiometric titrations. As a consequence, only weak (500 μM) binding is observed to DNA with no appreciable selectivity. An NMR structure of a thymine-bulge-containing hairpin shows that the thymine is extrahelical but rotated toward the major groove. NMR data for Zn(cy4q) bound to DNA containing a thymine bulge is consistent with binding of the zinc(II) complex to the thymine N3(-) and stacking of the quinoline on top of the thymine. The thymine-bulge bound zinc(II) complex is pointed into the major groove, and there are interactions with the guanine positioned 5' to the thymine bulge.

Genetic Structure and the North American Postglacial Expansion of the Barnacle, Semibalanus balanoides

PubMed Central

O’Brien, Megan A.; Schmidt, Paul S.; Rand, David M.

2012-01-01

Population genetic characteristics are shaped by the life-history traits of organisms and the geologic history of their habitat. This study provides a neutral framework for understanding the population dynamics and opportunities for selection in Semibalanus balanoides, a species that figures prominently in ecological and evolutionary studies in the Atlantic intertidal. We used mitochondrial DNA (mtDNA) control region (N = 131) and microsatellite markers (∼40 individuals/site/locus) to survey populations of the broadly dispersing acorn barnacle from 8 sites spanning 800 km of North American coast and 1 site in Europe. Patterns of mtDNA sequence evolution were consistent with larger population sizes in Europe and population expansion at the conclusion of the last ice age, approximately 20 000 years ago, in North America. A significant portion of mitochondrial diversity was partitioned between the continents (φST = 0.281), but there was only weak structure observed from mtDNA within North America. Microsatellites showed significant structuring between the continents (FST = 0.021) as well as within North America (FST = 0.013). Isolation by distance in North America was largely driven by a split between populations south of Cape Cod and all others (P < 10−4). The glacial events responsible for generating allelic diversity at mtDNA and microsatellites may also be responsible for generating selectable variation at metabolic enzymes in S. balanoides. PMID:21885571

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.

Sequence-specific binding of counterions to B-DNA

PubMed Central

Denisov, Vladimir P.; Halle, Bertil

2000-01-01

Recent studies by x-ray crystallography, NMR, and molecular simulations have suggested that monovalent counterions can penetrate deeply into the minor groove of B form DNA. Such groove-bound ions potentially could play an important role in AT-tract bending and groove narrowing, thereby modulating DNA function in vivo. To address this issue, we report here 23Na magnetic relaxation dispersion measurements on oligonucleotides, including difference experiments with the groove-binding drug netropsin. The exquisite sensitivity of this method to ions in long-lived and intimate association with DNA allows us to detect sequence-specific sodium ion binding in the minor groove AT tract of three B-DNA dodecamers. The sodium ion occupancy is only a few percent, however, and therefore is not likely to contribute importantly to the ensemble of B-DNA structures. We also report results of ion competition experiments, indicating that potassium, rubidium, and cesium ions bind to the minor groove with similarly weak affinity as sodium ions, whereas ammonium ion binding is somewhat stronger. The present findings are discussed in the light of previous NMR and diffraction studies of sequence-specific counterion binding to DNA. PMID:10639130

Characterization of DNA-protein interactions using high-throughput sequencing data from pulldown experiments

NASA Astrophysics Data System (ADS)

Moreland, Blythe; Oman, Kenji; Curfman, John; Yan, Pearlly; Bundschuh, Ralf

Methyl-binding domain (MBD) protein pulldown experiments have been a valuable tool in measuring the levels of methylated CpG dinucleotides. Due to the frequent use of this technique, high-throughput sequencing data sets are available that allow a detailed quantitative characterization of the underlying interaction between methylated DNA and MBD proteins. Analyzing such data sets, we first found that two such proteins cannot bind closer to each other than 2 bp, consistent with structural models of the DNA-protein interaction. Second, the large amount of sequencing data allowed us to find rather weak but nevertheless clearly statistically significant sequence preferences for several bases around the required CpG. These results demonstrate that pulldown sequencing is a high-precision tool in characterizing DNA-protein interactions. This material is based upon work supported by the National Science Foundation under Grant No. DMR-1410172.

Conformation-dependent DNA attraction.

PubMed

Li, Weifeng; Nordenskiöld, Lars; Zhou, Ruhong; Mu, Yuguang

2014-06-21

Understanding how DNA molecules interact with other biomolecules is related to how they utilize their functions and is therefore critical for understanding their structure-function relationships. For a long time, the existence of Z-form DNA (a left-handed double helical version of DNA, instead of the common right-handed B-form) has puzzled the scientists, and the definitive biological significance of Z-DNA has not yet been clarified. In this study, the effects of DNA conformation in DNA-DNA interactions are explored by molecular dynamics simulations. Using umbrella sampling, we find that for both B- and Z-form DNA, surrounding Mg(2+) ions always exert themselves to screen the Coulomb repulsion between DNA phosphates, resulting in very weak attractive force. On the contrary, a tight and stable bound state is discovered for Z-DNA in the presence of Mg(2+) or Na(+), benefiting from their hydrophobic nature. Based on the contact surface and a dewetting process analysis, a two-stage binding process of Z-DNA is outlined: two Z-DNA first attract each other through charge screening and Mg(2+) bridges to phosphate groups in the same way as that of B-DNA, after which hydrophobic contacts of the deoxyribose groups are formed via a dewetting effect, resulting in stable attraction between two Z-DNA molecules. The highlighted hydrophobic nature of Z-DNA interaction from the current study may help to understand the biological functions of Z-DNA in gene transcription.

Antiproliferative, DNA intercalation and redox cycling activities of dioxonaphtho[2,3-d]imidazolium analogs of YM155: A structure-activity relationship study.

PubMed

Ho, Si-Han Sherman; Sim, Mei-Yi; Yee, Wei-Loong Sherman; Yang, Tianming; Yuen, Shyi-Peng John; Go, Mei-Lin

2015-11-02

The anticancer agent YM155 is widely investigated as a specific survivin suppressant. More recently, YM155 was found to induce DNA damage and this has raised doubts as to whether survivin is its primary target. In an effort to assess the contribution of DNA damage to the anticancer activity of YM155, several analogs were prepared and evaluated for antiproliferative activity on malignant cells, participation in DNA intercalation and free radical generation by redox cycling. The intact positively charged scaffold was found to be essential for antiproliferative activity and intercalation but was less critical for redox cycling where the minimal requirement was a pared down bicyclic quinone. Side chain requirements at the N(1) and N(3) positions of the scaffold were more alike for redox cycling and intercalation than antiproliferative activity, underscoring yet again, the limited structural overlaps for these activities. Furthermore, antiproliferative activities were poorly correlated to DNA intercalation and redox cycling. Potent antiproliferative activity (IC50 9-23 nM), exceeding that of YM155, was found for a minimally substituted methyl analog AB7. Like YM155 and other dioxonaphthoimidazoliums, AB7 was a modest DNA intercalator but with weak redox cycling activity. Thus, the capacity of this scaffold to inflict direct DNA damage leading to cell death may not be significant and YM155 should not be routinely classified as a DNA damaging agent. Copyright © 2015 Elsevier Masson SAS. All rights reserved.

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.

Chicken fetal liver DNA damage and adduct formation by activation-dependent DNA-reactive carcinogens and related compounds of several structural classes.

PubMed

Williams, Gary M; Duan, Jian-Dong; Brunnemann, Klaus D; Iatropoulos, Michael J; Vock, Esther; Deschl, Ulrich

2014-09-01

The chicken egg genotoxicity assay (CEGA), which utilizes the liver of an intact and aseptic embryo-fetal test organism, was evaluated using four activation-dependent DNA-reactive carcinogens and four structurally related less potent carcinogens or non-carcinogens. In the assay, three daily doses of test substances were administered to eggs containing 9-11-day-old fetuses and the fetal livers were assessed for two endpoints, DNA breaks using the alkaline single cell gel electrophoresis (comet) assay and DNA adducts using the (32)P-nucleotide postlabeling (NPL) assay. The effects of four carcinogens of different structures requiring distinct pathways of bioactivation, i.e., 2-acetylaminofluorene (AAF), aflatoxin B1 (AFB1), benzo[a]pyrene (B[a]P), and diethylnitrosamine (DEN), were compared with structurally related non-carcinogens fluorene (FLU) and benzo[e]pyrene (B[e]P) or weak carcinogens, aflatoxin B2 (AFB2) and N-nitrosodiethanolamine (NDELA). The four carcinogens all produced DNA breaks at microgram or low milligram total doses, whereas less potent carcinogens and non-carcinogens yielded borderline or negative results, respectively, at higher doses. AAF and B[a]P produced DNA adducts, whereas none was found with the related comparators FLU or B[e]P, consistent with comet results. DEN and NDELA were also negative for adducts, as expected in the case of DEN for an alkylating agent in the standard NPL assay. Also, AFB1 and AFB2 were negative in NPL, as expected, due to the nature of ring opened aflatoxin adducts, which are resistant to enzymatic digestion. Thus, the CEGA, using comet and NPL, is capable of detection of the genotoxicity of diverse DNA-reactive carcinogens, while not yielding false positives for non-carcinogens. © The Author 2014. Published by Oxford University Press on behalf of the Society of Toxicology. All rights reserved. For permissions, please email: journals.permissions@oup.com.

Effects of cytotoxic cis- and trans-diammine monochlorido platinum(II) complexes on selenium-dependent redox enzymes and DNA.

PubMed

Lemmerhirt, Heidi; Behnisch, Steven; Bodtke, Anja; Lillig, Christopher H; Pazderova, Lucia; Kasparkova, Jana; Brabec, Viktor; Bednarski, Patrick J

2018-01-01

Here we present the preparation of 14 pairs of cis- and trans-diammine monochlorido platinum(II) complexes, coordinated to heterocycles (i.e., imidazole, 2-methylimidazole and pyrazole) and linked to various acylhydrazones, which were designed as potential inhibitors of the selenium-dependent enzymes glutathione peroxidase 1 (GPx-1) and thioredoxin reductase 1 (TrxR-1). However, no inhibition of bovine GPx-1 and only weak inhibition of murine TrxR-1 was observed in in vitro assays. Nonetheless, the cis configured diammine monochlorido Pt(II) complexes exhibited cytotoxic and apoptotic properties on various human cancer cell lines, whereas the trans configured complexes generally showed weaker potency with a few exceptions. On the other hand, the trans complexes were generally more likely to lack cross-resistance to cisplatin than the cis analogues. Platinum was found bound to the nuclear DNA of cancer cells treated with representative Pt complexes, suggesting that DNA might be a possible target. Thus, detailed in vitro binding experiments with DNA were conducted. Interactions of the compounds with calf thymus DNA were investigated, including Pt binding kinetics, circular dichroism (CD) spectral changes, changes in DNA melting temperatures, unwinding of supercoiled plasmids and ethidium bromide displacement in DNA. The CD results indicate that the most active cis configured pyrazole-derived complex causes unique structural changes in the DNA compared to the other complexes as well as to those caused by cisplatin, suggesting a denaturation of the DNA structure. This may be important for the antiproliferative activity of this compound in the cancer cells. Copyright © 2017. Published by Elsevier Inc.

Using DNA mechanics to predict in vitro nucleosome positions and formation energies

PubMed Central

Morozov, Alexandre V.; Fortney, Karissa; Gaykalova, Daria A.; Studitsky, Vasily M.; Widom, Jonathan; Siggia, Eric D.

2009-01-01

In eukaryotic genomes, nucleosomes function to compact DNA and to regulate access to it both by simple physical occlusion and by providing the substrate for numerous covalent epigenetic tags. While competition with other DNA-binding factors and action of chromatin remodeling enzymes significantly affect nucleosome formation in vivo, nucleosome positions in vitro are determined by steric exclusion and sequence alone. We have developed a biophysical model, DNABEND, for the sequence dependence of DNA bending energies, and validated it against a collection of in vitro free energies of nucleosome formation and a set of in vitro nucleosome positions mapped at high resolution. We have also made a first ab initio prediction of nucleosomal DNA geometries, and checked its accuracy against the nucleosome crystal structure. We have used DNABEND to design both strong and weak histone- binding sequences, and measured the corresponding free energies of nucleosome formation. We find that DNABEND can successfully predict in vitro nucleosome positions and free energies, providing a physical explanation for the intrinsic sequence dependence of histone–DNA interactions. PMID:19509309

Cryo-EM of dynamic protein complexes in eukaryotic DNA replication.

PubMed

Sun, Jingchuan; Yuan, Zuanning; Bai, Lin; Li, Huilin

2017-01-01

DNA replication in Eukaryotes is a highly dynamic process that involves several dozens of proteins. Some of these proteins form stable complexes that are amenable to high-resolution structure determination by cryo-EM, thanks to the recent advent of the direct electron detector and powerful image analysis algorithm. But many of these proteins associate only transiently and flexibly, precluding traditional biochemical purification. We found that direct mixing of the component proteins followed by 2D and 3D image sorting can capture some very weakly interacting complexes. Even at 2D average level and at low resolution, EM images of these flexible complexes can provide important biological insights. It is often necessary to positively identify the feature-of-interest in a low resolution EM structure. We found that systematically fusing or inserting maltose binding protein (MBP) to selected proteins is highly effective in these situations. In this chapter, we describe the EM studies of several protein complexes involved in the eukaryotic DNA replication over the past decade or so. We suggest that some of the approaches used in these studies may be applicable to structural analysis of other biological systems. © 2016 The Protein Society.

Weak nanoscale chaos and anomalous relaxation in DNA

NASA Astrophysics Data System (ADS)

Mazur, Alexey K.

2017-06-01

Anomalous nonexponential relaxation in hydrated biomolecules is commonly attributed to the complexity of the free-energy landscapes, similarly to polymers and glasses. It was found recently that the hydrogen-bond breathing of terminal DNA base pairs exhibits a slow power-law relaxation attributable to weak Hamiltonian chaos, with parameters similar to experimental data. Here, the relationship is studied between this motion and spectroscopic signals measured in DNA with a small molecular photoprobe inserted into the base-pair stack. To this end, the earlier computational approach in combination with an analytical theory is applied to the experimental DNA fragment. It is found that the intensity of breathing dynamics is strongly increased in the internal base pairs that flank the photoprobe, with anomalous relaxation quantitatively close to that in terminal base pairs. A physical mechanism is proposed to explain the coupling between the relaxation of base-pair breathing and the experimental response signal. It is concluded that the algebraic relaxation observed experimentally is very likely a manifestation of weakly chaotic dynamics of hydrogen-bond breathing in the base pairs stacked to the photoprobe and that the weak nanoscale chaos can represent an ubiquitous hidden source of nonexponential relaxation in ultrafast spectroscopy.

Weak nanoscale chaos and anomalous relaxation in DNA.

PubMed

Mazur, Alexey K

2017-06-01

Anomalous nonexponential relaxation in hydrated biomolecules is commonly attributed to the complexity of the free-energy landscapes, similarly to polymers and glasses. It was found recently that the hydrogen-bond breathing of terminal DNA base pairs exhibits a slow power-law relaxation attributable to weak Hamiltonian chaos, with parameters similar to experimental data. Here, the relationship is studied between this motion and spectroscopic signals measured in DNA with a small molecular photoprobe inserted into the base-pair stack. To this end, the earlier computational approach in combination with an analytical theory is applied to the experimental DNA fragment. It is found that the intensity of breathing dynamics is strongly increased in the internal base pairs that flank the photoprobe, with anomalous relaxation quantitatively close to that in terminal base pairs. A physical mechanism is proposed to explain the coupling between the relaxation of base-pair breathing and the experimental response signal. It is concluded that the algebraic relaxation observed experimentally is very likely a manifestation of weakly chaotic dynamics of hydrogen-bond breathing in the base pairs stacked to the photoprobe and that the weak nanoscale chaos can represent an ubiquitous hidden source of nonexponential relaxation in ultrafast spectroscopy.

Probing the electrostatics and pharmacologic modulation of sequence-specific binding by the DNA-binding domain of the ETS-family transcription factor PU.1: a binding affinity and kinetics investigation

PubMed Central

Munde, Manoj; Poon, Gregory M. K.; Wilson, W. David

2013-01-01

Members of the ETS family of transcription factors regulate a functionally diverse array of genes. All ETS proteins share a structurally-conserved but sequence-divergent DNA-binding domain, known as the ETS domain. Although the structure and thermodynamics of the ETS-DNA complexes are well known, little is known about the kinetics of sequence recognition, a facet that offers potential insight into its molecular mechanism. We have characterized DNA binding by the ETS domain of PU.1 by biosensor-surface plasmon resonance (SPR). SPR analysis revealed a striking kinetic profile for DNA binding by the PU.1 ETS domain. At low salt concentrations, it binds high-affinity cognate DNA with a very slow association rate constant (≤105 M−1 s−1), compensated by a correspondingly small dissociation rate constant. The kinetics are strongly salt-dependent but mutually balance to produce a relatively weak dependence in the equilibrium constant. This profile contrasts sharply with reported data for other ETS domains (e.g., Ets-1, TEL) for which high-affinity binding is driven by rapid association (>107 M−1 s−1). We interpret this difference in terms of the hydration properties of ETS-DNA binding and propose that at least two mechanisms of sequence recognition are employed by this family of DNA-binding domain. Additionally, we use SPR to demonstrate the potential for pharmacological inhibition of sequence-specific ETS-DNA binding, using the minor groove-binding distamycin as a model compound. Our work establishes SPR as a valuable technique for extending our understanding of the molecular mechanisms of ETS-DNA interactions as well as developing potential small-molecule agents for biotechnological and therapeutic purposes. PMID:23416556

DNA interaction with naturally occurring antioxidant flavonoids quercetin, kaempferol, and delphinidin.

PubMed

Kanakis, C D; Tarantilis, P A; Polissiou, M G; Diamantoglou, S; Tajmir-Riahi, H A

2005-06-01

Flavonoids are strong antioxidants that prevent DNA damage. The anticancer and antiviral activities of these natural products are implicated in their mechanism of actions. However, there has been no information on the interactions of these antioxidants with individual DNA at molecular level. This study was designed to examine the interaction of quercetin (que), kaempferol (kae), and delphinidin (del) with calf-thymus DNA in aqueous solution at physiological conditions, using constant DNA concentration (6.5 mmol) and various drug/DNA(phosphate) ratios of 1/65 to 1. FTIR and UV-Visible difference spectroscopic methods are used to determine the drug binding sites, the binding constants and the effects of drug complexation on the stability and conformation of DNA duplex. Structural analysis showed quercetin, kaempferol, and delphinidin bind weakly to adenine, guanine (major groove), and thymine (minor groove) bases, as well as to the backbone phosphate group with overall binding constants K(que) = 7.25 x 10(4)M(-1), K(kae) = 3.60 x 10(4)M(-1), and K(del) = 1.66 x 10(4)M(-1). The stability of adduct formation is in the order of que>kae>del. Delphinidin with a positive charge induces more stabilizing effect on DNA duplex than quercetin and kaempferol. A partial B to A-DNA transition occurs at high drug concentrations.

Electrostatics, structure prediction, and the energy landscapes for protein folding and binding.

PubMed

Tsai, Min-Yeh; Zheng, Weihua; Balamurugan, D; Schafer, Nicholas P; Kim, Bobby L; Cheung, Margaret S; Wolynes, Peter G

2016-01-01

While being long in range and therefore weakly specific, electrostatic interactions are able to modulate the stability and folding landscapes of some proteins. The relevance of electrostatic forces for steering the docking of proteins to each other is widely acknowledged, however, the role of electrostatics in establishing specifically funneled landscapes and their relevance for protein structure prediction are still not clear. By introducing Debye-Hückel potentials that mimic long-range electrostatic forces into the Associative memory, Water mediated, Structure, and Energy Model (AWSEM), a transferable protein model capable of predicting tertiary structures, we assess the effects of electrostatics on the landscapes of thirteen monomeric proteins and four dimers. For the monomers, we find that adding electrostatic interactions does not improve structure prediction. Simulations of ribosomal protein S6 show, however, that folding stability depends monotonically on electrostatic strength. The trend in predicted melting temperatures of the S6 variants agrees with experimental observations. Electrostatic effects can play a range of roles in binding. The binding of the protein complex KIX-pKID is largely assisted by electrostatic interactions, which provide direct charge-charge stabilization of the native state and contribute to the funneling of the binding landscape. In contrast, for several other proteins, including the DNA-binding protein FIS, electrostatics causes frustration in the DNA-binding region, which favors its binding with DNA but not with its protein partner. This study highlights the importance of long-range electrostatics in functional responses to problems where proteins interact with their charged partners, such as DNA, RNA, as well as membranes. © 2015 The Protein Society.

A remote palm domain residue of RB69 DNA polymerase is critical for enzyme activity and influences the conformation of the active site.

PubMed

Jacewicz, Agata; Trzemecka, Anna; Guja, Kip E; Plochocka, Danuta; Yakubovskaya, Elena; Bebenek, Anna; Garcia-Diaz, Miguel

2013-01-01

Non-conserved amino acids that are far removed from the active site can sometimes have an unexpected effect on enzyme catalysis. We have investigated the effects of alanine replacement of residues distant from the active site of the replicative RB69 DNA polymerase, and identified a substitution in a weakly conserved palm residue (D714A), that renders the enzyme incapable of sustaining phage replication in vivo. D714, located several angstroms away from the active site, does not contact the DNA or the incoming dNTP, and our apoenzyme and ternary crystal structures of the Pol(D714A) mutant demonstrate that D714A does not affect the overall structure of the protein. The structures reveal a conformational change of several amino acid side chains, which cascade out from the site of the substitution towards the catalytic center, substantially perturbing the geometry of the active site. Consistent with these structural observations, the mutant has a significantly reduced k pol for correct incorporation. We propose that the observed structural changes underlie the severe polymerization defect and thus D714 is a remote, non-catalytic residue that is nevertheless critical for maintaining an optimal active site conformation. This represents a striking example of an action-at-a-distance interaction.

Acid-Labile Poly(glycidyl methacrylate)-Based Star Gene Vectors.

PubMed

Yang, Yan-Yu; Hu, Hao; Wang, Xing; Yang, Fei; Shen, Hong; Xu, Fu-Jian; Wu, De-Cheng

2015-06-10

It was recently reported that ethanolamine-functionalized poly(glycidyl methacrylate) (PGEA) possesses great potential applications in gene therapy due to its good biocompatibility and high transfection efficiency. Importing responsivity into PGEA vectors would further improve their performances. Herein, a series of responsive star-shaped vectors, acetaled β-cyclodextrin-PGEAs (A-CD-PGEAs) consisting of a β-CD core and five PGEA arms linked by acid-labile acetal groups, were proposed and characterized as therapeutic pDNA vectors. The A-CD-PGEAs owned abundant hydroxyl groups to shield extra positive charges of A-CD-PGEAs/pDNA complexes, and the star structure could decrease charge density. The incorporation of acetal linkers endowed A-CD-PGEAs with pH responsivity and degradation. In weakly acidic endosome, the broken acetal linkers resulted in decomposition of A-CD-PGEAs and morphological transformation of A-CD-PGEAs/pDNA complexes, lowering cytotoxicity and accelerating release of pDNA. In comparison with control CD-PGEAs without acetal linkers, A-CD-PGEAs exhibited significantly better transfection performances.

A Water‐Soluble Tetraazaperopyrene Dye as Strong G‐Quadruplex DNA Binder

PubMed Central

Hahn, Lena

2016-01-01

Abstract The interactions of the water‐soluble tetraazaperopyrene dye 1 with ct‐DNA, duplex‐[(dAdT)12 ⋅(dAdT)12], duplex‐[(dGdC)12 ⋅(dGdC)12] as well as with two G‐quadruplex‐forming sequences, namely the human telomeric 22AG and the promotor sequence c‐myc, were investigated by means of UV/visible and fluorescence spectroscopy, isothermal titration calorimetry (ITC) and molecular docking studies. Dye 1 exhibits a high affinity for G‐quadruplex structures over duplex DNA structures. Furthermore, the ligand shows promising G‐quadruplex discrimination, with an affinity towards c‐myc of 2×107  m −1 (i.e., K d=50 nm), which is higher than for 22AG (4×106  m −1). The ITC data reveal that compound 1 interacts with c‐myc in a stoichiometric ratio of 1:1 but also indicate the presence of two identical lower affinity secondary binding sites per quadruplex. In 22AG, there are two high affinity binding sites per quadruplex, that is, one on each side, with a further four weaker binding sites. For both quadruplex structures, the high affinity interactions between compound 1 and the quadruplex‐forming nucleic acid structures are weakly endothermic. Molecular docking studies suggest an end‐stacking binding mode for compound 1 interacting with quadruplex structures, and a higher affinity for the parallel conformation of c‐myc than for the mixed‐hybrid conformation of 22AG. In addition, docking studies also suggest that the reduced affinity for duplex DNA structures is due to the non‐viability of an intercalative binding mode. PMID:26997208

First evidence of DNA methylation in insect Tribolium castaneum: environmental regulation of DNA methylation within heterochromatin.

PubMed

Feliciello, Isidoro; Parazajder, Josip; Akrap, Ivana; Ugarković, Durđica

2013-05-01

DNA methylation has been studied in many eukaryotic organisms, in particular vertebrates, and was implicated in developmental and phenotypic variations. Little is known about the role of DNA methylation in invertebrates, although insects are considered as excellent models for studying the evolution of DNA methylation. In the red flour beetle, Tribolium castaneum (Tenebrionidae, Coleoptera), no evidence of DNA methylation has been found till now. In this paper, a cytosine methylation in Tribolium castaneum embryos was detected by methylation sensitive restriction endonucleases and immuno-dot blot assay. DNA methylation in embryos is followed by a global demethylation in larvae, pupae and adults. DNA demethylation seems to proceed actively through 5-hydroxymethylcytosine, most probably by the action of TET enzyme. Bisulfite sequencing of a highly abundant satellite DNA located in pericentromeric heterochromatin revealed similar profile of cytosine methylation in adults and embryos. Cytosine methylation was not only restricted to CpG sites but was found at CpA, CpT and CpC sites. In addition, complete cytosine demethylation of heterochromatic satellite DNA was induced by heat stress. The results reveal existence of DNA methylation cycling in T. castaneum ranging from strong overall cytosine methylation in embryos to a weak DNA methylation in other developmental stages. Nevertheless, DNA methylation is preserved within heterochromatin during development, indicating its role in heterochromatin formation and maintenance. It is, however, strongly affected by heat stress, suggesting a role for DNA methylation in heterochromatin structure modulation during heat stress response.

Searching target sites on DNA by proteins: Role of DNA dynamics under confinement

PubMed Central

Mondal, Anupam; Bhattacherjee, Arnab

2015-01-01

DNA-binding proteins (DBPs) rapidly search and specifically bind to their target sites on genomic DNA in order to trigger many cellular regulatory processes. It has been suggested that the facilitation of search dynamics is achieved by combining 3D diffusion with one-dimensional sliding and hopping dynamics of interacting proteins. Although, recent studies have advanced the knowledge of molecular determinants that affect one-dimensional search efficiency, the role of DNA molecule is poorly understood. In this study, by using coarse-grained simulations, we propose that dynamics of DNA molecule and its degree of confinement due to cellular crowding concertedly regulate its groove geometry and modulate the inter-communication with DBPs. Under weak confinement, DNA dynamics promotes many short, rotation-decoupled sliding events interspersed by hopping dynamics. While this results in faster 1D diffusion, associated probability of missing targets by jumping over them increases. In contrast, strong confinement favours rotation-coupled sliding to locate targets but lacks structural flexibility to achieve desired specificity. By testing under physiological crowding, our study provides a plausible mechanism on how DNA molecule may help in maintaining an optimal balance between fast hopping and rotation-coupled sliding dynamics, to locate target sites rapidly and form specific complexes precisely. PMID:26400158

Specificity of the weak binding between the phage SPO1 transcription-inhibitory protein, TF1, and SPO1 DNA.

PubMed

Johnson, G G; Geiduschek, E P

1977-04-05

The interaction of the phage SPO1 protein transcription factor 1 (TF1), with DNA has been analyzed by membrane filter binding and by sedimentation methods. Substantially specific binding of TF1 to helical SPO1 DNA can be demonstrated by nitrocellulose filter-binding assays at relatively low ionic strength (0.08). However, TF1-DNA complexes dissociate and reequilibrate relatively rapidly and this makes filter-binding assays unsuitable for quantitative measurements of binding equilibra. Accordingly, the sedimentation properties of TF1-DNA complexes have been explored and a short-column centrifugation assay has been elaborated for quantitative measurements. Preferential binding of TF1 to the hydroxymethyluracil-containing SPO1 DNA has also been demonstrated by short-column centrifugation. TF1 binds relatively weakly and somewhat cooperatively to SPO1 DNA at many sites; TF1-DNA complexes dissociate and reequilibrate rapidly. At 20 degrees C in 0.01 M phosphate, pH 7.5, 0.15 KC1, one molecule of TF1 can bind to approximately every 60 nucleotide pairs of SPO1 DNA.

Methodological flaws introduce strong bias into molecular analysis of microbial populations.

PubMed

Krakat, N; Anjum, R; Demirel, B; Schröder, P

2017-02-01

In this study, we report how different cell disruption methods, PCR primers and in silico analyses can seriously bias results from microbial population studies, with consequences for the credibility and reproducibility of the findings. Our results emphasize the pitfalls of commonly used experimental methods that can seriously weaken the interpretation of results. Four different cell lysis methods, three commonly used primer pairs and various computer-based analyses were applied to investigate the microbial diversity of a fermentation sample composed of chicken dung. The fault-prone, but still frequently used, amplified rRNA gene restriction analysis was chosen to identify common weaknesses. In contrast to other studies, we focused on the complete analytical process, from cell disruption to in silico analysis, and identified potential error rates. This identified a wide disagreement of results between applied experimental approaches leading to very different community structures depending on the chosen approach. The interpretation of microbial diversity data remains a challenge. In order to accurately investigate the taxonomic diversity and structure of prokaryotic communities, we suggest a multi-level approach combining DNA-based and DNA-independent techniques. The identified weaknesses of commonly used methods to study microbial diversity can be overcome by a multi-level approach, which produces more reliable data about the fate and behaviour of microbial communities of engineered habitats such as biogas plants, so that the best performance can be ensured. © 2016 The Society for Applied Microbiology.

Myopathic mtDNA Depletion Syndrome Due to Mutation in TK2 Gene.

PubMed

Martín-Hernández, Elena; García-Silva, María Teresa; Quijada-Fraile, Pilar; Rodríguez-García, María Elena; Rivera, Henry; Hernández-Laín, Aurelio; Coca-Robinot, David; Fernández-Toral, Joaquín; Arenas, Joaquín; Martín, Miguel A; Martínez-Azorín, Francisco

2017-01-01

Whole-exome sequencing was used to identify the disease gene(s) in a Spanish girl with failure to thrive, muscle weakness, mild facial weakness, elevated creatine kinase, deficiency of mitochondrial complex III and depletion of mtDNA. With whole-exome sequencing data, it was possible to get the whole mtDNA sequencing and discard any pathogenic variant in this genome. The analysis of whole exome uncovered a homozygous pathogenic mutation in thymidine kinase 2 gene ( TK2; NM_004614.4:c.323 C>T, p.T108M). TK2 mutations have been identified mainly in patients with the myopathic form of mtDNA depletion syndromes. This patient presents an atypical TK2-related myopathic form of mtDNA depletion syndromes, because despite having a very low content of mtDNA (<20%), she presents a slower and less severe evolution of the disease. In conclusion, our data confirm the role of TK2 gene in mtDNA depletion syndromes and expanded the phenotypic spectrum.

Effects of poly(lactic-co-glycolic acid) on preparation and characteristics of plasmid DNA-loaded solid lipid nanoparticles.

PubMed

Zhu, L; Xie, S; Dong, Z; Wang, X; Wang, Y; Zhou, W

2011-09-01

Poly(lactic-co-glycolic acid) (PLGA) was used as a polymeric emulsifier to encapsulate plasmid DNA into hydrogenated castor oil (HCO)-solid lipid nanoparticles (SLN) by w/o/w double emulsion and solvent evaporation techniques. The effects of PLGA on the preparation, characteristics and transfection efficiency of DNA-loaded SLN were studied. The results showed that PLGA was essential to form the primary w/o emulsion and the stability of the emulsion was enhanced with the increase of PLGA content. DNA-loaded SLN were spherical with smooth surfaces. The SLN had a negative charge in weak acid and alkaline environment but acquired a positive charge in acidic pH and the cationisation capacity of the SLN increased with the increase of PLGA/HCO ratio. Agarose gel electrophoresis demonstrated that the majority of the DNA maintained its structural integrity after preparation and being extracted or released from DNA-loaded SLN. When PLGA/HCO ratio increased from 5 to 15%, the encapsulation efficiency, loading capacity and transfection efficiency of the nanoparticles increased significantly, whereas the changes of particle size and polydispersity index were insignificant. Cytotoxicity study in cell culture demonstrated that the SLN was not toxic.

Transcriptional effect of an Aframomum angustifolium seed extract on human cutaneous cells using low-density DNA chips.

PubMed

Bonnet-Duquennoy, Mathilde; Dumas, Marc; Debacker, Adeline; Lazou, Kristell; Talbourdet, Sylvie; Franchi, Jocelyne; Heusèle, Catherine; André, Patrice; Schnebert, Sylvianne; Bonté, Frédéric; Kurfürst, Robin

2007-06-01

Studying photoexposed and photoprotected skin biopsies from young and aged women, it has been found that a specific zone, composed of the basal layers of the epidermis, the dermal epidermal junction, and the superficial dermis, is major target of aging and reactive oxygen species. We showed that this zone is characterized by significant variations at a transcriptional and/or protein levels. Using low-density DNA chip technology, we evaluated the effect of a natural mixture of Aframomum angustifolium seed extract containing labdane diterpenoids on these aging markers. Expression profiles of normal human fibroblasts (NHF) were studied using a customized cDNA macroarray system containing genes covering dermal structure, inflammatory responses, and oxidative stress defense mechanisms. For normal human keratinocyte (NHK) investigations, we chose OLISA technique, a sensitive and quantitative method developed by BioMérieux specifically designed to investigate cell death, proliferation, epidermal structure, differentiation, and oxidative stress defense response. We observed that this extract strongly modified gene expression profiles of treated NHK, but weakly for NHF. This extract regulated antioxidant defenses, dermal-epidermal junction components, and epidermal renewal-related genes. Using low-density DNA chip technology, we identified new potential actions of A. angustifolium seed extract on skin aging.

Experimental Evidence of Weak Excluded Volume Effects for Nanochannel Confined DNA

NASA Astrophysics Data System (ADS)

Gupta, Damini; Miller, Jeremy J.; Muralidhar, Abhiram; Mahshid, Sara; Reisner, Walter; Dorfman, Kevin D.

In the classical de Gennes picture of weak polymer nanochannel confinement, the polymer contour is envisioned as divided into a series of isometric blobs. Strong excluded volume interactions are present both within a blob and between blobs. In contrast, for semiflexible polymers like DNA, excluded volume interactions are of borderline strength within a blob but appreciable between blobs, giving rise to a chain description consisting of a string of anisometric blobs. We present experimental validation of this subtle effect of excluded volume for DNA nanochannel confinement by performing measurements of variance in chain extension of T4 DNA molecules as a function of effective nanochannel size (305-453 nm). Additionally, we show an approach to systematically reduce the effect of molecular weight dispersity of DNA samples, a typical experimental artifact, by combining confinement spectroscopy with simulations.

Molecular recognition principles and stationary-phase characteristics of topoisomer-selective chemoaffinity materials for chromatographic separation of circular plasmid DNA topoisomers.

PubMed

Mahut, Marek; Lindner, Wolfgang; Lämmerhofer, Michael

2012-01-18

We recently discovered the molecular recognition capability of a quinine carbamate ligand attached to silica as a powerful chemoaffinity material for the chromatographic separation of circular plasmid topoisomers of different linking numbers. In this paper we develop structure-selectivity relationship studies to figure out the essential structural features for topoisomer recognition. By varying different moieties of the original cinchonan-derived selector, it was shown that intercalation by the quinoline moiety of the ligand as assumed initially as the working hypothesis is not an essential feature for topoisomer recognition during chromatography. We found that the key elements for topoisomer selectivity are the presence of a rigid weak anion-exchange site and a H-donor site separated from each other in a defined distance by a 4-atom spacer. Additionally, incorporation of the weak anion-exchange site into a cyclic ring structure provides greater rigidity of the ligand molecule and turned out to be advantageous, if not mandatory, for (close to) baseline separation. © 2011 American Chemical Society

Retrospective natural history of thymidine kinase 2 deficiency.

PubMed

Garone, Caterina; Taylor, Robert W; Nascimento, Andrés; Poulton, Joanna; Fratter, Carl; Domínguez-González, Cristina; Evans, Julie C; Loos, Mariana; Isohanni, Pirjo; Suomalainen, Anu; Ram, Dipak; Hughes, M Imelda; McFarland, Robert; Barca, Emanuele; Lopez Gomez, Carlos; Jayawant, Sandeep; Thomas, Neil D; Manzur, Adnan Y; Kleinsteuber, Karin; Martin, Miguel A; Kerr, Timothy; Gorman, Grainne S; Sommerville, Ewen W; Chinnery, Patrick F; Hofer, Monika; Karch, Christoph; Ralph, Jeffrey; Cámara, Yolanda; Madruga-Garrido, Marcos; Domínguez-Carral, Jana; Ortez, Carlos; Emperador, Sonia; Montoya, Julio; Chakrapani, Anupam; Kriger, Joshua F; Schoenaker, Robert; Levin, Bruce; Thompson, John L P; Long, Yuelin; Rahman, Shamima; Donati, Maria Alice; DiMauro, Salvatore; Hirano, Michio

2018-03-30

Thymine kinase 2 (TK2) is a mitochondrial matrix protein encoded in nuclear DNA and phosphorylates the pyrimidine nucleosides: thymidine and deoxycytidine. Autosomal recessive TK2 mutations cause a spectrum of disease from infantile onset to adult onset manifesting primarily as myopathy. To perform a retrospective natural history study of a large cohort of patients with TK2 deficiency. The study was conducted by 42 investigators across 31 academic medical centres. We identified 92 patients with genetically confirmed diagnoses of TK2 deficiency: 67 from literature review and 25 unreported cases. Based on clinical and molecular genetics findings, we recognised three phenotypes with divergent survival: (1) infantile-onset myopathy (42.4%) with severe mitochondrial DNA (mtDNA) depletion, frequent neurological involvement and rapid progression to early mortality (median post-onset survival (POS) 1.00, CI 0.58 to 2.33 years); (2) childhood-onset myopathy (40.2%) with mtDNA depletion, moderate-to-severe progression of generalised weakness and median POS at least 13 years; and (3) late-onset myopathy (17.4%) with mild limb weakness at onset and slow progression to respiratory insufficiency with median POS of 23 years. Ophthalmoparesis and facial weakness are frequent in adults. Muscle biopsies show multiple mtDNA deletions often with mtDNA depletion. In TK2 deficiency, age at onset, rate of weakness progression and POS are important variables that define three clinical subtypes. Nervous system involvement often complicates the clinical course of the infantile-onset form while extraocular muscle and facial involvement are characteristic of the late-onset form. Our observations provide essential information for planning future clinical trials in this disorder. © Article author(s) (or their employer(s) unless otherwise stated in the text of the article) 2018. All rights reserved. No commercial use is permitted unless otherwise expressly granted.

Facilitated dissociation of transcription factors from single DNA binding sites

PubMed Central

Kamar, Ramsey I.; Banigan, Edward J.; Erbas, Aykut; Giuntoli, Rebecca D.; Olvera de la Cruz, Monica; Johnson, Reid C.; Marko, John F.

2017-01-01

The binding of transcription factors (TFs) to DNA controls most aspects of cellular function, making the understanding of their binding kinetics imperative. The standard description of bimolecular interactions posits that TF off rates are independent of TF concentration in solution. However, recent observations have revealed that proteins in solution can accelerate the dissociation of DNA-bound proteins. To study the molecular basis of facilitated dissociation (FD), we have used single-molecule imaging to measure dissociation kinetics of Fis, a key Escherichia coli TF and major bacterial nucleoid protein, from single dsDNA binding sites. We observe a strong FD effect characterized by an exchange rate ∼1×104 M−1s−1, establishing that FD of Fis occurs at the single-binding site level, and we find that the off rate saturates at large Fis concentrations in solution. Although spontaneous (i.e., competitor-free) dissociation shows a strong salt dependence, we find that FD depends only weakly on salt. These results are quantitatively explained by a model in which partially dissociated bound proteins are susceptible to invasion by competitor proteins in solution. We also report FD of NHP6A, a yeast TF with structure that differs significantly from Fis. We further perform molecular dynamics simulations, which indicate that FD can occur for molecules that interact far more weakly than those that we have studied. Taken together, our results indicate that FD is a general mechanism assisting in the local removal of TFs from their binding sites and does not necessarily require cooperativity, clustering, or binding site overlap. PMID:28364020

Femtosecond pump/supercontinuum-probe spectroscopy: optimized setup and signal analysis for single-shot spectral referencing.

PubMed

Dobryakov, A L; Kovalenko, S A; Weigel, A; Pérez-Lustres, J L; Lange, J; Müller, A; Ernsting, N P

2010-11-01

A setup for pump/supercontinuum-probe spectroscopy is described which (i) is optimized to cancel fluctuations of the probe light by single-shot referencing, and (ii) extends the probe range into the near-uv (1000-270 nm). Reflective optics allow 50 μm spot size in the sample and upon entry into two separate spectrographs. The correlation γ(same) between sample and reference readings of probe light level at every pixel exceeds 0.99, compared to γ(consec)<0.92 reported for consecutive referencing. Statistical analysis provides the confidence interval of the induced optical density, ΔOD. For demonstration we first examine a dye (Hoechst 33258) bound in the minor groove of double-stranded DNA. A weak 1.1 ps spectral oscillation in the fluorescence region, assigned to DNA breathing, is shown to be significant. A second example concerns the weak vibrational structure around t=0 which reflects stimulated Raman processes. With 1% fluctuations of probe power, baseline noise for a transient absorption spectrum becomes 25 μOD rms in 1 s at 1 kHz, allowing to record resonance Raman spectra of flavine adenine dinucleotide in the S(0) and S(1) state.

DNA Is an Antimicrobial Component of Neutrophil Extracellular Traps

PubMed Central

Halverson, Tyler W.R.; Wilton, Mike; Poon, Karen K. H.; Petri, Björn; Lewenza, Shawn

2015-01-01

Neutrophil extracellular traps (NETs) comprise an ejected lattice of chromatin enmeshed with granular and nuclear proteins that are capable of capturing and killing microbial invaders. Although widely employed to combat infection, the antimicrobial mechanism of NETs remains enigmatic. Efforts to elucidate the bactericidal component of NETs have focused on the role of NET-bound proteins including histones, calprotectin and cathepsin G protease; however, exogenous and microbial derived deoxyribonuclease (DNase) remains the most potent inhibitor of NET function. DNA possesses a rapid bactericidal activity due to its ability to sequester surface bound cations, disrupt membrane integrity and lyse bacterial cells. Here we demonstrate that direct contact and the phosphodiester backbone are required for the cation chelating, antimicrobial property of DNA. By treating NETs with excess cations or phosphatase enzyme, the antimicrobial activity of NETs is neutralized, but NET structure, including the localization and function of NET-bound proteins, is maintained. Using intravital microscopy, we visualized NET-like structures in the skin of a mouse during infection with Pseudomonas aeruginosa. Relative to other bacteria, P. aeruginosa is a weak inducer of NETosis and is more resistant to NETs. During NET exposure, we demonstrate that P. aeruginosa responds by inducing the expression of surface modifications to defend against DNA-induced membrane destabilization and NET-mediated killing. Further, we show induction of this bacterial response to NETs is largely due to the bacterial detection of DNA. Therefore, we conclude that the DNA backbone contributes both to the antibacterial nature of NETs and as a signal perceived by microbes to elicit host-resistance strategies. PMID:25590621

Assessment of mitochondrial DNA damage in little brown bats (Myotis lucifugus) collected near a mercury-contaminated river

USGS Publications Warehouse

Karouna-Renier, Natalie K.; White, Carl; Perkins, Christopher R.; Schmerfeld, John J.; Yates, David

2014-01-01

Historical discharges of Hg into the South River near the town of Waynesboro, VA, USA, have resulted in persistently elevated Hg concentrations in sediment, surface water, ground water, soil, and wildlife downstream of the discharge site. In the present study, we examined mercury (Hg) levels in in little brown bats (Myotis lucifugus) from this location and assessed the utility of a non-destructively collected tissue sample (wing punch) for determining mitochondrial DNA (mtDNA) damage in Hg exposed bats. Bats captured 1 and 3 km from the South River, exhibited significantly higher levels of total Hg (THg) in blood and fur than those from the reference location. We compared levels of mtDNA damage using real-time quantitative PCR (qPCR) analysis of two distinct regions of mtDNA. Genotoxicity is among the many known toxic effects of Hg, resulting from direct interactions with DNA or from oxidative damage. Because it lacks many of the protective protein structures and repair mechanisms associated with nuclear DNA, mtDNA is more sensitive to the effects of genotoxic chemicals and therefore may be a useful biomarker in chronically exposed organisms. Significantly higher levels of damage were observed in both regions of mtDNA in bats captured 3 km from the river than in controls. However, levels of mtDNA damage exhibited weak correlations with fur and blood THg levels, suggesting that other factors may play a role in the site-specific differences.

Label-free detection of DNA using a light-addressable potentiometric sensor modified with a positively charged polyelectrolyte layer

NASA Astrophysics Data System (ADS)

Wu, Chunsheng; Bronder, Thomas; Poghossian, Arshak; Werner, Carl Frederik; Schöning, Michael J.

2015-03-01

A multi-spot (16 spots) light-addressable potentiometric sensor (MLAPS) consisting of an Al-p-Si-SiO2 structure modified with a weak polyelectrolyte layer of PAH (poly(allylamine hydrochloride)) was applied for the label-free electrical detection of DNA (deoxyribonucleic acid) immobilization and hybridization by the intrinsic molecular charge for the first time. To achieve a preferentially flat orientation of DNA strands and thus, to reduce the distance between the DNA charge and MLAPS surface, the negatively charged probe single-stranded DNAs (ssDNA) were electrostatically adsorbed onto the positively charged PAH layer using a simple layer-by-layer (LbL) technique. In this way, more DNA charge can be positioned within the Debye length, yielding a higher sensor signal. The surface potential changes in each spot induced due to the surface modification steps (PAH adsorption, probe ssDNA immobilization, hybridization with complementary target DNA (cDNA), non-specific adsorption of mismatched ssDNA) were determined from the shifts of photocurrent-voltage curves along the voltage axis. A high sensor signal of 83 mV was registered after immobilization of probe ssDNA onto the PAH layer. The hybridization signal increases from 5 mV to 32 mV with increasing the concentration of cDNA from 0.1 nM to 5 μM. In contrast, a small signal of 5 mV was recorded in the case of non-specific adsorption of fully mismatched ssDNA (5 μM). The obtained results demonstrate the potential of the MLAPS in combination with the simple and rapid LbL immobilization technique as a promising platform for the future development of multi-spot light-addressable label-free DNA chips with direct electrical readout.A multi-spot (16 spots) light-addressable potentiometric sensor (MLAPS) consisting of an Al-p-Si-SiO2 structure modified with a weak polyelectrolyte layer of PAH (poly(allylamine hydrochloride)) was applied for the label-free electrical detection of DNA (deoxyribonucleic acid) immobilization and hybridization by the intrinsic molecular charge for the first time. To achieve a preferentially flat orientation of DNA strands and thus, to reduce the distance between the DNA charge and MLAPS surface, the negatively charged probe single-stranded DNAs (ssDNA) were electrostatically adsorbed onto the positively charged PAH layer using a simple layer-by-layer (LbL) technique. In this way, more DNA charge can be positioned within the Debye length, yielding a higher sensor signal. The surface potential changes in each spot induced due to the surface modification steps (PAH adsorption, probe ssDNA immobilization, hybridization with complementary target DNA (cDNA), non-specific adsorption of mismatched ssDNA) were determined from the shifts of photocurrent-voltage curves along the voltage axis. A high sensor signal of 83 mV was registered after immobilization of probe ssDNA onto the PAH layer. The hybridization signal increases from 5 mV to 32 mV with increasing the concentration of cDNA from 0.1 nM to 5 μM. In contrast, a small signal of 5 mV was recorded in the case of non-specific adsorption of fully mismatched ssDNA (5 μM). The obtained results demonstrate the potential of the MLAPS in combination with the simple and rapid LbL immobilization technique as a promising platform for the future development of multi-spot light-addressable label-free DNA chips with direct electrical readout. Electronic supplementary information (ESI) available. See DOI: 10.1039/c4nr07225a

The expanding phenotype of mitochondrial myopathy.

PubMed

DiMauro, Salvatore; Gurgel-Giannetti, Juliana

2005-10-01

Our understanding of mitochondrial diseases (defined restrictively as defects in the mitochondrial respiratory chain) continues to progress apace. In this review we provide an update of information regarding disorders that predominantly or exclusively affect skeletal muscle. Most recently described mitochondrial myopathies are due to defects in nuclear DNA, including coenzyme Q10 deficiency, and mutations in genes that control mitochondrial DNA (mtDNA) abundance and structure such as POLG and TK2. Barth syndrome, an X-linked recessive mitochondrial myopathy/cardiopathy, is associated with altered lipid composition of the inner mitochondrial membrane, but a putative secondary impairment of the respiratory chain remains to be documented. Concerning the 'other genome', the role played by mutations in protein encoding genes of mtDNA in causing isolated myopathies has been confirmed. It has also been confirmed that mutations in tRNA genes of mtDNA can cause predominantly myopathic syndromes and - contrary to conventional wisdom - these mutations can be homoplasmic. Defects in the mitochondrial respiratory chain impair energy production and almost invariably involve skeletal muscle, causing exercise intolerance, myalgia, cramps, or fixed weakness, which often affects extraocular muscles and results in droopy eyelids (ptosis) and progressive external ophthalmoplegia.

Nucleosome Positioning and NDR Structure at RNA Polymerase III Promoters

NASA Astrophysics Data System (ADS)

Helbo, Alexandra Søgaard; Lay, Fides D.; Jones, Peter A.; Liang, Gangning; Grønbæk, Kirsten

2017-02-01

Chromatin is structurally involved in the transcriptional regulation of all genes. While the nucleosome positioning at RNA polymerase II (pol II) promoters has been extensively studied, less is known about the chromatin structure at pol III promoters in human cells. We use a high-resolution analysis to show substantial differences in chromatin structure of pol II and pol III promoters, and between subtypes of pol III genes. Notably, the nucleosome depleted region at the transcription start site of pol III genes extends past the termination sequences, resulting in nucleosome free gene bodies. The +1 nucleosome is located further downstream than at pol II genes and furthermore displays weak positioning. The variable position of the +1 location is seen not only within individual cell populations and between cell types, but also between different pol III promoter subtypes, suggesting that the +1 nucleosome may be involved in the transcriptional regulation of pol III genes. We find that expression and DNA methylation patterns correlate with distinct accessibility patterns, where DNA methylation associates with the silencing and inaccessibility at promoters. Taken together, this study provides the first high-resolution map of nucleosome positioning and occupancy at human pol III promoters at specific loci and genome wide.

Sequence requirements of oligonucleotide chiral selectors for the capillary electrophoresis resolution of low-affinity DNA binders.

PubMed

Tohala, Luma; Oukacine, Farid; Ravelet, Corinne; Peyrin, Eric

2017-05-01

We recently reported that a great variety of DNA oligonucleotides (ONs) used as chiral selectors in partial-filling capillary electrophoresis (CE) exhibited interesting enantioresolution properties toward low-affinity DNA binders. Herein, the sequence prerequisites of ONs for the CE enantioseparation process were studied. First, the chiral resolution properties of a series of homopolymeric sequences (Poly-dT) of different lengths (from 5 to 60-mer) were investigated. It was shown that the size increase-dependent random coil-like conformation of Poly-dT favorably acted on the apparent selectivity and resolution. The base-unpairing state constituted also an important factor in the chiral resolution ability of ONs as the switch from the single-stranded to double-stranded structure was responsible for a significant decrease in the analyte selectivity range. Finally, the chemical diversity enhanced the enantioresolution ability of single-stranded ONs. The present work could lay the foundation for the design of performant ON chiral selectors for the CE separation of weak DNA binder enantiomers. © 2017 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

Double-stranded DNA breaks hidden in the neutral Comet assay suggest a role of the sperm nuclear matrix in DNA integrity maintenance

PubMed Central

Ribas-Maynou, J.; Gawecka, J.E.; Benet, J.; Ward, W.S.

2014-01-01

We used a mouse model in which sperm DNA damage was induced to understand the relationship of double-stranded DNA (dsDNA) breaks to sperm chromatin structure and to the Comet assay. Sperm chromatin fragmentation (SCF) produces dsDNA breaks located on the matrix attachment regions, between protamine toroids. In this model, epididymal sperm induced to undergo SCF can religate dsDNA breaks while vas deferens sperm cannot. Here, we demonstrated that the conventional neutral Comet assay underestimates the epididymal SCF breaks because the broken DNA ends remain attached to the nuclear matrix, causing the DNA to remain associated with the dispersion halo, and the Comet tails to be weak. Therefore, we term these hidden dsDNA breaks. When the Comet assay was modified to include an additional incubation with sodium dodecyl sulfate (SDS) and dithiothreitol (DTT) after the conventional lysis, thereby solubilizing the nuclear matrix, the broken DNA was released from the matrix, which resulted in a reduction of the sperm head halo and an increase in the Comet tail length, exposing the hidden dsDNA breaks. Conversely, SCF-induced vas deferens sperm had small halos and long tails with the conventional neutral Comet assay, suggesting that the broken DNA ends were not tethered to the nuclear matrix. These results suggest that the attachment to the nuclear matrix is crucial for the religation of SCF-induced DNA breaks in sperm. Our data suggest that the neutral Comet assay identifies only dsDNA breaks that are released from the nuclear matrix and that the addition of an SDS treatment can reveal these hidden dsDNA breaks. PMID:24282283

Double-stranded DNA breaks hidden in the neutral Comet assay suggest a role of the sperm nuclear matrix in DNA integrity maintenance.

PubMed

Ribas-Maynou, J; Gawecka, J E; Benet, J; Ward, W S

2014-04-01

We used a mouse model in which sperm DNA damage was induced to understand the relationship of double-stranded DNA (dsDNA) breaks to sperm chromatin structure and to the Comet assay. Sperm chromatin fragmentation (SCF) produces dsDNA breaks located on the matrix attachment regions, between protamine toroids. In this model, epididymal sperm induced to undergo SCF can religate dsDNA breaks while vas deferens sperm cannot. Here, we demonstrated that the conventional neutral Comet assay underestimates the epididymal SCF breaks because the broken DNA ends remain attached to the nuclear matrix, causing the DNA to remain associated with the dispersion halo, and the Comet tails to be weak. Therefore, we term these hidden dsDNA breaks. When the Comet assay was modified to include an additional incubation with sodium dodecyl sulfate (SDS) and dithiothreitol (DTT) after the conventional lysis, thereby solubilizing the nuclear matrix, the broken DNA was released from the matrix, which resulted in a reduction of the sperm head halo and an increase in the Comet tail length, exposing the hidden dsDNA breaks. Conversely, SCF-induced vas deferens sperm had small halos and long tails with the conventional neutral Comet assay, suggesting that the broken DNA ends were not tethered to the nuclear matrix. These results suggest that the attachment to the nuclear matrix is crucial for the religation of SCF-induced DNA breaks in sperm. Our data suggest that the neutral Comet assay identifies only dsDNA breaks that are released from the nuclear matrix and that the addition of an SDS treatment can reveal these hidden dsDNA breaks.

Ultrathin Two-Dimensional Covalent Organic Framework Nanosheets: Preparation and Application in Highly Sensitive and Selective DNA Detection.

PubMed

Peng, Yongwu; Huang, Ying; Zhu, Yihan; Chen, Bo; Wang, Liying; Lai, Zhuangchai; Zhang, Zhicheng; Zhao, Meiting; Tan, Chaoliang; Yang, Nailiang; Shao, Fangwei; Han, Yu; Zhang, Hua

2017-06-28

The ability to prepare ultrathin two-dimensional (2D) covalent organic framework (COF) nanosheets (NSs) in high yield is of great importance for the further exploration of their unique properties and potential applications. Herein, by elaborately designing and choosing two flexible molecules with C 3v molecular symmetry as building units, a novel imine-linked COF, namely, TPA-COF, with a hexagonal layered structure and sheet-like morphology, is synthesized. Since the flexible building units are integrated into the COF skeletons, the interlayer stacking becomes weak, resulting in the easy exfoliation of TPA-COF into ultrathin 2D NSs. Impressively, for the first time, the detailed structural information, i.e., the pore channels and individual building units in the NSs, is clearly visualized by using the recently developed low-dose imaging technique of transmission electron microscopy (TEM). As a proof-of-concept application, the obtained ultrathin COF NSs are used as a novel fluorescence sensing platform for the highly sensitive and selective detection of DNA.

Additional hydrogen bonds and base-pair kinetics in the symmetrical AMP-DNA aptamer complex.

PubMed Central

Nonin-Lecomte, S; Lin, C H; Patel, D J

2001-01-01

The solution structure of an adenosine monophosphate (AMP)-DNA aptamer complex has been determined previously [Lin, C. H., and Patel, D. J. (1997) Chem. Biol. 4:817-832]. On a symmetrical aptamer complex containing the same binding loop, but with better resolved spectra, we have identified two additional hydrogen bond-mediated associations in the binding loop. One of these involves a rapidly exchanging G imino proton. The phosphate group of the AMP ligand was identified as the acceptor by comparison with other aptamer complexes. Imino proton exchange measurements also yielded the dissociation constants of the stem and binding loop base pairs. This study shows that nuclear magnetic resonance-based imino proton exchange is a good probe for detection of weak hydrogen-bond associations. PMID:11721004

Geography has a greater effect than Wolbachia infection on population genetic structure in the spider mite, Tetranychus pueraricola.

PubMed

Chen, Y-T; Zhang, Y-K; Du, W-X; Jin, P-Y; Hong, X-Y

2016-10-01

Wolbachia is an intracellular symbiotic bacterium that infects various spider mite species and is associated with alterations in host reproduction, which indicates the potential role in mite evolution. However, studies of Wolbachia infections in the spider mite Tetranychus pueraricola, a major agricultural pest, are limited. Here, we used multilocus sequence typing to determine Wolbachia infection status and examined the relationship between Wolbachia infection status and mitochondrial diversity in T. pueraricola from 12 populations in China. The prevalence of Wolbachia ranged from 2.8 to 50%, and three strains (wTpue1, wTpue2, and wTpue3) were identified. We also found double infections (wTpue1 + wTpue3) within the same individuals. Furthermore, the wTpue1 strain caused weak cytoplasmic incompatibility (CI) (egg hatchability ~55%), whereas another widespread strain, wTpue3, did not induce CI. There was no reduction in mitochondrial DNA (mtDNA) or nuclear DNA diversity among infected individuals, and mtDNA haplotypes did not correspond to specific Wolbachia strains. Phylogenetic analysis and analysis of molecular variance revealed that the distribution of mtDNA and nuclear DNA haplotypes were significantly associated with geography. These findings indicate that Wolbachia infection in T. pueraricola is complex, but T. pueraricola genetic differentiation likely resulted from substantial geographic isolation.

HDOCK: a web server for protein–protein and protein–DNA/RNA docking based on a hybrid strategy

PubMed Central

Yan, Yumeng; Zhang, Di; Zhou, Pei; Li, Botong

2017-01-01

Abstract Protein–protein and protein–DNA/RNA interactions play a fundamental role in a variety of biological processes. Determining the complex structures of these interactions is valuable, in which molecular docking has played an important role. To automatically make use of the binding information from the PDB in docking, here we have presented HDOCK, a novel web server of our hybrid docking algorithm of template-based modeling and free docking, in which cases with misleading templates can be rescued by the free docking protocol. The server supports protein–protein and protein–DNA/RNA docking and accepts both sequence and structure inputs for proteins. The docking process is fast and consumes about 10–20 min for a docking run. Tested on the cases with weakly homologous complexes of <30% sequence identity from five docking benchmarks, the HDOCK pipeline tied with template-based modeling on the protein–protein and protein–DNA benchmarks and performed better than template-based modeling on the three protein–RNA benchmarks when the top 10 predictions were considered. The performance of HDOCK became better when more predictions were considered. Combining the results of HDOCK and template-based modeling by ranking first of the template-based model further improved the predictive power of the server. The HDOCK web server is available at http://hdock.phys.hust.edu.cn/. PMID:28521030

Regulating DNA Self-assembly by DNA-Surface Interactions.

PubMed

Liu, Longfei; Li, Yulin; Wang, Yong; Zheng, Jianwei; Mao, Chengde

2017-12-14

DNA self-assembly provides a powerful approach for preparation of nanostructures. It is often studied in bulk solution and involves only DNA-DNA interactions. When confined to surfaces, DNA-surface interactions become an additional, important factor to DNA self-assembly. However, the way in which DNA-surface interactions influence DNA self-assembly is not well studied. In this study, we showed that weak DNA-DNA interactions could be stabilized by DNA-surface interactions to allow large DNA nanostructures to form. In addition, the assembly can be conducted isothermally at room temperature in as little as 5 seconds. © 2017 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim.

Sequence-Dependent Elasticity and Electrostatics of Single-Stranded DNA: Signatures of Base-Stacking

PubMed Central

McIntosh, Dustin B.; Duggan, Gina; Gouil, Quentin; Saleh, Omar A.

2014-01-01

Base-stacking is a key factor in the energetics that determines nucleic acid structure. We measure the tensile response of single-stranded DNA as a function of sequence and monovalent salt concentration to examine the effects of base-stacking on the mechanical and thermodynamic properties of single-stranded DNA. By comparing the elastic response of highly stacked poly(dA) and that of a polypyrimidine sequence with minimal stacking, we find that base-stacking in poly(dA) significantly enhances the polymer’s rigidity. The unstacking transition of poly(dA) at high force reveals that the intrinsic electrostatic tension on the molecule varies significantly more weakly on salt concentration than mean-field predictions. Further, we provide a model-independent estimate of the free energy difference between stacked poly(dA) and unstacked polypyrimidine, finding it to be ∼−0.25 kBT/base and nearly constant over three orders of magnitude in salt concentration. PMID:24507606

The yeast Saccharomyces cerevisiae DNA polymerase IV: possible involvement in double strand break DNA repair.

PubMed

Leem, S H; Ropp, P A; Sugino, A

1994-08-11

We identified and purified a new DNA polymerase (DNA polymerase IV), which is similar to mammalian DNA polymerase beta, from Saccharomyces cerevisiae and suggested that it is encoded by YCR14C (POLX) on chromosome III. Here, we provided a direct evidence that the purified DNA polymerase IV is indeed encoded by POLX. Strains harboring a pol4 deletion mutation exhibit neither mitotic growth defect nor a meiosis defect, suggesting that DNA polymerase IV participates in nonessential functions in DNA metabolism. The deletion strains did not exhibit UV-sensitivity. However, they did show weak sensitivity to MMS-treatment and exhibited a hyper-recombination phenotype when intragenic recombination was measured during meiosis. Furthermore, MAT alpha pol4 delta segregants had a higher frequency of illegitimate mating with a MAT alpha tester strain than that of wild-type cells. These results suggest that DNA polymerase IV participates in a double-strand break repair pathway. A 3.2kb of the POL4 transcript was weakly expressed in mitotically growing cells. During meiosis, a 2.2 kb POL4 transcript was greatly induced, while the 3.2 kb transcript stayed at constant levels. This induction was delayed in a swi4 delta strain during meiosis, while no effect was observed in a swi6 delta strain.

Genetic diversity and population structure in Bactrocera correcta (Diptera: Tephritidae) inferred from mtDNA cox1 and microsatellite markers

PubMed Central

Qin, Yu-Jia; Buahom, Nopparat; Krosch, Matthew N.; Du, Yu; Wu, Yi; Malacrida, Anna R.; Deng, Yu-Liang; Liu, Jia-Qi; Jiang, Xiao-Long; Li, Zhi-Hong

2016-01-01

Bactrocera correcta is one of the most destructive pests of horticultural crops in tropical and subtropical regions. Despite the economic risk, the population genetics of this pest have remained relatively unexplored. This study explores population genetic structure and contemporary gene flow in B. correcta in Chinese Yunnan Province and attempts to place observed patterns within the broader geographical context of the species’ total range. Based on combined data from mtDNA cox1 sequences and 12 microsatellite loci obtained from 793 individuals located in 7 countries, overall genetic structuring was low. The expansion history of this species, including likely human-mediated dispersal, may have played a role in shaping the observed weak structure. The study suggested a close relationship between Yunnan Province and adjacent countries, with evidence for Western and/or Southern Yunnan as the invasive origin of B. correcta within Yunnan Province. The information gleaned from this analysis of gene flow and population structure has broad implications for quarantine, trade and management of this pest, especially in China where it is expanding northward. Future studies should concentrate effort on sampling South Asian populations, which would enable better inferences of the ancestral location of B. correcta and its invasion history into and throughout Asia. PMID:27929126

Genetic structure and signatures of selection in grey reef sharks (Carcharhinus amblyrhynchos).

PubMed

Momigliano, P; Harcourt, R; Robbins, W D; Jaiteh, V; Mahardika, G N; Sembiring, A; Stow, A

2017-09-01

With overfishing reducing the abundance of marine predators in multiple marine ecosystems, knowledge of genetic structure and local adaptation may provide valuable information to assist sustainable management. Despite recent technological advances, most studies on sharks have used small sets of neutral markers to describe their genetic structure. We used 5517 nuclear single-nucleotide polymorphisms (SNPs) and a mitochondrial DNA (mtDNA) gene to characterize patterns of genetic structure and detect signatures of selection in grey reef sharks (Carcharhinus amblyrhynchos). Using samples from Australia, Indonesia and oceanic reefs in the Indian Ocean, we established that large oceanic distances represent barriers to gene flow, whereas genetic differentiation on continental shelves follows an isolation by distance model. In Australia and Indonesia differentiation at nuclear SNPs was weak, with coral reefs acting as stepping stones maintaining connectivity across large distances. Differentiation of mtDNA was stronger, and more pronounced in females, suggesting sex-biased dispersal. Four independent tests identified a set of loci putatively under selection, indicating that grey reef sharks in eastern Australia are likely under different selective pressures to those in western Australia and Indonesia. Genetic distances averaged across all loci were uncorrelated with genetic distances calculated from outlier loci, supporting the conclusion that different processes underpin genetic divergence in these two data sets. This pattern of heterogeneous genomic differentiation, suggestive of local adaptation, has implications for the conservation of grey reef sharks; furthermore, it highlights that marine species showing little genetic differentiation at neutral loci may exhibit patterns of cryptic genetic structure driven by local selection.

Myopathic mtDNA Depletion Syndrome Due to Mutation in TK2 Gene.

PubMed

Martín-Hernández, Elena; García-Silva, María Teresa; Quijada-Fraile, Pilar; Rodríguez-García, María Elena; Hernández-Laín, Aurelio; Coca-Robinot, David; Rivera, Henry; Fernández-Toral, Joaquín; Arenas, Joaquín; Martín, MiguelÁngel; Martínez-Azorín, Francisco

2016-02-29

Whole-exome sequencing (WES) was used to identify the disease gene(s) in a Spanish girl with failure to thrive, muscle weakness, mild facial weakness, elevated creatine kinase (CK), deficiency of mitochondrial complex III and depletion of mtDNA. With WES data, it was possible to get the whole mtDNA sequencing and discard any pathogenic variant in this genome. The analysis of whole exome uncovered a homozygous pathogenic mutation in Thymidine kinase 2 gene (TK2; NM_004614.4:c.323C>T, p.T108M). TK2 mutations have been identified mainly in patients with the myopathic form of mtDNA depletion syndromes (MDS). This patient presents an atypical TK2 related-myopathic form of MDS, because despite having a very low content of mtDNA (<20%), she presents a slower and less severe evolution of the disease. In conclusion, our data confirm the role of TK2 gene in MDS and expanded the phenotypic spectrum.

Genome-Wide Motif Statistics are Shaped by DNA Binding Proteins over Evolutionary Time Scales

NASA Astrophysics Data System (ADS)

Qian, Long; Kussell, Edo

The composition of genomes with respect to short DNA motifs impacts the ability of DNA binding proteins to locate and bind their target sites. Since nonfunctional DNA binding can be detrimental to cellular functions and ultimately to organismal fitness, organisms could benefit from reducing the number of nonfunctional binding sites genome wide. Using in vitro measurements of binding affinities for a large collection of DNA binding proteins, in multiple species, we detect a significant global avoidance of weak binding sites in genomes. The underlying evolutionary process leaves a distinct genomic hallmark in that similar words have correlated frequencies, which we detect in all species across domains of life. We hypothesize that natural selection against weak binding sites contributes to this process, and using an evolutionary model we show that the strength of selection needed to maintain global word compositions is on the order of point mutation rates. Alternative contributions may come from interference of protein-DNA binding with replication and mutational repair processes, which operates with similar rates. We conclude that genome-wide word compositions have been molded by DNA binding proteins through tiny evolutionary steps over timescales spanning millions of generations.

Impact of Heterogeneity and Lattice Bond Strength on DNA Triangle Crystal Growth.

PubMed

Stahl, Evi; Praetorius, Florian; de Oliveira Mann, Carina C; Hopfner, Karl-Peter; Dietz, Hendrik

2016-09-07

One key goal of DNA nanotechnology is the bottom-up construction of macroscopic crystalline materials. Beyond applications in fields such as photonics or plasmonics, DNA-based crystal matrices could possibly facilitate the diffraction-based structural analysis of guest molecules. Seeman and co-workers reported in 2009 the first designed crystal matrices based on a 38 kDa DNA triangle that was composed of seven chains. The crystal lattice was stabilized, unprecedentedly, by Watson-Crick base pairing. However, 3D crystallization of larger designed DNA objects that include more chains such as DNA origami remains an unsolved problem. Larger objects would offer more degrees of freedom and design options with respect to tailoring lattice geometry and for positioning other objects within a crystal lattice. The greater rigidity of multilayer DNA origami could also positively influence the diffractive properties of crystals composed of such particles. Here, we rationally explore the role of heterogeneity and Watson-Crick interaction strengths in crystal growth using 40 variants of the original DNA triangle as model multichain objects. Crystal growth of the triangle was remarkably robust despite massive chemical, geometrical, and thermodynamical sample heterogeneity that we introduced, but the crystal growth sensitively depended on the sequences of base pairs next to the Watson-Crick sticky ends of the triangle. Our results point to weak lattice interactions and high concentrations as decisive factors for achieving productive crystallization, while sample heterogeneity and impurities played a minor role.

DNA Immobilization and Hybridization Detection by the Intrinsic Molecular Charge Using Capacitive Field-Effect Sensors Modified with a Charged Weak Polyelectrolyte Layer.

PubMed

Bronder, Thomas S; Poghossian, Arshak; Scheja, Sabrina; Wu, Chunsheng; Keusgen, Michael; Mewes, Dieter; Schöning, Michael J

2015-09-16

Miniaturized setup, compatibility with advanced micro- and nanotechnologies, and ability to detect biomolecules by their intrinsic molecular charge favor the semiconductor field-effect platform as one of the most attractive approaches for the development of label-free DNA chips. In this work, a capacitive field-effect EIS (electrolyte-insulator-semiconductor) sensor covered with a layer-by-layer prepared, positively charged weak polyelectrolyte layer of PAH (poly(allylamine hydrochloride)) was used for the label-free electrical detection of DNA (deoxyribonucleic acid) immobilization and hybridization. The negatively charged probe single-stranded DNA (ssDNA) molecules were electrostatically adsorbed onto the positively charged PAH layer, resulting in a preferentially flat orientation of the ssDNA molecules within the Debye length, thus yielding a reduced charge-screening effect and a higher sensor signal. Each sensor-surface modification step (PAH adsorption, probe ssDNA immobilization, hybridization with complementary target DNA (cDNA), reducing an unspecific adsorption by a blocking agent, incubation with noncomplementary DNA (ncDNA) solution) was monitored by means of capacitance-voltage and constant-capacitance measurements. In addition, the surface morphology of the PAH layer was studied by atomic force microscopy and contact-angle measurements. High hybridization signals of 34 and 43 mV were recorded in low-ionic strength solutions of 10 and 1 mM, respectively. In contrast, a small signal of 4 mV was recorded in the case of unspecific adsorption of fully mismatched ncDNA. The density of probe ssDNA and dsDNA molecules as well as the hybridization efficiency was estimated using the experimentally measured DNA immobilization and hybridization signals and a simplified double-layer capacitor model. The results of field-effect experiments were supported by fluorescence measurements, verifying the DNA-immobilization and hybridization event.

Entropy of finite random binary sequences with weak long-range correlations.

PubMed

Melnik, S S; Usatenko, O V

2014-11-01

We study the N-step binary stationary ergodic Markov chain and analyze its differential entropy. Supposing that the correlations are weak we express the conditional probability function of the chain through the pair correlation function and represent the entropy as a functional of the pair correlator. Since the model uses the two-point correlators instead of the block probability, it makes it possible to calculate the entropy of strings at much longer distances than using standard methods. A fluctuation contribution to the entropy due to finiteness of random chains is examined. This contribution can be of the same order as its regular part even at the relatively short lengths of subsequences. A self-similar structure of entropy with respect to the decimation transformations is revealed for some specific forms of the pair correlation function. Application of the theory to the DNA sequence of the R3 chromosome of Drosophila melanogaster is presented.

Entropy of finite random binary sequences with weak long-range correlations

NASA Astrophysics Data System (ADS)

Melnik, S. S.; Usatenko, O. V.

2014-11-01

We study the N -step binary stationary ergodic Markov chain and analyze its differential entropy. Supposing that the correlations are weak we express the conditional probability function of the chain through the pair correlation function and represent the entropy as a functional of the pair correlator. Since the model uses the two-point correlators instead of the block probability, it makes it possible to calculate the entropy of strings at much longer distances than using standard methods. A fluctuation contribution to the entropy due to finiteness of random chains is examined. This contribution can be of the same order as its regular part even at the relatively short lengths of subsequences. A self-similar structure of entropy with respect to the decimation transformations is revealed for some specific forms of the pair correlation function. Application of the theory to the DNA sequence of the R3 chromosome of Drosophila melanogaster is presented.

Chromatin tethering effects of hNopp140 are involved in the spatial organization of nucleolus and the rRNA gene transcription

PubMed Central

Tsai, Yi-Tzang; Lin, Chen-I; Chen, Hung-Kai; Lee, Kuo-Ming; Hsu, Chia-Yi; Yang, Shun-Jen

2008-01-01

The short arms of five human acrocentric chromosomes contain ribosomal gene (rDNA) clusters where numerous mini-nucleoli arise at the exit of mitosis. These small nucleoli tend to coalesce into one or a few large nucleoli during interphase by unknown mechanisms. Here, we demonstrate that the N- and C-terminal domains of a nucleolar protein, hNopp140, bound respectively to α-satellite arrays and rDNA clusters of acrocentric chromosomes for nucleolar formation. The central acidic-and-basic repeated domain of hNopp140, possessing a weak self-self interacting ability, was indispensable for hNopp140 to build up a nucleolar round-shaped structure. The N- or the C-terminally truncated hNopp140 caused nucleolar segregation and was able to alter locations of the rDNA transcription, as mediated by detaching the rDNA repeats from the acrocentric α-satellite arrays. Interestingly, an hNopp140 mutant, made by joining the N- and C-terminal domains but excluding the entire central repeated region, induced nucleolar disruption and global chromatin condensation. Furthermore, RNAi knockdown of hNopp140 resulted in dispersion of the rDNA and acrocentric α-satellite sequences away from nucleolus that was accompanied by rDNA transcriptional silence. Our findings indicate that hNopp140, a scaffold protein, is involved in the nucleolar assembly, fusion, and maintenance. PMID:18253863

Transformation of Saccharomyces cerevisiae with linear DNA killer plasmids from Kluyveromyces lactis.

PubMed Central

Gunge, N; Murata, K; Sakaguchi, K

1982-01-01

Protoplasts of Saccharomyces cerevisiae were mixed with linear DNA plasmids, pGKl1 and pGKl2, isolated from a Kluyveromyces lactis killer strain and treated with polyethylene glycol. Out of 2,000 colonies regenerated on a nonselective medium, two killer transformants were obtained. The pGKl plasmids and the killer character were stably maintained in one (Pdh-1) of them. Another transformant, Pdl-1, was a weak killer, and the subclones consisted of a mixture of weak and nonkiller cells. The weak killers were characterized by the presence of pGKl1 in a decreased amount, and nonkillers were characterized by the absence of pGKl1. The occurrence of two new plasmids which migrated faster than pGKl1 in an agarose gel was observed in Pdl-1 and its subclones, whether weak or nonkillers. Staining with 4',6-diamidino-2-phenylindole revealed that the pGKl plasmids exist in the cytosol of transformant cells with numerous copy numbers. Images PMID:7045080

Theory of electron transfer and molecular state in DNA

NASA Astrophysics Data System (ADS)

Endres, Robert Gunter

2002-09-01

In this thesis, a mechanism for long-range electron transfer in DNA and a systematic search for high conductance DNA are developed. DNA is well known for containing the genetic code of all living species. On the other hand, there are some experimental indications that DNA can mediate effectively long-range electron transfer leading to the concept of chemistry at a distance. This can be important for DNA damage and healing. In the first part of the thesis, a possible mechanism for long-range electron transfer is introduced. The weak distance dependent electron transfer was experimentally observed using transition metal intercalators for donor and acceptor. In our model calculations, the transfer is mediated by the molecular analogue of a Kondo bound state well known from solid state physics of mixed-valence rare-earth compounds. We believe this is quite realistic, since localized d orbitals of the transition metal ions could function as an Anderson impurity embedded in a reservoir of rather delocalized molecular orbitals of the intercalator ligands and DNA pi orbitals. The effective Anderson model is solved with a physically intuitive variational ansatz as well as with the essentially exact DMRG method. The electronic transition matrix element, which is important because it contains the donor-acceptor distance dependence, is obtained with the Mulliken-Hush algorithm as well as from Born-Oppenheimer potential energy surfaces. Our possible explanation of long-range electron transfer is put in context to other more conventional mechanisms which also could lead to similar behavior. Another important issue of DNA is its possible use for nano-technology. Although DNA's mechanical properties are excellent, the question whether it can be conducting and be used for nano-wires is highly controversial. Experimentally, DNA shows conducting, semi-conducting and insulating properties. Motivated by these wide ranging experimental results on the conductivity of DNA, we have embarked on a theoretical effort to ascertain what conditions might induce such remarkable behavior. We use a combination of an ab initio density functional theory method and a parameterized Huckel-Slater-Koster model. Our focus here is to examine whether any likely DNA structures or environments can yield reduced activation gaps to conduction or enhanced electronic overlaps. In particular, we study a hypothetical stretched ribbon structure, A-, and B-form DNA, and the effects of counterions and humidity. Unlike solids, DNA and other molecules are considered soft condensed matter. Hence, we study the influence of vibrations upon the electronic structure of DNA. We calculate parameters for charge transfer rates between adjacent bases. We find good agreement between our estimated rates and recent experimental data assuming that torsional vibrations limit the charge transfer most significantly.

HDOCK: a web server for protein-protein and protein-DNA/RNA docking based on a hybrid strategy.

PubMed

Yan, Yumeng; Zhang, Di; Zhou, Pei; Li, Botong; Huang, Sheng-You

2017-07-03

Protein-protein and protein-DNA/RNA interactions play a fundamental role in a variety of biological processes. Determining the complex structures of these interactions is valuable, in which molecular docking has played an important role. To automatically make use of the binding information from the PDB in docking, here we have presented HDOCK, a novel web server of our hybrid docking algorithm of template-based modeling and free docking, in which cases with misleading templates can be rescued by the free docking protocol. The server supports protein-protein and protein-DNA/RNA docking and accepts both sequence and structure inputs for proteins. The docking process is fast and consumes about 10-20 min for a docking run. Tested on the cases with weakly homologous complexes of <30% sequence identity from five docking benchmarks, the HDOCK pipeline tied with template-based modeling on the protein-protein and protein-DNA benchmarks and performed better than template-based modeling on the three protein-RNA benchmarks when the top 10 predictions were considered. The performance of HDOCK became better when more predictions were considered. Combining the results of HDOCK and template-based modeling by ranking first of the template-based model further improved the predictive power of the server. The HDOCK web server is available at http://hdock.phys.hust.edu.cn/. © The Author(s) 2017. Published by Oxford University Press on behalf of Nucleic Acids Research.

DNA relatedness and biochemical features of Campylobacter spp. isolated in central and South Australia.

PubMed Central

Steele, T W; Sangster, N; Lanser, J A

1985-01-01

Investigations of the etiology of diarrhea in patients in South Australia and the Northern Territory showed that Campylobacter spp. other than Campylobacter jejuni and C. coli were common in children. Campylobacters which were hippurate positive, nitrate negative, and susceptible to cephalothin and polymyxins were shown to be closely related to C. jejuni by DNA studies. Thermotolerant catalase-negative campylobacters were also isolated. These were H2S negative and biochemically resembled the catalase-negative or weak strains found in dogs in Sweden. DNA studies showed these campylobacters to be distinct from C. sputorum subsp. sputorum and to form a homogeneous group distinct from the enteropathogenic catalase-positive campylobacters. Preliminary studies suggest that these campylobacters are related to the Swedish catalase-negative or weak strains. PMID:2991331

Variations in Nuclear Localization Strategies Among Pol X Family Enzymes.

PubMed

Kirby, Thomas W; Pedersen, Lars C; Gabel, Scott A; Gassman, Natalie R; London, Robert E

2018-06-22

Despite the essential roles of pol X family enzymes in DNA repair, information about the structural basis of their nuclear import is limited. Recent studies revealed the unexpected presence of a functional NLS in DNA polymerase β, indicating the importance of active nuclear targeting, even for enzymes likely to leak into and out of the nucleus. The current studies further explore the active nuclear transport of these enzymes by identifying and structurally characterizing the functional NLS sequences in the three remaining human pol X enzymes: terminal deoxynucleotidyl transferase (TdT), DNA polymerase μ (pol μ), and DNA polymerase λ (pol λ). NLS identifications are based on Importin α (Impα) binding affinity determined by fluorescence polarization of fluorescein-labeled NLS peptides, X-ray crystallographic analysis of the Impα∆IBB•NLS complexes, and fluorescence-based subcellular localization studies. All three polymerases use NLS sequences located near their N-terminus; TdT and pol μ utilize monopartite NLS sequences, while pol λ utilizes a bipartite sequence, unique among the pol X family members. The pol μ NLS has relatively weak measured affinity for Impα, due in part to its proximity to the N-terminus that limits non-specific interactions of flanking residues preceding the NLS. However, this effect is partially mitigated by an N-terminal sequence unsupportive of Met1 removal by methionine aminopeptidase, leading to a 3-fold increase in affinity when the N-terminal methionine is present. Nuclear targeting is unique to each pol X family enzyme with variations dependent on the structure and unique functional role of each polymerase. This article is protected by copyright. All rights reserved. This article is protected by copyright. All rights reserved.

Cytoplasmic DNA variation and biogeography of Larix Mill. in northeast Asia.

PubMed

Polezhaeva, Maria A; Lascoux, Martin; Semerikov, Vladimir L

2010-03-01

Range-wide variation in 54 populations of Dahurian larch (Larix gmelinii) and related taxa in Northeast Asia was assessed with four mitochondrial PCR-RFLP and five chloroplast SSR markers. Eleven mitotypes and 115 chlorotypes were detected. The highest diversity was observed in the southern Russian Far East where hybrids of L. gmelinii, L. olgensis and L. kamtschatica are distributed. In contrast, only two mitotypes occurred in L. cajanderi and L. gmelinii. The Japanese larch (L. kaempferi) was found to be closely related to populations of L. kamtschatica inhabiting the Kuril Islands and South Sakhalin, populations from the northern part of Sakhalin being more closely related to continental species. In general, both mitochondrial (G(ST) = 0.786; N(ST) = 0.823) and chloroplast (G(ST) = 0.144; R(ST) = 0.432) markers showed a strong phylogeographical structure and evidence of isolation-by-distance. Yet both markers did not allow a clear delineation of species borders. In particular, and contrary to expectations, cpDNA was not significantly better than mtDNA at delineating species borders. This lack of concordance between morphological species and molecular markers could reflect extensive ancestral haplotype sharing and past and ongoing introgression. Finally the distribution of mtDNA and cpDNA variation suggests the presence of several refugia during Pleistocene glacial intervals. In particular, mtDNA and cpDNA reveal weak but visible differentiation between L. gmelinii and L. cajanderi, suggesting independent glacial histories of these species.

Structural and Histone Binding Ability Characterizations of Human PWWP Domains

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

Wu, Hong; Zeng, Hong; Lam, Robert

2013-09-25

The PWWP domain was first identified as a structural motif of 100-130 amino acids in the WHSC1 protein and predicted to be a protein-protein interaction domain. It belongs to the Tudor domain 'Royal Family', which consists of Tudor, chromodomain, MBT and PWWP domains. While Tudor, chromodomain and MBT domains have long been known to bind methylated histones, PWWP was shown to exhibit histone binding ability only until recently. The PWWP domain has been shown to be a DNA binding domain, but sequence analysis and previous structural studies show that the PWWP domain exhibits significant similarity to other 'Royal Family' members,more » implying that the PWWP domain has the potential to bind histones. In order to further explore the function of the PWWP domain, we used the protein family approach to determine the crystal structures of the PWWP domains from seven different human proteins. Our fluorescence polarization binding studies show that PWWP domains have weak histone binding ability, which is also confirmed by our NMR titration experiments. Furthermore, we determined the crystal structures of the BRPF1 PWWP domain in complex with H3K36me3, and HDGF2 PWWP domain in complex with H3K79me3 and H4K20me3. PWWP proteins constitute a new family of methyl lysine histone binders. The PWWP domain consists of three motifs: a canonical {beta}-barrel core, an insertion motif between the second and third {beta}-strands and a C-terminal {alpha}-helix bundle. Both the canonical {beta}-barrel core and the insertion motif are directly involved in histone binding. The PWWP domain has been previously shown to be a DNA binding domain. Therefore, the PWWP domain exhibits dual functions: binding both DNA and methyllysine histones.« less

Identification of differentially methylated sites with weak methylation effect

USDA-ARS?s Scientific Manuscript database

DNA methylation is an epigenetic alteration crucial for regulating stress responses. Identifying large-scale DNA methylation at single nucleotide resolution is made possible by whole genome bisulfite sequencing. An essential task following the generation of bisulfite sequencing data is to detect dif...

Sensitivity of human cells expressing low-fidelity or weak-catalytic-activity variants of DNA polymerase ζ to genotoxic stresses.

PubMed

Suzuki, Tetsuya; Grúz, Petr; Honma, Masamitsu; Adachi, Noritaka; Nohmi, Takehiko

2016-09-01

Translesion DNA polymerases (TLS pols) play critical roles in defense mechanisms against genotoxic agents. The defects or mutations of TLS pols are predicted to result in hypersensitivity of cells to environmental mutagens. In this study, human cells expressing DNA polymerase ζ (Pol ζ) variants with low fidelity or weak catalytic activity have been established with Nalm-6-MSH+ cells and their sensitivity to mutagenicity and cytotoxicity of benzo[a]pyrene diol epoxide (BPDE) and ultraviolet-C light (UV-C) was examined. The low-fidelity mutants were engineered by knocking-in DNA sequences that direct changes of leucine 2618 to either phenylalanine (L2618F) or methionine (L2618M) of Pol ζ. The weak-catalytic-activity mutants were generated by knocking-in DNA sequences that direct changes of either tyrosine 2779 to phenylalanine (Y2779F) or aspartate 2781 to asparagine (D2781N). In addition, a +1 frameshift mutation, i.e., CCC to CCCC, was introduced in the coding region of the TK1 gene to measure the mutant frequencies. Doubling time and spontaneous TK mutant frequencies of the established cell lines were similar to those of the wild-type cells. The low-fidelity mutants displayed, however, higher sensitivity to the mutagenicity of BPDE and UV-C than the wild-type cells although their cytotoxic sensitivity was not changed. In contrast, the weak-catalytic-activity mutants were more sensitive to the cytotoxicity of BPDE and UV-C than the wild-type cells, and displayed much higher sensitivity to the clastogenicity of BPDE than the wild-type cells in an in vitro micronucleus assay. These results indicate that human Pol ζ is involved in TLS across DNA lesions induced by BPDE and UV-C and also that the TLS plays important roles in induction of mutations, clastogenicity and in cellular survival of the damaged human cells. Similarities and differences in in vivo roles of yeast and human Pol ζ in genome integrity are discussed. Copyright © 2016 Elsevier B.V. All rights reserved.

Chromium genotoxicity: a double-edged sword

PubMed Central

Nickens, Kristen P.; Patierno, Steven R.; Ceryak, Susan

2010-01-01

Certain forms of hexavalent chromium [Cr(VI)] are known respiratory carcinogens that induce a broad spectrum of DNA damage. Cr(VI)-carcinogenesis may be initiated or promoted through several mechanistic processes including, the intracellular metabolic reduction of Cr(VI) producing chromium species capable of interacting with DNA to yield genotoxic and mutagenic effects, Cr(VI)-induced inflammatory/immunological responses, and alteration of survival signaling pathways. Cr(VI) enters the cell through nonspecific anion channels, and is metabolically reduced by agents including ascorbate, glutathione, and cysteine to Cr(V), Cr(IV), and Cr(III). Cr(III) has a weak membrane permeability capacity and is unable to cross the cell membrane, thereby trapping it within the cell where it can bind to DNA and produce genetic damage leading to genomic instability. Structural genetic lesions produced by the intracellular reduction of Cr(VI) include DNA adducts, DNA strand breaks, DNA-protein crosslinks, oxidized bases, abasic sites, and DNA inter- and intrastrand crosslinks. The damage induced by Cr(VI) can lead to dysfunctional DNA replication and transcription, aberrant cell cycle checkpoints, dysregulated DNA repair mechanisms, microsatelite instability, inflammatory responses, and the disruption of key regulatory gene networks responsible for the balance of cell survival and cell death, which may all play an important role in Cr(VI) carcinogenesis. Several lines of evidence have indicated that neoplastic progression is a result of consecutive genetic/epigenetic changes that provide cellular survival advantages, and ultimately lead to the conversion of normal human cells to malignant cancer cells. This review is based on studies that provide a glimpse into Cr(VI) carcinogenicity via mechanisms including Cr(VI)-induced death-resistance, the involvement of DNA repair mechanisms in survival after chromium exposure, and the activation of survival signaling cascades in response to Cr(VI) genotoxicity. PMID:20430016

Overcoming the Coupling Dilemma in DNA-Programmable Nanoparticle Assemblies by "Ag+ Soldering".

PubMed

Wang, Huiqiao; Li, Yulin; Liu, Miao; Gong, Ming; Deng, Zhaoxiang

2015-05-20

Strong coupling between nanoparticles is critical for facilitating charge and energy transfers. Despite the great success of DNA-programmable nanoparticle assemblies, the very weak interparticle coupling represents a key barrier to various applications. Here, an extremely simple, fast, and highly efficient process combining DNA-programming and molecular/ionic bonding is developed to address this challenge, which exhibits a seamless fusion with DNA nanotechnology. © 2015 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

Genome-Wide Motif Statistics are Shaped by DNA Binding Proteins over Evolutionary Time Scales

NASA Astrophysics Data System (ADS)

Qian, Long; Kussell, Edo

2016-10-01

The composition of a genome with respect to all possible short DNA motifs impacts the ability of DNA binding proteins to locate and bind their target sites. Since nonfunctional DNA binding can be detrimental to cellular functions and ultimately to organismal fitness, organisms could benefit from reducing the number of nonfunctional DNA binding sites genome wide. Using in vitro measurements of binding affinities for a large collection of DNA binding proteins, in multiple species, we detect a significant global avoidance of weak binding sites in genomes. We demonstrate that the underlying evolutionary process leaves a distinct genomic hallmark in that similar words have correlated frequencies, a signal that we detect in all species across domains of life. We consider the possibility that natural selection against weak binding sites contributes to this process, and using an evolutionary model we show that the strength of selection needed to maintain global word compositions is on the order of point mutation rates. Likewise, we show that evolutionary mechanisms based on interference of protein-DNA binding with replication and mutational repair processes could yield similar results and operate with similar rates. On the basis of these modeling and bioinformatic results, we conclude that genome-wide word compositions have been molded by DNA binding proteins acting through tiny evolutionary steps over time scales spanning millions of generations.

The Carpathian range represents a weak genetic barrier in South-East Europe

PubMed Central

2014-01-01

Background In the present study we have assessed whether the Carpathian Mountains represent a genetic barrier in East Europe. Therefore, we have analyzed the mtDNA of 128 native individuals of Romania: 62 of them from the North of Romania, and 66 from South Romania. Results We have analyzed their mtDNA variability in the context of other European and Near Eastern populations through multivariate analyses. The results show that regarding the mtDNA haplogroup and haplotype distributions the Romanian groups living outside the Carpathian range (South Romania) displayed some degree of genetic differentiation compared to those living within the Carpahian range (North Romania). Conclusion The main differentiation between the mtDNA variability of the groups from North and South Romania can be attributed to the demographic movements from East to West (prehistoric or historic) that differently affected in these regions, suggesting that the Carpathian mountain range represents a weak genetic barrier in South-East Europe. PMID:24885208

Classification and assessment tools for structural motif discovery algorithms.

PubMed

Badr, Ghada; Al-Turaiki, Isra; Mathkour, Hassan

2013-01-01

Motif discovery is the problem of finding recurring patterns in biological data. Patterns can be sequential, mainly when discovered in DNA sequences. They can also be structural (e.g. when discovering RNA motifs). Finding common structural patterns helps to gain a better understanding of the mechanism of action (e.g. post-transcriptional regulation). Unlike DNA motifs, which are sequentially conserved, RNA motifs exhibit conservation in structure, which may be common even if the sequences are different. Over the past few years, hundreds of algorithms have been developed to solve the sequential motif discovery problem, while less work has been done for the structural case. In this paper, we survey, classify, and compare different algorithms that solve the structural motif discovery problem, where the underlying sequences may be different. We highlight their strengths and weaknesses. We start by proposing a benchmark dataset and a measurement tool that can be used to evaluate different motif discovery approaches. Then, we proceed by proposing our experimental setup. Finally, results are obtained using the proposed benchmark to compare available tools. To the best of our knowledge, this is the first attempt to compare tools solely designed for structural motif discovery. Results show that the accuracy of discovered motifs is relatively low. The results also suggest a complementary behavior among tools where some tools perform well on simple structures, while other tools are better for complex structures. We have classified and evaluated the performance of available structural motif discovery tools. In addition, we have proposed a benchmark dataset with tools that can be used to evaluate newly developed tools.

Origin, genetic diversity, and population structure of Chinese domestic sheep.

PubMed

Chen, Shan-Yuan; Duan, Zi-Yuan; Sha, Tao; Xiangyu, Jinggong; Wu, Shi-Fang; Zhang, Ya-Ping

2006-07-19

To characterize the origin, genetic diversity, and phylogeographic structure of Chinese domestic sheep, we here analyzed a 531-bp fragment of mtDNA control region of 449 Chinese autochthonous sheep from 19 breeds/populations from 13 geographic regions, together with previously reported 44 sequences from Chinese indigenous sheep. Phylogenetic analysis showed that all three previously defined lineages A, B, and C were found in all sampled Chinese sheep populations, except for the absence of lineage C in four populations. Network profiles revealed that the lineages B and C displayed a star-like phylogeny with the founder haplotype in the centre, and that two star-like subclades with two founder haplotypes were identified in lineage A. The pattern of genetic variation in lineage A, together with the divergence time between the two central founder haplotypes suggested that two independent domestication events have occurred in sheep lineage A. Considerable mitochondrial diversity was observed in Chinese sheep. Weak structuring was observed either among Chinese indigenous sheep populations or between Asian and European sheep and this can be attributable to long-term strong gene flow induced by historical human movements. The high levels of intra-population diversity in Chinese sheep and the weak phylogeographic structuring indicated three geographically independent domestication events have occurred and the domestication place was not only confined to the Near East, but also occurred in other regions.

Podoscyphic acid, a new inhibitor of avian myeloblastosis virus and Moloney murine leukemia virus reverse transcriptase from a Podoscypha species.

PubMed

Erkel, G; Anke, T; Velten, R; Steglich, W

1991-01-01

A novel enzyme inhibitor of RNA-directed DNA-polymerases of avian myeloblastosis and murine leukemia virus was isolated from fermentations of an tasmanian Podoscypha species. Its structure was elucidated by spectroscopic methods and oxidative degradation as (E)-4,5-dioxo-2-hexadecenoic acid (1). The enzyme inhibitor, which was named podoscyphic acid, did not inhibit DNA and RNA synthesis in permeabilized L 1210 cells nor did it affect RNA synthesis in isolated nuclei of L 1210 cells. 1 inhibits protein synthesis in whole L 1210 cells and rabbit reticulocyte lysate and shows very weak antimicrobial and cytotoxic properties. The testing of ethyl (E)-4,5-dioxo-2-hexadecenoate (2) and (E)-4-oxo-2-tetradecenoic acid (11) revealed the importance of the free gamma-oxoacrylic acid unit for the biological activities of 1.

Episodic weakness due to mitochondrial DNA MT-ATP6/8 mutations.

PubMed

Auré, Karine; Dubourg, Odile; Jardel, Claude; Clarysse, Lucie; Sternberg, Damien; Fournier, Emmanuel; Laforêt, Pascal; Streichenberger, Nathalie; Petiot, Philippe; Gervais-Bernard, Hélène; Vial, Christophe; Bedat-Millet, Anne-Laure; Drouin-Garraud, Valérie; Bouillaud, Frédéric; Vandier, Christophe; Fontaine, Bertrand; Lombès, Anne

2013-11-19

To report that homoplasmic deleterious mutations in the mitochondrial DNA MT-ATP6/8 genes may be responsible for acute episodes of limb weakness mimicking periodic paralysis due to channelopathies and dramatically responding to acetazolamide. Mitochondrial DNA sequencing and restriction PCR, oxidative phosphorylation functional assays, reactive oxygen species metabolism, and patch-clamp technique in cultured skin fibroblasts. Occurrence of a typical MELAS (mitochondrial encephalopathy with lactic acidosis and stroke-like episodes) syndrome in a single member of a large pedigree with episodic weakness associated with a later-onset distal motor neuropathy led to the disclosure of 2 deleterious mitochondrial DNA mutations. The MT-ATP6 m.9185T>C p.Leu220Pro mutation, previously associated with Leigh syndrome, was present in all family members, while the MT-TL1 m.3271T>C mutation, a known cause of MELAS syndrome, was observed in the sole patient with MELAS presentation. Significant defect of complexes V and I as well as oxidative stress were observed in both primary fibroblasts and cybrid cells with 100% m.9185T>C mutation. Permanent plasma membrane depolarization and altered permeability to K(+) in fibroblasts provided a link with the paralysis episodes. Screening of 9 patients, based on their clinical phenotype, identified 4 patients with similar deleterious MT-ATP6 mutations (twice m.9185T>C and once m.9176T>C or m.8893T>C). A fifth patient presented with an original potentially deleterious MT-ATP8 mutation (m.8403T>C). All mutations were associated with almost-normal complex V activity but significant oxidative stress and permanent plasma membrane depolarization. Homoplasmic mutations in the MT-ATP6/8 genes may cause episodic weakness responding to acetazolamide treatment.

Investigations on the Interactions of 5-Fluorouracil with Herring Sperm DNA: Steady State/Time Resolved and Molecular Modeling Studies

NASA Astrophysics Data System (ADS)

Chinnathambi, Shanmugavel; Karthikeyan, Subramani; Velmurugan, Devadasan; Hanagata, Nobutaka; Aruna, Prakasarao; Ganesan, Singaravelu

2015-04-01

In the present study, the interaction of 5-Fluorouracil with herring sperm DNA is reported using spectroscopic and molecular modeling techniques. This binding study of 5-FU with hs-DNA is of paramount importance in understanding chemico-biological interactions for drug design, pharmacy and biochemistry without altering the original structure. The challenge of the study was to find the exact binding mode of the drug 5-Fluorouracil with hs-DNA. From the absorption studies, a hyperchromic effect was observed for the herring sperm DNA in the presence of 5-Fluorouracil and a binding constant of 6.153 × 103 M-1 for 5-Fluorouracil reveals the existence of weak interaction between the 5-Fluorouracil and herring sperm DNA. Ethidium bromide loaded herring sperm DNA showed a quenching in the fluorescence intensity after the addition of 5-Fluorouracil. The binding constants for 5-Fluorouracil stranded DNA and competitive bindings of 5-FU interacting with DNA-EB systems were examined by fluorescence spectra. The Stern-Volmer plots and fluorescence lifetime results confirm the static quenching nature of the drug-DNA complex. The binding constant Kb was 2.5 × 104 L mol-1 and the number of binding sites are 1.17. The 5-FU on DNA system was calculated using double logarithmic plot. From the Forster nonradiative energy transfer study it has been found that the distance of 5-FU from DNA was 4.24 nm. In addition to the spectroscopic results, the molecular modeling studies also revealed the major groove binding as well as the partial intercalation mode of binding between the 5-Fluorouracil and herring sperm DNA. The binding energy and major groove binding as -6.04 kcal mol-1 and -6.31 kcal mol-1 were calculated from the modeling studies. All the testimonies manifested that binding modes between 5-Fluorouracil and DNA were evidenced to be groove binding and in partial intercalative mode.

Conjugation of Benzylvanillin and Benzimidazole Structure Improves DNA Binding with Enhanced Antileukemic Properties

PubMed Central

Al-Mudarris, Ban A.; Chen, Shih-Hsun; Liang, Po-Huang; Osman, Hasnah; Jamal Din, Shah Kamal Khan; Abdul Majid, Amin M. S.

2013-01-01

Benzyl-o-vanillin and benzimidazole nucleus serve as important pharmacophore in drug discovery. The benzyl vanillin (2-(benzyloxy)-3-methoxybenzaldehyde) compound shows anti-proliferative activity in HL60 leukemia cancer cells and can effect cell cycle progression at G2/M phase. Its apoptosis activity was due to disruption of mitochondrial functioning. In this study, we have studied a series of compounds consisting of benzyl vanillin and benzimidazole structures. We hypothesize that by fusing these two structures we can produce compounds that have better anticancer activity with improved specificity particularly towards the leukemia cell line. Here we explored the anticancer activity of three compounds namely 2-(2-benzyloxy-3-methoxyphenyl)-1H-benzimidazole, 2MP, N-1-(2-benzyloxy-3-methoxybenzyl)-2-(2-benzyloxy-3-methoxyphenyl)-1H-benzimidazole, 2XP, and (R) and (S)-1-(2-benzyloxy-3-methoxyphenyl)-2, 2, 2-trichloroethyl benzenesulfonate, 3BS and compared their activity to 2-benzyloxy-3-methoxybenzaldehyde, (Bn1), the parent compound. 2XP and 3BS induces cell death of U937 leukemic cell line through DNA fragmentation that lead to the intrinsic caspase 9 activation. DNA binding study primarily by the equilibrium binding titration assay followed by the Viscosity study reveal the DNA binding through groove region with intrinsic binding constant 7.39 µM/bp and 6.86 µM/bp for 3BS and 2XP respectively. 2XP and 3BS showed strong DNA binding activity by the UV titration method with the computational drug modeling showed that both 2XP and 3BS failed to form any electrostatic linkages except via hydrophobic interaction through the minor groove region of the nucleic acid. The benzylvanillin alone (Bn1) has weak anticancer activity even after it was combined with the benzimidazole (2MP), but after addition of another benzylvanillin structure (2XP), stronger activity was observed. Also, the combination of benzylvanillin with benzenesulfonate (3BS) significantly improved the anticancer activity of Bn1. The present study provides a new insight of benzyl vanillin derivatives as potential anti-leukemic agent. PMID:24260527

The Weak Shall Inherit: Bacteriocin-Mediated Interactions in Bacterial Populations

PubMed Central

Majeed, Hadeel; Lampert, Adam; Ghazaryan, Lusine; Gillor, Osnat

2013-01-01

Background Evolutionary arms race plays a major role in shaping biological diversity. In microbial systems, competition often involves chemical warfare and the production of bacteriocins, narrow-spectrum toxins aimed at killing closely related strains by forming pores in their target’s membrane or by degrading the target’s RNA or DNA. Although many empirical and theoretical studies describe competitive exclusion of bacteriocin-sensitive strains by producers of bacteriocins, the dynamics among producers are largely unknown. Methodology/Principal findings We used a reporter-gene assay to show that the bacterial response to bacteriocins’ treatment mirrors the inflicted damage Potent bacteriocins are lethal to competing strains, but at sublethal doses can serve as strong inducing agents, enhancing their antagonists’ bacteriocin production. In contrast, weaker bacteriocins are less toxic to their competitors and trigger mild bacteriocin expression. We used empirical and numerical models to explore the role of cross-induction in the arms race between bacteriocin-producing strains. We found that in well-mixed, unstructured environments where interactions are global, producers of weak bacteriocins are selectively advantageous and outcompete producers of potent bacteriocins. However, in spatially structured environments, where interactions are local, each producer occupies its own territory, and competition takes place only in “no man’s lands” between territories, resulting in much slower dynamics. Conclusion/Significance The models we present imply that producers of potent bacteriocins that trigger a strong response in neighboring bacteriocinogenic strains are doomed, while producers of weak bacteriocins that trigger a mild response in bacteriocinogenic strains flourish. This counter-intuitive outcome might explain the preponderance of weak bacteriocin producers in nature. However, the described scenario is prolonged in spatially structured environments thus promoting coexistence, allowing migration and evolution, and maintaining bacterial diversity. PMID:23704942

E-motif formed by extrahelical cytosine bases in DNA homoduplexes of trinucleotide and hexanucleotide repeats

PubMed Central

Pan, Feng; Zhang, Yuan; Man, Viet Hoang; Roland, Christopher

2018-01-01

Abstract Atypical DNA secondary structures play an important role in expandable trinucleotide repeat (TR) and hexanucleotide repeat (HR) diseases. The cytosine mismatches in C-rich homoduplexes and hairpin stems are weakly bonded; experiments show that for certain sequences these may flip out of the helix core, forming an unusual structure termed an ‘e-motif’. We have performed molecular dynamics simulations of C-rich TR and HR DNA homoduplexes in order to characterize the conformations, stability and dynamics of formation of the e-motif, where the mismatched cytosines symmetrically flip out in the minor groove, pointing their base moieties towards the 5′-direction in each strand. TRs have two non-equivalent reading frames, (GCC)n and (CCG)n; while HRs have three: (CCCGGC)n, (CGGCCC)n, (CCCCGG)n. We define three types of pseudo basepair steps related to the mismatches and show that the e-motif is only stable in (GCC)n and (CCCGGC)n homoduplexes due to the favorable stacking of pseudo GpC steps (whose nature depends on whether TRs or HRs are involved) and the formation of hydrogen bonds between the mismatched cytosine at position i and the cytosine (TRs) or guanine (HRs) at position i − 2 along the same strand. We also characterize the extended e-motif, where all mismatched cytosines are extruded, their extra-helical stacking additionally stabilizing the homoduplexes. PMID:29190385

Targeted Inactivation of DNA Photolyase Genes in Medaka Fish (Oryzias latipes).

PubMed

Ishikawa-Fujiwara, Tomoko; Shiraishi, Eri; Fujikawa, Yoshihiro; Mori, Toshio; Tsujimura, Tohru; Todo, Takeshi

2017-01-01

Proteins of the cryptochrome/photolyase family (CPF) exhibit sequence and structural conservation, but their functions are divergent. Photolyase is a DNA repair enzyme that catalyzes the light-dependent repair of ultraviolet (UV)-induced photoproducts, whereas cryptochrome acts as a photoreceptor or circadian clock protein. Two types of DNA photolyase exist: CPD photolyase, which repairs cyclobutane pyrimidine dimers (CPDs), and 6-4 photolyase, which repairs 6-4 pyrimidine-pyrimidone photoproducts (6-4PPs). Although the Cry-DASH protein is classified as a cryptochrome, it also has light-dependent DNA repair activity. To determine the significance of the three light-dependent repair enzymes in recovering from solar UV-induced DNA damage at the organismal level, we generated mutants in each gene in medaka using the CRISPR genome editing technique. The light-dependent repair activity of the mutants was examined in vitro in cultured cells and in vivo in skin tissue. Light-dependent repair of CPD was lost in the CPD photolyase-deficient mutant, whereas weak repair activity against 6-4PPs persisted in the 6-4 photolyase-deficient mutant. These results suggest the existence of a heretofore unknown 6-4PP repair pathway and thus improve our understanding of the mechanisms of defense against solar UV in vertebrates. © 2016 The Authors. Photochemistry and Photobiology published by Wiley Periodicals, Inc. on behalf of American Society for Photobiology.

Genetic Variation of Lactobacillus delbrueckii subsp. lactis Bacteriophages Isolated from Cheese Processing Plants in Finland

PubMed Central

Forsman, Päivi; Alatossava, Tapani

1991-01-01

The genomes of four Lactobacillus delbrueckii subsp. lactis bacteriophages were characterized by restriction endonuclease mapping, Southern hybridization, and heteroduplex analysis. The phages were isolated from different cheese processing plants in Finland between 1950 and 1972. All four phages had a small isometric head and a long noncontractile tail. Two different types of genome (double-stranded DNA) organization existed among the different phages, the pac type and the cos type, corresponding to alternative types of phage DNA packaging. Three phages belonged to the pac type, and a fourth was a cos-type phage. The pac-type phages were genetically closely related. In the genomes of the pac-type phages, three putative insertion/deletions (0.7 to 0.8 kb, 1.0 kb, and 1.5 kb) and one other region (0.9 kb) containing clustered base substitutions were discovered and localized. At the phenotype level, three main differences were observed among the pac-type phages. These concerned two minor structural proteins and the efficiency of phage DNA packaging. The genomes of the pac-type phages showed only weak homology with that of the cos-type phage. Phage-related DNA, probably a defective prophage, was located in the chromosome of the host strain sensitive to the cos-type phage. This DNA exhibited homology under stringent conditions to the pac-type phages. Images PMID:16348513

Lipid nanocapsule functionalization by lipopeptides derived from human papillomavirus type-16 capsid for nucleic acid delivery into cancer cells.

PubMed

Weyland, M; Griveau, A; Bejaud, J; Benoit, J-P; Coursaget, P; Garcion, E

2013-10-01

Plasmid DNA (pDNA) and small interfering RNAs (siRNAs) are very useful tools for the treatment of cancer. However, pDNA and siRNAs efficacy is restricted by their negative charge and susceptibility to degradation by endonucleases that prevent them penetrating tissue and cellular barriers such as the plasma and endolysosomal membranes. Viral vectors have some advantages but their use is largely limited by their immunogenicity. On the other hand, synthetic nanoparticles have advantage of being relatively non-immunogenic but their ability to deliver nucleic acids remains less efficient than their viral counterparts. The present study is focussed on the development and evaluation of biomimetic lipid nanocapsules (LNCs) functionalized with a L1 papillomavirus type-16 capsid-derived lipopeptide on their surface, for transfection of U87MG glioma cells and Caco-2 colorectal adenocarcinoma cells with pDNA or siRNAs. Since the L1-peptide has been described as a nuclear localization signal able to complex with nucleic acids and bind to heparan sulfate on the cell surface, the structure and function of L1-peptide bound to LNCs (L1-LNCs) were investigated. Although L1-LNCs were shown to complex with both pDNA and siRNAs, the pDNA-L1-LNC complexes showed only weak transfection efficiency. In contrast, siRNA-L1-LNC complexes appeared as effective repressors of targeted messengers. Copyright © 2013 Elsevier B.V. All rights reserved.

Weak Population Structure in European Roe Deer (Capreolus capreolus) and Evidence of Introgressive Hybridization with Siberian Roe Deer (C. pygargus) in Northeastern Poland

PubMed Central

Olano-Marin, Juanita; Plis, Kamila; Sönnichsen, Leif; Borowik, Tomasz; Niedziałkowska, Magdalena; Jędrzejewska, Bogumiła

2014-01-01

We investigated contemporary and historical influences on the pattern of genetic diversity of European roe deer (Capreolus capreolus). The study was conducted in northeastern Poland, a zone where vast areas of primeval forests are conserved and where the European roe deer was never driven to extinction. A total of 319 unique samples collected in three sampling areas were genotyped at 16 microsatellites and one fragment (610 bp) of mitochondrial DNA (mtDNA) control region. Genetic diversity was high, and a low degree of genetic differentiation among sampling areas was observed with both microsatellites and mtDNA. No evidence of genetic differentiation between roe deer inhabiting open fields and forested areas was found, indicating that the ability of the species to exploit these contrasting environments might be the result of its phenotypic plasticity. Half of the studied individuals carried an mtDNA haplotype that did not belong to C. capreolus, but to a related species that does not occur naturally in the area, the Siberian roe deer (C. pygargus). No differentiation between individuals with Siberian and European mtDNA haplotypes was detected at microsatellite loci. Introgression of mtDNA of Siberian roe deer into the genome of European roe deer has recently been detected in eastern Europe. Such introgression might be caused by human-mediated translocations of Siberian roe deer within the range of European roe deer or by natural hybridization between these species in the past. PMID:25271423

Fine-scale genetic structure and cryptic associations reveal evidence of kin-based sociality in the African forest elephant.

PubMed

Schuttler, Stephanie G; Philbrick, Jessica A; Jeffery, Kathryn J; Eggert, Lori S

2014-01-01

Spatial patterns of relatedness within animal populations are important in the evolution of mating and social systems, and have the potential to reveal information on species that are difficult to observe in the wild. This study examines the fine-scale genetic structure and connectivity of groups within African forest elephants, Loxodonta cyclotis, which are often difficult to observe due to forest habitat. We tested the hypothesis that genetic similarity will decline with increasing geographic distance, as we expect kin to be in closer proximity, using spatial autocorrelation analyses and Tau K(r) tests. Associations between individuals were investigated through a non-invasive genetic capture-recapture approach using network models, and were predicted to be more extensive than the small groups found in observational studies, similar to fission-fusion sociality found in African savanna (Loxodonta africana) and Asian (Elephas maximus) species. Dung samples were collected in Lopé National Park, Gabon in 2008 and 2010 and genotyped at 10 microsatellite loci, genetically sexed, and sequenced at the mitochondrial DNA control region. We conducted analyses on samples collected at three different temporal scales: a day, within six-day sampling sessions, and within each year. Spatial autocorrelation and Tau K(r) tests revealed genetic structure, but results were weak and inconsistent between sampling sessions. Positive spatial autocorrelation was found in distance classes of 0-5 km, and was strongest for the single day session. Despite weak genetic structure, individuals within groups were significantly more related to each other than to individuals between groups. Social networks revealed some components to have large, extensive groups of up to 22 individuals, and most groups were composed of individuals of the same matriline. Although fine-scale population genetic structure was weak, forest elephants are typically found in groups consisting of kin and based on matrilines, with some individuals having more associates than observed from group sizes alone.

Shallow Population Genetic Structures of Thread-sail Filefish (Stephanolepis cirrhifer) Populations from Korean Coastal Waters.

PubMed

Yoon, M; Park, W; Nam, Y K; Kim, D S

2012-02-01

Genetic diversities, population genetic structures and demographic histories of the thread-sail filefish Stephanolepis cirrhifer were investigated by nucleotide sequencing of 336 base pairs of the mitochondrial DNA (mtDNA) control region in 111 individuals collected from six populations in Korean coastal waters. A total of 70 haplotypes were defined by 58 variable nucleotide sites. The neighbor-joining tree of the 70 haplotypes was shallow and did not provide evidence of geographical associations. Expansion of S. cirrhifer populations began approximate 51,000 to 102,000 years before present, correlating with the period of sea level rise since the late Pleistocene glacial maximum. High levels of haplotype diversities (0.974±0.029 to 1.000±0.076) and nucleotide diversities (0.014 to 0.019), and low levels of genetic differentiation among populations inferred from pairwise population F ST values (-0.007 to 0.107), support an expansion of the S. cirrhifer population. Hierarchical analysis of molecular variance (AMOVA) revealed weak but significant genetic structures among three groups (F CT = 0.028, p<0.05), and no genetic variation within groups (0.53%; F SC = 0.005, p = 0.23). These results may help establish appropriate fishery management strategies for stocks of S. cirrhifer and related species.

Shallow Population Genetic Structures of Thread-sail Filefish (Stephanolepis cirrhifer) Populations from Korean Coastal Waters

PubMed Central

Yoon, M.; Park, W.; Nam, Y. K.; Kim, D. S.

2012-01-01

Genetic diversities, population genetic structures and demographic histories of the thread-sail filefish Stephanolepis cirrhifer were investigated by nucleotide sequencing of 336 base pairs of the mitochondrial DNA (mtDNA) control region in 111 individuals collected from six populations in Korean coastal waters. A total of 70 haplotypes were defined by 58 variable nucleotide sites. The neighbor-joining tree of the 70 haplotypes was shallow and did not provide evidence of geographical associations. Expansion of S. cirrhifer populations began approximate 51,000 to 102,000 years before present, correlating with the period of sea level rise since the late Pleistocene glacial maximum. High levels of haplotype diversities (0.974±0.029 to 1.000±0.076) and nucleotide diversities (0.014 to 0.019), and low levels of genetic differentiation among populations inferred from pairwise population FST values (−0.007 to 0.107), support an expansion of the S. cirrhifer population. Hierarchical analysis of molecular variance (AMOVA) revealed weak but significant genetic structures among three groups (FCT = 0.028, p<0.05), and no genetic variation within groups (0.53%; FSC = 0.005, p = 0.23). These results may help establish appropriate fishery management strategies for stocks of S. cirrhifer and related species. PMID:25049547

An Aryl Hydrocarbon Receptor from the Salamander Ambystoma mexicanum Exhibits Low Sensitivity to 2,3,7,8-Tetrachlorodibenzo-p-dioxin.

PubMed

Shoots, Jenny; Fraccalvieri, Domenico; Franks, Diana G; Denison, Michael S; Hahn, Mark E; Bonati, Laura; Powell, Wade H

2015-06-02

Structural features of the aryl hydrocarbon receptor (AHR) can underlie species- and population-specific differences in its affinity for 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD). These differences often explain variations in TCDD toxicity. Frogs are relatively insensitive to dioxin, and Xenopus AHRs bind TCDD with low affinity. Weak TCDD binding results from the combination of three residues in the ligand-binding domain: A354 and A370, and N325. Here we sought to determine whether this mechanism of weak TCDD binding is shared by other amphibian AHRs. We isolated an AHR cDNA from the Mexican axolotl (Ambystoma mexicanum). The encoded polypeptide contains identical residues at positions that confer low TCDD affinity to X. laevis AHRs (A364, A380, and N335), and homology modeling predicts they protrude into the binding cavity. Axolotl AHR bound one-tenth the TCDD of mouse AHR in velocity sedimentation analysis, and in transactivation assays, the EC50 for TCDD was 23 nM, similar to X. laevis AHR1β (27 nM) and greater than AHR containing the mouse ligand-binding domain (0.08 nM). Sequence, modeled structure, and function indicate that axolotl AHR binds TCDD weakly, predicting that A. mexicanum lacks sensitivity toTCDD toxicity. We hypothesize that this characteristic of axolotl and Xenopus AHRs arose in a common ancestor of the Caudata and Anura.

Exploring the diversity of Listeria monocytogenes biofilm architecture by high-throughput confocal laser scanning microscopy and the predominance of the honeycomb-like morphotype.

PubMed

Guilbaud, Morgan; Piveteau, Pascal; Desvaux, Mickaël; Brisse, Sylvain; Briandet, Romain

2015-03-01

Listeria monocytogenes is involved in food-borne illness with a high mortality rate. The persistence of the pathogen along the food chain can be associated with its ability to form biofilms on inert surfaces. While most of the phenotypes associated with biofilms are related to their spatial organization, most published data comparing biofilm formation by L. monocytogenes isolates are based on the quantitative crystal violet assay, which does not give access to structural information. Using a high-throughput confocal-imaging approach, the aim of this work was to decipher the structural diversity of biofilms formed by 96 L. monocytogenes strains isolated from various environments. Prior to large-scale analysis, an experimental design was created to improve L. monocytogenes biofilm formation in microscopic-grade microplates, with special emphasis on the growth medium composition. Microscopic analysis of biofilms formed under the selected conditions by the 96 isolates revealed only weak correlation between the genetic lineages of the isolates and the structural properties of the biofilms. However, a gradient in their geometric descriptors (biovolume, mean thickness, and roughness), ranging from flat multilayers to complex honeycomb-like structures, was shown. The dominant honeycomb-like morphotype was characterized by hollow voids hosting free-swimming cells and localized pockets containing mixtures of dead cells and extracellular DNA (eDNA). Copyright © 2015, American Society for Microbiology. All Rights Reserved.

Isolation of genomic DNA using magnetic cobalt ferrite and silica particles.

PubMed

Prodelalová, Jana; Rittich, Bohuslav; Spanová, Alena; Petrová, Katerina; Benes, Milan J

2004-11-12

Adsorption separation techniques as an alternative to laborious traditional methods (e.g., based on phenol extraction procedure) have been applied for DNA purification. In this work we used two types of particles: silica and cobalt ferrite (unmodified or modified with a reagent containing weakly basic aminoethyl groups, aminophenyl groups, or alginic acid). DNA from chicken erythrocytes and DNA isolated from bacteria Lactococcus lactis were used for testing of adsorption/desorption properties of particles. The cobalt ferrite particles modified with different reagents were used for isolation of PCR-ready bacterial DNA from different dairy products.

Dynamics of DNA breathing: weak noise analysis, finite time singularity, and mapping onto the quantum Coulomb problem.

PubMed

Fogedby, Hans C; Metzler, Ralf

2007-12-01

We study the dynamics of denaturation bubbles in double-stranded DNA on the basis of the Poland-Scheraga model. We show that long time distributions for the survival of DNA bubbles and the size autocorrelation function can be derived from an asymptotic weak noise approach. In particular, below the melting temperature the bubble closure corresponds to a noisy finite time singularity. We demonstrate that the associated Fokker-Planck equation is equivalent to a quantum Coulomb problem. Below the melting temperature, the bubble lifetime is associated with the continuum of scattering states of the repulsive Coulomb potential; at the melting temperature, the Coulomb potential vanishes and the underlying first exit dynamics exhibits a long time power law tail; above the melting temperature, corresponding to an attractive Coulomb potential, the long time dynamics is controlled by the lowest bound state. Correlations and finite size effects are discussed.

Deep-sea vent phage DNA polymerase specifically initiates DNA synthesis in the absence of primers.

PubMed

Zhu, Bin; Wang, Longfei; Mitsunobu, Hitoshi; Lu, Xueling; Hernandez, Alfredo J; Yoshida-Takashima, Yukari; Nunoura, Takuro; Tabor, Stanley; Richardson, Charles C

2017-03-21

A DNA polymerase is encoded by the deep-sea vent phage NrS-1. NrS-1 has a unique genome organization containing genes that are predicted to encode a helicase and a single-stranded DNA (ssDNA)-binding protein. The gene for an unknown protein shares weak homology with the bifunctional primase-polymerases (prim-pols) from archaeal plasmids but is missing the zinc-binding domain typically found in primases. We show that this gene product has efficient DNA polymerase activity and is processive in DNA synthesis in the presence of the NrS-1 helicase and ssDNA-binding protein. Remarkably, this NrS-1 DNA polymerase initiates DNA synthesis from a specific template DNA sequence in the absence of any primer. The de novo DNA polymerase activity resides in the N-terminal domain of the protein, whereas the C-terminal domain enhances DNA binding.

Structure, function, and tethering of DNA-binding domains in σ 54 transcriptional activators

DOE PAGES

Vidangos, Natasha; Maris, Ann E.; Young, Anisa; ...

2013-07-02

In this paper, we compare the structure, activity, and linkage of DNA-binding domains (DBDs) from σ 54 transcriptional activators and discuss how the properties of the DBDs and the linker to the neighboring domain are affected by the overall properties and requirements of the full proteins. These transcriptional activators bind upstream of specific promoters that utilize σ 54-polymerase. Upon receiving a signal the activators assemble into hexamers, which then, through adenosine triphosphate (ATP) hydrolysis, drive a conformational change in polymerase that enables transcription initiation. We present structures of the DBDs of activators nitrogen regulatory protein C 1 (NtrC1) and Nif-likemore » homolog 2 (Nlh2) from the thermophile Aquifex aeolicus. The structures of these domains and their relationship to other parts of the activators are discussed. These structures are compared with previously determined structures of the DBDs of NtrC4, NtrC, ZraR, and factor for inversion stimulation. The N-terminal linkers that connect the DBDs to the central domains in NtrC1 and Nlh2 were studied and found to be unstructured. Additionally, a crystal structure of full-length NtrC1 was solved, but density of the DBDs was extremely weak, further indicating that the linker between ATPase and DBDs functions as a flexible tether. Flexible linking of ATPase and DBDs is likely necessary to allow assembly of the active hexameric ATPase ring. Finally, the comparison of this set of activators also shows clearly that strong dimerization of the DBD only occurs when other domains do not dimerize strongly.« less

Microbial mutagenic effects of the DNA minor groove binder pibenzimol (Hoechst 33258) and a series of mustard analogues.

PubMed

Ferguson, L R; Denny, W A

1995-06-01

A series of aniline mustards and half-mustards targeted to DNA by linkage (through a polymethylene chain) to the bisbenzimidazole chromophore of pibenzimol (Hoechst 33258) have been evaluated for their mutagenic properties, as estimated in three strains of Salmonella typhimurium, and for their mitotic crossing-over and petite mutagenesis activities in Saccharomyces cerevisiae strain D5. Agarose gel electrophoresis studies showed that only the derivative with the longest linker chain cross-linked DNA, with the remaining compounds being monoalkylators. The parent (non-alkylator) minor groove binding ligand (Hoechst 33258) was inactive in the bacterial strains TA98 or TA100 but weakly mutagenic in TA102, and caused neither mitotic crossing-over nor 'petite' mutagenesis in yeast. Aniline half-mustard itself (monoalkylator) was an effective base-pair substitution mutagen (events in S. typhimurium strain TA100) with some frameshift mutagenesis activity in TA98, but showed only weak effects in the yeast assays, whereas aniline mustard (cross-linker) was inactive in these bacterial systems but caused substantial amounts of mitotic crossing-over in yeast. The composite molecules studied here showed effects more characteristic of the minor groove binding chromophore than of alkylating moieties. All showed weak mutagenic activity in TA102 and none in TA98. The only compound to show significant mitotic crossing-over ability was the long-chain derivative which cross-linked DNA. For most of the compounds, the mutagenicity data provided no supportive evidence for DNA alkylation. Since other evidence suggests this does occur readily, it is likely to have a different target to that seen with untargeted aniline mustards. The significant antitumor activity and low mutagenic potential shown by these compounds make them worthy of further study.

Lab-on-a-chip platform for high throughput drug discovery with DNA-encoded chemical libraries

NASA Astrophysics Data System (ADS)

Grünzner, S.; Reddavide, F. V.; Steinfelder, C.; Cui, M.; Busek, M.; Klotzbach, U.; Zhang, Y.; Sonntag, F.

2017-02-01

The fast development of DNA-encoded chemical libraries (DECL) in the past 10 years has received great attention from pharmaceutical industries. It applies the selection approach for small molecular drug discovery. Because of the limited choices of DNA-compatible chemical reactions, most DNA-encoded chemical libraries have a narrow structural diversity and low synthetic yield. There is also a poor correlation between the ranking of compounds resulted from analyzing the sequencing data and the affinity measured through biochemical assays. By combining DECL with dynamical chemical library, the resulting DNA-encoded dynamic library (EDCCL) explores the thermodynamic equilibrium of reversible reactions as well as the advantages of DNA encoded compounds for manipulation/detection, thus leads to enhanced signal-to-noise ratio of the selection process and higher library quality. However, the library dynamics are caused by the weak interactions between the DNA strands, which also result in relatively low affinity of the bidentate interaction, as compared to a stable DNA duplex. To take advantage of both stably assembled dual-pharmacophore libraries and EDCCLs, we extended the concept of EDCCLs to heat-induced EDCCLs (hi-EDCCLs), in which the heat-induced recombination process of stable DNA duplexes and affinity capture are carried out separately. To replace the extremely laborious and repetitive manual process, a fully automated device will facilitate the use of DECL in drug discovery. Herein we describe a novel lab-on-a-chip platform for high throughput drug discovery with hi-EDCCL. A microfluidic system with integrated actuation was designed which is able to provide a continuous sample circulation by reducing the volume to a minimum. It consists of a cooled and a heated chamber for constant circulation. The system is capable to generate stable temperatures above 75 °C in the heated chamber to melt the double strands of the DNA and less than 15 °C in the cooled chamber, to reanneal the reshuffled library. In the binding chamber (the cooled chamber) specific retaining structures are integrated. These hold back beads functionalized with the target protein, while the chamber is continuously flushed with library molecules. Afterwards the whole system can be flushed with buffer to wash out unspecific bound molecules. Finally the protein-loaded beads with attached molecules can be eluted for further investigation.

New dye-labeled terminators for improved DNA sequencing patterns.

PubMed Central

Rosenblum, B B; Lee, L G; Spurgeon, S L; Khan, S H; Menchen, S M; Heiner, C R; Chen, S M

1997-01-01

We have used two new dye sets for automated dye-labeled terminator DNA sequencing. One set consists of four, 4,7-dichlororhodamine dyes (d-rhodamines). The second set consists of energy-transfer dyes that use the 5-carboxy-d-rhodamine dyes as acceptor dyes and the 5- or 6-carboxy isomers of 4'-aminomethylfluorescein as the donor dye. Both dye sets utilize a new linker between the dye and the nucleotide, and both provide more even peak heights in terminator sequencing than the dye-terminators consisting of unsubstituted rhodamine dyes. The unsubstituted rhodamine terminators produced electropherograms in which weak G peaks are observed after A peaks and occasionally C peaks. The number of weak G peaks has been reduced or eliminated with the new dye terminators. The general improvement in peak evenness improves accuracy for the automated base-calling software. The improved signal-to-noise ratio of the energy-transfer dye-labeled terminators combined with more even peak heights results in successful sequencing of high molecular weight DNA templates such as bacterial artificial chromosome DNA. PMID:9358158

Spectroscopic Identification of Lipid, Protein and DNA Changes in Breast Cancer tissues

NASA Astrophysics Data System (ADS)

Badr, Y. A.; Hassab Elnaby, S. I.

2007-02-01

The FTIR spectroscopy, at the range 4000 - 6000 cm-1 showed a clear distinction between normal and cancer tissues. Normal tissues spectra contain a doublet structure at 4258 and 4332 cm-1. This structure is usually on top of a small band that extends from 3950 cm-1 to 4400 cm-1. This structure us also observed from pure lipid tissues from control patients. The origin of this structure could be attributed to combinations of lipid lines. This structure is completely absent in cancer tissues, instead a broad intense band appears from 5100 cm-1 to 5200 cm-1. The intensity of this band varies from one patient to another. The shape of this broad band indicates that it is the due to random orientation changes in the proteins. This band has a peak at 5164 cm-1, it contains another small kink at 4882 cm-1. This may lead also to the conclusion that this window band is associated with a short half life time energy levels. On The other hand the photoacoustic spectrum of the same tissues , shows that in normal tissues there are three very distinct peaks (namely 1097,1159 and 1232 cm-1) they disappear in malignant tissues and replaced by many weak ripples. Two peaks (1578, 1690 cm-1) changes their position in malignant tissues(1626, 1678 cm-1). A change in DNA markers was also noticed in the range 600-1700 cm-1.

Relationship of the Structure of Bacterial Lipopolysaccharides to Its Function in Mitogenesis and Adjuvanticity

PubMed Central

Chiller, Jacques M.; Skidmore, Barry J.; Morrison, David C.; Weigle, William O.

1973-01-01

The ability of bacterial lipopolysaccharides to induce lymphocyte mitogenesis and to act as an adjuvant of antibody formation was attributable to the lipid-A region of the molecule. Measured by induction of DNA synthesis, lipid A was mitogenic for bone marrow-derived lymphocytes obtained from spleens of congenitally athymic mice but not for thymocytes obtained from thymuses of normal mice. Adjuvanticity was demonstrated by the ability of lipid A to convert a tolerogenic regimen of antigen into one eliciting an immune response and by its ability to markedly enhance the antibody response to a weak antigen. PMID:4579014

Peace, a MYB-like transcription factor, regulates petal pigmentation in flowering peach ‘Genpei’ bearing variegated and fully pigmented flowers

PubMed Central

Uematsu, Chiyomi; Inagaki, Azusa

2014-01-01

Flowering peach Prunus persica cv. Genpei bears pink and variegated flowers on a single tree. The structural genes involved in anthocyanin biosynthesis were expressed strongly in pink petals but only very weakly or not at all in variegated petals. A cDNA clone encoding a MYB-like gene, isolated from pink petals was strongly expressed only in pink petals. Introduction of this gene, via biolistics gave magenta spots in the white areas of variegated petals, therefore this gene was named as Peace (peach anthocyanin colour enhancement). Differences in Peace expression determine the pattern of flower colouration in flowering peach. The R2R3 DNA-binding domain of Peace is similar to those of other plant MYBs regulating anthocyanin biosynthesis. Key amino acids for tertiary structure and the motif for interaction with bHLH proteins were conserved in Peace. Phylogenetic analysis indicates that Peace is closely related to AtMYB123 (TT2), which regulates proanthocyanidin biosynthesis in Arabidopsis, and to anthocyanin regulators in monocots rather than to regulators in dicots. This is the first report that a TT2-like R2R3 MYB has been shown to regulate anthocyanin biosynthesis. PMID:24453228

Inferring Population Genetic Structure in Widely and Continuously Distributed Carnivores: The Stone Marten (Martes foina) as a Case Study

PubMed Central

Vergara, María; Basto, Mafalda P.; Madeira, María José; Gómez-Moliner, Benjamín J.; Santos-Reis, Margarida; Fernandes, Carlos; Ruiz-González, Aritz

2015-01-01

The stone marten is a widely distributed mustelid in the Palaearctic region that exhibits variable habitat preferences in different parts of its range. The species is a Holocene immigrant from southwest Asia which, according to fossil remains, followed the expansion of the Neolithic farming cultures into Europe and possibly colonized the Iberian Peninsula during the Early Neolithic (ca. 7,000 years BP). However, the population genetic structure and historical biogeography of this generalist carnivore remains essentially unknown. In this study we have combined mitochondrial DNA (mtDNA) sequencing (621 bp) and microsatellite genotyping (23 polymorphic markers) to infer the population genetic structure of the stone marten within the Iberian Peninsula. The mtDNA data revealed low haplotype and nucleotide diversities and a lack of phylogeographic structure, most likely due to a recent colonization of the Iberian Peninsula by a few mtDNA lineages during the Early Neolithic. The microsatellite data set was analysed with a) spatial and non-spatial Bayesian individual-based clustering (IBC) approaches (STRUCTURE, TESS, BAPS and GENELAND), and b) multivariate methods [discriminant analysis of principal components (DAPC) and spatial principal component analysis (sPCA)]. Additionally, because isolation by distance (IBD) is a common spatial genetic pattern in mobile and continuously distributed species and it may represent a challenge to the performance of the above methods, the microsatellite data set was tested for its presence. Overall, the genetic structure of the stone marten in the Iberian Peninsula was characterized by a NE-SW spatial pattern of IBD, and this may explain the observed disagreement between clustering solutions obtained by the different IBC methods. However, there was significant indication for contemporary genetic structuring, albeit weak, into at least three different subpopulations. The detected subdivision could be attributed to the influence of the rivers Ebro, Tagus and Guadiana, suggesting that main watercourses in the Iberian Peninsula may act as semi-permeable barriers to gene flow in stone martens. To our knowledge, this is the first phylogeographic and population genetic study of the species at a broad regional scale. We also wanted to make the case for the importance and benefits of using and comparing multiple different clustering and multivariate methods in spatial genetic analyses of mobile and continuously distributed species. PMID:26222680

Inferring Population Genetic Structure in Widely and Continuously Distributed Carnivores: The Stone Marten (Martes foina) as a Case Study.

PubMed

Vergara, María; Basto, Mafalda P; Madeira, María José; Gómez-Moliner, Benjamín J; Santos-Reis, Margarida; Fernandes, Carlos; Ruiz-González, Aritz

2015-01-01

The stone marten is a widely distributed mustelid in the Palaearctic region that exhibits variable habitat preferences in different parts of its range. The species is a Holocene immigrant from southwest Asia which, according to fossil remains, followed the expansion of the Neolithic farming cultures into Europe and possibly colonized the Iberian Peninsula during the Early Neolithic (ca. 7,000 years BP). However, the population genetic structure and historical biogeography of this generalist carnivore remains essentially unknown. In this study we have combined mitochondrial DNA (mtDNA) sequencing (621 bp) and microsatellite genotyping (23 polymorphic markers) to infer the population genetic structure of the stone marten within the Iberian Peninsula. The mtDNA data revealed low haplotype and nucleotide diversities and a lack of phylogeographic structure, most likely due to a recent colonization of the Iberian Peninsula by a few mtDNA lineages during the Early Neolithic. The microsatellite data set was analysed with a) spatial and non-spatial Bayesian individual-based clustering (IBC) approaches (STRUCTURE, TESS, BAPS and GENELAND), and b) multivariate methods [discriminant analysis of principal components (DAPC) and spatial principal component analysis (sPCA)]. Additionally, because isolation by distance (IBD) is a common spatial genetic pattern in mobile and continuously distributed species and it may represent a challenge to the performance of the above methods, the microsatellite data set was tested for its presence. Overall, the genetic structure of the stone marten in the Iberian Peninsula was characterized by a NE-SW spatial pattern of IBD, and this may explain the observed disagreement between clustering solutions obtained by the different IBC methods. However, there was significant indication for contemporary genetic structuring, albeit weak, into at least three different subpopulations. The detected subdivision could be attributed to the influence of the rivers Ebro, Tagus and Guadiana, suggesting that main watercourses in the Iberian Peninsula may act as semi-permeable barriers to gene flow in stone martens. To our knowledge, this is the first phylogeographic and population genetic study of the species at a broad regional scale. We also wanted to make the case for the importance and benefits of using and comparing multiple different clustering and multivariate methods in spatial genetic analyses of mobile and continuously distributed species.

n→π* Non-Covalent Interaction is Weak but Strong in Action

NASA Astrophysics Data System (ADS)

Singh, Santosh Kumar; Das, Aloke

2017-06-01

n→π* interaction is a newly discovered non-covalent interaction which involves delocalization of lone pair (n) electrons of an electronegative atom into π* orbital of a carbonyl group or an aromatic ring. It is widely observed in materials, biomolecules (protein, DNA, RNA), amino acids, neurotransmitter and drugs. However, due to its weak strength and counterintuitive nature its existence is debatable. Such weak interactions are often masked by solvent effects in condense phase or physiological conditions thereby, making it difficult to prove the presence of such weak interactions. Therefore, we have used isolated gas phase spectroscopy in combination with quantum chemical calculations to study n→π* interaction in several molecules where, our molecular systems are free from solvent effects or any external forces. Herein I will be discussing two of the molecular systems (phenyl formate and salicin) where, we have observed the significance of n→π* interaction in determining the conformational specificity of the molecules. We have proved the existence of n→π* interaction for the first time through IR spectroscopy by probing the carbonyl stretching frequency of phenyl formate. Our study is further pursued on a drug named salicin where, we have observed that its conformational preferences is ruled by n→π* interaction even though a strong hydrogen bonding interaction is present in the molecule. Our results show that n→π* interaction, in spite of its weak strength, should not be overlooked as it existence can play an important role in governing the structures of molecules like other strong non-covalent interactions do.

Raman spectroscopy of DNA-metal complexes. I. Interactions and conformational effects of the divalent cations: Mg, Ca, Sr, Ba, Mn, Co, Ni, Cu, Pd, and Cd.

PubMed

Duguid, J; Bloomfield, V A; Benevides, J; Thomas, G J

1993-11-01

Interactions of divalent metal cations (Mg2+, Ca2+, Ba2+, Sr2+, Mn2+, Co2+, Ni2+, Cu2+, Pd2+, and Cd2+) with DNA have been investigated by laser Raman spectroscopy. Both genomic calf-thymus DNA (> 23 kilobase pairs) and mononucleosomal fragments (160 base pairs) were employed as targets of metal interaction in solutions containing 5 weight-% DNA and metal:phosphate molar ratios of 0.6:1. Raman difference spectra reveal that transition metal cations (Mn2+, Co2+, Ni2+, Cu2+, Pd2+, and Cd2+) induce the greatest structural changes in B-DNA. The Raman (vibrational) band differences are extensive and indicate partial disordering of the B-form backbone, reduction in base stacking, reduction in base pairing, and specific metal interaction with acceptor sites on the purine (N7) and pyrimidine (N3) rings. Many of the observed spectral changes parallel those accompanying thermal denaturation of B-DNA and suggest that the metals link the bases of denatured DNA. While exocyclic carbonyls of dT, dG, and dC may stabilize metal ligation, correlation plots show that perturbations of the carbonyls are mainly a consequence of metal-induced denaturation of the double helix. Transition metal interactions with the DNA phosphates are weak in comparison to interactions with the bases, except in the case of Cu2+, which strongly perturbs both base and phosphate group vibrations. On the other hand, the Raman signature of B-DNA is largely unperturbed by Mg2+, Ca2+, Sr2+, and Ba2+, suggesting much weaker interactions of the alkaline earth metals with both base and phosphate sites. A notable exception is a moderate perturbation by alkaline earths of purine N7 sites in 160-base pair DNA, with Ca2+ causing the greatest effect. Correlation plots demonstrate a strong interrelationship between perturbations of Raman bands assigned to ring vibrations of the bases and those of bands assigned to exocyclic carbonyls and backbone phosphodiester groups. However, strong correlations do not occur between the Raman phosphodioxy band (centered near 1092 cm-1) and other Raman bands, suggesting that the former is not highly sensitive to the structural changes induced by divalent metal cations. The structural perturbations induced by divalent cations are much greater for > 23-kilobase pair DNA than for 160-base pair DNA, as evidenced by both the Raman difference spectra and the tendency toward the formation of insoluble aggregates. In the presence of transition metals, aggregation of high-molecular-weight DNA is evident at temperatures as low as 11 degrees C. A relationship between DNA melting and aggregation is proposed in which initial metal binding at major groove sites locally destabilizes the B-DNA double helix, causing displacement of the bases away from one another and exposing additional metal binding sites. Metal cation linkage of two displaced bases would allow separate DNA strands to crosslink. Aggregation is proposed to result from the formation of an extended network of these crosslinks.

The polymer physics of single DNA confined in nanochannels.

PubMed

Dai, Liang; Renner, C Benjamin; Doyle, Patrick S

2016-06-01

In recent years, applications and experimental studies of DNA in nanochannels have stimulated the investigation of the polymer physics of DNA in confinement. Recent advances in the physics of confined polymers, using DNA as a model polymer, have moved beyond the classic Odijk theory for the strong confinement, and the classic blob theory for the weak confinement. In this review, we present the current understanding of the behaviors of confined polymers while briefly reviewing classic theories. Three aspects of confined DNA are presented: static, dynamic, and topological properties. The relevant simulation methods are also summarized. In addition, comparisons of confined DNA with DNA under tension and DNA in semidilute solution are made to emphasize universal behaviors. Finally, an outlook of the possible future research for confined DNA is given. Copyright © 2015 Elsevier B.V. All rights reserved.

Identification of Differentially Methylated Sites with Weak Methylation Effects

PubMed Central

Tran, Hong; Zhu, Hongxiao; Wu, Xiaowei; Kim, Gunjune; Clarke, Christopher R.; Larose, Hailey; Haak, David C.; Westwood, James H.; Zhang, Liqing

2018-01-01

Deoxyribonucleic acid (DNA) methylation is an epigenetic alteration crucial for regulating stress responses. Identifying large-scale DNA methylation at single nucleotide resolution is made possible by whole genome bisulfite sequencing. An essential task following the generation of bisulfite sequencing data is to detect differentially methylated cytosines (DMCs) among treatments. Most statistical methods for DMC detection do not consider the dependency of methylation patterns across the genome, thus possibly inflating type I error. Furthermore, small sample sizes and weak methylation effects among different phenotype categories make it difficult for these statistical methods to accurately detect DMCs. To address these issues, the wavelet-based functional mixed model (WFMM) was introduced to detect DMCs. To further examine the performance of WFMM in detecting weak differential methylation events, we used both simulated and empirical data and compare WFMM performance to a popular DMC detection tool methylKit. Analyses of simulated data that replicated the effects of the herbicide glyphosate on DNA methylation in Arabidopsis thaliana show that WFMM results in higher sensitivity and specificity in detecting DMCs compared to methylKit, especially when the methylation differences among phenotype groups are small. Moreover, the performance of WFMM is robust with respect to small sample sizes, making it particularly attractive considering the current high costs of bisulfite sequencing. Analysis of empirical Arabidopsis thaliana data under varying glyphosate dosages, and the analysis of monozygotic (MZ) twins who have different pain sensitivities—both datasets have weak methylation effects of <1%—show that WFMM can identify more relevant DMCs related to the phenotype of interest than methylKit. Differentially methylated regions (DMRs) are genomic regions with different DNA methylation status across biological samples. DMRs and DMCs are essentially the same concepts, with the only difference being how methylation information across the genome is summarized. If methylation levels are determined by grouping neighboring cytosine sites, then they are DMRs; if methylation levels are calculated based on single cytosines, they are DMCs. PMID:29419727

Alpha-crystallins are involved in specific interactions with the murine gamma D/E/F-crystallin-encoding gene.

PubMed

Pietrowski, D; Durante, M J; Liebstein, A; Schmitt-John, T; Werner, T; Graw, J

1994-07-08

The promoter of the murine gamma E-crystallin (gamma E-Cry) encoding gene (gamma E-cry) was analyzed for specific interactions with lenticular proteins in a gel-retardation assay. A 21-bp fragment immediately downstream of the transcription initiation site (DOTIS) is demonstrated to be responsible for specific interactions with lens extracts. The DOTIS-binding protein(s) accept only the sense DNA strand as target; anti-sense or double-stranded DNA do not interact with these proteins. The DOTIS sequence element is highly conserved among the murine gamma D-, gamma E- and gamma F-cry and is present at comparable positions in the orthologous rat genes. Only a weak or even no protein-binding activity is observed if a few particular bases are changed, as in the rat gamma A-, gamma C- and gamma E-cry elements. DOTIS-binding proteins were found in commercially available bovine alpha-Cry preparations. The essential participation of alpha-Cry in the DNA-binding protein complex was confirmed using alpha-Cry-specific monoclonal antibody. The results reported here point to a novel function of alpha-Cry besides the structural properties in the lens.

Yeast mitochondrial HMG proteins: DNA-binding properties of the most evolutionarily divergent component of mitochondrial nucleoids.

PubMed

Bakkaiova, Jana; Marini, Victoria; Willcox, Smaranda; Nosek, Jozef; Griffith, Jack D; Krejci, Lumir; Tomaska, Lubomir

2015-12-08

Yeast mtDNA is compacted into nucleoprotein structures called mitochondrial nucleoids (mt-nucleoids). The principal mediators of nucleoid formation are mitochondrial high-mobility group (HMG)-box containing (mtHMG) proteins. Although these proteins are some of the fastest evolving components of mt-nucleoids, it is not known whether the divergence of mtHMG proteins on the level of their amino acid sequences is accompanied by diversification of their biochemical properties. In the present study we performed a comparative biochemical analysis of yeast mtHMG proteins from Saccharomyces cerevisiae (ScAbf2p), Yarrowia lipolytica (YlMhb1p) and Candida parapsilosis (CpGcf1p). We found that all three proteins exhibit relatively weak binding to intact dsDNA. In fact, ScAbf2p and YlMhb1p bind quantitatively to this substrate only at very high protein to DNA ratios and CpGcf1p shows only negligible binding to dsDNA. In contrast, the proteins exhibit much higher preference for recombination intermediates such as Holliday junctions (HJ) and replication forks (RF). Therefore, we hypothesize that the roles of the yeast mtHMG proteins in maintenance and compaction of mtDNA in vivo are in large part mediated by their binding to recombination/replication intermediates. We also speculate that the distinct biochemical properties of CpGcf1p may represent one of the prerequisites for frequent evolutionary tinkering with the form of the mitochondrial genome in the CTG-clade of hemiascomycetous yeast species. © 2016 Authors.

INCURVATA2 Encodes the Catalytic Subunit of DNA Polymerase α and Interacts with Genes Involved in Chromatin-Mediated Cellular Memory in Arabidopsis thaliana

PubMed Central

Barrero, José María; González-Bayón, Rebeca; del Pozo, Juan Carlos; Ponce, María Rosa; Micol, José Luis

2007-01-01

Cell type–specific gene expression patterns are maintained by the stable inheritance of transcriptional states through mitosis, requiring the action of multiprotein complexes that remodel chromatin structure. Genetic and molecular interactions between chromatin remodeling factors and components of the DNA replication machinery have been identified in Schizosaccharomyces pombe, indicating that some epigenetic marks are replicated simultaneously to DNA with the participation of the DNA replication complexes. This model of epigenetic inheritance might be extended to the plant kingdom, as we report here with the positional cloning and characterization of INCURVATA2 (ICU2), which encodes the putative catalytic subunit of the DNA polymerase α of Arabidopsis thaliana. The strong icu2-2 and icu2-3 insertional alleles caused fully penetrant zygotic lethality when homozygous and incompletely penetrant gametophytic lethality, probably because of loss of DNA polymerase activity. The weak icu2-1 allele carried a point mutation and caused early flowering, leaf incurvature, and homeotic transformations of sepals into carpels and of petals into stamens. Further genetic analyses indicated that ICU2 interacts with TERMINAL FLOWER2, the ortholog of HETEROCHROMATIN PROTEIN1 of animals and yeasts, and with the Polycomb group (PcG) gene CURLY LEAF. Another PcG gene, EMBRYONIC FLOWER2, was found to be epistatic to ICU2. Quantitative RT-PCR analyses indicated that a number of regulatory genes were derepressed in the icu2-1 mutant, including genes associated with flowering time, floral meristem, and floral organ identity. PMID:17873092

Spatial genetic structure and asymmetrical gene flow within the Pacific walrus

USGS Publications Warehouse

Sonsthagen, Sarah A.; Jay, Chadwick V.; Fischbach, Anthony S.; Sage, George K.; Talbot, Sandra L.

2012-01-01

Pacific walruses (Odobenus rosmarus divergens) occupying shelf waters of Pacific Arctic seas migrate during spring and summer from 3 breeding areas in the Bering Sea to form sexually segregated nonbreeding aggregations. We assessed genetic relationships among 2 putative breeding populations and 6 nonbreeding aggregations. Analyses of mitochondrial DNA (mtDNA) control region sequence data suggest that males are distinct among breeding populations (ΦST=0.051), and between the eastern Chukchi and other nonbreeding aggregations (ΦST=0.336–0.449). Nonbreeding female aggregations were genetically distinct across marker types (microsatellite FST=0.019; mtDNA ΦST=0.313), as was eastern Chukchi and all other nonbreeding aggregations (microsatellite FST=0.019–0.035; mtDNA ΦST=0.386–0.389). Gene flow estimates are asymmetrical from St. Lawrence Island into the southeastern Bering breeding population for both sexes. Partitioning of haplotype frequencies among breeding populations suggests that individuals exhibit some degree of philopatry, although weak. High levels of genetic differentiation among eastern Chukchi and all other nonbreeding aggregations, but considerably lower genetic differentiation between breeding populations, suggest that at least 1 genetically distinct breeding population remained unsampled. Limited genetic structure at microsatellite loci between assayed breeding areas can emerge from several processes, including male-mediated gene flow, or population admixture following a decrease in census size (i.e., due to commercial harvest during 1880–1950s) and subsequent recovery. Nevertheless, high levels of genetic diversity in the Pacific walrus, which withstood prolonged decreases in census numbers with little impact on neutral genetic diversity, may reflect resiliency in the face of past environmental challenges.

Engineering nanometre-scale coherence in soft matter

NASA Astrophysics Data System (ADS)

Liu, Chaoren; Xiang, Limin; Zhang, Yuqi; Zhang, Peng; Beratan, David N.; Li, Yueqi; Tao, Nongjian

2016-10-01

Electronic delocalization in redox-active polymers may be disrupted by the heterogeneity of the environment that surrounds each monomer. When the differences in monomer redox-potential induced by the environment are small (as compared with the monomer-monomer electronic interactions), delocalization persists. Here we show that guanine (G) runs in double-stranded DNA support delocalization over 4-5 guanine bases. The weak interaction between delocalized G blocks on opposite DNA strands is known to support partially coherent long-range charge transport. The molecular-resolution model developed here finds that the coherence among these G blocks follows an even-odd orbital-symmetry rule and predicts that weakening the interaction between G blocks exaggerates the resistance oscillations. These findings indicate how sequence can be exploited to change the balance between coherent and incoherent transport. The predictions are tested and confirmed using break-junction experiments. Thus, tailored orbital symmetry and structural fluctuations may be used to produce coherent transport with a length scale of multiple nanometres in soft-matter assemblies, a length scale comparable to that of small proteins.

DNA Three-Way Junction for Differentiation of Single-Nucleotide Polymorphisms with Fluorescent Copper Nanoparticles.

PubMed

Sun, Feifei; You, Ying; Liu, Jie; Song, Quanwei; Shen, Xiaotong; Na, Na; Ouyang, Jin

2017-05-23

A label- and enzyme-free fluorescent sensor for the detection of single-nucleotide polymorphisms (SNPs) at room temperature is proposed, using new copper nanoparticles (CuNPs) as fluorescent reporters. The CuNPs were constructed by using a DNA three-way junction (3WJ) template. In this assay, two complementary adenine/thymine-rich probes can hybridize with the wild-type target simultaneously to construct a 3WJ structure, serving as an efficient scaffold for the generation of CuNPs. However, the CuNPs produce weak fluorescence when the probes bind with a mutant-type target. SNPs can be identified by the difference in fluorescence intensity of the CuNPs. This SNPs detection strategy is straightforward, cost-effective, and avoids the complicated procedures of labeling or enzymatic reactions. The fluorescent sensor is versatile and can be applied to all types of mutation because the probes are programmable. Moreover, the sensor exhibits good detection performance in biological samples. © 2017 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim.

Crowding-facilitated macromolecular transport in attractive micropost arrays.

PubMed

Chien, Fan-Tso; Lin, Po-Keng; Chien, Wei; Hung, Cheng-Hsiang; Yu, Ming-Hung; Chou, Chia-Fu; Chen, Yeng-Long

2017-05-02

Our study of DNA dynamics in weakly attractive nanofabricated post arrays revealed crowding enhances polymer transport, contrary to hindered transport in repulsive medium. The coupling of DNA diffusion and adsorption to the microposts results in more frequent cross-post hopping and increased long-term diffusivity with increased crowding density. We performed Langevin dynamics simulations and found maximum long-term diffusivity in post arrays with gap sizes comparable to the polymer radius of gyration. We found that macromolecular transport in weakly attractive post arrays is faster than in non-attractive dense medium. Furthermore, we employed hidden Markov analysis to determine the transition of macromolecular adsorption-desorption on posts and hopping between posts. The apparent free energy barriers are comparable to theoretical estimates determined from polymer conformational fluctuations.

Static and Dynamic Properties of DNA Confined in Nanochannels

NASA Astrophysics Data System (ADS)

Gupta, Damini

Next-generation sequencing (NGS) techniques have considerably reduced the cost of high-throughput DNA sequencing. However, it is challenging to detect large-scale genomic variations by NGS due to short read lengths. Genome mapping can easily detect large-scale structural variations because it operates on extremely large intact molecules of DNA with adequate resolution. One of the promising methods of genome mapping is based on confining large DNA molecules inside a nanochannel whose cross-sectional dimensions are approximately 50 nm. Even though this genome mapping technology has been commercialized, the current understanding of the polymer physics of DNA in nanochannel confinement is based on theories and lacks much needed experimental support. The results of this dissertation are aimed at providing a detailed experimental understanding of equilibrium properties of nanochannel-confined DNA molecules. The results are divided into three parts. In first part, we evaluate the role of channel shape on thermodynamic properties of channel confined DNA molecules using a combination of fluorescence microscopy and simulations. Specifically, we show that high aspect ratio of rectangular channels significantly alters the chain statistics as compared to an equivalent square channel with same cross-sectional area. In the second part, we present experimental evidence that weak excluded volume effects arise in DNA nanochannel confinement, which form the physical basis for the extended de Gennes regime. We also show how confinement spectroscopy and simulations can be combined to reduce molecular weight dispersity effects arising from shearing, photo-cleavage, and nonuniform staining of DNA. Finally, the third part of the thesis concerns the dynamic properties of nanochannel confined DNA. We directly measure the center-of-mass diffusivity of single DNA molecules in confinement and show that that it is necessary to modify the classical results of de Gennes to account for local chain stiffness of DNA in order to explain the experimental results. In the end, we believe that our findings from the experimental test of the phase diagram for channel-confined DNA, with careful control over molecular weight dispersity, channel geometry, and electrostatic interactions, will provide a firm foundation for the emerging genome mapping technology.

Large-scale variation in subsurface stream biofilms: a cross-regional comparison of metabolic function and community similarity.

PubMed

Findlay, S; Sinsabaugh, R L

2006-10-01

We examined bacterial metabolic activity and community similarity in shallow subsurface stream sediments distributed across three regions of the eastern United States to assess whether there were parallel changes in functional and structural attributes at this large scale. Bacterial growth, oxygen consumption, and a suite of extracellular enzyme activities were assayed to describe functional variability. Community similarity was assessed using randomly amplified polymorphic DNA (RAPD) patterns. There were significant differences in streamwater chemistry, metabolic activity, and bacterial growth among regions with, for instance, twofold higher bacterial production in streams near Baltimore, MD, compared to Hubbard Brook, NH. Five of eight extracellular enzymes showed significant differences among regions. Cluster analyses of individual streams by metabolic variables showed clear groups with significant differences in representation of sites from different regions among groups. Clustering of sites based on randomly amplified polymorphic DNA banding resulted in groups with generally less internal similarity although there were still differences in distribution of regional sites. There was a marginally significant (p = 0.09) association between patterns based on functional and structural variables. There were statistically significant but weak (r2 approximately 30%) associations between landcover and measures of both structure and function. These patterns imply a large-scale organization of biofilm communities and this structure may be imposed by factor(s) such as landcover and covariates such as nutrient concentrations, which are known to also cause differences in macrobiota of stream ecosystems.

Derivation of Reliable Geometries in QM Calculations of DNA Structures: Explicit Solvent QM/MM and Restrained Implicit Solvent QM Optimizations of G-Quadruplexes.

PubMed

Gkionis, Konstantinos; Kruse, Holger; Šponer, Jiří

2016-04-12

Modern dispersion-corrected DFT methods have made it possible to perform reliable QM studies on complete nucleic acid (NA) building blocks having hundreds of atoms. Such calculations, although still limited to investigations of potential energy surfaces, enhance the portfolio of computational methods applicable to NAs and offer considerably more accurate intrinsic descriptions of NAs than standard MM. However, in practice such calculations are hampered by the use of implicit solvent environments and truncation of the systems. Conventional QM optimizations are spoiled by spurious intramolecular interactions and severe structural deformations. Here we compare two approaches designed to suppress such artifacts: partially restrained continuum solvent QM and explicit solvent QM/MM optimizations. We report geometry relaxations of a set of diverse double-quartet guanine quadruplex (GQ) DNA stems. Both methods provide neat structures without major artifacts. However, each one also has distinct weaknesses. In restrained optimizations, all errors in the target geometries (i.e., low-resolution X-ray and NMR structures) are transferred to the optimized geometries. In QM/MM, the initial solvent configuration causes some heterogeneity in the geometries. Nevertheless, both approaches represent a decisive step forward compared to conventional optimizations. We refine earlier computations that revealed sizable differences in the relative energies of GQ stems computed with AMBER MM and QM. We also explore the dependence of the QM/MM results on the applied computational protocol.

The evolution and population structure of Lactobacillus fermentum from different naturally fermented products as determined by multilocus sequence typing (MLST).

PubMed

Dan, Tong; Liu, Wenjun; Song, Yuqin; Xu, Haiyan; Menghe, Bilige; Zhang, Heping; Sun, Zhihong

2015-05-20

Lactobacillus fermentum is economically important in the production and preservation of fermented foods. A repeatable and discriminative typing method was devised to characterize L. fermentum at the molecular level. The multilocus sequence typing (MLST) scheme developed was based on analysis of the internal sequence of 11 housekeeping gene fragments (clpX, dnaA, dnaK, groEL, murC, murE, pepX, pyrG, recA, rpoB, and uvrC). MLST analysis of 203 isolates of L. fermentum from Mongolia and seven provinces/ autonomous regions in China identified 57 sequence types (ST), 27 of which were represented by only a single isolate, indicating high genetic diversity. Phylogenetic analyses based on the sequence of the 11 housekeeping gene fragments indicated that the L. fermentum isolates analyzed belonged to two major groups. A standardized index of association (I A (S)) indicated a weak clonal population structure in L. fermentum. Split decomposition analysis indicated that recombination played an important role in generating the genetic diversity observed in L. fermentum. The results from the minimum spanning tree strongly suggested that evolution of L. fermentum STs was not correlated with geography or food-type. The MLST scheme developed will be valuable for further studies on the evolution and population structure of L. fermentum isolates used in food products.

Microarray Detection of Duplex and Triplex DNA Binders with DNA-Modified Gold Nanoparticles

PubMed Central

Lytton-Jean, Abigail K. R.; Han, Min Su; Mirkin, Chad A.

2008-01-01

We have designed a chip-based assay, using microarray technology, for determining the relative binding affinities of duplex and triplex DNA binders. This assay combines the high discrimination capabilities afforded by DNA-modified Au nanoparticles with the high-throughput capabilities of DNA microarrays. The detection and screening of duplex DNA binders are important because these molecules, in many cases, are potential anticancer agents as well as toxins. Triplex DNA binders are also promising drug candidates. These molecules, in conjunction with triplex forming oligonucleotides, could potentially be used to achieve control of gene expression by interfering with transcription factors that bind to DNA. Therefore, the ability to screen for these molecules in a high-throughput fashion could dramatically improve the drug screening process. The assay reported here provides excellent discrimination between strong, intermediate, and weak duplex and triplex DNA binders in a high-throughput fashion. PMID:17614366

Construction of cDNA library and preliminary analysis of expressed sequence tags from Siberian tiger

PubMed Central

Liu, Chang-Qing; Lu, Tao-Feng; Feng, Bao-Gang; Liu, Dan; Guan, Wei-Jun; Ma, Yue-Hui

2010-01-01

In this study we successfully constructed a full-length cDNA library from Siberian tiger, Panthera tigris altaica, the most well-known wild Animal. Total RNA was extracted from cultured Siberian tiger fibroblasts in vitro. The titers of primary and amplified libraries were 1.30×106 pfu/ml and 1.62×109 pfu/ml respectively. The proportion of recombinants from unamplified library was 90.5% and average length of exogenous inserts was 1.13 kb. A total of 282 individual ESTs with sizes ranging from 328 to 1,142bps were then analyzed the BLASTX score revealed that 53.9% of the sequences were classified as strong match, 38.6% as nominal and 7.4% as weak match. 28.0% of them were found to be related to enzyme/catalytic protein, 20.9% ESTs to metabolism, 13.1% ESTs to transport, 12.1% ESTs to signal transducer/cell communication, 9.9% ESTs to structure protein, 3.9% ESTs to immunity protein/defense metabolism, 3.2% ESTs to cell cycle, and 8.9 ESTs classified as novel genes. These results demonstrated that the reliability and representativeness of the cDNA library attained to the requirements of a standard cDNA library. This library provided a useful platform for the functional genomic research of Siberian tigers. PMID:20941376

[Cloning and expression regulation of 1-deoxy-D-xylulose-5-phosphate reductoisomerase cDNA from Alpinia officinarum].

PubMed

Zhang, Chun-Rong; Yang, Quan; Chen, Hu-Biao; Pang, Yu-Xin; Tang, Xiao-Min; Cheng, Xuan-Xuan; Wu, Wen-Ya; Chen, Shi-Min

2012-11-01

The rhizome of Alpinia officinarum is a widely used Chinese herbal medicine. The essential oil in A. officinarum rhizome is mainly composed of 1, 8-cineole and other monoterpenes, as the major bioactive ingredients. In plants, monoterpenes are synthesized through the methylerythritol phosphate (MEP) pathway in the plastids, and 1-deoxy-D-xylulose 5-phosphate reductoisomerase (DXR) is an enzyme catalyzing a committed step of the MEP pathway. In the present study, the full-length cDNA encoding DXR was cloned from the rhizome of A. officinarum, using homology-based RT-PCR and rapid amplification of cDNA ends (RACE) techniques. The new cDNA was designated as AoDXR and submitted to GenBank to be assigned with an accession number HQ874658. The full-length cDNA of AoDXR was 1 670 bp containing a 1 419 bp open reading frame encoding a polypeptide of 472 amino acids with a calculated molecular mass of 51.48 kDa and an isoelectric point of 6.15. Bioinformatic analyses revealed that AoDXR showed extensive homology with DXRs from other plant species and contained a conserved plastids transit peptide, a Pro-rich region and two highly conserved NADPH-binding motifs in its N-terminal region characterized by all plant DXRs. The phylogenetic analysis revealed that AoDXR belonged to angiosperm DXRs. The structural modeling of AoDXR showed that AoDXR had the typical V-shaped structure of DXR proteins. The tissue expression pattern analysis indicated that AoDXR expressed strongly in leaves, weak in rhizomes of A. officinarum. Exogenous methyl jasmonate (MeJA) could enhance the expression of AoDXR and the production of 1, 8-cineole in A. officinarum rhizomes. The cloning and characterization of AoDXR will be helpful to reveal the molecular regulation mechanism of monoterpene biosynthesis in A. officinarum and provides a candidate gene for metabolic engineering in improving the medicinal quality of A. officinarum rhizome.

454 Pyrosequencing to Describe Microbial Eukaryotic Community Composition, Diversity and Relative Abundance: A Test for Marine Haptophytes

PubMed Central

Egge, Elianne; Bittner, Lucie; Andersen, Tom; Audic, Stéphane; de Vargas, Colomban; Edvardsen, Bente

2013-01-01

Next generation sequencing of ribosomal DNA is increasingly used to assess the diversity and structure of microbial communities. Here we test the ability of 454 pyrosequencing to detect the number of species present, and assess the relative abundance in terms of cell numbers and biomass of protists in the phylum Haptophyta. We used a mock community consisting of equal number of cells of 11 haptophyte species and compared targeting DNA and RNA/cDNA, and two different V4 SSU rDNA haptophyte-biased primer pairs. Further, we tested four different bioinformatic filtering methods to reduce errors in the resulting sequence dataset. With sequencing depth of 11000–20000 reads and targeting cDNA with Haptophyta specific primers Hap454 we detected all 11 species. A rarefaction analysis of expected number of species recovered as a function of sampling depth suggested that minimum 1400 reads were required here to recover all species in the mock community. Relative read abundance did not correlate to relative cell numbers. Although the species represented with the largest biomass was also proportionally most abundant among the reads, there was generally a weak correlation between proportional read abundance and proportional biomass of the different species, both with DNA and cDNA as template. The 454 sequencing generated considerable spurious diversity, and more with cDNA than DNA as template. With initial filtering based only on match with barcode and primer we observed 100-fold more operational taxonomic units (OTUs) at 99% similarity than the number of species present in the mock community. Filtering based on quality scores, or denoising with PyroNoise resulted in ten times more OTU99% than the number of species. Denoising with AmpliconNoise reduced the number of OTU99% to match the number of species present in the mock community. Based on our analyses, we propose a strategy to more accurately depict haptophyte diversity using 454 pyrosequencing. PMID:24069303

Built-in adjuvanticity of genetically and protein-engineered chimeric molecules for targeting of influenza A peptide epitopes.

PubMed

Kerekov, Nikola S; Ivanova, Iva I; Mihaylova, Nikolina M; Nikolova, Maria; Prechl, Jozsef; Tchorbanov, Andrey I

2014-10-01

Highly purified, subunit, or synthetic viral antigens are known to be weakly immunogenic and potentate only the antibody, rather than cell-mediated immune responses. An alternative approach for inducing protective immunity with small viral peptides would be the direct targeting of viral epitopes to the immunocompetent cells by DNA vaccines encoding antibody fragments specific to activating cell surface co-receptor molecules. Here, we are exploring as a new genetic vaccine, a DNA chimeric molecule encoding a T and B cell epitope-containing influenza A virus hemagglutinin peptide joined to sequences encoding a single-chain variable fragment antibody fragment specific for the costimulatory B cell complement receptors 1 and 2. This recombinant DNA molecule was inserted into eukaryotic expression vector and used as a naked DNA vaccine in WT and CR1/2 KO mice. The intramuscular administration of the DNA construct resulted in the in vivo expression of an immunogenic chimeric protein, which cross-links cell surface receptors on influenza-specific B cells. The DNA vaccination was followed by prime-boosting with the protein-engineered replica of the DNA construct, thus delivering an activation intracellular signal. Immunization with an expression vector containing the described construct and boosting with the protein chimera induced a strong anti-influenza cytotoxic response, modulation of cytokine profile, and a weak antibody response in Balb/c mice. The same immunization scheme did not result in generation of influenza-specific response in mice lacking the target receptor, underlining the molecular adjuvant effect of receptor targeting.

DNA packaging intermediates of bacteriophage Φ174

PubMed Central

Music, Cynthia L; Cheng, R Holland; Bowen, Zorina; McKenna, Robert; Rossmann, Michael G; Baker, Timothy S; Incardona, Nino L

2014-01-01

Background Like many viruses, bacteriophage ΦX174 packages its I)NA genome into a procapsid that is assembled from structural intermediates and scaffolding proteins. The procapsid contains the structural proteins F, G and H, as well as the scaffolding proteins B and D. Provirions are formed by packaging of DNA together with the small internal J proteins, while losing at least some of the B scaffolding proteins. Eventually, loss of the I) scaffolding proteins and the remaining B proteins leads to the formation of mature virions. Results ΦX174 108S 'procapsids' have been purified in milligram quantities by removing 114S (mature virion) and 70S (abortive capsid) particles from crude lysates by differential precipitation with polyethylene glycol. 132S 'provirions' were purified on sucrose gradients in the presence of EDTA. Cryo-electron microscopy (cryo-EM) was used to obtain reconstructions of procapsids and provirions. Although these are very similar to each other, their structures differ greatly from that of the virion. The F and G proteins, whose atomic structures in virions were previously determined from X-ray crystallography, were fitted into the cryo-EM reconstructions. This showed that the pentamer of G proteins on each five-fold vertex changes its conformation only slightly during DNA packaging and maturation, whereas major tertiary and quaternary structural changes occur in the F protein. The procapsids and provirions were found to contain 120 copies of the I) protein arranged as tetramers on the twofold axes. IDNA might enter procapsids through one of the 30 Å diameter holes on the icosahedral three-fold axes. Conclusions Combining cryo-EM image reconstruction and X-ray crystallography has revealed the major conformational changes that can occur in viral assembly. The function of the scaffolding proteins may be, in part, to support weak interactions between the structural proteins in the procapsids and to cover surfaces that are subsequently required for subunit–subunit interaction in the virion. The structures presented here are, therefore, analogous to chaperone proteins complexed with folding intermediates of a substrate. PMID:7613866

Association of in vitro fertilization with global and IGF2/H19 methylation variation in newborn twins.

PubMed

Loke, Y J; Galati, J C; Saffery, R; Craig, J M

2015-04-01

In vitro fertilization (IVF) and its subset intracytoplasmic sperm injection (ICSI), are widely used medical treatments for conception. There has been controversy over whether IVF is associated with adverse short- and long-term health outcomes of offspring. As with other prenatal factors, epigenetic change is thought to be a molecular mediator of any in utero programming effects. Most studies focused on DNA methylation at gene-specific and genomic level, with only a few on associations between DNA methylation and IVF. Using buccal epithelium from 208 twin pairs from the Peri/Postnatal Epigenetic Twin Study (PETS), we investigated associations between IVF and DNA methylation on a global level, using the proxies of Alu and LINE-1 interspersed repeats in addition to two locus-specific regulatory regions within IGF2/H19, controlling for 13 potentially confounding factors. Using multiple correction testing, we found strong evidence that IVF-conceived twins have lower DNA methylation in Alu, and weak evidence of lower methylation in one of the two IGF2/H19 regulatory regions and LINE-1, compared with naturally conceived twins. Weak evidence of a relationship between ICSI and DNA methylation within IGF2/H19 regulatory region was found, suggesting that one or more of the processes associated with IVF/ICSI may contribute to these methylation differences. Lower within- and between-pair DNA methylation variation was also found in IVF-conceived twins for LINE-1, Alu and one IGF2/H19 regulatory region. Although larger sample sizes are needed, our results provide additional insight to the possible influence of IVF and ICSI on DNA methylation. To our knowledge, this is the largest study to date investigating the association of IVF and DNA methylation.

Emerging technologies for studying DNA methylation for the molecular diagnosis of cancer

PubMed Central

Marzese, Diego M.; Hoon, Dave S.B.

2015-01-01

DNA methylation is an epigenetic mechanism that plays a key role in regulating gene expression and other functions. Although this modification is seen in different sequence contexts, the most frequently detected DNA methylation in mammals involves cytosine-guanine dinucleotides. Pathological alterations in DNA methylation patterns are described in a variety of human diseases, including cancer. Unlike genetic changes, DNA methylation is heavily influenced by subtle modifications in the cellular microenvironment. In all cancers, aberrant DNA methylation is involved in the alteration of a large number of oncological pathways with relevant theranostic utility. Several technologies for DNA methylation mapping were recently developed and successfully applied in cancer studies. The scope of these technologies varies from assessing a single cytosine-guanine locus to genome-wide distribution of DNA methylation. Here, we review the strengths and weaknesses of these approaches in the context of clinical utility for the molecular diagnosis of human cancers. PMID:25797072

Population differentiation in the red-legged kittiwake (Rissa brevirostris) as revealed by mitochondrial DNA

USGS Publications Warehouse

Patirana, A.; Hatcher, S.A.; Friesen, Vicki L.

2002-01-01

Population decline in red-legged kittiwakes (Rissa brevirostris) over recent decades has necessitated the collection of information on the distribution of genetic variation within and among colonies for implementation of suitable management policies. Here we present a preliminary study of the extent of genetic structuring and gene flow among the three principal breeding locations of red-legged kittiwakes using the hypervariable Domain I of the mitochondrial control region. Genetic variation was high relative to other species of seabirds, and was similar among locations. Analysis of molecular variance indicated that population genetic structure was statistically significant, and nested clade analysis suggested that kittiwakes breeding on Bering Island maybe genetically isolated from those elsewhere. However, phylogeographic structure was weak. Although this analysis involved only a single locus and a small number of samples, it suggests that red-legged kittiwakes probably constitute a single evolutionary significant unit; the possibility that they constitute two management units requires further investigation.

Population structure and connectivity of tiger sharks (Galeocerdo cuvier) across the Indo-Pacific Ocean basin

PubMed Central

Williams, Samuel M.; Otway, Nicholas M.; Nielsen, Einar E.; Maher, Safia L.; Bennett, Mike B.; Ovenden, Jennifer R.

2017-01-01

Population genetic structure using nine polymorphic nuclear microsatellite loci was assessed for the tiger shark (Galeocerdo cuvier) at seven locations across the Indo-Pacific, and one location in the southern Atlantic. Genetic analyses revealed considerable genetic structuring (FST > 0.14, p < 0.001) between all Indo-Pacific locations and Brazil. By contrast, no significant genetic differences were observed between locations from within the Pacific or Indian Oceans, identifying an apparent large, single Indo-Pacific population. A lack of differentiation between tiger sharks sampled in Hawaii and other Indo-Pacific locations identified herein is in contrast to an earlier global tiger shark nDNA study. The results of our power analysis provide evidence to suggest that the larger sample sizes used here negated any weak population subdivision observed previously. These results further highlight the need for cross-jurisdictional efforts to manage the sustainable exploitation of large migratory sharks like G. cuvier. PMID:28791159

Population structure and connectivity of tiger sharks (Galeocerdo cuvier) across the Indo-Pacific Ocean basin.

PubMed

Holmes, Bonnie J; Williams, Samuel M; Otway, Nicholas M; Nielsen, Einar E; Maher, Safia L; Bennett, Mike B; Ovenden, Jennifer R

2017-07-01

Population genetic structure using nine polymorphic nuclear microsatellite loci was assessed for the tiger shark ( Galeocerdo cuvier ) at seven locations across the Indo-Pacific, and one location in the southern Atlantic. Genetic analyses revealed considerable genetic structuring ( F ST  > 0.14, p  < 0.001) between all Indo-Pacific locations and Brazil. By contrast, no significant genetic differences were observed between locations from within the Pacific or Indian Oceans, identifying an apparent large, single Indo-Pacific population. A lack of differentiation between tiger sharks sampled in Hawaii and other Indo-Pacific locations identified herein is in contrast to an earlier global tiger shark nDNA study. The results of our power analysis provide evidence to suggest that the larger sample sizes used here negated any weak population subdivision observed previously. These results further highlight the need for cross-jurisdictional efforts to manage the sustainable exploitation of large migratory sharks like G. cuvier .

The role of cytosine methylation on charge transport through a DNA strand

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

Qi, Jianqing, E-mail: jqqi@uw.edu; Anantram, M. P., E-mail: anantmp@uw.edu; Govind, Niranjan, E-mail: niri.govind@pnnl.gov

Cytosine methylation has been found to play a crucial role in various biological processes, including a number of human diseases. The detection of this small modification remains challenging. In this work, we computationally explore the possibility of detecting methylated DNA strands through direct electrical conductance measurements. Using density functional theory and the Landauer-Büttiker method, we study the electronic properties and charge transport through an eight base-pair methylated DNA strand and its native counterpart. We first analyze the effect of cytosine methylation on the tight-binding parameters of two DNA strands and then model the transmission of the electrons and conductance throughmore » the strands both with and without decoherence. We find that the main difference of the tight-binding parameters between the native DNA and the methylated DNA lies in the on-site energies of (methylated) cytosine bases. The intra- and inter-strand hopping integrals between two nearest neighboring guanine base and (methylated) cytosine base also change with the addition of the methyl groups. Our calculations show that in the phase-coherent limit, the transmission of the methylated strand is close to the native strand when the energy is nearby the highest occupied molecular orbital level and larger than the native strand by 5 times in the bandgap. The trend in transmission also holds in the presence of the decoherence with the same rate. The lower conductance for the methylated strand in the experiment is suggested to be caused by the more stable structure due to the introduction of the methyl groups. We also study the role of the exchange-correlation functional and the effect of contact coupling by choosing coupling strengths ranging from weak to strong coupling limit.« less

The role of cytosine methylation on charge transport through a DNA strand

NASA Astrophysics Data System (ADS)

Qi, Jianqing; Govind, Niranjan; Anantram, M. P.

2015-09-01

Cytosine methylation has been found to play a crucial role in various biological processes, including a number of human diseases. The detection of this small modification remains challenging. In this work, we computationally explore the possibility of detecting methylated DNA strands through direct electrical conductance measurements. Using density functional theory and the Landauer-Büttiker method, we study the electronic properties and charge transport through an eight base-pair methylated DNA strand and its native counterpart. We first analyze the effect of cytosine methylation on the tight-binding parameters of two DNA strands and then model the transmission of the electrons and conductance through the strands both with and without decoherence. We find that the main difference of the tight-binding parameters between the native DNA and the methylated DNA lies in the on-site energies of (methylated) cytosine bases. The intra- and inter-strand hopping integrals between two nearest neighboring guanine base and (methylated) cytosine base also change with the addition of the methyl groups. Our calculations show that in the phase-coherent limit, the transmission of the methylated strand is close to the native strand when the energy is nearby the highest occupied molecular orbital level and larger than the native strand by 5 times in the bandgap. The trend in transmission also holds in the presence of the decoherence with the same rate. The lower conductance for the methylated strand in the experiment is suggested to be caused by the more stable structure due to the introduction of the methyl groups. We also study the role of the exchange-correlation functional and the effect of contact coupling by choosing coupling strengths ranging from weak to strong coupling limit.

Phylogeographic patterns of Lygus pratensis (Hemiptera: Miridae): Evidence for weak genetic structure and recent expansion in northwest China.

PubMed

Zhang, Li-Juan; Cai, Wan-Zhi; Luo, Jun-Yu; Zhang, Shuai; Wang, Chun-Yi; Lv, Li-Min; Zhu, Xiang-Zhen; Wang, Li; Cui, Jin-Jie

2017-01-01

Lygus pratensis (L.) is an important cotton pest in China, especially in the northwest region. Nymphs and adults cause serious quality and yield losses. However, the genetic structure and geographic distribution of L. pratensis is not well known. We analyzed genetic diversity, geographical structure, gene flow, and population dynamics of L. pratensis in northwest China using mitochondrial and nuclear sequence datasets to study phylogeographical patterns and demographic history. L. pratensis (n = 286) were collected at sites across an area spanning 2,180,000 km2, including the Xinjiang and Gansu-Ningxia regions. Populations in the two regions could be distinguished based on mitochondrial criteria but the overall genetic structure was weak. The nuclear dataset revealed a lack of diagnostic genetic structure across sample areas. Phylogenetic analysis indicated a lack of population level monophyly that may have been caused by incomplete lineage sorting. The Mantel test showed a significant correlation between genetic and geographic distances among the populations based on the mtDNA data. However the nuclear dataset did not show significant correlation. A high level of gene flow among populations was indicated by migration analysis; human activities may have also facilitated insect movement. The availability of irrigation water and ample cotton hosts makes the Xinjiang region well suited for L. pratensis reproduction. Bayesian skyline plot analysis, star-shaped network, and neutrality tests all indicated that L. pratensis has experienced recent population expansion. Climatic changes and extensive areas occupied by host plants have led to population expansion of L. pratensis. In conclusion, the present distribution and phylogeographic pattern of L. pratensis was influenced by climate, human activities, and availability of plant hosts.

Population Genetic Structure and Demographic History of Atrina pectinata Based on Mitochondrial DNA and Microsatellite Markers

PubMed Central

Xue, Dong-Xiu; Wang, Hai-Yan; Zhang, Tao; Liu, Jin-Xian

2014-01-01

The pen shell, Atrina pectinata, is one of the commercial bivalves in East Asia and thought to be recently affected by anthropogenic pressure (habitat destruction and/or fishing pressure). Information on its population genetic structure is crucial for the conservation of A. pectinata. Considering its long pelagic larval duration and iteroparity with high fecundity, the genetic structure for A. pectinata could be expected to be weak at a fine scale. However, the unusual oceanography in the coasts of China and Korea suggests potential for restricted dispersal of pelagic larvae and geographical differentiation. In addition, environmental changes associated with Pleistocene sea level fluctuations on the East China Sea continental shelf may also have strongly influenced historical population demography and genetic diversity of marine organisms. Here, partial sequences of the mitochondrial Cytochrome c oxidase subunit I (COI) gene and seven microsatellite loci were used to estimate population genetic structure and demographic history of seven samples from Northern China coast and one sample from North Korea coast. Despite high levels of genetic diversity within samples, there was no genetic differentiation among samples from Northern China coast and low but significant genetic differentiation between some of the Chinese samples and the North Korean sample. A late Pleistocene population expansion, probably after the Last Glacial Maximum, was also demonstrated for A. pectinata samples. No recent genetic bottleneck was detected in any of the eight samples. We concluded that both historical recolonization (through population range expansion and demographic expansion in the late Pleistocene) and current gene flow (through larval dispersal) were responsible for the weak level of genetic structure detected in A. pectinata. PMID:24789175

How Conformational Dynamics of DNA Polymerase Select Correct Substrates: Experiments and Simulations

PubMed Central

Kirmizialtin, Serdal; Nguyen, Virginia; Johnson, Kenneth A.; Elber, Ron

2012-01-01

Summary Nearly every enzyme undergoes a significant change in structure after binding it’s substrate. New experimental and theoretical analyses of the role of changes in HIV reverse transcriptase structure in selecting a correct substrate are presented. Atomically detailed simulations using the Milestoning method predict a rate and free energy profile of the conformational change commensurate with experimental data. A large conformational change occurring on a ms timescale locks the correct nucleotide at the active site, but promotes release of a mismatched nucleotide. The positions along the reaction coordinate that decide the yield of the reaction are not determined by the chemical step. Rather, the initial steps of weak substrate binding and protein conformational transition significantly enrich the yield of a reaction with a correct substrate, while the same steps diminish the reaction probability of an incorrect substrate. PMID:22483109

Dynamic distribution patterns of ribosomal DNA and chromosomal evolution in Paphiopedilum, a lady's slipper orchid

PubMed Central

2011-01-01

Background Paphiopedilum is a horticulturally and ecologically important genus of ca. 80 species of lady's slipper orchids native to Southeast Asia. These plants have long been of interest regarding their chromosomal evolution, which involves a progressive aneuploid series based on either fission or fusion of centromeres. Chromosome number is positively correlated with genome size, so rearrangement processes must include either insertion or deletion of DNA segments. We have conducted Fluorescence In Situ Hybridization (FISH) studies using 5S and 25S ribosomal DNA (rDNA) probes to survey for rearrangements, duplications, and phylogenetically-correlated variation within Paphiopedilum. We further studied sequence variation of the non-transcribed spacers of 5S rDNA (5S-NTS) to examine their complex duplication history, including the possibility that concerted evolutionary forces may homogenize diversity. Results 5S and 25S rDNA loci among Paphiopedilum species, representing all key phylogenetic lineages, exhibit a considerable diversity that correlates well with recognized evolutionary groups. 25S rDNA signals range from 2 (representing 1 locus) to 9, the latter representing hemizygosity. 5S loci display extensive structural variation, and show from 2 specific signals to many, both major and minor and highly dispersed. The dispersed signals mainly occur at centromeric and subtelomeric positions, which are hotspots for chromosomal breakpoints. Phylogenetic analysis of cloned 5S rDNA non-transcribed spacer (5S-NTS) sequences showed evidence for both ancient and recent post-speciation duplication events, as well as interlocus and intralocus diversity. Conclusions Paphiopedilum species display many chromosomal rearrangements - for example, duplications, translocations, and inversions - but only weak concerted evolutionary forces among highly duplicated 5S arrays, which suggests that double-strand break repair processes are dynamic and ongoing. These results make the genus a model system for the study of complex chromosomal evolution in plants. PMID:21910890

Dynamic distribution patterns of ribosomal DNA and chromosomal evolution in Paphiopedilum, a lady's slipper orchid.

PubMed

Lan, Tianying; Albert, Victor A

2011-09-12

Paphiopedilum is a horticulturally and ecologically important genus of ca. 80 species of lady's slipper orchids native to Southeast Asia. These plants have long been of interest regarding their chromosomal evolution, which involves a progressive aneuploid series based on either fission or fusion of centromeres. Chromosome number is positively correlated with genome size, so rearrangement processes must include either insertion or deletion of DNA segments. We have conducted Fluorescence In Situ Hybridization (FISH) studies using 5S and 25S ribosomal DNA (rDNA) probes to survey for rearrangements, duplications, and phylogenetically-correlated variation within Paphiopedilum. We further studied sequence variation of the non-transcribed spacers of 5S rDNA (5S-NTS) to examine their complex duplication history, including the possibility that concerted evolutionary forces may homogenize diversity. 5S and 25S rDNA loci among Paphiopedilum species, representing all key phylogenetic lineages, exhibit a considerable diversity that correlates well with recognized evolutionary groups. 25S rDNA signals range from 2 (representing 1 locus) to 9, the latter representing hemizygosity. 5S loci display extensive structural variation, and show from 2 specific signals to many, both major and minor and highly dispersed. The dispersed signals mainly occur at centromeric and subtelomeric positions, which are hotspots for chromosomal breakpoints. Phylogenetic analysis of cloned 5S rDNA non-transcribed spacer (5S-NTS) sequences showed evidence for both ancient and recent post-speciation duplication events, as well as interlocus and intralocus diversity. Paphiopedilum species display many chromosomal rearrangements--for example, duplications, translocations, and inversions--but only weak concerted evolutionary forces among highly duplicated 5S arrays, which suggests that double-strand break repair processes are dynamic and ongoing. These results make the genus a model system for the study of complex chromosomal evolution in plants.

Structural Insights Into DNA Repair by RNase T—An Exonuclease Processing 3′ End of Structured DNA in Repair Pathways

PubMed Central

Hsiao, Yu-Yuan; Fang, Woei-Horng; Lee, Chia-Chia; Chen, Yi-Ping; Yuan, Hanna S.

2014-01-01

DNA repair mechanisms are essential for preservation of genome integrity. However, it is not clear how DNA are selected and processed at broken ends by exonucleases during repair pathways. Here we show that the DnaQ-like exonuclease RNase T is critical for Escherichia coli resistance to various DNA-damaging agents and UV radiation. RNase T specifically trims the 3′ end of structured DNA, including bulge, bubble, and Y-structured DNA, and it can work with Endonuclease V to restore the deaminated base in an inosine-containing heteroduplex DNA. Crystal structure analyses further reveal how RNase T recognizes the bulge DNA by inserting a phenylalanine into the bulge, and as a result the 3′ end of blunt-end bulge DNA can be digested by RNase T. In contrast, the homodimeric RNase T interacts with the Y-structured DNA by a different binding mode via a single protomer so that the 3′ overhang of the Y-structured DNA can be trimmed closely to the duplex region. Our data suggest that RNase T likely processes bulge and bubble DNA in the Endonuclease V–dependent DNA repair, whereas it processes Y-structured DNA in UV-induced and various other DNA repair pathways. This study thus provides mechanistic insights for RNase T and thousands of DnaQ-like exonucleases in DNA 3′-end processing. PMID:24594808

DNA damaging potential of Ganoderma lucidum extracts.

PubMed

Gurovic, María Soledad Vela; Viceconte, Fátima R; Pereyra, Marcelo T; Bidegain, Maximiliano A; Cubitto, María Amelia

2018-05-10

Ganoderma lucidum (Lingzhi or Reishi) is a medicinal mushroom historically used in Asian countries to treat a wide variety of diseases and prolong life. In the last years, G. lucidum has been internationally recognized as an effective adjuvant in cancer treatment. Among active components, the most recent research indicates that polysaccharides modulate the immune response favoring the recovery from toxicity of chemo and radiotherapy while triterpenes are cytotoxic to tumoral cells mainly by altering gene expression. Beyond this body of evidence on the efficacy of G. lucidum in cancer treatment, it is not yet understood whether these extracts exert the same mechanisms of action than current antitumoral drugs. In this study, we tested the DNA damaging potential of G. lucidum extracts by the β-galactosidase biochemical prophage induction assay (BIA) using doxorubicin, a DNA intercalating agent, as a positive control. This assay was traditionally used to screen microbial metabolites towards antitumoral agents. Here, we used this bacterial assay for the first time to assess DNA damage of herbal drugs. After a bioguided assay, only a purified fraction of G. lucidum containing a mixture of C16 and C18:1 fatty acids exerted weak activity which could not be attributed to direct interaction with DNA. At the same concentrations, the induction observed for doxorubicin was clearly contrasting. The micro BIA assay could be successfully used to demonstrate differences in cellular effects between G. lucidum extracts and doxorubicin. These results showed that G. lucidum extracts display weak DNA damaging potential. Since DNA injury promotes aging and cancer, our results substantiate the traditional use of this mushroom to prolong life. Copyright © 2018 Elsevier B.V. All rights reserved.

Recognition of Local DNA Structures by p53 Protein

PubMed Central

Brázda, Václav; Coufal, Jan

2017-01-01

p53 plays critical roles in regulating cell cycle, apoptosis, senescence and metabolism and is commonly mutated in human cancer. These roles are achieved by interaction with other proteins, but particularly by interaction with DNA. As a transcription factor, p53 is well known to bind consensus target sequences in linear B-DNA. Recent findings indicate that p53 binds with higher affinity to target sequences that form cruciform DNA structure. Moreover, p53 binds very tightly to non-B DNA structures and local DNA structures are increasingly recognized to influence the activity of wild-type and mutant p53. Apart from cruciform structures, p53 binds to quadruplex DNA, triplex DNA, DNA loops, bulged DNA and hemicatenane DNA. In this review, we describe local DNA structures and summarize information about interactions of p53 with these structural DNA motifs. These recent data provide important insights into the complexity of the p53 pathway and the functional consequences of wild-type and mutant p53 activation in normal and tumor cells. PMID:28208646

In vivo genotoxicity assessment of acrylamide and glycidyl methacrylate.

PubMed

Dobrovolsky, Vasily N; Pacheco-Martinez, M Monserrat; McDaniel, L Patrice; Pearce, Mason G; Ding, Wei

2016-01-01

Acrylamide (ACR) and glycidyl methacrylate (GMA) are structurally related compounds used for making polymers with various properties. Both chemicals can be present in food either as a byproduct of processing or a constituent of packaging. We performed a comprehensive evaluation of ACR and GMA genotoxicity in Fisher 344 rats using repeated gavage administrations. Clastogenicity was measured by scoring micronucleated (MN) erythrocytes from peripheral blood, DNA damage in liver, bone marrow and kidneys was measured using the Comet assay, and gene mutation was measured using the red blood cell (RBC) and reticulocyte Pig-a assay. A limited histopathology evaluation was performed in order to determine levels of cytotoxicity. Doses of up to 20 mg/kg/day of ACR and up to 250 mg/kg/day of GMA were used. ACR treatment resulted in DNA damage in the liver, but not in the bone marrow. While ACR was not a clastogen, it was a weak (equivocal) mutagen in the cells of bone marrow. GMA caused DNA damage in the cells of bone marrow, liver and kidney, and induced MN reticulocytes and Pig-a mutant RBCs in a dose-dependent manner. Collectively, our data suggest that both compounds are in vivo genotoxins, but the genotoxicity of ACR is tissue specific. Published by Elsevier Ltd.

Use of a small molecule as an initiator for interchain staudinger reaction: A new ATP sensing platform using product fluorescence.

PubMed

Yu, Huan; Zheng, Jing; Yang, Sheng; Asiri, Abdullah M; Alamry, Khalid A; Sun, Mingtai; Zhang, Kui; Wang, Suhua; Yang, Ronghua

2018-02-01

We demonstrated that a small molecule induced interchain Staudinger reaction can be employed for highly selective detection of adenosine triphosphate (ATP), an important energy-storage biomolecule. A designed ATP split aptamer (A1) was first functionalized with a weakly fluorescent coumarin derivative due to an azide group (azido-coumarin). The second DNA strand (A2) was covalently linked with triphenylphosphine, which could selectively and efficiently reduce azido to amino group through the Staudinger reaction. The A2 was then hybridized with a half of another designed longer DNA strand (T1). The second half of T1 was a split aptamer and selectively recognized ATP with A1 to form a sandwich structure. The specific interaction between ATP and the aptamers drew the two functionalized DNA strands (A1 and A2) together to initiate the interchain Staudinger reduction at fmol-nmol concentration level, hence produced fluorescent 7-aminocoumarin which could be used as an indicator for the presence of trace ATP. The reaction process had a concentration dependent manner with ATP in a large concentration range. Such a strategy of interchain Staudinger reaction can be extended to construct biosensors for other small functional molecules on the basis of judiciously designed aptamers. Copyright © 2017 Elsevier B.V. All rights reserved.

Structural Stability of Mathematical Models of National Economy

NASA Astrophysics Data System (ADS)

Ashimov, Abdykappar A.; Sultanov, Bahyt T.; Borovskiy, Yuriy V.; Adilov, Zheksenbek M.; Ashimov, Askar A.

2011-12-01

In the paper we test robustness of particular dynamic systems in a compact regions of a plane and a weak structural stability of one dynamic system of high order in a compact region of its phase space. The test was carried out based on the fundamental theory of dynamical systems on a plane and based on the conditions for weak structural stability of high order dynamic systems. A numerical algorithm for testing the weak structural stability of high order dynamic systems has been proposed. Based on this algorithm we assess the weak structural stability of one computable general equilibrium model.

Spectroscopic studies of the binding of Cu(II) complexes of oxicam NSAIDs to alternating G-C and homopolymeric G-C sequences

NASA Astrophysics Data System (ADS)

Chakraborty, Sreeja; Bose, Madhuparna; Sarkar, Munna

2014-03-01

Drugs belonging to the Non-steroidal anti-inflammatory (NSAID) group are not only used as anti-inflammatory, analgesic and anti-pyretic agents, but also show anti-cancer effects. Complexing them with a bioactive metal like copper, show an enhancement in their anti-cancer effects compared to the bare drugs, whose exact mechanism of action is not yet fully understood. For the first time, it was shown by our group that Cu(II)-NSAIDs can directly bind to the DNA backbone. The ability of the copper complexes of NSAIDs namely meloxicam and piroxicam to bind to the DNA backbone could be a possible molecular mechanism behind their enhanced anticancer effects. Elucidating base sequence specific interaction of Cu(II)-NSAIDs to the DNA will provide information on their possible binding sites in the genome sequence. In this work, we present how these complexes respond to differences in structure and hydration pattern of GC rich sequences. For this, binding studies of Cu(II) complexes of piroxicam [Cu(II)-(Px)2 (L)2] and meloxicam [Cu(II)-(Mx)2 (L)] with alternating GC (polydG-dC) and homopolymeric GC (polydG-polydC) sequences were carried out using a combination of spectroscopic techniques that include UV-Vis absorption, fluorescence and circular dichroism (CD) spectroscopy. The Cu(II)-NSAIDs show strong binding affinity to both polydG-dC and polydG-polydC. The role reversal of Cu(II)-meloxicam from a strong binder of polydG-dC (Kb = 11.5 × 103 M-1) to a weak binder of polydG-polydC (Kb = 5.02 × 103 M-1), while Cu(II)-piroxicam changes from a strong binder of polydG-polydC (Kb = 8.18 × 103 M-1) to a weak one of polydG-dC (Kb = 2.18 × 103 M-1), point to the sensitivity of these complexes to changes in the backbone structures/hydration. Changes in the profiles of UV absorption band and CD difference spectra, upon complex binding to polynucleotides and the results of competitive binding assay using ethidium bromide (EtBr) fluorescence indicate different binding modes in each case.

Overproduction, purification, and ATPase activity of the Escherichia coli RuvB protein involved in DNA repair.

PubMed Central

Iwasaki, H; Shiba, T; Makino, K; Nakata, A; Shinagawa, H

1989-01-01

The ruvA and ruvB genes of Escherichia coli constitute an operon which belongs to the SOS regulon. Genetic evidence suggests that the products of the ruv operon are involved in DNA repair and recombination. To begin biochemical characterization of these proteins, we developed a plasmid system that overproduced RuvB protein to 20% of total cell protein. Starting from the overproducing system, we purified RuvB protein. The purified RuvB protein behaved like a monomer in gel filtration chromatography and had an apparent relative molecular mass of 38 kilodaltons in sodium dodecyl sulfate-polyacrylamide gel electrophoresis, which agrees with the value predicted from the DNA sequence. The amino acid sequence of the amino-terminal region of the purified protein was analyzed, and the sequence agreed with the one deduced from the DNA sequence. Since the deduced sequence of RuvB protein contained the consensus sequence for ATP-binding proteins, we examined the ATP-binding and ATPase activities of the purified RuvB protein. RuvB protein had a stronger affinity to ADP than to ATP and weak ATPase activity. The results suggest that the weak ATPase activity of RuvB protein is at least partly due to end product inhibition by ADP. Images PMID:2529252

Exploring DNA-binding Proteins with In Vivo Chemical Cross-linking and Mass Spectrometry

PubMed Central

Qiu, Haibo; Wang, Yinsheng

2009-01-01

DNA-binding proteins are very important constituents of proteomes of all species and play crucial roles in transcription, DNA replication, recombination, repair and other activities associated with DNA. Although a number of DNA-binding proteins have been identified, many proteins involved in gene regulation and DNA repair are likely still unknown because of their dynamic and/or weak interactions with DNA. In this report, we described an approach for the comprehensive identification of DNA-binding proteins with in vivo formaldehyde cross-linking and LC-MS/MS. DNA-binding proteins could be purified via the isolation of DNA-protein complexes and released from the complexes by reversing the cross-linking. By using this method, we were able to identify more than one hundred DNA-binding proteins, such as proteins involved in transcription, gene regulation, DNA replication and repair, and a large number of proteins which are potentially associated with DNA and DNA-binding proteins. This method should be generally applicable to the investigation of other nucleic acid-binding proteins, and hold great potential in the comprehensive study of gene regulation, DNA damage response and repair, as well as many other critical biological processes at proteomic level. PMID:19714816

Making the Bend: DNA Tertiary Structure and Protein-DNA Interactions

PubMed Central

Harteis, Sabrina; Schneider, Sabine

2014-01-01

DNA structure functions as an overlapping code to the DNA sequence. Rapid progress in understanding the role of DNA structure in gene regulation, DNA damage recognition and genome stability has been made. The three dimensional structure of both proteins and DNA plays a crucial role for their specific interaction, and proteins can recognise the chemical signature of DNA sequence (“base readout”) as well as the intrinsic DNA structure (“shape recognition”). These recognition mechanisms do not exist in isolation but, depending on the individual interaction partners, are combined to various extents. Driving force for the interaction between protein and DNA remain the unique thermodynamics of each individual DNA-protein pair. In this review we focus on the structures and conformations adopted by DNA, both influenced by and influencing the specific interaction with the corresponding protein binding partner, as well as their underlying thermodynamics. PMID:25026169

Relations between Shannon entropy and genome order index in segmenting DNA sequences.

PubMed

Zhang, Yi

2009-04-01

Shannon entropy H and genome order index S are used in segmenting DNA sequences. Zhang [Phys. Rev. E 72, 041917 (2005)] found that the two schemes are equivalent when a DNA sequence is converted to a binary sequence of S (strong H bond) and W (weak H bond). They left the mathematical proof to mathematicians who are interested in this issue. In this paper, a possible mathematical explanation is given. Moreover, we find that Chargaff parity rule 2 is the necessary condition of the equivalence, and the equivalence disappears when a DNA sequence is regarded as a four-symbol sequence. At last, we propose that S-2(-H) may be related to species evolution.

Structural Transformation of Wireframe DNA Origami via DNA Polymerase Assisted Gap-Filling.

PubMed

Agarwal, Nayan P; Matthies, Michael; Joffroy, Bastian; Schmidt, Thorsten L

2018-03-27

The programmability of DNA enables constructing nanostructures with almost any arbitrary shape, which can be decorated with many functional materials. Moreover, dynamic structures can be realized such as molecular motors and walkers. In this work, we have explored the possibility to synthesize the complementary sequences to single-stranded gap regions in the DNA origami scaffold cost effectively by a DNA polymerase rather than by a DNA synthesizer. For this purpose, four different wireframe DNA origami structures were designed to have single-stranded gap regions. This reduced the number of staple strands needed to determine the shape and size of the final structure after gap filling. For this, several DNA polymerases and single-stranded binding (SSB) proteins were tested, with T4 DNA polymerase being the best fit. The structures could be folded in as little as 6 min, and the subsequent optimized gap-filling reaction was completed in less than 3 min. The introduction of flexible gap regions results in fully collapsed or partially bent structures due to entropic spring effects. Finally, we demonstrated structural transformations of such deformed wireframe DNA origami structures with DNA polymerases including the expansion of collapsed structures and the straightening of curved tubes. We anticipate that this approach will become a powerful tool to build DNA wireframe structures more material-efficiently, and to quickly prototype and test new wireframe designs that can be expanded, rigidified, or mechanically switched. Mechanical force generation and structural transitions will enable applications in structural DNA nanotechnology, plasmonics, or single-molecule biophysics.

Mechanism for recognition of polyubiquitin chains: balancing affinity through interplay between multivalent binding and dynamics.

PubMed

Markin, Craig J; Xiao, Wei; Spyracopoulos, Leo

2010-08-18

RAP80 plays a key role in signal transduction in the DNA damage response by recruiting proteins to DNA damage foci by binding K63-polyubiquitin chains with two tandem ubiquitin-interacting motifs (tUIM). It is generally recognized that the typically weak interaction between ubiquitin (Ub) and various recognition motifs is intensified by themes such as tandem recognition motifs and Ub polymerization to achieve biological relevance. However, it remains an intricate problem to develop a detailed molecular mechanism to describe the process that leads to amplification of the Ub signal. A battery of solution-state NMR methods and molecular dynamics simulations were used to demonstrate that RAP80-tUIM employs mono- and multivalent interactions with polyUb chains to achieve enhanced affinity in comparison to monoUb interactions for signal amplification. The enhanced affinity is balanced by unfavorable entropic effects that include partial quenching of rapid reorientation between individual UIM domains and individual Ub domains in the bound state. For the RAP80-tUIM-polyUb interaction, increases in affinity with increasing chain length are a result of increased numbers of mono- and multivalent binding sites in the longer polyUb chains. The mono- and multivalent interactions are characterized by intrinsically weak binding and fast off-rates; these weak interactions with fast kinetics may be an important factor underlying the transient nature of protein-protein interactions that comprise DNA damage foci.

The critical protein interactions and structures that elicit growth deregulation in cancer and viral replication

PubMed Central

Ou, Horng D.; May, Andrew P.

2010-01-01

One of the greatest challenges in biomedicine is to define the critical targets and network interactions that are subverted to elicit growth deregulation in human cells. Understanding and developing rational treatments for cancer requires a definition of the key molecular targets and how they interact to elicit the complex growth deregulation phenotype. Viral proteins provide discerning and powerful probes to understand both how cells work and how they can be manipulated using a minimal number of components. The small DNA viruses have evolved to target inherent weaknesses in cellular protein interaction networks to hijack the cellular DNA and protein replication machinery. In the battle to escape the inevitability of senescence and programmed cell death, cancers have converged on similar mechanisms, through the acquisition and selection of somatic mutations that drive unchecked cellular replication in tumors. Understanding the dynamic mechanisms through which a minimal number of viral proteins promote host cells to undergo unscheduled and pathological replication is a powerful strategy to identify critical targets that are also disrupted in cancer. Viruses can therefore be used as tools to probe the system-wide protein-protein interactions and structures that drive growth deregulation in human cells. Ultimately this can provide a path for developing system context-dependent therapeutics. This review will describe ongoing experimental approaches using viruses to study pathways deregulated in cancer, with a particular focus on viral cellular protein-protein interactions and structures. PMID:21061422

Effect of C(60) fullerene on the duplex formation of i-motif DNA with complementary DNA in solution.

PubMed

Jin, Kyeong Sik; Shin, Su Ryon; Ahn, Byungcheol; Jin, Sangwoo; Rho, Yecheol; Kim, Heesoo; Kim, Seon Jeong; Ree, Moonhor

2010-04-15

The structural effects of fullerene on i-motif DNA were investigated by characterizing the structures of fullerene-free and fullerene-bound i-motif DNA, in the presence of cDNA and in solutions of varying pH, using circular dichroism and synchrotron small-angle X-ray scattering. To facilitate a direct structural comparison between the i-motif and duplex structures in response to pH stimulus, we developed atomic scale structural models for the duplex and i-motif DNA structures, and for the C(60)/i-motif DNA hybrid associated with the cDNA strand, assuming that the DNA strands are present in an ideal right-handed helical conformation. We found that fullerene shifted the pH-induced conformational transition between the i-motif and the duplex structure, possibly due to the hydrophobic interactions between the terminal fullerenes and between the terminal fullerenes and an internal TAA loop in the DNA strand. The hybrid structure showed a dramatic reduction in cyclic hysteresis.

Alternative DNA structure formation in the mutagenic human c-MYC promoter

PubMed Central

del Mundo, Imee Marie A.; Zewail-Foote, Maha; Kerwin, Sean M.

2017-01-01

Abstract Mutation ‘hotspot’ regions in the genome are susceptible to genetic instability, implicating them in diseases. These hotspots are not random and often co-localize with DNA sequences potentially capable of adopting alternative DNA structures (non-B DNA, e.g. H-DNA and G4-DNA), which have been identified as endogenous sources of genomic instability. There are regions that contain overlapping sequences that may form more than one non-B DNA structure. The extent to which one structure impacts the formation/stability of another, within the sequence, is not fully understood. To address this issue, we investigated the folding preferences of oligonucleotides from a chromosomal breakpoint hotspot in the human c-MYC oncogene containing both potential G4-forming and H-DNA-forming elements. We characterized the structures formed in the presence of G4-DNA-stabilizing K+ ions or H-DNA-stabilizing Mg2+ ions using multiple techniques. We found that under conditions favorable for H-DNA formation, a stable intramolecular triplex DNA structure predominated; whereas, under K+-rich, G4-DNA-forming conditions, a plurality of unfolded and folded species were present. Thus, within a limited region containing sequences with the potential to adopt multiple structures, only one structure predominates under a given condition. The predominance of H-DNA implicates this structure in the instability associated with the human c-MYC oncogene. PMID:28334873

STAT1:DNA sequence-dependent binding modulation by phosphorylation, protein:protein interactions and small-molecule inhibition

PubMed Central

Bonham, Andrew J.; Wenta, Nikola; Osslund, Leah M.; Prussin, Aaron J.; Vinkemeier, Uwe; Reich, Norbert O.

2013-01-01

The DNA-binding specificity and affinity of the dimeric human transcription factor (TF) STAT1, were assessed by total internal reflectance fluorescence protein-binding microarrays (TIRF-PBM) to evaluate the effects of protein phosphorylation, higher-order polymerization and small-molecule inhibition. Active, phosphorylated STAT1 showed binding preferences consistent with prior characterization, whereas unphosphorylated STAT1 showed a weak-binding preference for one-half of the GAS consensus site, consistent with recent models of STAT1 structure and function in response to phosphorylation. This altered-binding preference was further tested by use of the inhibitor LLL3, which we show to disrupt STAT1 binding in a sequence-dependent fashion. To determine if this sequence-dependence is specific to STAT1 and not a general feature of human TF biology, the TF Myc/Max was analysed and tested with the inhibitor Mycro3. Myc/Max inhibition by Mycro3 is sequence independent, suggesting that the sequence-dependent inhibition of STAT1 may be specific to this system and a useful target for future inhibitor design. PMID:23180800

Global structure of forked DNA in solution revealed by high-resolution single-molecule FRET.

PubMed

Sabir, Tara; Schröder, Gunnar F; Toulmin, Anita; McGlynn, Peter; Magennis, Steven W

2011-02-09

Branched DNA structures play critical roles in DNA replication, repair, and recombination in addition to being key building blocks for DNA nanotechnology. Here we combine single-molecule multiparameter fluorescence detection and molecular dynamics simulations to give a general approach to global structure determination of branched DNA in solution. We reveal an open, planar structure of a forked DNA molecule with three duplex arms and demonstrate an ion-induced conformational change. This structure will serve as a benchmark for DNA-protein interaction studies.

Electrostatic theory of the assembly of PAMAM dendrimers and DNA.

PubMed

Perico, Angelo

2016-05-01

The electrostatic interactions mediated by counterions between a cationic PAMAM dendrimer, modelized as a sphere of radius and cationic surface charge highly increasing with generation, and a DNA, modelized as an anionic elastic line, are analytically calculated in the framework of condensation theory. Under these interactions the DNA is wrapped around the sphere. For excess phosphates relative to dendrimer primary amines, the free energy of the DNA-dendrimer complex displays an absolute minimum when the complex is weakly negatively overcharged. This overcharging opposes gene delivery. For a highly positive dendrimer and a DNA fixed by experimental conditions to a number of phosphates less than the number of dendrimer primary amines, excess amine charges, the dendrimer may at the same time bind stably DNA and interact with negative cell membranes to activate cell transfection in fair agreement with molecular simulations and experiments. © 2016 Wiley Periodicals, Inc.

Antioxidant and prooxidant effects of polyphenol compounds on copper-mediated DNA damage.

PubMed

Perron, Nathan R; García, Carla R; Pinzón, Julio R; Chaur, Manuel N; Brumaghim, Julia L

2011-05-01

Inhibition of copper-mediated DNA damage has been determined for several polyphenol compounds. The 50% inhibition concentration values (IC(50)) for most of the tested polyphenols are between 8 and 480 μM for copper-mediated DNA damage prevention. Although most tested polyphenols were antioxidants under these conditions, they generally inhibited Cu(I)-mediated DNA damage less effectively than Fe(II)-mediated damage, and some polyphenols also displayed prooxidant activity. Because semiquinone radicals and hydroxyl radical adducts were detected by EPR spectroscopy in solutions of polyphenols, Cu(I), and H(2)O(2), it is likely that weak polyphenol-Cu(I) interactions permit a redox-cycling mechanism, whereby the necessary reactants to cause DNA damage (Cu(I), H(2)O(2), and reducing agents) are regenerated. The polyphenol compounds that prevent copper-mediated DNA damage likely follow a radical scavenging pathway as determined by EPR spectroscopy. Copyright © 2011 Elsevier Inc. All rights reserved.

Alternative DNA structure formation in the mutagenic human c-MYC promoter.

PubMed

Del Mundo, Imee Marie A; Zewail-Foote, Maha; Kerwin, Sean M; Vasquez, Karen M

2017-05-05

Mutation 'hotspot' regions in the genome are susceptible to genetic instability, implicating them in diseases. These hotspots are not random and often co-localize with DNA sequences potentially capable of adopting alternative DNA structures (non-B DNA, e.g. H-DNA and G4-DNA), which have been identified as endogenous sources of genomic instability. There are regions that contain overlapping sequences that may form more than one non-B DNA structure. The extent to which one structure impacts the formation/stability of another, within the sequence, is not fully understood. To address this issue, we investigated the folding preferences of oligonucleotides from a chromosomal breakpoint hotspot in the human c-MYC oncogene containing both potential G4-forming and H-DNA-forming elements. We characterized the structures formed in the presence of G4-DNA-stabilizing K+ ions or H-DNA-stabilizing Mg2+ ions using multiple techniques. We found that under conditions favorable for H-DNA formation, a stable intramolecular triplex DNA structure predominated; whereas, under K+-rich, G4-DNA-forming conditions, a plurality of unfolded and folded species were present. Thus, within a limited region containing sequences with the potential to adopt multiple structures, only one structure predominates under a given condition. The predominance of H-DNA implicates this structure in the instability associated with the human c-MYC oncogene. © The Author(s) 2017. Published by Oxford University Press on behalf of Nucleic Acids Research.

Measuring Sperm DNA Fragmentation and Clinical Outcomes of Medically Assisted Reproduction: A Systematic Review and Meta-Analysis.

PubMed

Cissen, Maartje; Wely, Madelon van; Scholten, Irma; Mansell, Steven; Bruin, Jan Peter de; Mol, Ben Willem; Braat, Didi; Repping, Sjoerd; Hamer, Geert

2016-01-01

Sperm DNA fragmentation has been associated with reduced fertilization rates, embryo quality, pregnancy rates and increased miscarriage rates. Various methods exist to test sperm DNA fragmentation such as the sperm chromatin structure assay (SCSA), the sperm chromatin dispersion (SCD) test, the terminal deoxynucleotidyl transferase mediated deoxyuridine triphosphate nick end labelling (TUNEL) assay and the single cell gel electrophoresis (Comet) assay. We performed a systematic review and meta-analysis to assess the value of measuring sperm DNA fragmentation in predicting chance of ongoing pregnancy with IVF or ICSI. Out of 658 unique studies, 30 had extractable data and were thus included in the meta-analysis. Overall, the sperm DNA fragmentation tests had a reasonable to good sensitivity. A wide variety of other factors may also affect the IVF/ICSI outcome, reflected by limited to very low specificity. The constructed hierarchical summary receiver operating characteristic (HSROC) curve indicated a fair discriminatory capacity of the TUNEL assay (area under the curve (AUC) of 0.71; 95% CI 0.66 to 0.74) and Comet assay (AUC of 0.73; 95% CI 0.19 to 0.97). The SCSA and the SCD test had poor predictive capacity. Importantly, for the TUNEL assay, SCD test and Comet assay, meta-regression showed no differences in predictive value between IVF and ICSI. For the SCSA meta-regression indicated the predictive values for IVF and ICSI were different. The present review suggests that current sperm DNA fragmentation tests have limited capacity to predict the chance of pregnancy in the context of MAR. Furthermore, sperm DNA fragmentation tests have little or no difference in predictive value between IVF and ICSI. At this moment, there is insufficient evidence to recommend the routine use of sperm DNA fragmentation tests in couples undergoing MAR both for the prediction of pregnancy and for the choice of treatment. Given the significant limitations of the evidence and the methodological weakness and design of the included studies, we do urge for further research on the predictive value of sperm DNA fragmentation for the chance of pregnancy after MAR, also in comparison with other predictors of pregnancy after MAR.

Tracing the history of goat pastoralism: new clues from mitochondrial and Y chromosome DNA in North Africa.

PubMed

Pereira, Filipe; Queirós, Sara; Gusmão, Leonor; Nijman, Isäac J; Cuppen, Edwin; Lenstra, Johannes A; Davis, Simon J M; Nejmeddine, Fouad; Amorim, António

2009-12-01

Valuable insights into the history of human populations have been obtained by studying the genetic composition of their domesticated species. Here we address some of the long-standing questions about the origin and subsequent movements of goat pastoralism in Northern Africa. We present the first study combining results from mitochondrial DNA (mtDNA) and Y chromosome loci for the genetic characterization of a domestic goat population. Our analyses indicate a remarkably high diversity of maternal and paternal lineages in a sample of indigenous goats from the northwestern fringe of the African continent. Median-joining networks and a multidimensional scaling of ours and almost 2000 published mtDNA sequences revealed a considerable genetic affinity between goat populations from the Maghreb (Northwest Africa) and the Near East. It has been previously shown that goats have a weak phylogeographic structure compatible with high levels of gene flow, as demonstrated by the worldwide dispersal of the predominant mtDNA haplogroup A. In contrast, our results revealed a strong correlation between genetic and geographical distances in 20 populations from different regions of the world. The distribution of Y chromosome haplotypes in Maghrebi goats indicates a common origin for goat patrilines in both Mediterranean coastal regions. Taken together, these results suggest that the colonization and subsequent dispersal of domestic goats in Northern Africa was influenced by the maritime diffusion throughout the Mediterranean Sea and its coastal regions of pastoralist societies whose economy included goat herding. Finally, we also detected traces of gene flow between goat populations from the Maghreb and the Iberian Peninsula corroborating evidence of past cultural and commercial contacts across the Strait of Gibraltar.

Max-E47, a Designed Minimalist Protein that Targets the E-Box DNA Site In Vivo and In Vitro

PubMed Central

Xu, Jing; Chen, Gang; De Jong, Antonia T.; Shahravan, S. Hesam; Shin, Jumi A.

2009-01-01

Max-E47 is a designed hybrid protein comprising the Max DNA-binding basic region and E47 HLH dimerization subdomain. In the yeast one-hybrid system (Y1H), Max-E47 shows strong transcriptional activation from the E-box site, 5'-CACGTG, targeted by the Myc/Max/Mad network of transcription factors; two mutants, Max-E47Y and Max-E47YF, activate more weakly from the E-box in the Y1H. Quantitative fluorescence anisotropy titrations to gain free energies of protein:DNA binding gave low nM Kd values for the native MaxbHLHZ, Max-E47, and the Y and YF mutants binding to the E-box site (14 nM, 15 nM, 9 nM, and 6 nM, respectively), with no detectable binding to a nonspecific control duplex. Because these minimalist, E-box-binding hybrids have no activation domain and no interactions with the c-MycbHLHZ, as shown by the yeast two-hybrid assay, they can potentially serve as dominant-negative inhibitors that suppress activation of E-box-responsive genes targeted by transcription factors including the c-Myc/Max complex. As proof-of-principle, we used our modified Y1H, which allows direct competition between two proteins vying for a DNA target, to show that Max-E47 effectively outcompetes the native MaxbHLHZ for the E-box; weaker competition is observed from the two mutants, consistent with Y1H results. These hybrids provide a minimalist scaffold for further exploration of the relationship between protein structure and DNA-binding function and may have applications as protein therapeutics or biochemical probes capable of targeting the E-box site. PMID:19449889

Monoketone analogs of curcumin, a new class of Fanconi anemia pathway inhibitors.

PubMed

Landais, Igor; Hiddingh, Sanne; McCarroll, Matthew; Yang, Chao; Sun, Aiming; Turker, Mitchell S; Snyder, James P; Hoatlin, Maureen E

2009-12-31

The Fanconi anemia (FA) pathway is a multigene DNA damage response network implicated in the repair of DNA lesions that arise during replication or after exogenous DNA damage. The FA pathway displays synthetic lethal relationship with certain DNA repair genes such as ATM (Ataxia Telangectasia Mutated) that are frequently mutated in tumors. Thus, inhibition of FANCD2 monoubiquitylation (FANCD2-Ub), a key step in the FA pathway, might target tumor cells defective in ATM through synthetic lethal interaction. Curcumin was previously identified as a weak inhibitor of FANCD2-Ub. The aim of this study is to identify derivatives of curcumin with better activity and specificity. Using a replication-free assay in Xenopus extracts, we screened monoketone analogs of curcumin for inhibition of FANCD2-Ub and identified analog EF24 as a strong inhibitor. Mechanistic studies suggest that EF24 targets the FA pathway through inhibition of the NF-kB pathway kinase IKK. In HeLa cells, nanomolar concentrations of EF24 inhibited hydroxyurea (HU)-induced FANCD2-Ub and foci in a cell-cycle independent manner. Survival assays revealed that EF24 specifically sensitizes FA-competent cells to the DNA crosslinking agent mitomycin C (MMC). In addition, in contrast with curcumin, ATM-deficient cells are twofold more sensitive to EF24 than matched wild-type cells, consistent with a synthetic lethal effect between FA pathway inhibition and ATM deficiency. An independent screen identified 4H-TTD, a compound structurally related to EF24 that displays similar activity in egg extracts and in cells. These results suggest that monoketone analogs of curcumin are potent inhibitors of the FA pathway and constitute a promising new class of targeted anticancer compounds.

Monoketone analogs of curcumin, a new class of Fanconi anemia pathway inhibitors

PubMed Central

2009-01-01

Background The Fanconi anemia (FA) pathway is a multigene DNA damage response network implicated in the repair of DNA lesions that arise during replication or after exogenous DNA damage. The FA pathway displays synthetic lethal relationship with certain DNA repair genes such as ATM (Ataxia Telangectasia Mutated) that are frequently mutated in tumors. Thus, inhibition of FANCD2 monoubiquitylation (FANCD2-Ub), a key step in the FA pathway, might target tumor cells defective in ATM through synthetic lethal interaction. Curcumin was previously identified as a weak inhibitor of FANCD2-Ub. The aim of this study is to identify derivatives of curcumin with better activity and specificity. Results Using a replication-free assay in Xenopus extracts, we screened monoketone analogs of curcumin for inhibition of FANCD2-Ub and identified analog EF24 as a strong inhibitor. Mechanistic studies suggest that EF24 targets the FA pathway through inhibition of the NF-kB pathway kinase IKK. In HeLa cells, nanomolar concentrations of EF24 inhibited hydroxyurea (HU)-induced FANCD2-Ub and foci in a cell-cycle independent manner. Survival assays revealed that EF24 specifically sensitizes FA-competent cells to the DNA crosslinking agent mitomycin C (MMC). In addition, in contrast with curcumin, ATM-deficient cells are twofold more sensitive to EF24 than matched wild-type cells, consistent with a synthetic lethal effect between FA pathway inhibition and ATM deficiency. An independent screen identified 4H-TTD, a compound structurally related to EF24 that displays similar activity in egg extracts and in cells. Conclusions These results suggest that monoketone analogs of curcumin are potent inhibitors of the FA pathway and constitute a promising new class of targeted anticancer compounds. PMID:20043851

Structure and stability of the consecutive stereoregulated chiral phosphorothioate DNA duplex.

PubMed

Kanaori, K; Tamura, Y; Wada, T; Nishi, M; Kanehara, H; Morii, T; Tajima, K; Makino, K

1999-12-07

The duplex structures of the stereoregulated phosphorothioate DNAs, [R(p),R(p)]- and [S(p),S(p)]-[d(GC(ps)T(ps)ACG)] (ps, phosphorothioate; PS-DNA), with their complementary RNA have been investigated by combined use of (1)H NMR and restrained molecular dynamics calculation. Compared to those obtained for the unmodified duplex structures (PO-DNA.RNA), the NOE cross-peak intensities are virtually identical for the PS-DNA.RNA hybrid duplexes. The structural analysis on the basis of the NOE restraints reveals that all of the three DNA.RNA duplexes take a A-form conformation and that there is no significant difference in the base stacking for the DNA.RNA hybrid duplexes. On the other hand, the NOE cross-peak intensities of the protons around the central T(ps)A step of the PS-DNA.DNA duplexes are apparently different from those of PO-DNA. DNA. The chemical shifts of H8/6 and H1' at the T(ps)A step are also largely different among PS-DNA.DNAs and PO-DNA.DNA, suggesting that the DNA.DNA structure is readily changed by the introduction of the phosphorothioate groups to the central T(p)A step. The structure calculations indicate that all of these DNA.DNA duplexes are B-form although there exist some small differences in helical parameters between the [R(p),R(p)]- and [S(p),S(p)]PS-DNA.DNA duplexes. The melting temperatures (T(m)) were determined for all of the duplexes by plotting the chemical shift change of isolated peaks as a function of temperature. For the PS-DNA.RNA hybrid duplexes, the [S(p),S(p)] isomer is less stable than the [R(p),R(p)] isomer while this trend is reversed for the PS-DNA.DNA duplexes. Consequently, although the PS-DNA.RNA duplexes take the similar A-form structure, the duplex stability is different between PS-DNA.RNA duplexes. The stability of the DNA.RNA duplexes may not be governed by the A-form structure itself but by some other factors such as the hydration around the phosphorothioate backbone, although the T(m) difference of the DNA.DNA duplexes could be explained by the structural factor.

Interaction of proliferating cell nuclear antigen with PMS2 is required for MutLα activation and function in mismatch repair

PubMed Central

Genschel, Jochen; Kadyrova, Lyudmila Y.; Iyer, Ravi R.; Dahal, Basanta K.; Kadyrov, Farid A.; Modrich, Paul

2017-01-01

Eukaryotic MutLα (mammalian MLH1–PMS2 heterodimer; MLH1–PMS1 in yeast) functions in early steps of mismatch repair as a latent endonuclease that requires a mismatch, MutSα/β, and DNA-loaded proliferating cell nuclear antigen (PCNA) for activation. We show here that human PCNA and MutLα interact specifically but weakly in solution to form a complex of approximately 1:1 stoichiometry that depends on PCNA interaction with the C-terminal endonuclease domain of the MutLα PMS2 subunit. Amino acid substitution mutations within a PMS2 C-terminal 721QRLIAP motif attenuate or abolish human MutLα interaction with PCNA, as well as PCNA-dependent activation of MutLα endonuclease, PCNA- and DNA-dependent activation of MutLα ATPase, and MutLα function in in vitro mismatch repair. Amino acid substitution mutations within the corresponding yeast PMS1 motif (723QKLIIP) reduce or abolish mismatch repair in vivo. Coupling of a weak allele within this motif (723AKLIIP) with an exo1Δ null mutation, which individually confer only weak mutator phenotypes, inactivates mismatch repair in the yeast cell. PMID:28439008

Interaction of proliferating cell nuclear antigen with PMS2 is required for MutLα activation and function in mismatch repair.

PubMed

Genschel, Jochen; Kadyrova, Lyudmila Y; Iyer, Ravi R; Dahal, Basanta K; Kadyrov, Farid A; Modrich, Paul

2017-05-09

Eukaryotic MutLα (mammalian MLH1-PMS2 heterodimer; MLH1-PMS1 in yeast) functions in early steps of mismatch repair as a latent endonuclease that requires a mismatch, MutSα/β, and DNA-loaded proliferating cell nuclear antigen (PCNA) for activation. We show here that human PCNA and MutLα interact specifically but weakly in solution to form a complex of approximately 1:1 stoichiometry that depends on PCNA interaction with the C-terminal endonuclease domain of the MutLα PMS2 subunit. Amino acid substitution mutations within a PMS2 C-terminal 721 QRLIAP motif attenuate or abolish human MutLα interaction with PCNA, as well as PCNA-dependent activation of MutLα endonuclease, PCNA- and DNA-dependent activation of MutLα ATPase, and MutLα function in in vitro mismatch repair. Amino acid substitution mutations within the corresponding yeast PMS1 motif ( 723 QKLIIP) reduce or abolish mismatch repair in vivo. Coupling of a weak allele within this motif ( 723 AKLIIP) with an exo1 Δ null mutation, which individually confer only weak mutator phenotypes, inactivates mismatch repair in the yeast cell.

A soluble RecN homologue provides means for biochemical and genetic analysis of DNA double-strand break repair in Escherichia coli.

PubMed

Grove, Jane I; Wood, Stuart R; Briggs, Geoffrey S; Oldham, Neil J; Lloyd, Robert G

2009-12-03

RecN is a highly conserved, SMC-like protein in bacteria. It plays an important role in the repair of DNA double-strand breaks and is therefore a key factor in maintaining genome integrity. The insolubility of Escherichia coli RecN has limited efforts to unravel its function. We overcame this limitation by replacing the resident coding sequence with that of Haemophilus influenzae RecN. The heterologous construct expresses Haemophilus RecN from the SOS-inducible E. coli promoter. The hybrid gene is fully functional, promoting survival after I-SceI induced DNA breakage, gamma irradiation or exposure to mitomycin C as effectively as the native gene, indicating that the repair activity is conserved between these two species. H. influenzae RecN is quite soluble, even when expressed at high levels, and is readily purified. Its analysis by ionisation-mass spectrometry, gel filtration and glutaraldehyde crosslinking indicates that it is probably a dimer under physiological conditions, although a higher multimer cannot be excluded. The purified protein displays a weak ATPase activity that is essential for its DNA repair function in vivo. However, no DNA-binding activity was detected, which contrasts with RecN from Bacillus subtilis. RecN proteins from Aquifex aeolicus and Bacteriodes fragilis also proved soluble. Neither binds DNA, but the Aquifex RecN has weak ATPase activity. Our findings support studies indicating that RecN, and the SOS response in general, behave differently in E. coli and B. subtilis. The hybrid recN reported provides new opportunities to study the genetics and biochemistry of how RecN operates in E. coli.

Imaging genes, chromosomes, and nuclear structures using laser-scanning confocal microscopy

NASA Astrophysics Data System (ADS)

Ballard, Stephen G.

1990-08-01

For 350 years, the optical microscope has had a powerful symbiotic relationship with biology. Until this century, optical microscopy was the only means of examining cellular structure; in return, biologists have contributed greatly to the evolution of microscope design and technique. Recent advances in the detection and processing of optical images, together with methods for labelling specific biological molecules, have brought about a resurgence in the application of optical microscopy to the biological sciences. One of the areas in which optical microscopy is breaking new ground is in elucidating the large scale organization of chromatin in chromosomes and cell nuclei. Nevertheless, imaging the contents of the cell nucleus is a difficult challenge for light microscopy, for two principal reasons. First, the dimensions of all but the largest nuclear structures (nucleoli, vacuoles) are close to or below the resolving power of far field optics. Second, the native optical contrast properties of many important chromatin structures (eg. chromosome domains, centromere regions) are very weak, or essentially zero. As an extreme example, individual genes probably have nothing to distinguish them other than their sequence of DNA bases, which cannot be directly visualized with any current form of microscopy. Similarly, the interphase nucleus shows no direct visible evidence of focal chromatin domains. Thus, imaging of such entities depends heavily on contrast enhancement methods. The most promising of these is labelling DNA in situ using sequence-specific probes that may be visualized using fluorescent dyes. We have applied this method to detecting individual genes in metaphase chromosomes and interphase nuclei, and to imaging a number of DNA-containing structures including chromosome domains, metaphase chromosomes and centromere regions. We have also demonstrated the applicability of in situ fluorescent labelling to detecting numerical and structural abnormalities both in condensed metaphase chromosomes and in interphase nuclei. The ability to image the loci of fluorescent-labelled gene probes hybridized to chromosomes and to interphase nuclei will play a major role in the mapping of the human genome. This presentation is an overview of our laboratory's efforts to use confocal imaging to address fundamental questions about the structure and organization of genes, chromosomes and cell nuclei, and to develop applications useful in clinical diagnosis of inherited diseases.

In situ structure and dynamics of DNA origami determined through molecular dynamics simulations

PubMed Central

Yoo, Jejoong; Aksimentiev, Aleksei

2013-01-01

The DNA origami method permits folding of long single-stranded DNA into complex 3D structures with subnanometer precision. Transmission electron microscopy, atomic force microscopy, and recently cryo-EM tomography have been used to characterize the properties of such DNA origami objects, however their microscopic structures and dynamics have remained unknown. Here, we report the results of all-atom molecular dynamics simulations that characterized the structural and mechanical properties of DNA origami objects in unprecedented microscopic detail. When simulated in an aqueous environment, the structures of DNA origami objects depart from their idealized targets as a result of steric, electrostatic, and solvent-mediated forces. Whereas the global structural features of such relaxed conformations conform to the target designs, local deformations are abundant and vary in magnitude along the structures. In contrast to their free-solution conformation, the Holliday junctions in the DNA origami structures adopt a left-handed antiparallel conformation. We find the DNA origami structures undergo considerable temporal fluctuations on both local and global scales. Analysis of such structural fluctuations reveals the local mechanical properties of the DNA origami objects. The lattice type of the structures considerably affects global mechanical properties such as bending rigidity. Our study demonstrates the potential of all-atom molecular dynamics simulations to play a considerable role in future development of the DNA origami field by providing accurate, quantitative assessment of local and global structural and mechanical properties of DNA origami objects. PMID:24277840

In situ structure and dynamics of DNA origami determined through molecular dynamics simulations.

PubMed

Yoo, Jejoong; Aksimentiev, Aleksei

2013-12-10

The DNA origami method permits folding of long single-stranded DNA into complex 3D structures with subnanometer precision. Transmission electron microscopy, atomic force microscopy, and recently cryo-EM tomography have been used to characterize the properties of such DNA origami objects, however their microscopic structures and dynamics have remained unknown. Here, we report the results of all-atom molecular dynamics simulations that characterized the structural and mechanical properties of DNA origami objects in unprecedented microscopic detail. When simulated in an aqueous environment, the structures of DNA origami objects depart from their idealized targets as a result of steric, electrostatic, and solvent-mediated forces. Whereas the global structural features of such relaxed conformations conform to the target designs, local deformations are abundant and vary in magnitude along the structures. In contrast to their free-solution conformation, the Holliday junctions in the DNA origami structures adopt a left-handed antiparallel conformation. We find the DNA origami structures undergo considerable temporal fluctuations on both local and global scales. Analysis of such structural fluctuations reveals the local mechanical properties of the DNA origami objects. The lattice type of the structures considerably affects global mechanical properties such as bending rigidity. Our study demonstrates the potential of all-atom molecular dynamics simulations to play a considerable role in future development of the DNA origami field by providing accurate, quantitative assessment of local and global structural and mechanical properties of DNA origami objects.

Secondary structure prediction and structure-specific sequence analysis of single-stranded DNA.

PubMed

Dong, F; Allawi, H T; Anderson, T; Neri, B P; Lyamichev, V I

2001-08-01

DNA sequence analysis by oligonucleotide binding is often affected by interference with the secondary structure of the target DNA. Here we describe an approach that improves DNA secondary structure prediction by combining enzymatic probing of DNA by structure-specific 5'-nucleases with an energy minimization algorithm that utilizes the 5'-nuclease cleavage sites as constraints. The method can identify structural differences between two DNA molecules caused by minor sequence variations such as a single nucleotide mutation. It also demonstrates the existence of long-range interactions between DNA regions separated by >300 nt and the formation of multiple alternative structures by a 244 nt DNA molecule. The differences in the secondary structure of DNA molecules revealed by 5'-nuclease probing were used to design structure-specific probes for mutation discrimination that target the regions of structural, rather than sequence, differences. We also demonstrate the performance of structure-specific 'bridge' probes complementary to non-contiguous regions of the target molecule. The structure-specific probes do not require the high stringency binding conditions necessary for methods based on mismatch formation and permit mutation detection at temperatures from 4 to 37 degrees C. Structure-specific sequence analysis is applied for mutation detection in the Mycobacterium tuberculosis katG gene and for genotyping of the hepatitis C virus.

Phylogeography of the antilopine wallaroo (Macropus antilopinus) across tropical northern Australia.

PubMed

Wadley, Jessica J; Fordham, Damien A; Thomson, Vicki A; Ritchie, Euan G; Austin, Jeremy J

2016-11-01

The distribution of antilopine wallaroo, Macropus antilopinus , is marked by a break in the species' range between Queensland and the Northern Territory, coinciding with the Carpentarian barrier. Previous work on M. antilopinus revealed limited genetic differentiation between the Northern Territory and Queensland M. antilopinus populations across this barrier. The study also identified a number of divergent lineages in the Northern Territory, but was unable to elucidate any geographic structure. Here, we re-examine these results to (1) determine phylogeographic patterns across the range of M. antilopinus and (2) infer the biogeographic barriers associated with these patterns. The tropical savannahs of northern Australia: from the Cape York Peninsula in the east, to the Kimberley in the west. We examined phylogeographic patterns in M. antilopinus using a larger number of samples and three mtDNA genes: NADH dehydrogenase subunit 2, cytochrome b, and the control region. Two datasets were generated and analyzed: (1) a subset of samples with all three mtDNA regions concatenated together and (2) all samples for just control region sequences that included samples from the previous study. Analysis included generating phylogenetic trees based on Bayesian analysis and intraspecific median-joining networks. The contemporary spatial structure of M. antilopinus mtDNA lineages revealed five shallow clades and a sixth, divergent lineage. The genetic differences that we found between Queensland and Northern Territory M. antilopinus samples confirmed the split in the geographic distribution of the species. We also found weak genetic differentiation between Northern Territory samples and those from the Kimberley region of Western Australia, possibly due to the Kimberley Plateau-Arnhem Land barrier. Within the Northern Territory, two clades appear to be parapatric in the west, while another two clades are broadly sympatric across the Northern Territory. MtDNA diversity of M. antilopinus revealed an unexpectedly complex evolutionary history involving multiple sympatric and parapatric mtDNA clades across northern Australia. These phylogeographic patterns highlight the importance of investigating genetic variation across distributions of species and integrating this information into biodiversity conservation.

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.

Unique structural modulation of a non-native substrate by cochaperone DnaJ.

PubMed

Tiwari, Satyam; Kumar, Vignesh; Jayaraj, Gopal Gunanathan; Maiti, Souvik; Mapa, Koyeli

2013-02-12

The role of bacterial DnaJ protein as a cochaperone of DnaK is strongly appreciated. Although DnaJ unaccompanied by DnaK can bind unfolded as well as native substrate proteins, its role as an individual chaperone remains elusive. In this study, we demonstrate that DnaJ binds a model non-native substrate with a low nanomolar dissociation constant and, more importantly, modulates the structure of its non-native state. The structural modulation achieved by DnaJ is different compared to that achieved by the DnaK-DnaJ complex. The nature of structural modulation exerted by DnaJ is suggestive of a unique unfolding activity on the non-native substrate by the chaperone. Furthermore, we demonstrate that the zinc binding motif along with the C-terminal substrate binding domain of DnaJ is necessary and sufficient for binding and the subsequent binding-induced structural alterations of the non-native substrate. We hypothesize that this hitherto unknown structural alteration of non-native states by DnaJ might be important for its chaperoning activity by removing kinetic traps of the folding intermediates.

Optimisation of intradermal DNA electrotransfer for immunisation.

PubMed

Vandermeulen, Gaëlle; Staes, Edith; Vanderhaeghen, Marie Lise; Bureau, Michel Francis; Scherman, Daniel; Préat, Véronique

2007-12-04

The development of DNA vaccines requires appropriate delivery technologies. Electrotransfer is one of the most efficient methods of non-viral gene transfer. In the present study, intradermal DNA electrotransfer was first optimised. Strong effects of the injection method and the dose of DNA on luciferase expression were demonstrated. Pre-treatments were evaluated to enhance DNA diffusion in the skin but neither hyaluronidase injection nor iontophoresis improved efficiency of intradermal DNA electrotransfer. Then, DNA immunisation with a weakly immunogenic model antigen, luciferase, was investigated. After intradermal injection of the plasmid encoding luciferase, electrotransfer (HV 700 V/cm 100 micros, LV 200 V/cm 400 ms) was required to induce immune response. The response was Th1-shifted compared to immunisation with the luciferase recombinant protein. Finally, DNA electrotransfer in the skin, the muscle or the ear pinna was compared. Muscle DNA electrotransfer resulted in the highest luciferase expression and the best IgG response. Nevertheless electrotransfer into the skin, the muscle and the ear pinna all resulted in IFN-gamma secretion by luciferase-stimulated splenocytes suggesting that an efficient Th1 response was induced in all case.

Role of DNA secondary structures in fragile site breakage along human chromosome 10

PubMed Central

Dillon, Laura W.; Pierce, Levi C. T.; Ng, Maggie C. Y.; Wang, Yuh-Hwa

2013-01-01

The formation of alternative DNA secondary structures can result in DNA breakage leading to cancer and other diseases. Chromosomal fragile sites, which are regions of the genome that exhibit chromosomal breakage under conditions of mild replication stress, are predicted to form stable DNA secondary structures. DNA breakage at fragile sites is associated with regions that are deleted, amplified or rearranged in cancer. Despite the correlation, unbiased examination of the ability to form secondary structures has not been evaluated in fragile sites. Here, using the Mfold program, we predict potential DNA secondary structure formation on the human chromosome 10 sequence, and utilize this analysis to compare fragile and non-fragile DNA. We found that aphidicolin (APH)-induced common fragile sites contain more sequence segments with potential high secondary structure-forming ability, and these segments clustered more densely than those in non-fragile DNA. Additionally, using a threshold of secondary structure-forming ability, we refined legitimate fragile sites within the cytogenetically defined boundaries, and identified potential fragile regions within non-fragile DNA. In vitro detection of alternative DNA structure formation and a DNA breakage cell assay were used to validate the computational predictions. Many of the regions identified by our analysis coincide with genes mutated in various diseases and regions of copy number alteration in cancer. This study supports the role of DNA secondary structures in common fragile site instability, provides a systematic method for their identification and suggests a mechanism by which DNA secondary structures can lead to human disease. PMID:23297364

Binding of undamaged double stranded DNA to vaccinia virus uracil-DNA glycosylase

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

Schormann, Norbert; Banerjee, Surajit; Ricciardi, Robert

Background: Uracil-DNA glycosylases are evolutionarily conserved DNA repair enzymes. However, vaccinia virus uracil-DNA glycosylase (known as D4), also serves as an intrinsic and essential component of the processive DNA polymerase complex during DNA replication. In this complex D4 binds to a unique poxvirus specific protein A20 which tethers it to the DNA polymerase. At the replication fork the DNA scanning and repair function of D4 is coupled with DNA replication. So far, DNA-binding to D4 has not been structurally characterized. Results: This manuscript describes the first structure of a DNA-complex of a uracil-DNA glycosylase from the poxvirus family. This alsomore » represents the first structure of a uracil DNA glycosylase in complex with an undamaged DNA. In the asymmetric unit two D4 subunits bind simultaneously to complementary strands of the DNA double helix. Each D4 subunit interacts mainly with the central region of one strand. DNA binds to the opposite side of the A20-binding surface on D4. In comparison of the present structure with the structure of uracil-containing DNA-bound human uracil-DNA glycosylase suggests that for DNA binding and uracil removal D4 employs a unique set of residues and motifs that are highly conserved within the poxvirus family but different in other organisms. Conclusion: The first structure of D4 bound to a truly non-specific undamaged double-stranded DNA suggests that initial binding of DNA may involve multiple non-specific interactions between the protein and the phosphate backbone.« less

Binding of undamaged double stranded DNA to vaccinia virus uracil-DNA glycosylase

DOE PAGES

Schormann, Norbert; Banerjee, Surajit; Ricciardi, Robert; ...

2015-06-02

Background: Uracil-DNA glycosylases are evolutionarily conserved DNA repair enzymes. However, vaccinia virus uracil-DNA glycosylase (known as D4), also serves as an intrinsic and essential component of the processive DNA polymerase complex during DNA replication. In this complex D4 binds to a unique poxvirus specific protein A20 which tethers it to the DNA polymerase. At the replication fork the DNA scanning and repair function of D4 is coupled with DNA replication. So far, DNA-binding to D4 has not been structurally characterized. Results: This manuscript describes the first structure of a DNA-complex of a uracil-DNA glycosylase from the poxvirus family. This alsomore » represents the first structure of a uracil DNA glycosylase in complex with an undamaged DNA. In the asymmetric unit two D4 subunits bind simultaneously to complementary strands of the DNA double helix. Each D4 subunit interacts mainly with the central region of one strand. DNA binds to the opposite side of the A20-binding surface on D4. In comparison of the present structure with the structure of uracil-containing DNA-bound human uracil-DNA glycosylase suggests that for DNA binding and uracil removal D4 employs a unique set of residues and motifs that are highly conserved within the poxvirus family but different in other organisms. Conclusion: The first structure of D4 bound to a truly non-specific undamaged double-stranded DNA suggests that initial binding of DNA may involve multiple non-specific interactions between the protein and the phosphate backbone.« less

DNA mimic proteins: functions, structures, and bioinformatic analysis.

PubMed

Wang, Hao-Ching; Ho, Chun-Han; Hsu, Kai-Cheng; Yang, Jinn-Moon; Wang, Andrew H-J

2014-05-13

DNA mimic proteins have DNA-like negative surface charge distributions, and they function by occupying the DNA binding sites of DNA binding proteins to prevent these sites from being accessed by DNA. DNA mimic proteins control the activities of a variety of DNA binding proteins and are involved in a wide range of cellular mechanisms such as chromatin assembly, DNA repair, transcription regulation, and gene recombination. However, the sequences and structures of DNA mimic proteins are diverse, making them difficult to predict by bioinformatic search. To date, only a few DNA mimic proteins have been reported. These DNA mimics were not found by searching for functional motifs in their sequences but were revealed only by structural analysis of their charge distribution. This review highlights the biological roles and structures of 16 reported DNA mimic proteins. We also discuss approaches that might be used to discover new DNA mimic proteins.

SNBRFinder: A Sequence-Based Hybrid Algorithm for Enhanced Prediction of Nucleic Acid-Binding Residues.

PubMed

Yang, Xiaoxia; Wang, Jia; Sun, Jun; Liu, Rong

2015-01-01

Protein-nucleic acid interactions are central to various fundamental biological processes. Automated methods capable of reliably identifying DNA- and RNA-binding residues in protein sequence are assuming ever-increasing importance. The majority of current algorithms rely on feature-based prediction, but their accuracy remains to be further improved. Here we propose a sequence-based hybrid algorithm SNBRFinder (Sequence-based Nucleic acid-Binding Residue Finder) by merging a feature predictor SNBRFinderF and a template predictor SNBRFinderT. SNBRFinderF was established using the support vector machine whose inputs include sequence profile and other complementary sequence descriptors, while SNBRFinderT was implemented with the sequence alignment algorithm based on profile hidden Markov models to capture the weakly homologous template of query sequence. Experimental results show that SNBRFinderF was clearly superior to the commonly used sequence profile-based predictor and SNBRFinderT can achieve comparable performance to the structure-based template methods. Leveraging the complementary relationship between these two predictors, SNBRFinder reasonably improved the performance of both DNA- and RNA-binding residue predictions. More importantly, the sequence-based hybrid prediction reached competitive performance relative to our previous structure-based counterpart. Our extensive and stringent comparisons show that SNBRFinder has obvious advantages over the existing sequence-based prediction algorithms. The value of our algorithm is highlighted by establishing an easy-to-use web server that is freely accessible at http://ibi.hzau.edu.cn/SNBRFinder.

Structural Analysis of HMGD-DNA Complexes Reveal Influence of Intercalation on Sequence Selectivity and DNA Bending

PubMed Central

Churchill, Mair E.A.; Klass, Janet; Zoetewey, David L.

2010-01-01

The ubiquitous eukaryotic High-Mobility-Group-Box (HMGB) chromosomal proteins promote many chromatin-mediated cellular activities through their non-sequence-specific binding and bending of DNA. Minor groove DNA binding by the HMG box results in substantial DNA bending toward the major groove owing to electrostatic interactions, shape complementarity and DNA intercalation that occurs at two sites. Here, the structures of the complexes formed with DNA by a partially DNA intercalation-deficient mutant of Drosophila melanogaster HMGD have been determined by X-ray crystallography at a resolution of 2.85 Å. The six proteins and fifty base pairs of DNA in the crystal structure revealed a variety of bound conformations. All of the proteins bound in the minor groove, bridging DNA molecules, presumably because these DNA regions are easily deformed. The loss of the primary site of DNA intercalation decreased overall DNA bending and shape complementarity. However, DNA bending at the secondary site of intercalation was retained and most protein-DNA contacts were preserved. The mode of binding resembles the HMGB1-boxA-cisplatin-DNA complex, which also lacks a primary intercalating residue. This study provides new insights into the binding mechanisms used by HMG boxes to recognize varied DNA structures and sequences as well as modulate DNA structure and DNA bending. PMID:20800069

Quantification of transcription factor-DNA binding affinity in a living cell

PubMed Central

Belikov, Sergey; Berg, Otto G.; Wrange, Örjan

2016-01-01

The apparent dissociation constant (Kd) for specific binding of glucocorticoid receptor (GR) and androgen receptor (AR) to DNA was determined in vivo in Xenopus oocytes. The total nuclear receptor concentration was quantified as specifically retained [3H]-hormone in manually isolated oocyte nuclei. DNA was introduced by nuclear microinjection of single stranded phagemid DNA, chromatin is then formed during second strand synthesis. The fraction of DNA sites occupied by the expressed receptor was determined by dimethylsulphate in vivo footprinting and used for calculation of the receptor-DNA binding affinity. The forkhead transcription factor FoxA1 enhanced the DNA binding by GR with an apparent Kd of ∼1 μM and dramatically stimulated DNA binding by AR with an apparent Kd of ∼0.13 μM at a composite androgen responsive DNA element containing one FoxA1 binding site and one palindromic hormone receptor binding site known to bind one receptor homodimer. FoxA1 exerted a weak constitutive- and strongly cooperative DNA binding together with AR but had a less prominent effect with GR, the difference reflecting the licensing function of FoxA1 at this androgen responsive DNA element. PMID:26657626

Surface-assisted DNA self-assembly: An enzyme-free strategy towards formation of branched DNA lattice

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

Bhanjadeo, Madhabi M.; Academy of Scientific and Innovative Research; Nayak, Ashok K.

DNA based self-assembled nanostructures and DNA origami has proven useful for organizing nanomaterials with firm precision. However, for advanced applications like nanoelectronics and photonics, large-scale organization of self-assembled branched DNA (bDNA) into periodic lattices is desired. In this communication for the first time we report a facile method of self-assembly of Y-shaped bDNA nanostructures on the cationic surface of Aluminum (Al) foil to prepare periodic two dimensional (2D) bDNA lattice. Particularly those Y-shaped bDNA structures having smaller overhangs and unable to self-assemble in solution, they are easily assembled on the surface of Al foil in the absence of ligase. Fieldmore » emission scanning electron microscopy (FESEM) analysis shows homogenous distribution of two-dimensional bDNA lattices across the Al foil. When the assembled bDNA structures were recovered from the Al foil and electrophoresed in nPAGE only higher order polymeric bDNA structures were observed without a trace of monomeric structures which confirms the stability and high yield of the bDNA lattices. Therefore, this enzyme-free economic and efficient strategy for developing bDNA lattices can be utilized in assembling various nanomaterials for functional molecular components towards development of DNA based self-assembled nanodevices. - Highlights: • Al foil surface-assisted self-assembly of monomeric structures into larger branched DNA lattice. • FESEM study confirms the uniform distribution of two-dimensional bDNA lattice structures across the surface of Al foil. • Enzyme-free and economic strategy to prepare higher order structures from simpler DNA nanostructures have been confirmed by recovery assay. • Use of well proven sequences for the preparation of pure Y-shaped monomeric DNA nanostructure with high yield.« less

Shock Structure Analysis and Aerodynamics in a Weakly Ionized Gas Flow

NASA Technical Reports Server (NTRS)

Saeks, R.; Popovic, S.; Chow, A. S.

2006-01-01

The structure of a shock wave propagating through a weakly ionized gas is analyzed using an electrofluid dynamics model composed of classical conservation laws and Gauss Law. A viscosity model is included to correctly model the spatial scale of the shock structure, and quasi-neutrality is not assumed. A detailed analysis of the structure of a shock wave propagating in a weakly ionized gas is presented, together with a discussion of the physics underlying the key features of the shock structure. A model for the flow behind a shock wave propagating through a weakly ionized gas is developed and used to analyze the effect of the ionization on the aerodynamics and performance of a two-dimensional hypersonic lifting body.

Antibody recognition of melphalan adducts characterized using immobilized DNA: enhanced alkylation of G-Rich regions in cells compared to in vitro.

PubMed

McCartney, H; Martin, A M; Middleton, P G; Tilby, M J

2001-01-01

The bifunctional alkylating agent, melphalan, forms adducts on DNA that are recognized by two previously described monoclonal antibodies, MP5/73 and Amp4/42. Immunoreactivity to MP5/73 was lost when alkylated DNA was exposed to alkaline pH, while Amp4/42 only recognized the structures formed after the alkali treatment. Competitive enzyme-linked immunoadsorbent assays (ELISAs) indicated that in 0.01 and 0.1 M NaOH, loss of immunoreactivity to MP5/73 occurred with half-lives that were at least 2-fold longer than half-lives for gain of immunoreactivity to Amp4/42. This supported previously published evidence that Amp4/42 did not simply recognize all the products formed by alkali treatment of adducts that were initially recognized by MP5/73. Adducts recognized by MP5/73 on RNA were considerably more stable at 100 degrees C and pH 7 than adducts on DNA. This was consistent with the hypothesis that immunorecognition involved N7 guanine adducts and ruled out the involvement of phosphotriesters in immunoreactivity. Synthetic oligodeoxyribonucleotides, covalently immobilized onto 96-well plates, were reacted with melphalan and incubated for various periods with alkali, and then the levels of adducts recognized by each antibody in replicate wells were assayed by a direct binding ELISA. Adducts formed on oligodeoxyguanylic acid were recognized very weakly by Amp4/42, unlike other DNA sequences that were tested. Retention of immobilized DNA during alkali treatment was confirmed by immunoassay of cisplatin adducts. Poor recognition by Amp4/42 of adducts in oligodeoxyguanylic acid was confirmed by a competitive ELISA. Amp4/42, unlike MP5/73, efficiently recognized adducts resulting from alkylation of DNA with chlorambucil. It is concluded that the two antibodies recognized melphalan adducts in different DNA sequence environments and that this explains (a) the different alkali stability of immunoreactive adducts and (b) previous results which showed that, in DNA from melphalan-treated cells, adducts recognized by Amp4/42 formed a smaller proportion of total adducts compared to DNA alkylated in vitro. The results presented here indicate that this was caused by a marked cellular influence on the overall sequence-dependent pattern of DNA alkylation or repair.

Powering the programmed nanostructure and function of gold nanoparticles with catenated DNA machines

NASA Astrophysics Data System (ADS)

Elbaz, Johann; Cecconello, Alessandro; Fan, Zhiyuan; Govorov, Alexander O.; Willner, Itamar

2013-06-01

DNA nanotechnology is a rapidly developing research area in nanoscience. It includes the development of DNA machines, tailoring of DNA nanostructures, application of DNA nanostructures for computing, and more. Different DNA machines were reported in the past and DNA-guided assembly of nanoparticles represents an active research effort in DNA nanotechnology. Several DNA-dictated nanoparticle structures were reported, including a tetrahedron, a triangle or linear nanoengineered nanoparticle structures; however, the programmed, dynamic reversible switching of nanoparticle structures and, particularly, the dictated switchable functions emerging from the nanostructures, are missing elements in DNA nanotechnology. Here we introduce DNA catenane systems (interlocked DNA rings) as molecular DNA machines for the programmed, reversible and switchable arrangement of different-sized gold nanoparticles. We further demonstrate that the machine-powered gold nanoparticle structures reveal unique emerging switchable spectroscopic features, such as plasmonic coupling or surface-enhanced fluorescence.

The helical structure of DNA facilitates binding

NASA Astrophysics Data System (ADS)

Berg, Otto G.; Mahmutovic, Anel; Marklund, Emil; Elf, Johan

2016-09-01

The helical structure of DNA imposes constraints on the rate of diffusion-limited protein binding. Here we solve the reaction-diffusion equations for DNA-like geometries and extend with simulations when necessary. We find that the helical structure can make binding to the DNA more than twice as fast compared to a case where DNA would be reactive only along one side. We also find that this rate advantage remains when the contributions from steric constraints and rotational diffusion of the DNA-binding protein are included. Furthermore, we find that the association rate is insensitive to changes in the steric constraints on the DNA in the helix geometry, while it is much more dependent on the steric constraints on the DNA-binding protein. We conclude that the helical structure of DNA facilitates the nonspecific binding of transcription factors and structural DNA-binding proteins in general.

Transcription blockage by stable H-DNA analogs in vitro

PubMed Central

Pandey, Shristi; Ogloblina, Anna M.; Belotserkovskii, Boris P.; Dolinnaya, Nina G.; Yakubovskaya, Marianna G.; Mirkin, Sergei M.; Hanawalt, Philip C.

2015-01-01

DNA sequences that can form unusual secondary structures are implicated in regulating gene expression and causing genomic instability. H-palindromes are an important class of such DNA sequences that can form an intramolecular triplex structure, H-DNA. Within an H-palindrome, the H-DNA and canonical B-DNA are in a dynamic equilibrium that shifts toward H-DNA with increased negative supercoiling. The interplay between H- and B-DNA and the fact that the process of transcription affects supercoiling makes it difficult to elucidate the effects of H-DNA upon transcription. We constructed a stable structural analog of H-DNA that cannot flip into B-DNA, and studied the effects of this structure on transcription by T7 RNA polymerase in vitro. We found multiple transcription blockage sites adjacent to and within sequences engaged in this triplex structure. Triplex-mediated transcription blockage varied significantly with changes in ambient conditions: it was exacerbated in the presence of Mn2+ or by increased concentrations of K+ and Li+. Analysis of the detailed pattern of the blockage suggests that RNA polymerase is sterically hindered by H-DNA and has difficulties in unwinding triplex DNA. The implications of these findings for the biological roles of triple-stranded DNA structures are discussed. PMID:26101261

Enhanced transgene expression in rice following selection controlled by weak promoters.

PubMed

Zhou, Jie; Yang, Yong; Wang, Xuming; Yu, Feibo; Yu, Chulang; Chen, Juan; Cheng, Ye; Yan, Chenqi; Chen, Jianping

2013-03-27

Techniques that enable high levels of transgene expression in plants are attractive for the commercial production of plant-made recombinant pharmaceutical proteins or other gene transfer related strategies. The conventional way to increase the yield of desired transgenic products is to use strong promoters to control the expression of the transgene. Although many such promoters have been identified and characterized, the increase obtainable from a single promoter is ultimately limited to a certain extent. In this study, we report a method to magnify the effect of a single promoter by using a weak promoter-based selection system in transgenic rice. tCUP1, a fragment derived from the tobacco cryptic promoter (tCUP), was tested for its activity in rice by fusion to both a β-glucuronidase (GUS) reporter and a hygromycin phosphotransferase (HPT) selectable marker. The tCUP1 promoter allowed the recovery of transformed rice plants and conferred tissue specific expression of the GUS reporter, but was much weaker than the CaMV 35S promoter in driving a selectable marker for growth of resistant calli. However, in the resistant calli and regenerated transgenic plants selected by the use of tCUP1, the constitutive expression of green fluorescent protein (GFP) was dramatically increased as a result of the additive effect of multiple T-DNA insertions. The correlation between attenuated selection by a weak promoter and elevation of copy number and foreign gene expression was confirmed by using another relatively weak promoter from nopaline synthase (Nos). The use of weak promoter derived selectable markers leads to a high T-DNA copy number and then greatly increases the expression of the foreign gene. The method described here provides an effective approach to robustly enhance the expression of heterogenous transgenes through copy number manipulation in rice.

Paternal phylogeographic structure of the brown bear (Ursus arctos) in northeastern Asia and the effect of male-mediated gene flow to insular populations.

PubMed

Hirata, Daisuke; Mano, Tsutomu; Abramov, Alexei V; Baryshnikov, Gennady F; Kosintsev, Pavel A; Murata, Koichi; Masuda, Ryuichi

2017-01-01

Sex-biased dispersal is widespread among mammals, including the brown bear ( Ursus arctos ). Previous phylogeographic studies of the brown bear based on maternally inherited mitochondrial DNA have shown intraspecific genetic structuring around the northern hemisphere. The brown bears on Hokkaido Island, northern Japan, comprise three distinct maternal lineages that presumably immigrated to the island from the continent in three different periods. Here, we investigate the paternal genetic structure across northeastern Asia and assess the connectivity among and within intraspecific populations in terms of male-mediated gene flow. We analyzed paternally inherited Y-chromosomal DNA sequence data and Y-linked microsatellite data of 124 brown bears from Hokkaido, the southern Kuril Islands (Kunashiri and Etorofu), Sakhalin, and continental Eurasia (Kamchatka Peninsula, Ural Mountains, European Russia, and Tibet). The Hokkaido brown bear population is paternally differentiated from, and lacked recent genetic connectivity with, the continental Eurasian and North American populations. We detected weak spatial genetic structuring of the paternal lineages on Hokkaido, which may have arisen through male-mediated gene flow among natal populations. In addition, our results suggest that the different dispersal patterns between male and female brown bears, combined with the founder effect and subsequent genetic drift, contributed to the makeup of the Etorofu Island population, in which the maternal and paternal lineages show different origins. Brown bears on Hokkaido and the adjacent southern Kuril Islands experienced different maternal and paternal evolutionary histories. Our results indicate that sex-biased dispersal has played a significant role in the evolutionary history of the brown bear in continental populations and in peripheral insular populations, such as on Hokkaido, the southern Kuril Islands, and Sakhalin.

An affinity-structure database of helix-turn-helix: DNA complexes with a universal coordinate system.

PubMed

AlQuraishi, Mohammed; Tang, Shengdong; Xia, Xide

2015-11-19

Molecular interactions between proteins and DNA molecules underlie many cellular processes, including transcriptional regulation, chromosome replication, and nucleosome positioning. Computational analyses of protein-DNA interactions rely on experimental data characterizing known protein-DNA interactions structurally and biochemically. While many databases exist that contain either structural or biochemical data, few integrate these two data sources in a unified fashion. Such integration is becoming increasingly critical with the rapid growth of structural and biochemical data, and the emergence of algorithms that rely on the synthesis of multiple data types to derive computational models of molecular interactions. We have developed an integrated affinity-structure database in which the experimental and quantitative DNA binding affinities of helix-turn-helix proteins are mapped onto the crystal structures of the corresponding protein-DNA complexes. This database provides access to: (i) protein-DNA structures, (ii) quantitative summaries of protein-DNA binding affinities using position weight matrices, and (iii) raw experimental data of protein-DNA binding instances. Critically, this database establishes a correspondence between experimental structural data and quantitative binding affinity data at the single basepair level. Furthermore, we present a novel alignment algorithm that structurally aligns the protein-DNA complexes in the database and creates a unified residue-level coordinate system for comparing the physico-chemical environments at the interface between complexes. Using this unified coordinate system, we compute the statistics of atomic interactions at the protein-DNA interface of helix-turn-helix proteins. We provide an interactive website for visualization, querying, and analyzing this database, and a downloadable version to facilitate programmatic analysis. This database will facilitate the analysis of protein-DNA interactions and the development of programmatic computational methods that capitalize on integration of structural and biochemical datasets. The database can be accessed at http://ProteinDNA.hms.harvard.edu.

IFI16 Preferentially Binds to DNA with Quadruplex Structure and Enhances DNA Quadruplex Formation.

PubMed

Hároníková, Lucia; Coufal, Jan; Kejnovská, Iva; Jagelská, Eva B; Fojta, Miroslav; Dvořáková, Petra; Muller, Petr; Vojtesek, Borivoj; Brázda, Václav

2016-01-01

Interferon-inducible protein 16 (IFI16) is a member of the HIN-200 protein family, containing two HIN domains and one PYRIN domain. IFI16 acts as a sensor of viral and bacterial DNA and is important for innate immune responses. IFI16 binds DNA and binding has been described to be DNA length-dependent, but a preference for supercoiled DNA has also been demonstrated. Here we report a specific preference of IFI16 for binding to quadruplex DNA compared to other DNA structures. IFI16 binds to quadruplex DNA with significantly higher affinity than to the same sequence in double stranded DNA. By circular dichroism (CD) spectroscopy we also demonstrated the ability of IFI16 to stabilize quadruplex structures with quadruplex-forming oligonucleotides derived from human telomere (HTEL) sequences and the MYC promotor. A novel H/D exchange mass spectrometry approach was developed to assess protein interactions with quadruplex DNA. Quadruplex DNA changed the IFI16 deuteration profile in parts of the PYRIN domain (aa 0-80) and in structurally identical parts of both HIN domains (aa 271-302 and aa 586-617) compared to single stranded or double stranded DNAs, supporting the preferential affinity of IFI16 for structured DNA. Our results reveal the importance of quadruplex DNA structure in IFI16 binding and improve our understanding of how IFI16 senses DNA. IFI16 selectivity for quadruplex structure provides a mechanistic framework for IFI16 in immunity and cellular processes including DNA damage responses and cell proliferation.

Near-atomic structural model for bacterial DNA replication initiation complex and its functional insights.

PubMed

Shimizu, Masahiro; Noguchi, Yasunori; Sakiyama, Yukari; Kawakami, Hironori; Katayama, Tsutomu; Takada, Shoji

2016-12-13

Upon DNA replication initiation in Escherichia coli, the initiator protein DnaA forms higher-order complexes with the chromosomal origin oriC and a DNA-bending protein IHF. Although tertiary structures of DnaA and IHF have previously been elucidated, dynamic structures of oriC-DnaA-IHF complexes remain unknown. Here, combining computer simulations with biochemical assays, we obtained models at almost-atomic resolution for the central part of the oriC-DnaA-IHF complex. This complex can be divided into three subcomplexes; the left and right subcomplexes include pentameric DnaA bound in a head-to-tail manner and the middle subcomplex contains only a single DnaA. In the left and right subcomplexes, DnaA ATPases associated with various cellular activities (AAA+) domain III formed helices with specific structural differences in interdomain orientations, provoking a bend in the bound DNA. In the left subcomplex a continuous DnaA chain exists, including insertion of IHF into the DNA looping, consistent with the DNA unwinding function of the complex. The intervening spaces in those subcomplexes are crucial for DNA unwinding and loading of DnaB helicases. Taken together, this model provides a reasonable near-atomic level structural solution of the initiation complex, including the dynamic conformations and spatial arrangements of DnaA subcomplexes.

Structure of the Tetrameric p53 Tumor Suppressor Bound to DNA

DTIC Science & Technology

2002-05-01

been unable to prepare suitable p53/DNA cocrystals for structure determination. Nonetheless, we have successfully determined the medium resolution (2.7A... cocrystallization with longer DNA targets or DNA targets assembled into nucleosome core particles. The structure of tetrameric p53 bound to DNA will provide

DNA bending-induced phase transition of encapsidated genome in phage λ

PubMed Central

Lander, Gabriel C.; Johnson, John E.; Rau, Donald C.; Potter, Clinton S.; Carragher, Bridget; Evilevitch, Alex

2013-01-01

The DNA structure in phage capsids is determined by DNA–DNA interactions and bending energy. The effects of repulsive interactions on DNA interaxial distance were previously investigated, but not the effect of DNA bending on its structure in viral capsids. By varying packaged DNA length and through addition of spermine ions, we transform the interaction energy from net repulsive to net attractive. This allowed us to isolate the effect of bending on the resulting DNA structure. We used single particle cryo-electron microscopy reconstruction analysis to determine the interstrand spacing of double-stranded DNA encapsidated in phage λ capsids. The data reveal that stress and packing defects, both resulting from DNA bending in the capsid, are able to induce a long-range phase transition in the encapsidated DNA genome from a hexagonal to a cholesteric packing structure. This structural observation suggests significant changes in genome fluidity as a result of a phase transition affecting the rates of viral DNA ejection and packaging. PMID:23449219

Topological impact of noncanonical DNA structures on Klenow fragment of DNA polymerase.

PubMed

Takahashi, Shuntaro; Brazier, John A; Sugimoto, Naoki

2017-09-05

Noncanonical DNA structures that stall DNA replication can cause errors in genomic DNA. Here, we investigated how the noncanonical structures formed by sequences in genes associated with a number of diseases impacted DNA polymerization by the Klenow fragment of DNA polymerase. Replication of a DNA sequence forming an i-motif from a telomere, hypoxia-induced transcription factor, and an insulin-linked polymorphic region was effectively inhibited. On the other hand, replication of a mixed-type G-quadruplex (G4) from a telomere was less inhibited than that of the antiparallel type or parallel type. Interestingly, the i-motif was a better inhibitor of replication than were mixed-type G4s or hairpin structures, even though all had similar thermodynamic stabilities. These results indicate that both the stability and topology of structures formed in DNA templates impact the processivity of a DNA polymerase. This suggests that i-motif formation may trigger genomic instability by stalling the replication of DNA, causing intractable diseases.

Topological impact of noncanonical DNA structures on Klenow fragment of DNA polymerase

PubMed Central

Takahashi, Shuntaro; Brazier, John A.; Sugimoto, Naoki

2017-01-01

Noncanonical DNA structures that stall DNA replication can cause errors in genomic DNA. Here, we investigated how the noncanonical structures formed by sequences in genes associated with a number of diseases impacted DNA polymerization by the Klenow fragment of DNA polymerase. Replication of a DNA sequence forming an i-motif from a telomere, hypoxia-induced transcription factor, and an insulin-linked polymorphic region was effectively inhibited. On the other hand, replication of a mixed-type G-quadruplex (G4) from a telomere was less inhibited than that of the antiparallel type or parallel type. Interestingly, the i-motif was a better inhibitor of replication than were mixed-type G4s or hairpin structures, even though all had similar thermodynamic stabilities. These results indicate that both the stability and topology of structures formed in DNA templates impact the processivity of a DNA polymerase. This suggests that i-motif formation may trigger genomic instability by stalling the replication of DNA, causing intractable diseases. PMID:28827350

Molecular analysis of a phytohemagglutinin-defective cultivar of Phaseolus vulgaris L.

PubMed

Vitale, A; Ceriotti, A; Bollini, R

1985-10-01

The seeds of Phaseolus vulgaris cv. Pinto III are known to lack detectable amounts of phytohemagglutinin (PHA) and to accumulate very reduced levels of PHA mRNA compared with normal cultivars. Using PHA complementary-DNA clones and monospecific antibodies we analyzed cv. Pinto III genomic DNA and cotyledonary proteins synthesized both in vitro and in vivo. We detected genomic DNA sequences that hybridize with complementary-DNA clones for the two different classes of PHA polypeptides (PHA-E and PHA-L), at levels comparable to a normal bean cultivar. This indicates that the cv. Pinto III phenotype is not the result of a large deletion of the PHA structural genes. Messenger RNA isolated from cv. Pinto III developing cotyledons synthesizes in vitro very small amounts of a protein which is recognized by antibodies specific for PHA, and gives, on sodium dodecyl sulfate-polyacrylamide gel electrophoresis, a single band with molecular weight similar but not identical to that of PHA-L polypeptides. This protein is also synthesized in vivo at a very reduced level, less than 1% compared with PHA in normal cultivars, and has mitogenic activity comparable to that of the PHA-L subunit, while it shows very weak erythroagglutinating activity. The initial steps in the synthesis and processing of this protein are identical to those already identified for PHA polypeptides. The cv. Pinto III protein could be either a PHA-L polypeptide whose synthesis is not affected by the mutation or a PHA-like lectin present normally at low levels in P. vulgaris.

Gene conversion events and variable degree of homogenization of rDNA loci in cultivars of Brassica napus

PubMed Central

Sochorová, Jana; Coriton, Olivier; Kuderová, Alena; Lunerová, Jana; Chèvre, Anne-Marie; Kovařík, Aleš

2017-01-01

Background and aims Brassica napus (AACC, 2n = 38, oilseed rape) is a relatively recent allotetraploid species derived from the putative progenitor diploid species Brassica rapa (AA, 2n = 20) and Brassica oleracea (CC, 2n = 18). To determine the influence of intensive breeding conditions on the evolution of its genome, we analysed structure and copy number of rDNA in 21 cultivars of B. napus, representative of genetic diversity. Methods We used next-generation sequencing genomic approaches, Southern blot hybridization, expression analysis and fluorescence in situ hybridization (FISH). Subgenome-specific sequences derived from rDNA intergenic spacers (IGS) were used as probes for identification of loci composition on chromosomes. Key Results Most B. napus cultivars (18/21, 86 %) had more A-genome than C-genome rDNA copies. Three cultivars analysed by FISH (‘Darmor’, ‘Yudal’ and ‘Asparagus kale’) harboured the same number (12 per diploid set) of loci. In B. napus ‘Darmor’, the A-genome-specific rDNA probe hybridized to all 12 rDNA loci (eight on the A-genome and four on the C-genome) while the C-genome-specific probe showed weak signals on the C-genome loci only. Deep sequencing revealed high homogeneity of arrays suggesting that the C-genome genes were largely overwritten by the A-genome variants in B. napus ‘Darmor’. In contrast, B. napus ‘Yudal’ showed a lack of gene conversion evidenced by additive inheritance of progenitor rDNA variants and highly localized hybridization signals of subgenome-specific probes on chromosomes. Brassica napus ‘Asparagus kale’ showed an intermediate pattern to ‘Darmor’ and ‘Yudal’. At the expression level, most cultivars (95 %) exhibited stable A-genome nucleolar dominance while one cultivar (‘Norin 9’) showed co-dominance. Conclusions The B. napus cultivars differ in the degree and direction of rDNA homogenization. The prevalent direction of gene conversion (towards the A-genome) correlates with the direction of expression dominance indicating that gene activity may be needed for interlocus gene conversion. PMID:27707747

Electromagnetic and optical characteristics of Nb5+-doped double-crossover and salmon DNA thin films

NASA Astrophysics Data System (ADS)

Babu Mitta, Sekhar; Reddy Dugasani, Sreekantha; Jung, Soon-Gil; Vellampatti, Srivithya; Park, Tuson; Park, Sung Ha

2017-10-01

We report the fabrication and physical characteristics of niobium ion (Nb5+)-doped double-crossover DNA (DX-DNA) and salmon DNA (SDNA) thin films. Different concentrations of Nb5+ ([Nb5+]) are coordinated into the DNA molecules, and the thin films are fabricated via substrate-assisted growth (DX-DNA) and drop-casting (SDNA) on oxygen plasma treated substrates. We conducted atomic force microscopy to estimate the optimum concentration of Nb5+ ([Nb5+]O = 0.08 mM) in Nb5+-doped DX-DNA thin films, up to which the DX-DNA lattices maintain their structures without deformation. X-ray photoelectron spectroscopy (XPS) was performed to probe the chemical nature of the intercalated Nb5+ in the SDNA thin films. The change in peak intensities and the shift in binding energy were witnessed in XPS spectra to explicate the binding and charge transfer mechanisms between Nb5+ and SDNA molecules. UV-visible, Raman, and photoluminescence (PL) spectra were measured to determine the optical properties and thus investigate the binding modes, Nb5+ coordination sites in Nb5+-doped SDNA thin films, and energy transfer mechanisms, respectively. As [Nb5+] increases, the absorbance peak intensities monotonically increase until ˜[Nb5+]O and then decrease. However, from the Raman measurements, the peak intensities gradually decrease with an increase in [Nb5+] to reveal the binding mechanism and binding sites of metal ions in the SDNA molecules. From the PL, we observe the emission intensities to reduce them at up to ˜[Nb5+]O and then increase after that, expecting the energy transfer between the Nb5+ and SDNA molecules. The current-voltage measurement shows a significant increase in the current observed as [Nb5+] increases in the SDNA thin films when compared to that of pristine SDNA thin films. Finally, we investigate the temperature dependent magnetization in which the Nb5+-doped SDNA thin films reveal weak ferromagnetism due to the existence of tiny magnetic dipoles in the Nb5+-doped SDNA complex.

ZmXTH1, a new xyloglucan endotransglucosylase/hydrolase in maize, affects cell wall structure and composition in Arabidopsis thaliana.

PubMed

Genovesi, Valeria; Fornalé, Silvia; Fry, Stephen C; Ruel, Katia; Ferrer, Pau; Encina, Antonio; Sonbol, Fathi-Mohamed; Bosch, Josep; Puigdomènech, Pere; Rigau, Joan; Caparrós-Ruiz, David

2008-01-01

Xyloglucan endotransglucosylase/hydrolases (XTHs; EC 2.4.1.207 and/or EC 3.2.1.151) are enzymes involved in the modification of cell wall structure by cleaving and, often, also re-joining xyloglucan molecules in primary plant cell walls. Using a pool of antibodies raised against an enriched cell wall protein fraction, a new XTH cDNA in maize, ZmXTH1, has been isolated from a cDNA expression library obtained from the elongation zone of the maize root. The predicted protein has a putative N-terminal signal peptide and possesses the typical domains of this enzyme family, such as a catalytic domain that is homologous to that of Bacillus macerans beta-glucanase, a putative N-glycosylation motif, and four cysteine residues in the central and C terminal regions of the ZmXTH1 protein. Phylogenetic analysis of ZmXTH1 reveals that it belongs to subgroup 4, so far only reported from Poaceae monocot species. ZmXTH1 has been expressed in Pichia pastoris (a methylotrophic yeast) and the recombinant enzyme showed xyloglucan endotransglucosylase but not xyloglucan endohydrolase activity, representing the first enzyme belonging to subgroup 4 characterized in maize so far. Expression data indicate that ZmXTH1 is expressed in elongating tissues, modulated by culture conditions, and induced by gibberellins. Transient expression assays in onion cells reveal that ZmXTH1 is directed to the cell wall, although weakly bound. Finally, Arabidopsis thaliana plants expressing ZmXTH1 show slightly increased xyloglucan endohydrolase activity and alterations in the cell wall structure and composition.

Identification of Moraxella bovis by qualitative genetic transformation and nutritional assays.

PubMed Central

Juni, E; Heym, G A; Newcomb, R D

1988-01-01

Strains of Moraxella bovis were identified definitively through the combined use of a qualitative genetic transformation assay and determination of the ability of the organism under examination to grow in a defined medium (medium MB). Except for weak transformation by DNA from strains of M. lacunata, M. nonliquefaciens, and M. (Branhamella) ovis, DNA samples from all other members of the genus Moraxella failed to transform either of the two M. bovis auxotrophs used in this study. Images PMID:3389822

Recovery of Weak Factor Loadings When Adding the Mean Structure in Confirmatory Factor Analysis: A Simulation Study

PubMed Central

Ximénez, Carmen

2016-01-01

This article extends previous research on the recovery of weak factor loadings in confirmatory factor analysis (CFA) by exploring the effects of adding the mean structure. This issue has not been examined in previous research. This study is based on the framework of Yung and Bentler (1999) and aims to examine the conditions that affect the recovery of weak factor loadings when the model includes the mean structure, compared to analyzing the covariance structure alone. A simulation study was conducted in which several constraints were defined for one-, two-, and three-factor models. Results show that adding the mean structure improves the recovery of weak factor loadings and reduces the asymptotic variances for the factor loadings, particularly for the models with a smaller number of factors and a small sample size. Therefore, under certain circumstances, modeling the means should be seriously considered for covariance models containing weak factor loadings. PMID:26779071

Asymmetry at the molecular level in biology

NASA Astrophysics Data System (ADS)

Johnson, Louise N.

2005-10-01

Naturally occurring biological molecules are made of homochiral building blocks. Proteins are composed of L-amino acids (and not D-amino acids); nucleic acids such as DNA have D-ribose sugars (and not L-ribose sugars). It is not clear why nature selected a particular chirality. Selection could have occurred by chance or as a consequence of basic physical chemistry. Possible proposals, including the contribution of the parity violating the weak nuclear force, are discussed together with the mechanisms by which this very small contribution might be amplified. Homochirality of the amino acids has consequences for protein structure. Helices are right handed and beta sheets have a left-hand twist. When incorporated into the tertiary structure of a protein these chiralities limit the topologies of connections between helices and sheets. Polypeptides comprised of D-amino acids can be synthesized chemically and have been shown to adopt stable structures that are the mirror image of the naturally occurring L-amino acid polypeptides. Chirality is important in drug design. Three examples are discussed: penicillin; the CD4 antagonistic peptides; and thalidomide. The absolute hand of a biological structure can only be established by X-ray crystallographic methods using the technique of anomalous scattering.

The role of His-83 of yeast apurinic/apyrimidinic endonuclease Apn1 in catalytic incision of abasic sites in DNA.

PubMed

Dyakonova, Elena S; Koval, Vladimir V; Lomzov, Alexander A; Ishchenko, Alexander A; Fedorova, Olga S

2015-06-01

The apurinic/apyrimidinic (AP) endonuclease Apn1 from Saccharomyces cerevisiae is a key enzyme involved in the base excision repair (BER) at the cleavage stage of abasic sites (AP sites) in DNA. The crystal structure of Apn1 from S. cerevisiae is unresolved. Based on its high amino acid homology to Escherichia coli Endo IV, His-83 is believed to coordinate one of three Zn2+ ions in Apn1's active site similar to His-69 in Endo IV. Substituting His-83 with Ala is proposed to decrease the AP endonuclease activity of Apn1 owing to weak coordination of Zn2+ ions involved in enzymatic catalysis. The kinetics of recognition, binding, and incision of DNA substrates with the H83A Apn1 mutant was investigated. The stopped-flow method detecting fluorescence intensity changes of 2-aminopurine (2-aPu) was used to monitor the conformational dynamics of DNA at pre-steady-state conditions. We found substituting His-83 with Ala influenced catalytic complex formation and further incision of the damaged DNA strand. The H83A Apn1 catalysis depends not only on the location of the mismatch relative to the abasic site in DNA, but also on the nature of damage. We consider His-83 properly coordinates the active site Zn2+ ion playing a crucial role in catalytic incision stage. Our data prove suppressed enzymatic activity of H83A Apn1 results from the reduced number of active site Zn2+ ions. Our study provides insights into mechanistic specialty of AP site repair by yeast AP endonuclease Apn1 of Endo IV family, which members are not found in mammals, but are present in many microorganisms. The results will provide useful guidelines for design of new anti-fungal and anti-malarial agents. Copyright © 2015 Elsevier B.V. All rights reserved.

DHX9 helicase is involved in preventing genomic instability induced by alternatively structured DNA in human cells

PubMed Central

Jain, Aklank; Bacolla, Albino; del Mundo, Imee M.; Zhao, Junhua; Wang, Guliang; Vasquez, Karen M.

2013-01-01

Sequences that have the capacity to adopt alternative (i.e. non-B) DNA structures in the human genome have been implicated in stimulating genomic instability. Previously, we found that a naturally occurring intra-molecular triplex (H-DNA) caused genetic instability in mammals largely in the form of DNA double-strand breaks. Thus, it is of interest to determine the mechanism(s) involved in processing H-DNA. Recently, we demonstrated that human DHX9 helicase preferentially unwinds inter-molecular triplex DNA in vitro. Herein, we used a mutation-reporter system containing H-DNA to examine the relevance of DHX9 activity on naturally occurring H-DNA structures in human cells. We found that H-DNA significantly increased mutagenesis in small-interfering siRNA-treated, DHX9-depleted cells, affecting mostly deletions. Moreover, DHX9 associated with H-DNA in the context of supercoiled plasmids. To further investigate the role of DHX9 in the recognition/processing of H-DNA, we performed binding assays in vitro and chromatin immunoprecipitation assays in U2OS cells. DHX9 recognized H-DNA, as evidenced by its binding to the H-DNA structure and enrichment at the H-DNA region compared with a control region in human cells. These composite data implicate DHX9 in processing H-DNA structures in vivo and support its role in the overall maintenance of genomic stability at sites of alternatively structured DNA. PMID:24049074

DHX9 helicase is involved in preventing genomic instability induced by alternatively structured DNA in human cells.

PubMed

Jain, Aklank; Bacolla, Albino; Del Mundo, Imee M; Zhao, Junhua; Wang, Guliang; Vasquez, Karen M

2013-12-01

Sequences that have the capacity to adopt alternative (i.e. non-B) DNA structures in the human genome have been implicated in stimulating genomic instability. Previously, we found that a naturally occurring intra-molecular triplex (H-DNA) caused genetic instability in mammals largely in the form of DNA double-strand breaks. Thus, it is of interest to determine the mechanism(s) involved in processing H-DNA. Recently, we demonstrated that human DHX9 helicase preferentially unwinds inter-molecular triplex DNA in vitro. Herein, we used a mutation-reporter system containing H-DNA to examine the relevance of DHX9 activity on naturally occurring H-DNA structures in human cells. We found that H-DNA significantly increased mutagenesis in small-interfering siRNA-treated, DHX9-depleted cells, affecting mostly deletions. Moreover, DHX9 associated with H-DNA in the context of supercoiled plasmids. To further investigate the role of DHX9 in the recognition/processing of H-DNA, we performed binding assays in vitro and chromatin immunoprecipitation assays in U2OS cells. DHX9 recognized H-DNA, as evidenced by its binding to the H-DNA structure and enrichment at the H-DNA region compared with a control region in human cells. These composite data implicate DHX9 in processing H-DNA structures in vivo and support its role in the overall maintenance of genomic stability at sites of alternatively structured DNA.

Enzyme-adenylate structure of a bacterial ATP-dependent DNA ligase with a minimized DNA-binding surface.

PubMed

Williamson, Adele; Rothweiler, Ulli; Leiros, Hanna Kirsti Schrøder

2014-11-01

DNA ligases are a structurally diverse class of enzymes which share a common catalytic core and seal breaks in the phosphodiester backbone of double-stranded DNA via an adenylated intermediate. Here, the structure and activity of a recombinantly produced ATP-dependent DNA ligase from the bacterium Psychromonas sp. strain SP041 is described. This minimal-type ligase, like its close homologues, is able to ligate singly nicked double-stranded DNA with high efficiency and to join cohesive-ended and blunt-ended substrates to a more limited extent. The 1.65 Å resolution crystal structure of the enzyme-adenylate complex reveals no unstructured loops or segments, and suggests that this enzyme binds the DNA without requiring full encirclement of the DNA duplex. This is in contrast to previously characterized minimal DNA ligases from viruses, which use flexible loop regions for DNA interaction. The Psychromonas sp. enzyme is the first structure available for the minimal type of bacterial DNA ligases and is the smallest DNA ligase to be crystallized to date.

Method and apparatus for evaluating structural weakness in polymer matrix composites

DOEpatents

Wachter, E.A.; Fisher, W.G.

1996-01-09

A method and apparatus for evaluating structural weaknesses in polymer matrix composites is described. An object to be studied is illuminated with laser radiation and fluorescence emanating therefrom is collected and filtered. The fluorescence is then imaged and the image is studied to determine fluorescence intensity over the surface of the object being studied and the wavelength of maximum fluorescent intensity. Such images provide a map of the structural integrity of the part being studied and weaknesses, particularly weaknesses created by exposure of the object to heat, are readily visible in the image. 6 figs.

Method and apparatus for evaluating structural weakness in polymer matrix composites

DOEpatents

Wachter, Eric A.; Fisher, Walter G.

1996-01-01

A method and apparatus for evaluating structural weaknesses in polymer matrix composites is described. An object to be studied is illuminated with laser radiation and fluorescence emanating therefrom is collected and filtered. The fluorescence is then imaged and the image is studied to determine fluorescence intensity over the surface of the object being studied and the wavelength of maximum fluorescent intensity. Such images provide a map of the structural integrity of the part being studied and weaknesses, particularly weaknesses created by exposure of the object to heat, are readily visible in the image.

An overview of the structures of protein-DNA complexes

PubMed Central

Luscombe, Nicholas M; Austin, Susan E; Berman , Helen M; Thornton, Janet M

2000-01-01

On the basis of a structural analysis of 240 protein-DNA complexes contained in the Protein Data Bank (PDB), we have classified the DNA-binding proteins involved into eight different structural/functional groups, which are further classified into 54 structural families. Here we present this classification and review the functions, structures and binding interactions of these protein-DNA complexes. PMID:11104519

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

Structure of the adenylation domain of NAD[superscript +]-dependent DNA ligase from Staphylococcus aureus

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

Han, Seungil; Chang, Jeanne S.; Griffor, Matt

DNA ligase catalyzes phosphodiester-bond formation between immediately adjacent 5'-phosphate and 3''-hydroxyl groups in double-stranded DNA and plays a central role in many cellular and biochemical processes, including DNA replication, repair and recombination. Bacterial NAD{sup +}-dependent DNA ligases have been extensively characterized as potential antibacterial targets because of their essentiality and their structural distinction from human ATP-dependent DNA ligases. The high-resolution structure of the adenylation domain of Staphylococcus aureus NAD{sup +}-dependent DNA ligase establishes the conserved domain architecture with other bacterial adenylation domains. Two apo crystal structures revealed that the active site possesses the preformed NAD{sup +}-binding pocket and the 'C2more » tunnel' lined with hydrophobic residues: Leu80, Phe224, Leu287, Phe295 and Trp302. The C2 tunnel is unique to bacterial DNA ligases and the Leu80 side chain at the mouth of the tunnel points inside the tunnel and forms a narrow funnel in the S. aureus DNA ligase structure. Taken together with other DNA ligase structures, the S. aureus DNA ligase structure provides a basis for a more integrated understanding of substrate recognition and catalysis and will be also be of help in the development of small-molecule inhibitors.« less

MD simulations of papillomavirus DNA-E2 protein complexes hints at a protein structural code for DNA deformation.

PubMed

Falconi, M; Oteri, F; Eliseo, T; Cicero, D O; Desideri, A

2008-08-01

The structural dynamics of the DNA binding domains of the human papillomavirus strain 16 and the bovine papillomavirus strain 1, complexed with their DNA targets, has been investigated by modeling, molecular dynamics simulations, and nuclear magnetic resonance analysis. The simulations underline different dynamical features of the protein scaffolds and a different mechanical interaction of the two proteins with DNA. The two protein structures, although very similar, show differences in the relative mobility of secondary structure elements. Protein structural analyses, principal component analysis, and geometrical and energetic DNA analyses indicate that the two transcription factors utilize a different strategy in DNA recognition and deformation. Results show that the protein indirect DNA readout is not only addressable to the DNA molecule flexibility but it is finely tuned by the mechanical and dynamical properties of the protein scaffold involved in the interaction.

Barcode extension for analysis and reconstruction of structures

NASA Astrophysics Data System (ADS)

Myhrvold, Cameron; Baym, Michael; Hanikel, Nikita; Ong, Luvena L.; Gootenberg, Jonathan S.; Yin, Peng

2017-03-01

Collections of DNA sequences can be rationally designed to self-assemble into predictable three-dimensional structures. The geometric and functional diversity of DNA nanostructures created to date has been enhanced by improvements in DNA synthesis and computational design. However, existing methods for structure characterization typically image the final product or laboriously determine the presence of individual, labelled strands using gel electrophoresis. Here we introduce a new method of structure characterization that uses barcode extension and next-generation DNA sequencing to quantitatively measure the incorporation of every strand into a DNA nanostructure. By quantifying the relative abundances of distinct DNA species in product and monomer bands, we can study the influence of geometry and sequence on assembly. We have tested our method using 2D and 3D DNA brick and DNA origami structures. Our method is general and should be extensible to a wide variety of DNA nanostructures.

Barcode extension for analysis and reconstruction of structures.

PubMed

Myhrvold, Cameron; Baym, Michael; Hanikel, Nikita; Ong, Luvena L; Gootenberg, Jonathan S; Yin, Peng

2017-03-13

Collections of DNA sequences can be rationally designed to self-assemble into predictable three-dimensional structures. The geometric and functional diversity of DNA nanostructures created to date has been enhanced by improvements in DNA synthesis and computational design. However, existing methods for structure characterization typically image the final product or laboriously determine the presence of individual, labelled strands using gel electrophoresis. Here we introduce a new method of structure characterization that uses barcode extension and next-generation DNA sequencing to quantitatively measure the incorporation of every strand into a DNA nanostructure. By quantifying the relative abundances of distinct DNA species in product and monomer bands, we can study the influence of geometry and sequence on assembly. We have tested our method using 2D and 3D DNA brick and DNA origami structures. Our method is general and should be extensible to a wide variety of DNA nanostructures.

Barcode extension for analysis and reconstruction of structures

PubMed Central

Myhrvold, Cameron; Baym, Michael; Hanikel, Nikita; Ong, Luvena L; Gootenberg, Jonathan S; Yin, Peng

2017-01-01

Collections of DNA sequences can be rationally designed to self-assemble into predictable three-dimensional structures. The geometric and functional diversity of DNA nanostructures created to date has been enhanced by improvements in DNA synthesis and computational design. However, existing methods for structure characterization typically image the final product or laboriously determine the presence of individual, labelled strands using gel electrophoresis. Here we introduce a new method of structure characterization that uses barcode extension and next-generation DNA sequencing to quantitatively measure the incorporation of every strand into a DNA nanostructure. By quantifying the relative abundances of distinct DNA species in product and monomer bands, we can study the influence of geometry and sequence on assembly. We have tested our method using 2D and 3D DNA brick and DNA origami structures. Our method is general and should be extensible to a wide variety of DNA nanostructures. PMID:28287117

[DNA structure from A to Z--biological implications of structural diversity of DNA].

PubMed

Bukowiecka-Matusiak, Małgorzata; Woźniak, Lucyna A

2006-01-01

Deoxyribonucleic acid (DNA) is a biopolymer of nucleotides, usually adopting a double-stranded helical form in cells, with complementary base pairing holding the two strands together. The most stable is B-DNA conformation, although numerous other double helical structures can occur under specific conditions (A-DNA, Z-DNA, P-DNA). The existence of multiple-stranded (triplex, tetraplex) forms in vivo and their biological function in cells are subject of intensive studies.

Reducing Electroosmotic Flow Enables DNA Separations in Ultrathin Channels.

DTIC Science & Technology

1998-08-01

Chemical structure of DNA bases 2 Figure 1-2: Schematic diagram of DNA base pairing 5 Figure 1-3: Schematic diagram of the capillary and the...hydrogen atoms near one of the Figure 1-1: A. Chemical structure of the DNA backbone. B. Chemical structure of DNA bases . The DNA backbone consists...of pentose sugar (deoxyribose) held together by phosphodiester bonds. The DNA bases that are derivatives of purine are adenine (A) and guanine (G

Transcription blockage by stable H-DNA analogs in vitro.

PubMed

Pandey, Shristi; Ogloblina, Anna M; Belotserkovskii, Boris P; Dolinnaya, Nina G; Yakubovskaya, Marianna G; Mirkin, Sergei M; Hanawalt, Philip C

2015-08-18

DNA sequences that can form unusual secondary structures are implicated in regulating gene expression and causing genomic instability. H-palindromes are an important class of such DNA sequences that can form an intramolecular triplex structure, H-DNA. Within an H-palindrome, the H-DNA and canonical B-DNA are in a dynamic equilibrium that shifts toward H-DNA with increased negative supercoiling. The interplay between H- and B-DNA and the fact that the process of transcription affects supercoiling makes it difficult to elucidate the effects of H-DNA upon transcription. We constructed a stable structural analog of H-DNA that cannot flip into B-DNA, and studied the effects of this structure on transcription by T7 RNA polymerase in vitro. We found multiple transcription blockage sites adjacent to and within sequences engaged in this triplex structure. Triplex-mediated transcription blockage varied significantly with changes in ambient conditions: it was exacerbated in the presence of Mn(2+) or by increased concentrations of K(+) and Li(+). Analysis of the detailed pattern of the blockage suggests that RNA polymerase is sterically hindered by H-DNA and has difficulties in unwinding triplex DNA. The implications of these findings for the biological roles of triple-stranded DNA structures are discussed. © The Author(s) 2015. Published by Oxford University Press on behalf of Nucleic Acids Research.

Assembly and structural analysis of a covalently closed nano-scale DNA cage

PubMed Central

Andersen, Felicie F.; Knudsen, Bjarne; Oliveira, Cristiano Luis Pinto; Frøhlich, Rikke F.; Krüger, Dinna; Bungert, Jörg; Agbandje-McKenna, Mavis; McKenna, Robert; Juul, Sissel; Veigaard, Christopher; Koch, Jørn; Rubinstein, John L.; Guldbrandtsen, Bernt; Hede, Marianne S.; Karlsson, Göran; Andersen, Anni H.; Pedersen, Jan Skov; Knudsen, Birgitta R.

2008-01-01

The inherent properties of DNA as a stable polymer with unique affinity for partner molecules determined by the specific Watson–Crick base pairing makes it an ideal component in self-assembling structures. This has been exploited for decades in the design of a variety of artificial substrates for investigations of DNA-interacting enzymes. More recently, strategies for synthesis of more complex two-dimensional (2D) and 3D DNA structures have emerged. However, the building of such structures is still in progress and more experiences from different research groups and different fields of expertise are necessary before complex DNA structures can be routinely designed for the use in basal science and/or biotechnology. Here we present the design, construction and structural analysis of a covalently closed and stable 3D DNA structure with the connectivity of an octahedron, as defined by the double-stranded DNA helices that assembles from eight oligonucleotides with a yield of ∼30%. As demonstrated by Small Angle X-ray Scattering and cryo-Transmission Electron Microscopy analyses the eight-stranded DNA structure has a central cavity larger than the apertures in the surrounding DNA lattice and can be described as a nano-scale DNA cage, Hence, in theory it could hold proteins or other bio-molecules to enable their investigation in certain harmful environments or even allow their organization into higher order structures. PMID:18096620

Lactose-modified DNA tile nanostructures as drug carriers.

PubMed

Akkus Sut, Pinar; Tunc, Cansu Umran; Culha, Mustafa

2016-09-01

DNA hybridization allows the preparation of nanoscale DNA structures with desired shape and size. DNA structures using simple base pairing can be used for the delivery of drug molecules into the cells. Since DNA carries multiple negative charges, their cellular uptake efficiency is low. Thus, the modification of the DNA structures with molecules that may enhance the cellular internalization may be an option. The objective of this study is to construct DNA-based nanocarrier system and to investigate the cellular uptake of DNA tile with/without lactose modification. Doxorubicin was intercalated to DNA tile and cellular uptake of drug-loaded DNA-based carrier with/without lactose modification was investigated in vitro. HeLa, BT-474, and MDA-MB-231 cancer cells were used for cellular uptake studies and cytotoxicity assays. Using fluorescence spectroscopy, flow cytometry, and confocal microscopy, cellular uptake behavior of DNA tile was investigated. The cytotoxicity of DNA tile structures was determined with WST-1 assay. The results show that modification with lactose effectively increases the intracellular uptake of doxorubicin loaded DNA tile structure by cancer cells compared with the unmodified DNA tile. The findings of this study suggest that DNA-based nanostructures modified with carbohydrates can be used as suitable multifunctional nanocarriers with simple chemical modifications.

A New Protein Architecture for Processing Alkylation Damaged DNA: The Crystal Structure of DNA Glycosylase AlkD

PubMed Central

Rubinson, Emily H.; Metz, Audrey H.; O'Quin, Jami; Eichman, Brandt F.

2013-01-01

Summary DNA glycosylases safeguard the genome by locating and excising chemically modified bases from DNA. AlkD is a recently discovered bacterial DNA glycosylase that removes positively charged methylpurines from DNA, and was predicted to adopt a protein fold distinct from other DNA repair proteins. The crystal structure of Bacillus cereus AlkD presented here shows that the protein is composed exclusively of helical HEAT-like repeats, which form a solenoid perfectly shaped to accommodate a DNA duplex on the concave surface. Structural analysis of the variant HEAT repeats in AlkD provides a rationale for how this protein scaffolding motif has been modified to bind DNA. We report 7mG excision and DNA binding activities of AlkD mutants, along with a comparison of alkylpurine DNA glycosylase structures. Together, these data provide important insight into the requirements for alkylation repair within DNA and suggest that AlkD utilizes a novel strategy to manipulate DNA in its search for alkylpurine bases. PMID:18585735

The contribution of co-transcriptional RNA:DNA hybrid structures to DNA damage and genome instability

PubMed Central

Hamperl, Stephan; Cimprich, Karlene A.

2014-01-01

Accurate DNA replication and DNA repair are crucial for the maintenance of genome stability, and it is generally accepted that failure of these processes is a major source of DNA damage in cells. Intriguingly, recent evidence suggests that DNA damage is more likely to occur at genomic loci with high transcriptional activity. Furthermore, loss of certain RNA processing factors in eukaryotic cells is associated with increased formation of co-transcriptional RNA:DNA hybrid structures known as R-loops, resulting in double-strand breaks (DSBs) and DNA damage. However, the molecular mechanisms by which R-loop structures ultimately lead to DNA breaks and genome instability is not well understood. In this review, we summarize the current knowledge about the formation, recognition and processing of RNA:DNA hybrids, and discuss possible mechanisms by which these structures contribute to DNA damage and genome instability in the cell. PMID:24746923

Supercoil Formation During DNA Melting

NASA Astrophysics Data System (ADS)

Sayar, Mehmet; Avsaroglu, Baris; Kabakcioglu, Alkan

2009-03-01

Supercoil formation plays a key role in determining the structure-function relationship in DNA. Biological and technological processes, such as protein synthesis, polymerase chain reaction, and microarrays relys on separation of the two strands in DNA, which is coupled to the unwinding of the supercoiled structure. This problem has been studied theoretically via Peyrard-Bishop and Poland-Scheraga type models, which include a simple representation of the DNA structural properties. In recent years, computational models, which provide a more realtistic representaion of DNA molecule, have been used to study the melting behavior of short DNA chains. Here, we will present a new coarse-grained model of DNA which is capable of simulating sufficiently long DNA chains for studying the supercoil formation during melting, without sacrificing the local structural properties. Our coarse-grained model successfully reproduces the local geometry of the DNA molecule, such as the 3'-5' directionality, major-minor groove structure, and the helical pitch. We will present our initial results on the dynamics of supercoiling during DNA melting.

Method for sequencing DNA base pairs

DOEpatents

Sessler, Andrew M.; Dawson, John

1993-01-01

The base pairs of a DNA structure are sequenced with the use of a scanning tunneling microscope (STM). The DNA structure is scanned by the STM probe tip, and, as it is being scanned, the DNA structure is separately subjected to a sequence of infrared radiation from four different sources, each source being selected to preferentially excite one of the four different bases in the DNA structure. Each particular base being scanned is subjected to such sequence of infrared radiation from the four different sources as that particular base is being scanned. The DNA structure as a whole is separately imaged for each subjection thereof to radiation from one only of each source.

Mechanism for circularization of linear DNAs: circular parvovirus MVM DNA is formed by a "noose" sliding in a "lasso"-like DNA structure.

PubMed

Bratosin, S; Laub, O; Tal, J; Aloni, Y

1979-09-01

During an electron-microscopic survey with the aim of identifying the parvovirus MVM transcription template, we observed previously unidentified structures of MVM DNA in lysates of virus-infected cells. These included double-stranded "lasso"-like structures and relaxed circles. Both structures were of unit length MVM DNA, indicating that they were not intermediates formed during replication; they each represented about 5% of the total nuclear MVM DNA. The proportion of these structures was unchanged after digestion with sodium dodecyl sulfate/Pronase and RNase and after mild denaturation treatment. Cleavage of the "lasso" structures with EcoRI restriction endonuclease indicated that the "noose" part of the "lasso" structure is located on the 5' side of the genomic single-stranded MVM DNA. A model is presented for the molecular nature of the circularization process of MVM DNA in which the "lasso" structures are identified as intermediates during circle formation. This model proposes a mechanism for circularization of linear DNAs.

3DNALandscapes: a database for exploring the conformational features of DNA.

PubMed

Zheng, Guohui; Colasanti, Andrew V; Lu, Xiang-Jun; Olson, Wilma K

2010-01-01

3DNALandscapes, located at: http://3DNAscapes.rutgers.edu, is a new database for exploring the conformational features of DNA. In contrast to most structural databases, which archive the Cartesian coordinates and/or derived parameters and images for individual structures, 3DNALandscapes enables searches of conformational information across multiple structures. The database contains a wide variety of structural parameters and molecular images, computed with the 3DNA software package and known to be useful for characterizing and understanding the sequence-dependent spatial arrangements of the DNA sugar-phosphate backbone, sugar-base side groups, base pairs, base-pair steps, groove structure, etc. The data comprise all DNA-containing structures--both free and bound to proteins, drugs and other ligands--currently available in the Protein Data Bank. The web interface allows the user to link, report, plot and analyze this information from numerous perspectives and thereby gain insight into DNA conformation, deformability and interactions in different sequence and structural contexts. The data accumulated from known, well-resolved DNA structures can serve as useful benchmarks for the analysis and simulation of new structures. The collective data can also help to understand how DNA deforms in response to proteins and other molecules and undergoes conformational rearrangements.

Strong FANCA/FANCG but weak FANCA/FANCC interaction in the yeast 2-hybrid system.

PubMed

Reuter, T; Herterich, S; Bernhard, O; Hoehn, H; Gross, H J

2000-01-15

Three of at least 8 Fanconi anemia (FA) genes have been cloned (FANCA, FANCC, FANCG), but their functions remain unknown. Using the yeast 2-hybrid system and full-length cDNA, the authors found a strong interaction between FANCA and FANCG proteins. They also obtained evidence for a weak interaction between FANCA and FANCC. Neither FANCA nor FANCC was found to interact with itself. These results support the notion of a functional association between the FA gene products. (Blood. 2000;95:719-720)

DNA secondary structures: stability and function of G-quadruplex structures

PubMed Central

Bochman, Matthew L.; Paeschke, Katrin; Zakian, Virginia A.

2013-01-01

In addition to the canonical double helix, DNA can fold into various other inter- and intramolecular secondary structures. Although many such structures were long thought to be in vitro artefacts, bioinformatics demonstrates that DNA sequences capable of forming these structures are conserved throughout evolution, suggesting the existence of non-B-form DNA in vivo. In addition, genes whose products promote formation or resolution of these structures are found in diverse organisms, and a growing body of work suggests that the resolution of DNA secondary structures is critical for genome integrity. This Review focuses on emerging evidence relating to the characteristics of G-quadruplex structures and the possible influence of such structures on genomic stability and cellular processes, such as transcription. PMID:23032257

A Natural Polymorphism in rDNA Replication Origins Links Origin Activation with Calorie Restriction and Lifespan

PubMed Central

Kwan, Elizabeth X.; Foss, Eric J.; Tsuchiyama, Scott; Alvino, Gina M.; Kruglyak, Leonid; Kaeberlein, Matt; Raghuraman, M. K.; Brewer, Bonita J.; Kennedy, Brian K.; Bedalov, Antonio

2013-01-01

Aging and longevity are complex traits influenced by genetic and environmental factors. To identify quantitative trait loci (QTLs) that control replicative lifespan, we employed an outbred Saccharomyces cerevisiae model, generated by crossing a vineyard and a laboratory strain. The predominant QTL mapped to the rDNA, with the vineyard rDNA conferring a lifespan increase of 41%. The lifespan extension was independent of Sir2 and Fob1, but depended on a polymorphism in the rDNA origin of replication from the vineyard strain that reduced origin activation relative to the laboratory origin. Strains carrying vineyard rDNA origins have increased capacity for replication initiation at weak plasmid and genomic origins, suggesting that inability to complete genome replication presents a major impediment to replicative lifespan. Calorie restriction, a conserved mediator of lifespan extension that is also independent of Sir2 and Fob1, reduces rDNA origin firing in both laboratory and vineyard rDNA. Our results are consistent with the possibility that calorie restriction, similarly to the vineyard rDNA polymorphism, modulates replicative lifespan through control of rDNA origin activation, which in turn affects genome replication dynamics. PMID:23505383

A natural polymorphism in rDNA replication origins links origin activation with calorie restriction and lifespan.

PubMed

Kwan, Elizabeth X; Foss, Eric J; Tsuchiyama, Scott; Alvino, Gina M; Kruglyak, Leonid; Kaeberlein, Matt; Raghuraman, M K; Brewer, Bonita J; Kennedy, Brian K; Bedalov, Antonio

2013-01-01

Aging and longevity are complex traits influenced by genetic and environmental factors. To identify quantitative trait loci (QTLs) that control replicative lifespan, we employed an outbred Saccharomyces cerevisiae model, generated by crossing a vineyard and a laboratory strain. The predominant QTL mapped to the rDNA, with the vineyard rDNA conferring a lifespan increase of 41%. The lifespan extension was independent of Sir2 and Fob1, but depended on a polymorphism in the rDNA origin of replication from the vineyard strain that reduced origin activation relative to the laboratory origin. Strains carrying vineyard rDNA origins have increased capacity for replication initiation at weak plasmid and genomic origins, suggesting that inability to complete genome replication presents a major impediment to replicative lifespan. Calorie restriction, a conserved mediator of lifespan extension that is also independent of Sir2 and Fob1, reduces rDNA origin firing in both laboratory and vineyard rDNA. Our results are consistent with the possibility that calorie restriction, similarly to the vineyard rDNA polymorphism, modulates replicative lifespan through control of rDNA origin activation, which in turn affects genome replication dynamics.

An affinity-structure database of helix-turn-helix: DNA complexes with a universal coordinate system

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

AlQuraishi, Mohammed; Tang, Shengdong; Xia, Xide

Molecular interactions between proteins and DNA molecules underlie many cellular processes, including transcriptional regulation, chromosome replication, and nucleosome positioning. Computational analyses of protein-DNA interactions rely on experimental data characterizing known protein-DNA interactions structurally and biochemically. While many databases exist that contain either structural or biochemical data, few integrate these two data sources in a unified fashion. Such integration is becoming increasingly critical with the rapid growth of structural and biochemical data, and the emergence of algorithms that rely on the synthesis of multiple data types to derive computational models of molecular interactions. We have developed an integrated affinity-structure database inmore » which the experimental and quantitative DNA binding affinities of helix-turn-helix proteins are mapped onto the crystal structures of the corresponding protein-DNA complexes. This database provides access to: (i) protein-DNA structures, (ii) quantitative summaries of protein-DNA binding affinities using position weight matrices, and (iii) raw experimental data of protein-DNA binding instances. Critically, this database establishes a correspondence between experimental structural data and quantitative binding affinity data at the single basepair level. Furthermore, we present a novel alignment algorithm that structurally aligns the protein-DNA complexes in the database and creates a unified residue-level coordinate system for comparing the physico-chemical environments at the interface between complexes. Using this unified coordinate system, we compute the statistics of atomic interactions at the protein-DNA interface of helix-turn-helix proteins. We provide an interactive website for visualization, querying, and analyzing this database, and a downloadable version to facilitate programmatic analysis. Lastly, this database will facilitate the analysis of protein-DNA interactions and the development of programmatic computational methods that capitalize on integration of structural and biochemical datasets. The database can be accessed at http://ProteinDNA.hms.harvard.edu.« less

An affinity-structure database of helix-turn-helix: DNA complexes with a universal coordinate system

DOE PAGES

AlQuraishi, Mohammed; Tang, Shengdong; Xia, Xide

2015-11-19

Molecular interactions between proteins and DNA molecules underlie many cellular processes, including transcriptional regulation, chromosome replication, and nucleosome positioning. Computational analyses of protein-DNA interactions rely on experimental data characterizing known protein-DNA interactions structurally and biochemically. While many databases exist that contain either structural or biochemical data, few integrate these two data sources in a unified fashion. Such integration is becoming increasingly critical with the rapid growth of structural and biochemical data, and the emergence of algorithms that rely on the synthesis of multiple data types to derive computational models of molecular interactions. We have developed an integrated affinity-structure database inmore » which the experimental and quantitative DNA binding affinities of helix-turn-helix proteins are mapped onto the crystal structures of the corresponding protein-DNA complexes. This database provides access to: (i) protein-DNA structures, (ii) quantitative summaries of protein-DNA binding affinities using position weight matrices, and (iii) raw experimental data of protein-DNA binding instances. Critically, this database establishes a correspondence between experimental structural data and quantitative binding affinity data at the single basepair level. Furthermore, we present a novel alignment algorithm that structurally aligns the protein-DNA complexes in the database and creates a unified residue-level coordinate system for comparing the physico-chemical environments at the interface between complexes. Using this unified coordinate system, we compute the statistics of atomic interactions at the protein-DNA interface of helix-turn-helix proteins. We provide an interactive website for visualization, querying, and analyzing this database, and a downloadable version to facilitate programmatic analysis. Lastly, this database will facilitate the analysis of protein-DNA interactions and the development of programmatic computational methods that capitalize on integration of structural and biochemical datasets. The database can be accessed at http://ProteinDNA.hms.harvard.edu.« less

A DNA Origami Mechanical Device for the Regulation of Microcosmic Structural Rigidity.

PubMed

Wan, Neng; Hong, Zhouping; Wang, Huading; Fu, Xin; Zhang, Ziyue; Li, Chao; Xia, Han; Fang, Yan; Li, Maoteng; Zhan, Yi; Yang, Xiangliang

2017-11-01

DNA origami makes it feasible to fabricate a tremendous number of DNA nanostructures with various geometries, dimensions, and functionalities. Moreover, an increasing amount of research on DNA nanostructures is focused on biological and biomedical applications. Here, the reversible regulation of microcosmic structural rigidity is accomplished using a DNA origami device in vitro. The designed DNA origami monomer is composed of an internal central axis and an external sliding tube. Due to the external tube sliding, the device transforms between flexible and rigid states. By transporting the device into the liposome, the conformational change of the origami device induces a structural change in the liposome. The results obtained demonstrate that the programmed DNA origami device can be applied to regulate the microcosmic structural rigidity of liposomes. Because microcosmic structural rigidity is important to cell proliferation and function, the results obtained potentially provide a foundation for the regulation of cell microcosmic structural rigidity using DNA nanostructures. © 2017 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

Structure of the EndoMS-DNA Complex as Mismatch Restriction Endonuclease.

PubMed

Nakae, Setsu; Hijikata, Atsushi; Tsuji, Toshiyuki; Yonezawa, Kouki; Kouyama, Ken-Ichi; Mayanagi, Kouta; Ishino, Sonoko; Ishino, Yoshizumi; Shirai, Tsuyoshi

2016-11-01

Archaeal NucS nuclease was thought to degrade the single-stranded region of branched DNA, which contains flapped and splayed DNA. However, recent findings indicated that EndoMS, the orthologous enzyme of NucS, specifically cleaves double-stranded DNA (dsDNA) containing mismatched bases. In this study, we determined the structure of the EndoMS-DNA complex. The complex structure of the EndoMS dimer with dsDNA unexpectedly revealed that the mismatched bases were flipped out into binding sites, and the overall architecture most resembled that of restriction enzymes. The structure of the apo form was similar to the reported structure of Pyrococcus abyssi NucS, indicating that movement of the C-terminal domain from the resting state was required for activity. In addition, a model of the EndoMS-PCNA-DNA complex was preliminarily verified with electron microscopy. The structures strongly support the idea that EndoMS acts in a mismatch repair pathway. Copyright © 2016 Elsevier Ltd. All rights reserved.

The impact of sperm DNA damage in assisted conception and beyond: recent advances in diagnosis and treatment.

PubMed

Lewis, Sheena E M; John Aitken, R; Conner, Sarah J; Iuliis, Geoffry De; Evenson, Donald P; Henkel, Ralph; Giwercman, Aleksander; Gharagozloo, Parviz

2013-10-01

Sperm DNA damage is a useful biomarker for male infertility diagnosis and prediction of assisted reproduction outcomes. It is associated with reduced fertilization rates, embryo quality and pregnancy rates, and higher rates of spontaneous miscarriage and childhood diseases. This review provides a synopsis of the most recent studies from each of the authors, all of whom have major track records in the field of sperm DNA damage in the clinical setting. It explores current laboratory tests and the accumulating body of knowledge concerning the relationship between sperm DNA damage and clinical outcomes. The paper proceeds to discuss the strengths, weaknesses and clinical applicability of current sperm DNA tests. Next, the biological significance of DNA damage in the male germ line is considered. Finally, as sperm DNA damage is often the result of oxidative stress in the male reproductive tract, the potential contribution of antioxidant therapy in the clinical management of this condition is discussed. DNA damage in human spermatozoa is an important attribute of semen quality. It should be part of the clinical work up and properly controlled trials addressing the effectiveness of antioxidant therapy should be undertaken as a matter of urgency. Sperm DNA damage is a useful biomarker for male infertility diagnosis and prediction of assisted reproduction outcomes. It is associated with reduced fertilization rates, embryo quality and pregnancy rates, and higher rates of spontaneous miscarriage and childhood diseases. With all of these fertility check points, it shows more promise than conventional semen parameters from a diagnostic perspective. Despite this, few infertility clinics use it routinely. This review provides a synopsis of the most recent studies from each of the authors, all of whom have major track records in the field of sperm DNA damage in the clinical setting. It explores current laboratory tests and the accumulating body of knowledge concerning the relationship between sperm DNA damage and clinical outcomes. The paper proceeds to discuss the strengths and weaknesses and clinical applicability of current sperm DNA fragmentation tests. Next, the biological significance of DNA damage in the male germ line is considered. Finally, as sperm DNA damage is often the result of increased oxidative stress in the male reproductive tract, the potential contribution of antioxidant therapy in the clinical management of this condition is discussed. As those working in this field of clinical research, we conclude that DNA damage in human spermatozoa is an important attribute of semen quality which should be carefully assessed in the clinical work up of infertile couples and that properly controlled trials addressing the effectiveness of antioxidant therapy should be undertaken as a matter of urgency. Copyright © 2013 Reproductive Healthcare Ltd. Published by Elsevier Ltd. All rights reserved.

Association of health symptoms with low-level exposure to organophosphates, DNA damage, AChE activity, and occupational knowledge and practice among rice, corn, and double-crop farmers.

PubMed

Hongsibsong, Surat; Sittitoon, Nalin; Sapbamrer, Ratana

2017-03-28

This study aims to determine (1) total dialkylphosphate (ΣDAP) levels, occupational knowledge and practice, DNA damage, AChE activity, and health symptoms in rice, corn, and double-crop farmers; (2) the association of health symptoms with ΣDAP levels, occupational knowledge and practice, DNA damage, and AChE activity in farmers; and (3) the prevalence of health symptoms between farmers and non-farmers. A cross-sectional study was conducted by interviewing as well as analyzing urine and blood samples during July to August 2014. There were no differences in ΣDAP levels, AChE activity, and occupational knowledge and practice scores among all farmer groups. In terms of health symptoms related to ΣDAP, AChE activity, DNA damage, and occupational knowledge and practice, pesticide-related symptoms were determined, including breathlessness, chest pain, dry throat, numbness, muscle weakness, cramp, headache, dizziness, eye irritation, white/red rash, and white/red pimple, which were classified as respiratory, muscle, nervous, and epithelial symptoms. A remarkable finding was that farmers had a significantly higher prevalence of muscle weakness (odds ratio (OR)=3.79) and numbness (OR=3.45) as compared with non-farmers. Our findings, therefore, suggest that a long-term low-level exposure to organophosphates (OPs) may be associated with an increasing prevalence of muscle symptoms. However, a further cohort study incorporating sensitive health outcomes and measurement of multiple pesticides monitoring on a larger scale is warranted.

ExpandplusCrystal Structures of Poly(ADP-ribose) Polymerase-1 (PARP-1) Zinc Fingers Bound to DNA

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

M Langelier; J Planck; S Roy

2011-12-31

Poly(ADP-ribose) polymerase-1 (PARP-1) has two homologous zinc finger domains, Zn1 and Zn2, that bind to a variety of DNA structures to stimulate poly(ADP-ribose) synthesis activity and to mediate PARP-1 interaction with chromatin. The structural basis for interaction with DNA is unknown, which limits our understanding of PARP-1 regulation and involvement in DNA repair and transcription. Here, we have determined crystal structures for the individual Zn1 and Zn2 domains in complex with a DNA double strand break, providing the first views of PARP-1 zinc fingers bound to DNA. The Zn1-DNA and Zn2-DNA structures establish a novel, bipartite mode of sequence-independent DNAmore » interaction that engages a continuous region of the phosphodiester backbone and the hydrophobic faces of exposed nucleotide bases. Biochemical and cell biological analysis indicate that the Zn1 and Zn2 domains perform distinct functions. The Zn2 domain exhibits high binding affinity to DNA compared with the Zn1 domain. However, the Zn1 domain is essential for DNA-dependent PARP-1 activity in vitro and in vivo, whereas the Zn2 domain is not strictly required. Structural differences between the Zn1-DNA and Zn2-DNA complexes, combined with mutational and structural analysis, indicate that a specialized region of the Zn1 domain is re-configured through the hydrophobic interaction with exposed nucleotide bases to initiate PARP-1 activation.« less

Fructosylation induced structural changes in mammalian DNA examined by biophysical techniques

NASA Astrophysics Data System (ADS)

Zaman, Asif; Arif, Zarina; Alam, Khursheed

2017-03-01

Glycosylation of DNA, proteins, lipids, etc. by reducing sugars, can lead to the formation of advanced glycation end products (AGEs). These products may accumulate and involve in the pathogenesis of a number of diseases, contributing to tissue injury via several mechanisms. In this study, fructosylation of calf thymus dsDNA was carried out with varying concentrations of fructose. The neo-structure of fructosylated-DNA was studied by various biophysical techniques and morphological characterization. Fructosylated-DNA showed hyperchromicity, increase in fluorescence intensity and decrease in melting temperature. The CD signal of modified-DNA shifted in the direction of higher wavelength indicative of structural changes in DNA. FTIR results indicated shift in specific band positions in fructosylated-DNA. Morphological characterization of fructosylated-DNA exhibited strand breakage and aggregation. The results suggest that the structure and conformation of DNA may be altered under high concentrations of fructose.

Engineering the Structure and Properties of DNA-Nanoparticle Superstructures Using Polyvalent Counterions.

PubMed

Chou, Leo Y T; Song, Fayi; Chan, Warren C W

2016-04-06

DNA assembly of nanoparticles is a powerful approach to control their properties and prototype new materials. However, the structure and properties of DNA-assembled nanoparticles are labile and sensitive to interactions with counterions, which vary with processing and application environment. Here we show that substituting polyamines in place of elemental counterions significantly enhanced the structural rigidity and plasmonic properties of DNA-assembled metal nanoparticles. These effects arose from the ability of polyamines to condense DNA and cross-link DNA-coated nanoparticles. We further used polyamine wrapped DNA nanostructures as structural templates to seed the growth of polymer multilayers via layer-by-layer assembly, and controlled the degree of DNA condensation, plasmon coupling efficiency, and material responsiveness to environmental stimuli by varying polyelectrolyte composition. These results highlight counterion engineering as a versatile strategy to tailor the properties of DNA-nanoparticle assemblies for various applications, and should be applicable to other classes of DNA nanostructures.

Active substrates improving sensitivity in biomedical fluorescence microscopy

NASA Astrophysics Data System (ADS)

Le Moal, E.; Leveque-Fort, S.; Fort, E.; Lacharme, J.-P.; Fontaine-Aupart, M.-P.; Ricolleau, C.

2005-08-01

Fluorescence is widely used as a spectroscopic tool or for biomedical imaging, in particular for DNA chips. In some cases, detection of very low molecular concentrations and precise localization of biomarkers are limited by the weakness of the fluorescence signal. We present a new method based on sample substrates that improve fluorescence detection sensitivity. These active substrates consist in glass slides covered with metal (gold or silver) and dielectric (alumina) films and can directly be used with common microscope set-up. Fluorescence enhancement affects both excitation and decay rates and is strongly dependant on the distance to the metal surface. Furthermore, fluorescence collection is improved since fluorophore emission lobes are advantageously modified close to a reflective surface. Finally, additional improvements are achieved by structuring the metallic layer. Substrates morphology was mapped by Atomic Force Microscopy (AFM). Substrates optical properties were studied using mono- and bi-photonic fluorescence microscopy with time resolution. An original set-up was implemented for spatial radiation pattern's measurement. Detection improvement was then tested on commercial devices. Several biomedical applications are presented. Enhancement by two orders of magnitude are achieved for DNA chips and signal-to-noise ratio is greatly increased for cells imaging.

Molecular cloning of rock bream (Oplegnathus fasciatus) tumor necrosis factor-alpha and its effect on the respiratory burst activity of phagocytes.

PubMed

Kim, Min Sun; Hwang, Yoon Jung; Yoon, Ki Joon; Zenke, Kosuke; Nam, Yoon Kwon; Kim, Sung Koo; Kim, Ki Hong

2009-11-01

Rock bream (Oplegnathus fasciatus) tumor necrosis factor-alpha (rbTNF-alpha) gene was cloned, recombinantly produced, and the effect of the recombinant rbTNF-alpha on the respiratory burst activity of rock bream phagocytes was analyzed. Structurally, genomic DNA of rbTNF-alpha was comprised with four exons and three introns, and deduced amino acid sequence of its cDNA possessed the TNF family signature, a transmembrane domain, a protease cleavage site, and two cysteine residues, which are the typical characteristics of TNF-alpha gene in mammals and fish. The chemiluminescent (CL) response of rock bream phagocytes was significantly enhanced by pre-incubation with recombinant rbTNF-alpha, when opsonized zymosan was used as a stimulant of the respiratory burst. However, CL enhancing effect of the recombinant rbTNF-alpha was very weak when the respiratory burst activity of phagocytes was triggered with phorbol-12-myristate-13-acetate (PMA) instead of zymosan. These results suggest that rock bream TNF-alpha might have an ability to prime the respiratory burst activity of phagocytes against receptor-mediated phagocytosis inducing stimulants, such as zymosan, but have little ability against stimulants not accompanying receptor-mediated phagocytosis.

Disintegration of cruciform and G-quadruplex structures during the course of helicase-dependent amplification (HDA).

PubMed

Li, Dawei; Lv, Bei; Zhang, Hao; Lee, Jasmine Yiqin; Li, Tianhu

2015-04-15

Unlike chemical damages on DNA, physical alterations of B-form of DNA occur commonly in organisms that serve as signals for specified cellular events. Although the modes of action for repairing of chemically damaged DNA have been well studied nowadays, the repairing mechanisms for physically altered DNA structures have not yet been understood. Our current in vitro studies show that both breakdown of stable non-B DNA structures and resumption of canonical B-conformation of DNA can take place during the courses of isothermal helicase-dependent amplification (HDA). The pathway that makes the non-B DNA structures repairable is presumably the relieving of the accumulated torsional stress that was caused by the positive supercoiling. Our new findings suggest that living organisms might have evolved this distinct and economical pathway for repairing their physically altered DNA structures. Copyright © 2015 Elsevier Ltd. All rights reserved.

Crystal structure of the Msx-1 homeodomain/DNA complex.

PubMed

Hovde, S; Abate-Shen, C; Geiger, J H

2001-10-09

The Msx-1 homeodomain protein plays a crucial role in craniofacial, limb, and nervous system development. Homeodomain DNA-binding domains are comprised of 60 amino acids that show a high degree of evolutionary conservation. We have determined the structure of the Msx-1 homeodomain complexed to DNA at 2.2 A resolution. The structure has an unusually well-ordered N-terminal arm with a unique trajectory across the minor groove of the DNA. DNA specificity conferred by bases flanking the core TAAT sequence is explained by well ordered water-mediated interactions at Q50. Most interactions seen at the TAAT sequence are typical of the interactions seen in other homeodomain structures. Comparison of the Msx-1-HD structure to all other high resolution HD-DNA complex structures indicate a remarkably well-conserved sphere of hydration between the DNA and protein in these complexes.

Electrotransfection of Polyamine Folded DNA Origami Structures.

PubMed

Chopra, Aradhana; Krishnan, Swati; Simmel, Friedrich C

2016-10-12

DNA origami structures are artificial molecular nanostructures in which DNA double helices are forced into a closely packed configuration by a multitude of DNA strand crossovers. We show that three different types of origami structures (a flat sheet, a hollow tube, and a compact origami block) can be formed in magnesium-free buffer solutions containing low (<1 mM) concentrations of the condensing agent spermidine. Much like in DNA condensation, the amount of spermidine required for origami folding is proportional to the DNA concentration. At excessive amounts, the structures aggregate and precipitate. In contrast to origami structures formed in conventional buffers, the resulting structures are stable in the presence of high electric field pulses, such as those commonly used for electrotransfection experiments. We demonstrate that spermidine-stabilized structures are stable in cell lysate and can be delivered into mammalian cells via electroporation.

Method for sequencing DNA base pairs

DOEpatents

Sessler, A.M.; Dawson, J.

1993-12-14

The base pairs of a DNA structure are sequenced with the use of a scanning tunneling microscope (STM). The DNA structure is scanned by the STM probe tip, and, as it is being scanned, the DNA structure is separately subjected to a sequence of infrared radiation from four different sources, each source being selected to preferentially excite one of the four different bases in the DNA structure. Each particular base being scanned is subjected to such sequence of infrared radiation from the four different sources as that particular base is being scanned. The DNA structure as a whole is separately imaged for each subjection thereof to radiation from one only of each source. 6 figures.

Structural anatomy of telomere OB proteins.

PubMed

Horvath, Martin P

2011-10-01

Telomere DNA-binding proteins protect the ends of chromosomes in eukaryotes. A subset of these proteins are constructed with one or more OB folds and bind with G+T-rich single-stranded DNA found at the extreme termini. The resulting DNA-OB protein complex interacts with other telomere components to coordinate critical telomere functions of DNA protection and DNA synthesis. While the first crystal and NMR structures readily explained protection of telomere ends, the picture of how single-stranded DNA becomes available to serve as primer and template for synthesis of new telomere DNA is only recently coming into focus. New structures of telomere OB fold proteins alongside insights from genetic and biochemical experiments have made significant contributions towards understanding how protein-binding OB proteins collaborate with DNA-binding OB proteins to recruit telomerase and DNA polymerase for telomere homeostasis. This review surveys telomere OB protein structures alongside highly comparable structures derived from replication protein A (RPA) components, with the goal of providing a molecular context for understanding telomere OB protein evolution and mechanism of action in protection and synthesis of telomere DNA.

Structural anatomy of telomere OB proteins

PubMed Central

Horvath, Martin P.

2015-01-01

Telomere DNA-binding proteins protect the ends of chromosomes in eukaryotes. A subset of these proteins are constructed with one or more OB folds and bind with G+T-rich single-stranded DNA found at the extreme termini. The resulting DNA-OB protein complex interacts with other telomere components to coordinate critical telomere functions of DNA protection and DNA synthesis. While the first crystal and NMR structures readily explained protection of telomere ends, the picture of how single-stranded DNA becomes available to serve as primer and template for synthesis of new telomere DNA is only recently coming into focus. New structures of telomere OB fold proteins alongside insights from genetic and biochemical experiments have made significant contributions towards understanding how protein-binding OB proteins collaborate with DNA-binding OB proteins to recruit telomerase and DNA polymerase for telomere homeostasis. This review surveys telomere OB protein structures alongside highly comparable structures derived from replication protein A (RPA) components, with the goal of providing a molecular context for understanding telomere OB protein evolution and mechanism of action in protection and synthesis of telomere DNA. PMID:21950380

Combined effects of metal complexation and size expansion in the electronic structure of DNA base pairs

NASA Astrophysics Data System (ADS)

Brancolini, Giorgia; Di Felice, Rosa

2011-05-01

Novel DNA derivatives have been recently investigated in the pursuit of modified DNA duplexes to tune the electronic structure of DNA-based assemblies for nanotechnology applications. Size-expanded DNAs (e.g., xDNA) and metalated DNAs (M-DNA) may enhance stacking interactions and induce metallic conductivity, respectively. Here we explore possible ways of tailoring the DNA electronic structure by combining the aromatic size expansion with the metal-doping. We select the salient structures from our recent study on natural DNA pairs complexed with transition metal ions and consider the equivalent model configurations for xDNA pairs. We present the results of density functional theory electronic structure calculations of the metalated expanded base-pairs with various localized basis sets and exchange-correlation functionals. Implicit solvent and coordination water molecules are also included. Our results indicate that the effect of base expansion is largest in Ag-xGC complexes, while Cu-xGC complexes are the most promising candidates for nanowires with enhanced electron transfer and also for on-purpose modification of the DNA double-helix for signal detection.

Dna2 nuclease-helicase structure, mechanism and regulation by Rpa.

PubMed

Zhou, Chun; Pourmal, Sergei; Pavletich, Nikola P

2015-11-02

The Dna2 nuclease-helicase maintains genomic integrity by processing DNA double-strand breaks, Okazaki fragments and stalled replication forks. Dna2 requires ssDNA ends, and is dependent on the ssDNA-binding protein Rpa, which controls cleavage polarity. Here we present the 2.3 Å structure of intact mouse Dna2 bound to a 15-nucleotide ssDNA. The nuclease active site is embedded in a long, narrow tunnel through which the DNA has to thread. The helicase domain is required for DNA binding but not threading. We also present the structure of a flexibly-tethered Dna2-Rpa interaction that recruits Dna2 to Rpa-coated DNA. We establish that a second Dna2-Rpa interaction is mutually exclusive with Rpa-DNA interactions and mediates the displacement of Rpa from ssDNA. This interaction occurs at the nuclease tunnel entrance and the 5' end of the Rpa-DNA complex. Hence, it only displaces Rpa from the 5' but not 3' end, explaining how Rpa regulates cleavage polarity.

Structural insight into the specificity of the B3 DNA-binding domains provided by the co-crystal structure of the C-terminal fragment of BfiI restriction enzyme

PubMed Central

Golovenko, Dmitrij; Manakova, Elena; Zakrys, Linas; Zaremba, Mindaugas; Sasnauskas, Giedrius; Gražulis, Saulius; Siksnys, Virginijus

2014-01-01

The B3 DNA-binding domains (DBDs) of plant transcription factors (TF) and DBDs of EcoRII and BfiI restriction endonucleases (EcoRII-N and BfiI-C) share a common structural fold, classified as the DNA-binding pseudobarrel. The B3 DBDs in the plant TFs recognize a diverse set of target sequences. The only available co-crystal structure of the B3-like DBD is that of EcoRII-N (recognition sequence 5′-CCTGG-3′). In order to understand the structural and molecular mechanisms of specificity of B3 DBDs, we have solved the crystal structure of BfiI-C (recognition sequence 5′-ACTGGG-3′) complexed with 12-bp cognate oligoduplex. Structural comparison of BfiI-C–DNA and EcoRII-N–DNA complexes reveals a conserved DNA-binding mode and a conserved pattern of interactions with the phosphodiester backbone. The determinants of the target specificity are located in the loops that emanate from the conserved structural core. The BfiI-C–DNA structure presented here expands a range of templates for modeling of the DNA-bound complexes of the B3 family of plant TFs. PMID:24423868

Knot soliton in DNA and geometric structure of its free-energy density.

PubMed

Wang, Ying; Shi, Xuguang

2018-03-01

In general, the geometric structure of DNA is characterized using an elastic rod model. The Landau model provides us a new theory to study the geometric structure of DNA. By using the decomposition of the arc unit in the helical axis of DNA, we find that the free-energy density of DNA is similar to the free-energy density of a two-condensate superconductor. By using the φ-mapping topological current theory, the torus knot soliton hidden in DNA is demonstrated. We show the relation between the geometric structure and free-energy density of DNA and the Frenet equations in differential geometry theory are considered. Therefore, the free-energy density of DNA can be expressed by the curvature and torsion of the helical axis.

Structural basis of DNA folding and recognition in an AMP-DNA aptamer complex: distinct architectures but common recognition motifs for DNA and RNA aptamers complexed to AMP.

PubMed

Lin, C H; Patel, D J

1997-11-01

Structural studies by nuclear magnetic resonance (NMR) of RNA and DNA aptamer complexes identified through in vitro selection and amplification have provided a wealth of information on RNA and DNA tertiary structure and molecular recognition in solution. The RNA and DNA aptamers that target ATP (and AMP) with micromolar affinity exhibit distinct binding site sequences and secondary structures. We report below on the tertiary structure of the AMP-DNA aptamer complex in solution and compare it with the previously reported tertiary structure of the AMP-RNA aptamer complex in solution. The solution structure of the AMP-DNA aptamer complex shows, surprisingly, that two AMP molecules are intercalated at adjacent sites within a rectangular widened minor groove. Complex formation involves adaptive binding where the asymmetric internal bubble of the free DNA aptamer zippers up through formation of a continuous six-base mismatch segment which includes a pair of adjacent three-base platforms. The AMP molecules pair through their Watson-Crick edges with the minor groove edges of guanine residues. These recognition G.A mismatches are flanked by sheared G.A and reversed Hoogsteen G.G mismatch pairs. The AMP-DNA aptamer and AMP-RNA aptamer complexes have distinct tertiary structures and binding stoichiometries. Nevertheless, both complexes have similar structural features and recognition alignments in their binding pockets. Specifically, AMP targets both DNA and RNA aptamers by intercalating between purine bases and through identical G.A mismatch formation. The recognition G.A mismatch stacks with a reversed Hoogsteen G.G mismatch in one direction and with an adenine base in the other direction in both complexes. It is striking that DNA and RNA aptamers selected independently from libraries of 10(14) molecules in each case utilize identical mismatch alignments for molecular recognition with micromolar affinity within binding-site pockets containing common structural elements.

Crystallographic Phasing from Weak Anomalous Signals

PubMed Central

Liu, Qun; Hendrickson, Wayne A.

2015-01-01

The exploitation of anomalous signals for biological structural solution is maturing. Single-wavelength anomalous diffraction (SAD) is dominant in de novo structure analysis. Nevertheless, for challenging structures where the resolution is low (dmin ≥ 3.5 Å) or where only lighter atoms (Z ≤ 20) are present, as for native macromolecules, solved SAD structures are still scarce. With the recent rapid development in crystal handling, beamline instrumentation, optimization of data collection strategies, use of multiple crystals and structure determination technologies, the weak anomalous diffraction signals are now robustly measured and should be used for routine SAD structure determination. The review covers these recent advances on weak anomalous signals measurement, analysis and utilization. PMID:26432413

Crystallographic phasing from weak anomalous signals.

PubMed

Liu, Qun; Hendrickson, Wayne A

2015-10-01

The exploitation of anomalous signals for biological structural solution is maturing. Single-wavelength anomalous diffraction (SAD) is dominant in de novo structure analysis. Nevertheless, for challenging structures where the resolution is low (dmin≥3.5Å) or where only lighter atoms (Z≤20) are present, as for native macromolecules, solved SAD structures are still scarce. With the recent rapid development in crystal handling, beamline instrumentation, optimization of data collection strategies, use of multiple crystals and structure determination technologies, the weak anomalous diffraction signals are now robustly measured and should be used for routine SAD structure determination. The review covers these recent advances on weak anomalous signals measurement, analysis and utilization. Copyright © 2015 Elsevier Ltd. All rights reserved.

Structural changes induced by binding of the high-mobility group I protein to a mouse satellite DNA sequence.

PubMed Central

Slama-Schwok, A; Zakrzewska, K; Léger, G; Leroux, Y; Takahashi, M; Käs, E; Debey, P

2000-01-01

Using spectroscopic methods, we have studied the structural changes induced in both protein and DNA upon binding of the High-Mobility Group I (HMG-I) protein to a 21-bp sequence derived from mouse satellite DNA. We show that these structural changes depend on the stoichiometry of the protein/DNA complexes formed, as determined by Job plots derived from experiments using pyrene-labeled duplexes. Circular dichroism and melting temperature experiments extended in the far ultraviolet range show that while native HMG-I is mainly random coiled in solution, it adopts a beta-turn conformation upon forming a 1:1 complex in which the protein first binds to one of two dA.dT stretches present in the duplex. HMG-I structure in the 1:1 complex is dependent on the sequence of its DNA target. A 3:1 HMG-I/DNA complex can also form and is characterized by a small increase in the DNA natural bend and/or compaction coupled to a change in the protein conformation, as determined from fluorescence resonance energy transfer (FRET) experiments. In addition, a peptide corresponding to an extended DNA-binding domain of HMG-I induces an ordered condensation of DNA duplexes. Based on the constraints derived from pyrene excimer measurements, we present a model of these nucleated structures. Our results illustrate an extreme case of protein structure induced by DNA conformation that may bear on the evolutionary conservation of the DNA-binding motifs of HMG-I. We discuss the functional relevance of the structural flexibility of HMG-I associated with the nature of its DNA targets and the implications of the binding stoichiometry for several aspects of chromatin structure and gene regulation. PMID:10777751

Genome-wide DNA polymorphism in the indica rice varieties RGD-7S and Taifeng B as revealed by whole genome re-sequencing.

PubMed

Fu, Chong-Yun; Liu, Wu-Ge; Liu, Di-Lin; Li, Ji-Hua; Zhu, Man-Shan; Liao, Yi-Long; Liu, Zhen-Rong; Zeng, Xue-Qin; Wang, Feng

2016-03-01

Next-generation sequencing technologies provide opportunities to further understand genetic variation, even within closely related cultivars. We performed whole genome resequencing of two elite indica rice varieties, RGD-7S and Taifeng B, whose F1 progeny showed hybrid weakness and hybrid vigor when grown in the early- and late-cropping seasons, respectively. Approximately 150 million 100-bp pair-end reads were generated, which covered ∼86% of the rice (Oryza sativa L. japonica 'Nipponbare') reference genome. A total of 2,758,740 polymorphic sites including 2,408,845 SNPs and 349,895 InDels were detected in RGD-7S and Taifeng B, respectively. Applying stringent parameters, we identified 961,791 SNPs and 46,640 InDels between RGD-7S and Taifeng B (RGD-7S/Taifeng B). The density of DNA polymorphisms was 256.8 SNPs and 12.5 InDels per 100 kb for RGD-7S/Taifeng B. Copy number variations (CNVs) were also investigated. In RGD-7S, 1989 of 2727 CNVs were overlapped in 218 genes, and 1231 of 2010 CNVs were annotated in 175 genes in Taifeng B. In addition, we verified a subset of InDels in the interval of hybrid weakness genes, Hw3 and Hw4, and obtained some polymorphic InDel markers, which will provide a sound foundation for cloning hybrid weakness genes. Analysis of genomic variations will also contribute to understanding the genetic basis of hybrid weakness and heterosis.

Integrating De Novo Transcriptome Assembly and Cloning to Obtain Chicken Ovocleidin-17 Full-Length cDNA

PubMed Central

Ning, ZhongHua; Hincke, Maxwell T.; Yang, Ning; Hou, ZhuoCheng

2014-01-01

Efficiently obtaining full-length cDNA for a target gene is the key step for functional studies and probing genetic variations. However, almost all sequenced domestic animal genomes are not ‘finished’. Many functionally important genes are located in these gapped regions. It can be difficult to obtain full-length cDNA for which only partial amino acid/EST sequences exist. In this study we report a general pipeline to obtain full-length cDNA, and illustrate this approach for one important gene (Ovocleidin-17, OC-17) that is associated with chicken eggshell biomineralization. Chicken OC-17 is one of the best candidates to control and regulate the deposition of calcium carbonate in the calcified eggshell layer. OC-17 protein has been purified, sequenced, and has had its three-dimensional structure solved. However, researchers still cannot conduct OC-17 mRNA related studies because the mRNA sequence is unknown and the gene is absent from the current chicken genome. We used RNA-Seq to obtain the entire transcriptome of the adult hen uterus, and then conducted de novo transcriptome assembling with bioinformatics analysis to obtain candidate OC-17 transcripts. Based on this sequence, we used RACE and PCR cloning methods to successfully obtain the full-length OC-17 cDNA. Temporal and spatial OC-17 mRNA expression analyses were also performed to demonstrate that OC-17 is predominantly expressed in the adult hen uterus during the laying cycle and barely at immature developmental stages. Differential uterine expression of OC-17 was observed in hens laying eggs with weak versus strong eggshell, confirming its important role in the regulation of eggshell mineralization and providing a new tool for genetic selection for eggshell quality parameters. This study is the first one to report the full-length OC-17 cDNA sequence, and builds a foundation for OC-17 mRNA related studies. We provide a general method for biologists experiencing difficulty in obtaining candidate gene full-length cDNA sequences. PMID:24676480

Integrating de novo transcriptome assembly and cloning to obtain chicken Ovocleidin-17 full-length cDNA.

PubMed

Zhang, Quan; Liu, Long; Zhu, Feng; Ning, ZhongHua; Hincke, Maxwell T; Yang, Ning; Hou, ZhuoCheng

2014-01-01

Efficiently obtaining full-length cDNA for a target gene is the key step for functional studies and probing genetic variations. However, almost all sequenced domestic animal genomes are not 'finished'. Many functionally important genes are located in these gapped regions. It can be difficult to obtain full-length cDNA for which only partial amino acid/EST sequences exist. In this study we report a general pipeline to obtain full-length cDNA, and illustrate this approach for one important gene (Ovocleidin-17, OC-17) that is associated with chicken eggshell biomineralization. Chicken OC-17 is one of the best candidates to control and regulate the deposition of calcium carbonate in the calcified eggshell layer. OC-17 protein has been purified, sequenced, and has had its three-dimensional structure solved. However, researchers still cannot conduct OC-17 mRNA related studies because the mRNA sequence is unknown and the gene is absent from the current chicken genome. We used RNA-Seq to obtain the entire transcriptome of the adult hen uterus, and then conducted de novo transcriptome assembling with bioinformatics analysis to obtain candidate OC-17 transcripts. Based on this sequence, we used RACE and PCR cloning methods to successfully obtain the full-length OC-17 cDNA. Temporal and spatial OC-17 mRNA expression analyses were also performed to demonstrate that OC-17 is predominantly expressed in the adult hen uterus during the laying cycle and barely at immature developmental stages. Differential uterine expression of OC-17 was observed in hens laying eggs with weak versus strong eggshell, confirming its important role in the regulation of eggshell mineralization and providing a new tool for genetic selection for eggshell quality parameters. This study is the first one to report the full-length OC-17 cDNA sequence, and builds a foundation for OC-17 mRNA related studies. We provide a general method for biologists experiencing difficulty in obtaining candidate gene full-length cDNA sequences.

Assessing pathogenicity for novel mutation/sequence variants: the value of healthy older individuals.

PubMed

Zatz, Mayana; Pavanello, Rita de Cassia M; Lourenço, Naila Cristina V; Cerqueira, Antonia; Lazar, Monize; Vainzof, Mariz

2012-12-01

Improvement in DNA technology is increasingly revealing unexpected/unknown mutations in healthy persons and generating anxiety due to their still unknown health consequences. We report a 44-year-old healthy father of a 10-year-old daughter with bilateral coloboma and hearing loss, but without muscle weakness, in whom a whole-genome CGH revealed a deletion of exons 38-44 in the dystrophin gene. This mutation was inherited from her asymptomatic father, who was further clinically and molecularly evaluated for prognosis and genetic counseling (GC). This deletion was never identified by us in 982 Duchenne/Becker patients. To assess whether the present case represents a rare case of non-penetrance, and aiming to obtain more information for prognosis and GC, we suggested that healthy older relatives submit their DNA for analysis, to which several complied. Mutation analysis revealed that his mother, brother, and 56-year-old maternal uncle also carry the 38-44 deletion, suggesting it an unlikely cause of muscle weakness. Genome sequencing will disclose mutations and variants whose health impact are still unknown, raising important problems in interpreting results, defining prognosis, and discussing GC. We suggest that, in addition to family history, keeping the DNA of older relatives could be very informative, in particular for those interested in having their genome sequenced.

Underwound DNA under Tension: Structure, Elasticity, and Sequence-Dependent Behaviors

NASA Astrophysics Data System (ADS)

Sheinin, Maxim Y.; Forth, Scott; Marko, John F.; Wang, Michelle D.

2011-09-01

DNA melting under torsion plays an important role in a wide variety of cellular processes. In the present Letter, we have investigated DNA melting at the single-molecule level using an angular optical trap. By directly measuring force, extension, torque, and angle of DNA, we determined the structural and elastic parameters of torsionally melted DNA. Our data reveal that under moderate forces, the melted DNA assumes a left-handed structure as opposed to an open bubble conformation and is highly torsionally compliant. We have also discovered that at low forces melted DNA properties are highly dependent on DNA sequence. These results provide a more comprehensive picture of the global DNA force-torque phase diagram.

Structure and DNA-Binding Sites of the SWI1 AT-rich Interaction Domain (ARID) Suggest Determinants for Sequence-Specific DNA Recognition

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

Kim, Suhkmann; Zhang, Ziming; Upchurch, Sean

2004-04-16

2 ARID is a homologous family of DNA-binding domains that occur in DNA binding proteins from a wide variety of species, ranging from yeast to nematodes, insects, mammals and plants. SWI1, a member of the SWI/SNF protein complex that is involved in chromatin remodeling during transcription, contains the ARID motif. The ARID domain of human SWI1 (also known as p270) does not select for a specific DNA sequence from a random sequence pool. The lack of sequence specificity shown by the SWI1 ARID domain stands in contrast to the other characterized ARID domains, which recognize specific AT-rich sequences. We havemore » solved the three-dimensional structure of human SWI1 ARID using solution NMR methods. In addition, we have characterized non-specific DNA-binding by the SWI1 ARID domain. Results from this study indicate that a flexible long internal loop in ARID motif is likely to be important for sequence specific DNA-recognition. The structure of human SWI1 ARID domain also represents a distinct structural subfamily. Studies of ARID indicate that boundary of the DNA binding structural and functional domains can extend beyond the sequence homologous region in a homologous family of proteins. Structural studies of homologous domains such as ARID family of DNA-binding domains should provide information to better predict the boundary of structural and functional domains in structural genomic studies. Key Words: ARID, SWI1, NMR, structural genomics, protein-DNA interaction.« less

Non-B-DNA structures on the interferon-beta promoter?

PubMed

Robbe, K; Bonnefoy, E

1998-01-01

The high mobility group (HMG) I protein intervenes as an essential factor during the virus induced expression of the interferon-beta (IFN-beta) gene. It is a non-histone chromatine associated protein that has the dual capacity of binding to a non-B-DNA structure such as cruciform-DNA as well as to AT rich B-DNA sequences. In this work we compare the binding affinity of HMGI for a synthetic cruciform-DNA to its binding affinity for the HMGI-binding-site present in the positive regulatory domain II (PRDII) of the IFN-beta promoter. Using gel retardation experiments, we show that HMGI protein binds with at least ten times more affinity to the synthetic cruciform-DNA structure than to the PRDII B-DNA sequence. DNA hairpin sequences are present in both the human and the murine PRDII-DNAs. We discuss in this work the presence of, yet putative, non-B-DNA structures in the IFN-beta promoter.

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

From structure to mechanism—understanding initiation of DNA replication

PubMed Central

Riera, Alberto; Barbon, Marta; Noguchi, Yasunori; Reuter, L. Maximilian; Schneider, Sarah; Speck, Christian

2017-01-01

DNA replication results in the doubling of the genome prior to cell division. This process requires the assembly of 50 or more protein factors into a replication fork. Here, we review recent structural and biochemical insights that start to explain how specific proteins recognize DNA replication origins, load the replicative helicase on DNA, unwind DNA, synthesize new DNA strands, and reassemble chromatin. We focus on the minichromosome maintenance (MCM2–7) proteins, which form the core of the eukaryotic replication fork, as this complex undergoes major structural rearrangements in order to engage with DNA, regulate its DNA-unwinding activity, and maintain genome stability. PMID:28717046

Sequence and Structure Dependent DNA-DNA Interactions

NASA Astrophysics Data System (ADS)

Kopchick, Benjamin; Qiu, Xiangyun

Molecular forces between dsDNA strands are largely dominated by electrostatics and have been extensively studied. Quantitative knowledge has been accumulated on how DNA-DNA interactions are modulated by varied biological constituents such as ions, cationic ligands, and proteins. Despite its central role in biology, the sequence of DNA has not received substantial attention and ``random'' DNA sequences are typically used in biophysical studies. However, ~50% of human genome is composed of non-random-sequence DNAs, particularly repetitive sequences. Furthermore, covalent modifications of DNA such as methylation play key roles in gene functions. Such DNAs with specific sequences or modifications often take on structures other than the canonical B-form. Here we present series of quantitative measurements of the DNA-DNA forces with the osmotic stress method on different DNA sequences, from short repeats to the most frequent sequences in genome, and to modifications such as bromination and methylation. We observe peculiar behaviors that appear to be strongly correlated with the incurred structural changes. We speculate the causalities in terms of the differences in hydration shell and DNA surface structures.

Abasic and oxidized ribonucleotides embedded in DNA are processed by human APE1 and not by RNase H2

PubMed Central

Malfatti, Matilde Clarissa; Balachander, Sathya; Antoniali, Giulia; Koh, Kyung Duk; Saint-Pierre, Christine; Gasparutto, Didier; Chon, Hyongi; Crouch, Robert J.

2017-01-01

Abstract Ribonucleoside 5′-monophosphates (rNMPs) are the most common non-standard nucleotides found in DNA of eukaryotic cells, with over 100 million rNMPs transiently incorporated in the mammalian genome per cell cycle. Human ribonuclease (RNase) H2 is the principal enzyme able to cleave rNMPs in DNA. Whether RNase H2 may process abasic or oxidized rNMPs incorporated in DNA is unknown. The base excision repair (BER) pathway is mainly responsible for repairing oxidized and abasic sites into DNA. Here we show that human RNase H2 is unable to process an abasic rNMP (rAP site) or a ribose 8oxoG (r8oxoG) site embedded in DNA. On the contrary, we found that recombinant purified human apurinic/apyrimidinic endonuclease-1 (APE1) and APE1 from human cell extracts efficiently process an rAP site in DNA and have weak endoribonuclease and 3′-exonuclease activities on r8oxoG substrate. Using biochemical assays, our results provide evidence of a human enzyme able to recognize and process abasic and oxidized ribonucleotides embedded in DNA. PMID:28977421

Cloning, sequencing, and expression of dnaK-operon proteins from the thermophilic bacterium Thermus thermophilus.

PubMed

Osipiuk, J; Joachimiak, A

1997-09-12

We propose that the dnaK operon of Thermus thermophilus HB8 is composed of three functionally linked genes: dnaK, grpE, and dnaJ. The dnaK and dnaJ gene products are most closely related to their cyanobacterial homologs. The DnaK protein sequence places T. thermophilus in the plastid Hsp70 subfamily. In contrast, the grpE translated sequence is most similar to GrpE from Clostridium acetobutylicum, a Gram-positive anaerobic bacterium. A single promoter region, with homology to the Escherichia coli consensus promoter sequences recognized by the sigma70 and sigma32 transcription factors, precedes the postulated operon. This promoter is heat-shock inducible. The dnaK mRNA level increased more than 30 times upon 10 min of heat shock (from 70 degrees C to 85 degrees C). A strong transcription terminating sequence was found between the dnaK and grpE genes. The individual genes were cloned into pET expression vectors and the thermophilic proteins were overproduced at high levels in E. coli and purified to homogeneity. The recombinant T. thermophilus DnaK protein was shown to have a weak ATP-hydrolytic activity, with an optimum at 90 degrees C. The ATPase was stimulated by the presence of GrpE and DnaJ. Another open reading frame, coding for ClpB heat-shock protein, was found downstream of the dnaK operon.

Molecular structure of r/GCG/d/TATACGC/ - A DNA-RNA hybrid helix joined to double helical DNA

NASA Technical Reports Server (NTRS)

Wang, A. H.-J.; Fujii, S.; Rich, A.; Van Boom, J. H.; Van Der Marel, G. A.; Van Boeckel, S. A. A.

1982-01-01

The molecule r(GCG)d(TATACGC) is self-complementary and forms two DNA-RNA hybrid segments surrounding a central region of double helical DNA; its molecular structure has been solved by X-ray analysis. All three parts of the molecule adopt a conformation which is close to that seen in the 11-fold RNA double helix. The conformation of the ribonucleotides is partly determined by water molecules bridging between the ribose O2' hydroxyl group and cytosine O2. The hybrid-DNA duplex junction contains no structural discontinuities. However, the central DNA TATA sequence has some structural irregularities.

DNA Secondary Structure at Chromosomal Fragile Sites in Human Disease

PubMed Central

Thys, Ryan G; Lehman, Christine E; Pierce, Levi C. T; Wang, Yuh-Hwa

2015-01-01

DNA has the ability to form a variety of secondary structures that can interfere with normal cellular processes, and many of these structures have been associated with neurological diseases and cancer. Secondary structure-forming sequences are often found at chromosomal fragile sites, which are hotspots for sister chromatid exchange, chromosomal translocations, and deletions. Structures formed at fragile sites can lead to instability by disrupting normal cellular processes such as DNA replication and transcription. The instability caused by disruption of replication and transcription can lead to DNA breakage, resulting in gene rearrangements and deletions that cause disease. In this review, we discuss the role of DNA secondary structure at fragile sites in human disease. PMID:25937814

Emerging Biomimetic Applications of DNA Nanotechnology.

PubMed

Shen, Haijing; Wang, Yingqian; Wang, Jie; Li, Zhihao; Yuan, Quan

2018-06-25

Re-engineering cellular components and biological processes has received great interest and promised compelling advantages in applications ranging from basic cell biology to biomedicine. With the advent of DNA nanotechnology, the programmable self-assembly ability makes DNA an appealing candidate for rational design of artificial components with different structures and functions. This Forum Article summarizes recent developments of DNA nanotechnology in mimicking the structures and functions of existing cellular components. We highlight key successes in the achievements of DNA-based biomimetic membrane proteins and discuss the assembly behavior of these artificial proteins. Then, we focus on the construction of higher-order structures by DNA nanotechnology to recreate cell-like structures. Finally, we explore the current challenges and speculate on future directions of DNA nanotechnology in biomimetics.

Peliosis hepatis in a dog infected with Bartonella henselae.

PubMed

Kitchell, B E; Fan, T M; Kordick, D; Breitschwerdt, E B; Wollenberg, G; Lichtensteiger, C A

2000-02-15

A 6-year-old spayed female Golden Retriever was examined because of generalized weakness and abdominal distention. Abdominal ultrasonography revealed a large quantity of peritoneal fluid. In addition, the liver appeared larger than normal and contained multiple, small, nodular masses and cyst-like structures. Abdominal exploratory surgery was performed, and 5 L of serosanguineous peritoneal fluid was removed. Gross lesions were not found in the stomach, kidneys, intestines, adrenal glands, or urinary bladder. There were diffuse cystic nodules in all liver lobes. The dog did not recover from anesthesia. A diagnosis of peliosis hepatis was made on the basis of gross and histologic appearance of the liver. A polymerase chain reaction assay revealed Bartonella henselae DNA in liver specimens. To our knowledge, this is the first report of molecular evidence of B henselae infection in a dog with peliosis hepatis.

Structural Insights into the HIV-1 Minus-strand Strong-stop DNA*

PubMed Central

Chen, Yingying; Maskri, Ouerdia; Chaminade, Françoise; René, Brigitte; Benkaroun, Jessica; Godet, Julien; Mély, Yves; Mauffret, Olivier; Fossé, Philippe

2016-01-01

An essential step of human immunodeficiency virus type 1 (HIV-1) reverse transcription is the first strand transfer that requires base pairing of the R region at the 3′-end of the genomic RNA with the complementary r region at the 3′-end of minus-strand strong-stop DNA (ssDNA). HIV-1 nucleocapsid protein (NC) facilitates this annealing process. Determination of the ssDNA structure is needed to understand the molecular basis of NC-mediated genomic RNA-ssDNA annealing. For this purpose, we investigated ssDNA using structural probes (nucleases and potassium permanganate). This study is the first to determine the secondary structure of the full-length HIV-1 ssDNA in the absence or presence of NC. The probing data and phylogenetic analysis support the folding of ssDNA into three stem-loop structures and the presence of four high-affinity binding sites for NC. Our results support a model for the NC-mediated annealing process in which the preferential binding of NC to four sites triggers unfolding of the three-dimensional structure of ssDNA, thus facilitating interaction of the r sequence of ssDNA with the R sequence of the genomic RNA. In addition, using gel retardation assays and ssDNA mutants, we show that the NC-mediated annealing process does not rely on a single pathway (zipper intermediate or kissing complex). PMID:26668324

Atomic Force Microscopy Studies on DNA Structural Changes Induced by Vincristine Sulfate and Aspirin

NASA Astrophysics Data System (ADS)

Zhu, Yi; Zeng, Hu; Xie, Jianming; Ba, Long; Gao, Xiang; Lu, Zuhong

2004-04-01

We report that atomic force microscopy (AFM) studies on structural variations of a linear plasmid DNA interact with various concentrations of vincristine sulfate and aspirin. The different binding images show that vincrinstine sulfate binding DNA chains caused some loops and cleavages of the DNA fragments, whereas aspirin interaction caused the width changes and conformational transition of the DNA fragments. Two different DNA structural alternations could be explained by the different mechanisms of the interactions with these two components. Our work indicates that the AFM is a powerful tool in studying the interaction between DNA and small molecules.

Structure of an XPF endonuclease with and without DNA suggests a model for substrate recognition

PubMed Central

Newman, Matthew; Murray-Rust, Judith; Lally, John; Rudolf, Jana; Fadden, Andrew; Knowles, Philip P; White, Malcolm F; McDonald, Neil Q

2005-01-01

The XPF/Mus81 structure-specific endonucleases cleave double-stranded DNA (dsDNA) within asymmetric branched DNA substrates and play an essential role in nucleotide excision repair, recombination and genome integrity. We report the structure of an archaeal XPF homodimer alone and bound to dsDNA. Superposition of these structures reveals a large domain movement upon binding DNA, indicating how the (HhH)2 domain and the nuclease domain are coupled to allow the recognition of double-stranded/single-stranded DNA junctions. We identify two nonequivalent DNA-binding sites and propose a model in which XPF distorts the 3′ flap substrate in order to engage both binding sites and promote strand cleavage. The model rationalises published biochemical data and implies a novel role for the ERCC1 subunit of eukaryotic XPF complexes. PMID:15719018

[A fluoride-sensor for kink structure in DNA condensation process].

PubMed

Liu, Yan-Hui; Zhang, Jing; Chen, Ying-Bing; Li, Yu-Pu; Hu, Lin

2014-01-01

Bloomfield has pointed out that the kink structure occurs for sharp bending during DNA condensation process, until now, which has not been proved by experiments. Using UV Spectrophotometer, the effects of fluoride and chlorine on the polyamine-DNA condensation system can be detected. Fluoride and chlorine both belong to the halogen family, but their effects on spermine-DNA condensation system are totally different. Fluoride ions make blue-shift and hyperchromicity appear in the spermine-DNA condensation system, but chlorine ions only make insignificant hyperchromicity happen in this system. Both fluoride ions and chlorine ions only make insignificant hyperchromicity happen in spermidine-DNA condensation system. Based on the distinguished character of fluoride, a fluoride-sensor for "kink" structure in DNA condensation was developed and the second kind of "kink" structure only appear in the spermine-DNA condensation system.

Overstretching of B-DNA with various pulling protocols: Appearance of structural polymorphism and S-DNA

NASA Astrophysics Data System (ADS)

Garai, Ashok; Mogurampelly, Santosh; Bag, Saientan; Maiti, Prabal K.

2017-12-01

We report a structural polymorphism of the S-DNA when a canonical B-DNA is stretched under different pulling protocols and provide a fundamental molecular understanding of the DNA stretching mechanism. Extensive all atom molecular dynamics simulations reveal a clear formation of S-DNA when the B-DNA is stretched along the 3' directions of the opposite strands (OS3) and is characterized by the changes in the number of H-bonds, entropy, and free energy. Stretching along the 5' directions of the opposite strands (OS5) leads to force induced melting form of the DNA. Interestingly, stretching along the opposite ends of the same strand leads to a coexistence of both the S- and melted M-DNA structures. We also do the structural characterization of the S-DNA by calculating various helical parameters. We find that the S-DNA has a twist of ˜10° which corresponds to a helical repeat length of ˜36 base pairs in close agreement with the previous experimental results. Moreover, we find that the free energy barrier between the canonical and overstretched states of DNA is higher for the same termini pulling protocol in comparison to all other protocols considered in this work. Overall, our observations not only reconcile with the available experimental results qualitatively but also enhance the understanding of different overstretched DNA structures.

Mitochondrial DNA phylogeography of the Norway rat.

PubMed

Song, Ying; Lan, Zhenjiang; Kohn, Michael H

2014-01-01

Central Eastern Asia, foremost the area bordering northern China and Mongolia, has been thought to be the geographic region where Norway rats (Rattus norvegicus) have originated. However recent fossil analyses pointed to their origin in southern China. Moreover, whereas analyses of fossils dated the species' origin as ∼ 1.2-1.6 million years ago (Mya), molecular analyses yielded ∼ 0.5-2.9 Mya. Here, to study the geographic origin of the Norway rat and its spread across the globe we analyzed new and all published mitochondrial DNA cytochrome-b (cyt-b; N = 156) and D-loop (N = 212) sequences representing wild rats from four continents and select inbred strains. Our results are consistent with an origin of the Norway rat in southern China ∼ 1.3 Mya, subsequent prehistoric differentiation and spread in China and Asia from an initially weakly structured ancestral population, followed by further spread and differentiation across the globe during historic times. The recent spreading occurred mostly from derived European populations rather than from archaic Asian populations. We trace laboratory strains to wild lineages from Europe and North America and these represent a subset of the diversity of the rat; leaving Asian lineages largely untapped as a resource for biomedical models. By studying rats from Europe we made the observation that mtDNA diversity cannot be interpreted without consideration of pest control and, possibly, the evolution of rodenticide resistance. However, demographic models explored by forward-time simulations cannot fully explain the low mtDNA diversity of European rats and lack of haplotype sharing with their source from Asia. Comprehensive nuclear marker analyses of a larger sample of Norway rats representing the world are needed to better resolve the evolutionary history of wild rats and of laboratory rats, as well as to better understand the evolution of anticoagulant resistance.

Using classical population genetics tools with heterochroneous data: time matters!

PubMed

Depaulis, Frantz; Orlando, Ludovic; Hänni, Catherine

2009-01-01

New polymorphism datasets from heterochroneous data have arisen thanks to recent advances in experimental and microbial molecular evolution, and the sequencing of ancient DNA (aDNA). However, classical tools for population genetics analyses do not take into account heterochrony between subsets, despite potential bias on neutrality and population structure tests. Here, we characterize the extent of such possible biases using serial coalescent simulations. We first use a coalescent framework to generate datasets assuming no or different levels of heterochrony and contrast most classical population genetic statistics. We show that even weak levels of heterochrony ( approximately 10% of the average depth of a standard population tree) affect the distribution of polymorphism substantially, leading to overestimate the level of polymorphism theta, to star like trees, with an excess of rare mutations and a deficit of linkage disequilibrium, which are the hallmark of e.g. population expansion (possibly after a drastic bottleneck). Substantial departures of the tests are detected in the opposite direction for more heterochroneous and equilibrated datasets, with balanced trees mimicking in particular population contraction, balancing selection, and population differentiation. We therefore introduce simple corrections to classical estimators of polymorphism and of the genetic distance between populations, in order to remove heterochrony-driven bias. Finally, we show that these effects do occur on real aDNA datasets, taking advantage of the currently available sequence data for Cave Bears (Ursus spelaeus), for which large mtDNA haplotypes have been reported over a substantial time period (22-130 thousand years ago (KYA)). Considering serial sampling changed the conclusion of several tests, indicating that neglecting heterochrony could provide significant support for false past history of populations and inappropriate conservation decisions. We therefore argue for systematically considering heterochroneous models when analyzing heterochroneous samples covering a large time scale.

Design pattern mining using distributed learning automata and DNA sequence alignment.

PubMed

Esmaeilpour, Mansour; Naderifar, Vahideh; Shukur, Zarina

2014-01-01

Over the last decade, design patterns have been used extensively to generate reusable solutions to frequently encountered problems in software engineering and object oriented programming. A design pattern is a repeatable software design solution that provides a template for solving various instances of a general problem. This paper describes a new method for pattern mining, isolating design patterns and relationship between them; and a related tool, DLA-DNA for all implemented pattern and all projects used for evaluation. DLA-DNA achieves acceptable precision and recall instead of other evaluated tools based on distributed learning automata (DLA) and deoxyribonucleic acid (DNA) sequences alignment. The proposed method mines structural design patterns in the object oriented source code and extracts the strong and weak relationships between them, enabling analyzers and programmers to determine the dependency rate of each object, component, and other section of the code for parameter passing and modular programming. The proposed model can detect design patterns better that available other tools those are Pinot, PTIDEJ and DPJF; and the strengths of their relationships. The result demonstrate that whenever the source code is build standard and non-standard, based on the design patterns, then the result of the proposed method is near to DPJF and better that Pinot and PTIDEJ. The proposed model is tested on the several source codes and is compared with other related models and available tools those the results show the precision and recall of the proposed method, averagely 20% and 9.6% are more than Pinot, 27% and 31% are more than PTIDEJ and 3.3% and 2% are more than DPJF respectively. The primary idea of the proposed method is organized in two following steps: the first step, elemental design patterns are identified, while at the second step, is composed to recognize actual design patterns.

Culture creates genetic structure in the Caucasus: Autosomal, mitochondrial, and Y-chromosomal variation in Daghestan

PubMed Central

Marchani, Elizabeth E; Watkins, W Scott; Bulayeva, Kazima; Harpending, Henry C; Jorde, Lynn B

2008-01-01

Background Near the junction of three major continents, the Caucasus region has been an important thoroughfare for human migration. While the Caucasus Mountains have diverted human traffic to the few lowland regions that provide a gateway from north to south between the Caspian and Black Seas, highland populations have been isolated by their remote geographic location and their practice of patrilocal endogamy. We investigate how these cultural and historical differences between highland and lowland populations have affected patterns of genetic diversity. We test 1) whether the highland practice of patrilocal endogamy has generated sex-specific population relationships, and 2) whether the history of migration and military conquest associated with the lowland populations has left Central Asian genes in the Caucasus, by comparing genetic diversity and pairwise population relationships between Daghestani populations and reference populations throughout Europe and Asia for autosomal, mitochondrial, and Y-chromosomal markers. Results We found that the highland Daghestani populations had contrasting histories for the mitochondrial DNA and Y-chromosome data sets. Y-chromosomal haplogroup diversity was reduced among highland Daghestani populations when compared to other populations and to highland Daghestani mitochondrial DNA haplogroup diversity. Lowland Daghestani populations showed Turkish and Central Asian affinities for both mitochondrial and Y-chromosomal data sets. Autosomal population histories are strongly correlated to the pattern observed for the mitochondrial DNA data set, while the correlation between the mitochondrial DNA and Y-chromosome distance matrices was weak and not significant. Conclusion The reduced Y-chromosomal diversity exhibited by highland Daghestani populations is consistent with genetic drift caused by patrilocal endogamy. Mitochondrial and Y-chromosomal phylogeographic comparisons indicate a common Near Eastern origin of highland populations. Lowland Daghestani populations show varying influence from Near Eastern and Central Asian populations. PMID:18637195

Strong and stable geographic differentiation of swamp buffalo maternal and paternal lineages indicates domestication in the China/Indochina border region.

PubMed

Zhang, Yi; Lu, Yongfang; Yindee, Marnoch; Li, Kuan-Yi; Kuo, Hsiao-Yun; Ju, Yu-Ten; Ye, Shaohui; Faruque, Md Omar; Li, Qiang; Wang, Yachun; Cuong, Vu Chi; Pham, Lan Doan; Bouahom, Bounthong; Yang, Bingzhuang; Liang, Xianwei; Cai, Zhihua; Vankan, Dianne; Manatchaiworakul, Wallaya; Kowlim, Nonglid; Duangchantrasiri, Somphot; Wajjwalku, Worawidh; Colenbrander, Ben; Zhang, Yuan; Beerli, Peter; Lenstra, Johannes A; Barker, J Stuart F

2016-04-01

The swamp type of the Asian water buffalo is assumed to have been domesticated by about 4000 years BP, following the introduction of rice cultivation. Previous localizations of the domestication site were based on mitochondrial DNA (mtDNA) variation within China, accounting only for the maternal lineage. We carried out a comprehensive sampling of China, Taiwan, Vietnam, Laos, Thailand, Nepal and Bangladesh and sequenced the mtDNA Cytochrome b gene and control region and the Y-chromosomal ZFY, SRY and DBY sequences. Swamp buffalo has a higher diversity of both maternal and paternal lineages than river buffalo, with also a remarkable contrast between a weak phylogeographic structure of river buffalo and a strong geographic differentiation of swamp buffalo. The highest diversity of the swamp buffalo maternal lineages was found in south China and north Indochina on both banks of the Mekong River, while the highest diversity in paternal lineages was in the China/Indochina border region. We propose that domestication in this region was later followed by introgressive capture of wild cows west of the Mekong. Migration to the north followed the Yangtze valley as well as a more eastern route, but also involved translocations of both cows and bulls over large distances with a minor influence of river buffaloes in recent decades. Bayesian analyses of various migration models also supported domestication in the China/Indochina border region. Coalescence analysis yielded consistent estimates for the expansion of the major swamp buffalo haplogroups with a credibility interval of 900 to 3900 years BP. The spatial differentiation of mtDNA and Y-chromosomal haplotype distributions indicates a lack of gene flow between established populations that is unprecedented in livestock. © 2015 John Wiley & Sons Ltd.

Sequence-specific activation of the DNA sensor cGAS by Y-form DNA structures as found in primary HIV-1 cDNA.

PubMed

Herzner, Anna-Maria; Hagmann, Cristina Amparo; Goldeck, Marion; Wolter, Steven; Kübler, Kirsten; Wittmann, Sabine; Gramberg, Thomas; Andreeva, Liudmila; Hopfner, Karl-Peter; Mertens, Christina; Zillinger, Thomas; Jin, Tengchuan; Xiao, Tsan Sam; Bartok, Eva; Coch, Christoph; Ackermann, Damian; Hornung, Veit; Ludwig, Janos; Barchet, Winfried; Hartmann, Gunther; Schlee, Martin

2015-10-01

Cytosolic DNA that emerges during infection with a retrovirus or DNA virus triggers antiviral type I interferon responses. So far, only double-stranded DNA (dsDNA) over 40 base pairs (bp) in length has been considered immunostimulatory. Here we found that unpaired DNA nucleotides flanking short base-paired DNA stretches, as in stem-loop structures of single-stranded DNA (ssDNA) derived from human immunodeficiency virus type 1 (HIV-1), activated the type I interferon-inducing DNA sensor cGAS in a sequence-dependent manner. DNA structures containing unpaired guanosines flanking short (12- to 20-bp) dsDNA (Y-form DNA) were highly stimulatory and specifically enhanced the enzymatic activity of cGAS. Furthermore, we found that primary HIV-1 reverse transcripts represented the predominant viral cytosolic DNA species during early infection of macrophages and that these ssDNAs were highly immunostimulatory. Collectively, our study identifies unpaired guanosines in Y-form DNA as a highly active, minimal cGAS recognition motif that enables detection of HIV-1 ssDNA.

Optical Voltage Sensing Using DNA Origami

PubMed Central

2018-01-01

We explore the potential of DNA nanotechnology for developing novel optical voltage sensing nanodevices that convert a local change of electric potential into optical signals. As a proof-of-concept of the sensing mechanism, we assembled voltage responsive DNA origami structures labeled with a single pair of FRET dyes. The DNA structures were reversibly immobilized on a nanocapillary tip and underwent controlled structural changes upon application of an electric field. The applied field was monitored through a change in FRET efficiency. By exchanging the position of a single dye, we could tune the voltage sensitivity of our DNA origami structure, demonstrating the flexibility and versatility of our approach. The experimental studies were complemented by coarse-grained simulations that characterized voltage-dependent elastic deformation of the DNA nanostructures and the associated change in the distance between the FRET pair. Our work opens a novel pathway for determining the mechanical properties of DNA origami structures and highlights potential applications of dynamic DNA nanostructures as voltage sensors. PMID:29430924

Crystal structure of MboIIA methyltransferase.

PubMed

Osipiuk, Jerzy; Walsh, Martin A; Joachimiak, Andrzej

2003-09-15

DNA methyltransferases (MTases) are sequence-specific enzymes which transfer a methyl group from S-adenosyl-L-methionine (AdoMet) to the amino group of either cytosine or adenine within a recognized DNA sequence. Methylation of a base in a specific DNA sequence protects DNA from nucleolytic cleavage by restriction enzymes recognizing the same DNA sequence. We have determined at 1.74 A resolution the crystal structure of a beta-class DNA MTase MboIIA (M.MboIIA) from the bacterium Moraxella bovis, the smallest DNA MTase determined to date. M.MboIIA methylates the 3' adenine of the pentanucleotide sequence 5'-GAAGA-3'. The protein crystallizes with two molecules in the asymmetric unit which we propose to resemble the dimer when M.MboIIA is not bound to DNA. The overall structure of the enzyme closely resembles that of M.RsrI. However, the cofactor-binding pocket in M.MboIIA forms a closed structure which is in contrast to the open-form structures of other known MTases.

Evidence of pervasive biologically functional secondary structures within the genomes of eukaryotic single-stranded DNA viruses.

PubMed

Muhire, Brejnev Muhizi; Golden, Michael; Murrell, Ben; Lefeuvre, Pierre; Lett, Jean-Michel; Gray, Alistair; Poon, Art Y F; Ngandu, Nobubelo Kwanele; Semegni, Yves; Tanov, Emil Pavlov; Monjane, Adérito Luis; Harkins, Gordon William; Varsani, Arvind; Shepherd, Dionne Natalie; Martin, Darren Patrick

2014-02-01

Single-stranded DNA (ssDNA) viruses have genomes that are potentially capable of forming complex secondary structures through Watson-Crick base pairing between their constituent nucleotides. A few of the structural elements formed by such base pairings are, in fact, known to have important functions during the replication of many ssDNA viruses. Unknown, however, are (i) whether numerous additional ssDNA virus genomic structural elements predicted to exist by computational DNA folding methods actually exist and (ii) whether those structures that do exist have any biological relevance. We therefore computationally inferred lists of the most evolutionarily conserved structures within a diverse selection of animal- and plant-infecting ssDNA viruses drawn from the families Circoviridae, Anelloviridae, Parvoviridae, Nanoviridae, and Geminiviridae and analyzed these for evidence of natural selection favoring the maintenance of these structures. While we find evidence that is consistent with purifying selection being stronger at nucleotide sites that are predicted to be base paired than at sites predicted to be unpaired, we also find strong associations between sites that are predicted to pair with one another and site pairs that are apparently coevolving in a complementary fashion. Collectively, these results indicate that natural selection actively preserves much of the pervasive secondary structure that is evident within eukaryote-infecting ssDNA virus genomes and, therefore, that much of this structure is biologically functional. Lastly, we provide examples of various highly conserved but completely uncharacterized structural elements that likely have important functions within some of the ssDNA virus genomes analyzed here.

Evidence of Pervasive Biologically Functional Secondary Structures within the Genomes of Eukaryotic Single-Stranded DNA Viruses

PubMed Central

Muhire, Brejnev Muhizi; Golden, Michael; Murrell, Ben; Lefeuvre, Pierre; Lett, Jean-Michel; Gray, Alistair; Poon, Art Y. F.; Ngandu, Nobubelo Kwanele; Semegni, Yves; Tanov, Emil Pavlov; Monjane, Adérito Luis; Harkins, Gordon William; Varsani, Arvind; Shepherd, Dionne Natalie

2014-01-01

Single-stranded DNA (ssDNA) viruses have genomes that are potentially capable of forming complex secondary structures through Watson-Crick base pairing between their constituent nucleotides. A few of the structural elements formed by such base pairings are, in fact, known to have important functions during the replication of many ssDNA viruses. Unknown, however, are (i) whether numerous additional ssDNA virus genomic structural elements predicted to exist by computational DNA folding methods actually exist and (ii) whether those structures that do exist have any biological relevance. We therefore computationally inferred lists of the most evolutionarily conserved structures within a diverse selection of animal- and plant-infecting ssDNA viruses drawn from the families Circoviridae, Anelloviridae, Parvoviridae, Nanoviridae, and Geminiviridae and analyzed these for evidence of natural selection favoring the maintenance of these structures. While we find evidence that is consistent with purifying selection being stronger at nucleotide sites that are predicted to be base paired than at sites predicted to be unpaired, we also find strong associations between sites that are predicted to pair with one another and site pairs that are apparently coevolving in a complementary fashion. Collectively, these results indicate that natural selection actively preserves much of the pervasive secondary structure that is evident within eukaryote-infecting ssDNA virus genomes and, therefore, that much of this structure is biologically functional. Lastly, we provide examples of various highly conserved but completely uncharacterized structural elements that likely have important functions within some of the ssDNA virus genomes analyzed here. PMID:24284329

Less Pollen-Mediated Gene Flow for More Signatures of Glacial Lineages: Congruent Evidence from Balsam Fir cpDNA and mtDNA for Multiple Refugia in Eastern and Central North America

PubMed Central

Cinget, Benjamin; Gérardi, Sébastien; Beaulieu, Jean; Bousquet, Jean

2015-01-01

The phylogeographic structure and postglacial history of balsam fir (Abies balsamea), a transcontinental North American boreal conifer, was inferred using mitochondrial DNA (mtDNA) and chloroplast DNA (cpDNA) markers. Genetic structure among 107 populations (mtDNA data) and 75 populations (cpDNA data) was analyzed using Bayesian and genetic distance approaches. Population differentiation was high for mtDNA (dispersed by seeds only), but also for cpDNA (dispersed by seeds and pollen), indicating that pollen gene flow is more restricted in balsam fir than in other boreal conifers. Low cpDNA gene flow in balsam fir may relate to low pollen production due to the inherent biology of the species and populations being decimated by recurrent spruce budworm epidemics, and/or to low dispersal of pollen grains due to their peculiar structural properties. Accordingly, a phylogeographic structure was detected using both mtDNA and cpDNA markers and population structure analyses supported the existence of at least five genetically distinct glacial lineages in central and eastern North America. Four of these would originate from glacial refugia located south of the Laurentide ice sheet, while the last one would have persisted in the northern Labrador region. As expected due to reduced pollen-mediated gene flow, congruence between the geographic distribution of mtDNA and cpDNA lineages was higher than in other North American conifers. However, concordance was not complete, reflecting that restricted but nonetheless detectable cpDNA gene flow among glacial lineages occurred during the Holocene. As a result, new cpDNA and mtDNA genome combinations indicative of cytoplasmic genome capture were observed. PMID:25849816

Surface-assisted DNA self-assembly: An enzyme-free strategy towards formation of branched DNA lattice.

PubMed

Bhanjadeo, Madhabi M; Nayak, Ashok K; Subudhi, Umakanta

2017-04-01

DNA based self-assembled nanostructures and DNA origami has proven useful for organizing nanomaterials with firm precision. However, for advanced applications like nanoelectronics and photonics, large-scale organization of self-assembled branched DNA (bDNA) into periodic lattices is desired. In this communication for the first time we report a facile method of self-assembly of Y-shaped bDNA nanostructures on the cationic surface of Aluminum (Al) foil to prepare periodic two dimensional (2D) bDNA lattice. Particularly those Y-shaped bDNA structures having smaller overhangs and unable to self-assemble in solution, they are easily assembled on the surface of Al foil in the absence of ligase. Field emission scanning electron microscopy (FESEM) analysis shows homogenous distribution of two-dimensional bDNA lattices across the Al foil. When the assembled bDNA structures were recovered from the Al foil and electrophoresed in nPAGE only higher order polymeric bDNA structures were observed without a trace of monomeric structures which confirms the stability and high yield of the bDNA lattices. Therefore, this enzyme-free economic and efficient strategy for developing bDNA lattices can be utilized in assembling various nanomaterials for functional molecular components towards development of DNA based self-assembled nanodevices. Copyright © 2017 Elsevier Inc. All rights reserved.

Genetic Structure of Wild Bonobo Populations: Diversity of Mitochondrial DNA and Geographical Distribution

PubMed Central

Higuchi, Shoko; Sakamaki, Tetsuya; Hart, John A.; Hart, Terese B.; Tokuyama, Nahoko; Reinartz, Gay E.; Guislain, Patrick; Dupain, Jef; Cobden, Amy K.; Mulavwa, Mbangi N.; Yangozene, Kumugo; Darroze, Serge; Devos, Céline; Furuichi, Takeshi

2013-01-01

Bonobos (Pan paniscus) inhabit regions south of the Congo River including all areas between its southerly tributaries. To investigate the genetic diversity and evolutionary relationship among bonobo populations, we sequenced mitochondrial DNA from 376 fecal samples collected in seven study populations located within the eastern and western limits of the species’ range. In 136 effective samples from different individuals (range: 7–37 per population), we distinguished 54 haplotypes in six clades (A1, A2, B1, B2, C, D), which included a newly identified clade (D). MtDNA haplotypes were regionally clustered; 83 percent of haplotypes were locality-specific. The distribution of haplotypes across populations and the genetic diversity within populations thus showed highly geographical patterns. Using population distance measures, seven populations were categorized in three clusters: the east, central, and west cohorts. Although further elucidation of historical changes in the geological setting is required, the geographical patterns of genetic diversity seem to be shaped by paleoenvironmental changes during the Pleistocene. The present day riverine barriers appeared to have a weak effect on gene flow among populations, except for the Lomami River, which separates the TL2 population from the others. The central cohort preserves a high genetic diversity, and two unique clades of haplotypes were found in the Wamba/Iyondji populations in the central cohort and in the TL2 population in the eastern cohort respectively. This knowledge may contribute to the planning of bonobo conservation. PMID:23544084

DNA methylation levels in candidate genes associated with chronological age in mammals are not conserved in a long-lived seabird

PubMed Central

Polanowski, Andrea M.; McMahon, Clive; Deagle, Bruce E.; Dickinson, Joanne L.; Hindell, Mark A.; Jarman, Simon N.

2017-01-01

Most seabirds do not have any outward identifiers of their chronological age, so estimation of seabird population age structure generally requires expensive, long-term banding studies. We investigated the potential to use a molecular age biomarker to estimate age in short-tailed shearwaters (Ardenna tenuirostris). We quantified DNA methylation in several A. tenuirostris genes that have shown age-related methylation changes in mammals. In birds ranging from chicks to 21 years of age, bisulphite treated blood and feather DNA was sequenced and methylation levels analysed in 67 CpG sites in 13 target gene regions. From blood samples, five of the top relationships with age were identified in KCNC3 loci (CpG66: R2 = 0.325, p = 0.019). In feather samples ELOVL2 (CpG42: R2 = 0.285, p = 0.00048) and EDARADD (CpG46: R2 = 0.168, p = 0.0067) were also weakly correlated with age. However, the majority of markers had no clear association with age (of 131 comparisons only 12 had a p-value < 0.05) and statistical analysis using a penalised lasso approach did not produce an accurate ageing model. Our data indicate that some age-related signatures identified in orthologous mammalian genes are not conserved in the long-lived short tailed shearwater. Alternative molecular approaches will be required to identify a reliable biomarker of chronological age in these seabirds. PMID:29216256

Design and analysis of linear cascade DNA hybridization chain reactions using DNA hairpins

NASA Astrophysics Data System (ADS)

Bui, Hieu; Garg, Sudhanshu; Miao, Vincent; Song, Tianqi; Mokhtar, Reem; Reif, John

2017-01-01

DNA self-assembly has been employed non-conventionally to construct nanoscale structures and dynamic nanoscale machines. The technique of hybridization chain reactions by triggered self-assembly has been shown to form various interesting nanoscale structures ranging from simple linear DNA oligomers to dendritic DNA structures. Inspired by earlier triggered self-assembly works, we present a system for controlled self-assembly of linear cascade DNA hybridization chain reactions using nine distinct DNA hairpins. NUPACK is employed to assist in designing DNA sequences and Matlab has been used to simulate DNA hairpin interactions. Gel electrophoresis and ensemble fluorescence reaction kinetics data indicate strong evidence of linear cascade DNA hybridization chain reactions. The half-time completion of the proposed linear cascade reactions indicates a linear dependency on the number of hairpins.

DNA methylation profiling using HpaII tiny fragment enrichment by ligation-mediated PCR (HELP)

PubMed Central

Suzuki, Masako; Greally, John M.

2010-01-01

The HELP assay is a technique that allows genome-wide analysis of cytosine methylation. Here we describe the assay, its relative strengths and weaknesses, and the transition of the assay from a microarray to massively-parallel sequencing-based foundation. PMID:20434563

Characterization of the geometry and topology of DNA pictured as a discrete collection of atoms

PubMed Central

Olson, Wilma K.

2014-01-01

The structural and physical properties of DNA are closely related to its geometry and topology. The classical mathematical treatment of DNA geometry and topology in terms of ideal smooth space curves was not designed to characterize the spatial arrangements of atoms found in high-resolution and simulated double-helical structures. We present here new and rigorous numerical methods for the rapid and accurate assessment of the geometry and topology of double-helical DNA structures in terms of the constituent atoms. These methods are well designed for large DNA datasets obtained in detailed numerical simulations or determined experimentally at high-resolution. We illustrate the usefulness of our methodology by applying it to the analysis of three canonical double-helical DNA chains, a 65-bp minicircle obtained in recent molecular dynamics simulations, and a crystallographic array of protein-bound DNA duplexes. Although we focus on fully base-paired DNA structures, our methods can be extended to treat the geometry and topology of melted DNA structures as well as to characterize the folding of arbitrary molecules such as RNA and cyclic peptides. PMID:24791158

Structure of human DNA polymerase iota and the mechanism of DNA synthesis.

PubMed

Makarova, A V; Kulbachinskiy, A V

2012-06-01

Cellular DNA polymerases belong to several families and carry out different functions. Highly accurate replicative DNA polymerases play the major role in cell genome replication. A number of new specialized DNA polymerases were discovered at the turn of XX-XXI centuries and have been intensively studied during the last decade. Due to the special structure of the active site, these enzymes efficiently perform synthesis on damaged DNA but are characterized by low fidelity. Human DNA polymerase iota (Pol ι) belongs to the Y-family of specialized DNA polymerases and is one of the most error-prone enzymes involved in DNA synthesis. In contrast to other DNA polymerases, Pol ι is able to use noncanonical Hoogsteen interactions for nucleotide base pairing. This allows it to incorporate nucleotides opposite various lesions in the DNA template that impair Watson-Crick interactions. Based on the data of X-ray structural analysis of Pol ι in complexes with various DNA templates and dNTP substrates, we consider the structural peculiarities of the Pol ι active site and discuss possible mechanisms that ensure the unique behavior of the enzyme on damaged and undamaged DNA.

Effects of seminal plasma and flash-freezing on DNA structure of stallion epididymal sperm exposed to different potentiators of DNA damage.

PubMed

Serafini, R; Varner, D D; Blanchard, T L; Teague, S R; LaCaze, K; Love, C C

2018-05-24

The tolerance of sperm DNA structure to seminal plasma and freezing conditions has both clinical and basic biologic relevance. In this study, fresh (FS) or flash-frozen (FZ) stallion epididymal sperm were exposed (SP + ) or unexposed (SP - ) to seminal plasma. Sperm were then evaluated to monitor the degree of change in DNA structure following challenge with chemical (dithiothreitol-DTT), oxidative (iron sulfate; FeSO 4 ) or enzymatic (DNase I) potentiators of DNA damage. For sperm not treated with potentiators (controls), there was no effect of SP treatment (SP - vs. SP + ) or freezing treatment (FS vs. FZ; non-significant) on measures of any DNA assays (i.e., 8-hydroxy, 2'deoxyguanosine [8OHdG], TUNEL, or sperm chromatin structure [SCSA] assays). Group FZ was more susceptible than Group FS to potentiators of DNA damage. Percent 8OHdG-positive sperm was higher in Group FZ/SP - treated with FeSO 4 than all other groups (P < 0.05). Percent TUNEL-positive sperm was similar among FZ/SP - groups treated with DTT, FeSO 4 , or DNase (non-significant) and was higher in these groups than all other treatments (P < 0.05). Percent COMP-α t was higher following treatment with DNase or DTT, as compared to their respective controls, regardless of prior exposure to SP (P < 0.05). Overall, sperm DNA structure was unaffected by seminal plasma or freezing treatment when samples were not exposed to potentiators of sperm DNA damage; however, marked differences were identified in DNA structure when sperm were challenged with chemical, oxidative or enzymatic treatments. These results highlight the importance of challenging DNA structure prior to analysis. The use of potentiators of DNA damage provided a model to evaluate sperm DNA structure following exposure of sperm to various experimental treatments. Copyright © 2018 Elsevier Inc. All rights reserved.

From Structure-Function Analyses to Protein Engineering for Practical Applications of DNA Ligase

PubMed Central

Tanabe, Maiko; Nishida, Hirokazu

2015-01-01

DNA ligases are indispensable in all living cells and ubiquitous in all organs. DNA ligases are broadly utilized in molecular biology research fields, such as genetic engineering and DNA sequencing technologies. Here we review the utilization of DNA ligases in a variety of in vitro gene manipulations, developed over the past several decades. During this period, fewer protein engineering attempts for DNA ligases have been made, as compared to those for DNA polymerases. We summarize the recent progress in the elucidation of the DNA ligation mechanisms obtained from the tertiary structures solved thus far, in each step of the ligation reaction scheme. We also present some examples of engineered DNA ligases, developed from the viewpoint of their three-dimensional structures. PMID:26508902

Dna2 nuclease-helicase structure, mechanism and regulation by Rpa

PubMed Central

Zhou, Chun; Pourmal, Sergei; Pavletich, Nikola P

2015-01-01

The Dna2 nuclease-helicase maintains genomic integrity by processing DNA double-strand breaks, Okazaki fragments and stalled replication forks. Dna2 requires ssDNA ends, and is dependent on the ssDNA-binding protein Rpa, which controls cleavage polarity. Here we present the 2.3 Å structure of intact mouse Dna2 bound to a 15-nucleotide ssDNA. The nuclease active site is embedded in a long, narrow tunnel through which the DNA has to thread. The helicase domain is required for DNA binding but not threading. We also present the structure of a flexibly-tethered Dna2-Rpa interaction that recruits Dna2 to Rpa-coated DNA. We establish that a second Dna2-Rpa interaction is mutually exclusive with Rpa-DNA interactions and mediates the displacement of Rpa from ssDNA. This interaction occurs at the nuclease tunnel entrance and the 5’ end of the Rpa-DNA complex. Hence, it only displaces Rpa from the 5’ but not 3’ end, explaining how Rpa regulates cleavage polarity. DOI: http://dx.doi.org/10.7554/eLife.09832.001 PMID:26491943

Crystal Structure of the Chromodomain Helicase DNA-binding Protein 1 (Chd1) DNA-binding Domain in Complex with DNA*

PubMed Central

Sharma, Amit; Jenkins, Katherine R.; Héroux, Annie; Bowman, Gregory D.

2011-01-01

Chromatin remodelers are ATP-dependent machines that dynamically alter the chromatin packaging of eukaryotic genomes by assembling, sliding, and displacing nucleosomes. The Chd1 chromatin remodeler possesses a C-terminal DNA-binding domain that is required for efficient nucleosome sliding and believed to be essential for sensing the length of DNA flanking the nucleosome core. The structure of the Chd1 DNA-binding domain was recently shown to consist of a SANT and SLIDE domain, analogous to the DNA-binding domain of the ISWI family, yet the details of how Chd1 recognized DNA were not known. Here we present the crystal structure of the Saccharomyces cerevisiae Chd1 DNA-binding domain in complex with a DNA duplex. The bound DNA duplex is straight, consistent with the preference exhibited by the Chd1 DNA-binding domain for extranucleosomal DNA. Comparison of this structure with the recently solved ISW1a DNA-binding domain bound to DNA reveals that DNA lays across each protein at a distinct angle, yet contacts similar surfaces on the SANT and SLIDE domains. In contrast to the minor groove binding seen for Isw1 and predicted for Chd1, the SLIDE domain of the Chd1 DNA-binding domain contacts the DNA major groove. The majority of direct contacts with the phosphate backbone occur only on one DNA strand, suggesting that Chd1 may not strongly discriminate between major and minor grooves. PMID:22033927

From structure to mechanism-understanding initiation of DNA replication.

PubMed

Riera, Alberto; Barbon, Marta; Noguchi, Yasunori; Reuter, L Maximilian; Schneider, Sarah; Speck, Christian

2017-06-01

DNA replication results in the doubling of the genome prior to cell division. This process requires the assembly of 50 or more protein factors into a replication fork. Here, we review recent structural and biochemical insights that start to explain how specific proteins recognize DNA replication origins, load the replicative helicase on DNA, unwind DNA, synthesize new DNA strands, and reassemble chromatin. We focus on the minichromosome maintenance (MCM2-7) proteins, which form the core of the eukaryotic replication fork, as this complex undergoes major structural rearrangements in order to engage with DNA, regulate its DNA-unwinding activity, and maintain genome stability. © 2017 Riera et al.; Published by Cold Spring Harbor Laboratory Press.

Structure-function correlation of chloroquine and analogues as transgene expression enhancers in nonviral gene delivery.

PubMed

Cheng, Jianjun; Zeidan, Ryan; Mishra, Swaroop; Liu, Aijie; Pun, Suzie H; Kulkarni, Rajan P; Jensen, Gregory S; Bellocq, Nathalie C; Davis, Mark E

2006-11-02

To understand how chloroquine (CQ) enhances transgene expression in polycation-based, nonviral gene delivery systems, a number of CQ analogues with variations in the aliphatic amino side chain or in the aromatic ring are synthesized and investigated. Our studies indicate that the aliphatic amino moiety of CQ is essential to provide increased gene expression. Further, the enhancements are more dramatically affected by changes to the aromatic ring and are positively correlated to the strength of intercalation between DNA and the CQ analogues. Quinacrine (QC), a CQ analogue with a fused acridinyl structure that can strongly intercalate DNA, enhances transfection similarly to CQ at a concentration 10 times lower, while N(4)-(4-pyridinyl)-N(1),N(1)-diethyl-1,4-pentanediamine (CP), a CQ analogue that has a weakly intercalating pyridinyl ring, shows no effect on gene expression. Subtle change on the 7-substituent of the chloroquine aromatic structure can also greatly affect the ability of the CQ analogues to enhance transgene expression. Transfection in the presence of N(4)-(7-trifluoromethyl-4-quinolinyl)-N(1),N(1)-diethyl-1,4-pentanediamin e (CQ7a) shows expression efficiency 10 times higher than in the presence of CQ at same concentration, while transfection in the presence of N(4)-(4-quinolinyl)-N(1),N(1)-diethyl-1,4-pentanediamine (CQ7b) does not reveal any enhancing effects on expression. Through a number of comparative studies with CQ and its analogues, we conclude that there are at least three mechanistic features of CQ that lead to the enhancement in gene expression: (i) pH buffering in endocytic vesicles, (ii) displacement of polycations from the nucleic acids in polyplexes, and (iii) alteration of the biophysical properties of the released nucleic acid.

Structure solution of DNA-binding proteins and complexes with ARCIMBOLDO libraries

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

Pröpper, Kevin; Instituto de Biologia Molecular de Barcelona; Meindl, Kathrin

2014-06-01

The structure solution of DNA-binding protein structures and complexes based on the combination of location of DNA-binding protein motif fragments with density modification in a multi-solution frame is described. Protein–DNA interactions play a major role in all aspects of genetic activity within an organism, such as transcription, packaging, rearrangement, replication and repair. The molecular detail of protein–DNA interactions can be best visualized through crystallography, and structures emphasizing insight into the principles of binding and base-sequence recognition are essential to understanding the subtleties of the underlying mechanisms. An increasing number of high-quality DNA-binding protein structure determinations have been witnessed despite themore » fact that the crystallographic particularities of nucleic acids tend to pose specific challenges to methods primarily developed for proteins. Crystallographic structure solution of protein–DNA complexes therefore remains a challenging area that is in need of optimized experimental and computational methods. The potential of the structure-solution program ARCIMBOLDO for the solution of protein–DNA complexes has therefore been assessed. The method is based on the combination of locating small, very accurate fragments using the program Phaser and density modification with the program SHELXE. Whereas for typical proteins main-chain α-helices provide the ideal, almost ubiquitous, small fragments to start searches, in the case of DNA complexes the binding motifs and DNA double helix constitute suitable search fragments. The aim of this work is to provide an effective library of search fragments as well as to determine the optimal ARCIMBOLDO strategy for the solution of this class of structures.« less

DNA Based Molecular Scale Nanofabrication

DTIC Science & Technology

2015-12-04

structure. We developed a method to produce nanoscale patterns on SAM. (d) Studied the molecular imprinting of DNA origami structure using polymer...to produce nanoscale patterns on SAM. (d) Studied the molecular imprinting of DNA origami structure using polymer substrates. Developed a high... imprinting using DNA nanostructure templates. Soft lithography uses polymeric stamps with certain features to transfer the pattern for printing

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.

DNA Origami Reorganizes upon Interaction with Graphite: Implications for High-Resolution DNA Directed Protein Patterning

PubMed Central

Rahman, Masudur; Neff, David; Green, Nathaniel; Norton, Michael L.

2016-01-01

Although there is a long history of the study of the interaction of DNA with carbon surfaces, limited information exists regarding the interaction of complex DNA-based nanostructures with the important material graphite, which is closely related to graphene. In view of the capacity of DNA to direct the assembly of proteins and optical and electronic nanoparticles, the potential for combining DNA-based materials with graphite, which is an ultra-flat, conductive carbon substrate, requires evaluation. A series of imaging studies utilizing Atomic Force Microscopy has been applied in order to provide a unified picture of this important interaction of structured DNA and graphite. For the test structure examined, we observe a rapid destabilization of the complex DNA origami structure, consistent with a strong interaction of single-stranded DNA with the carbon surface. This destabilizing interaction can be obscured by an intentional or unintentional primary intervening layer of single-stranded DNA. Because the interaction of origami with graphite is not completely dissociative, and because the frustrated, expanded structure is relatively stable over time in solution, it is demonstrated that organized structures of pairs of the model protein streptavidin can be produced on carbon surfaces using DNA origami as the directing material. PMID:28335324

Structural Organization and Strain Variation in the Genome of Varicella Zoster Virus

DTIC Science & Technology

1984-10-23

Zoster 6 Growth of VZV in tissue culture 9 Structure and proteins of VZV 15 Structure of HSV DNA 20 Classification of herpesviruses based on DNA...structure 28 Strain variation in herpesvirus DNA 31 VZV DNA 33 Specific aims 36 II. MATERIALS AND METHODS 38 Cells and viruses 38 Isolation of virus...endonuclease fragments by colony hybridization 106 21. Selected methods of restriction endonuclease mapping .... 109 22. Identification of

Structure of fluorescent metal clusters on a DNA template.

NASA Astrophysics Data System (ADS)

Vdovichev, A. A.; Sych, T. S.; Reveguk, Z. V.; Smirnova, A. A.; Maksimov, D. A.; Ramazanov, R. R.; Kononov, A. I.

2016-08-01

Luminescent metal clusters are a subject of growing interest in recent years due to their bright emission from visible to near infrared range. Detailed structure of the fluorescent complexes of Ag and other metal clusters with ligands still remains a challenging task. In this joint experimental and theoretical study we synthesized Ag-DNA complexes on a DNA oligonucleotide emitting in violet- green spectral range. The structure of DNA template was determined by means of various spectral measurements (CD, MS, XPS). Comparison of the experimental fluorescent excitation spectra and calculated absorption spectra for different QM/MM optimized structures allowed us to determine the detailed structure of the green cluster containing three silver atoms in the stem of the DNA hairpin structure stabilized by cytosine-Ag+-cytosine bonds.

Aldehydes with high and low toxicities inactivate cells by damaging distinct cellular targets.

PubMed

Xie, Ming-Zhang; Shoulkamy, Mahmoud I; Salem, Amir M H; Oba, Shunya; Goda, Mizuki; Nakano, Toshiaki; Ide, Hiroshi

2016-04-01

Aldehydes are genotoxic and cytotoxic molecules and have received considerable attention for their associations with the pathogenesis of various human diseases. In addition, exposure to anthropogenic aldehydes increases human health risks. The general mechanism of aldehyde toxicity involves adduct formation with biomolecules such as DNA and proteins. Although the genotoxic effects of aldehydes such as mutations and chromosomal aberrations are directly related to DNA damage, the role of DNA damage in the cytotoxic effects of aldehydes is poorly understood because concurrent protein damage by aldehydes has similar effects. In this study, we have analysed how saturated and α,β-unsaturated aldehydes exert cytotoxic effects through DNA and protein damage. Interestingly, DNA repair is essential for alleviating the cytotoxic effect of weakly toxic aldehydes such as saturated aldehydes but not highly toxic aldehydes such as long α,β-unsaturated aldehydes. Thus, highly toxic aldehydes inactivate cells exclusively by protein damage. Our data suggest that DNA interstrand crosslinks, but not DNA-protein crosslinks and DNA double-strand breaks, are the critical cytotoxic DNA damage induced by aldehydes. Further, we show that the depletion of intracellular glutathione and the oxidation of thioredoxin 1 partially account for the DNA damage-independent cytotoxicity of aldehydes. On the basis of these findings, we have proposed a mechanistic model of aldehyde cytotoxicity mediated by DNA and protein damage. Copyright © 2016 Elsevier B.V. All rights reserved.

No Evidence for Infection of UK Prostate Cancer Patients with XMRV, BK Virus, Trichomonas vaginalis or Human Papilloma Viruses

PubMed Central

Groom, Harriet C. T.; Warren, Anne Y.; Neal, David E.; Bishop, Kate N.

2012-01-01

The prevalence of specific infections in UK prostate cancer patients was investigated. Serum from 84 patients and 62 controls was tested for neutralisation of xenotropic murine leukaemia virus-related virus (XMRV) Envelope. No reactivity was found in the patient samples. In addition, a further 100 prostate DNA samples were tested for XMRV, BK virus, Trichomonas vaginalis and human papilloma viruses by nucleic acid detection techniques. Despite demonstrating DNA integrity and assay sensitivity, we failed to detect the presence of any of these agents in DNA samples, bar one sample that was weakly positive for HPV16. Therefore we conclude that these infections are absent in this typical cohort of men with prostate cancer. PMID:22470540

No evidence for infection of UK prostate cancer patients with XMRV, BK virus, Trichomonas vaginalis or human papilloma viruses.

PubMed

Groom, Harriet C T; Warren, Anne Y; Neal, David E; Bishop, Kate N

2012-01-01

The prevalence of specific infections in UK prostate cancer patients was investigated. Serum from 84 patients and 62 controls was tested for neutralisation of xenotropic murine leukaemia virus-related virus (XMRV) Envelope. No reactivity was found in the patient samples. In addition, a further 100 prostate DNA samples were tested for XMRV, BK virus, Trichomonas vaginalis and human papilloma viruses by nucleic acid detection techniques. Despite demonstrating DNA integrity and assay sensitivity, we failed to detect the presence of any of these agents in DNA samples, bar one sample that was weakly positive for HPV16. Therefore we conclude that these infections are absent in this typical cohort of men with prostate cancer.

'Mitominis': multiplex PCR analysis of reduced size amplicons for compound sequence analysis of the entire mtDNA control region in highly degraded samples.

PubMed

Eichmann, Cordula; Parson, Walther

2008-09-01

The traditional protocol for forensic mitochondrial DNA (mtDNA) analyses involves the amplification and sequencing of the two hypervariable segments HVS-I and HVS-II of the mtDNA control region. The primers usually span fragment sizes of 300-400 bp each region, which may result in weak or failed amplification in highly degraded samples. Here we introduce an improved and more stable approach using shortened amplicons in the fragment range between 144 and 237 bp. Ten such amplicons were required to produce overlapping fragments that cover the entire human mtDNA control region. These were co-amplified in two multiplex polymerase chain reactions and sequenced with the individual amplification primers. The primers were carefully selected to minimize binding on homoplasic and haplogroup-specific sites that would otherwise result in loss of amplification due to mis-priming. The multiplexes have successfully been applied to ancient and forensic samples such as bones and teeth that showed a high degree of degradation.

Mechanism of inhibition of adenovirus DNA replication by the acyclic nucleoside triphosphate analogue (S)-HPMPApp: influence of the adenovirus DNA binding protein.

PubMed Central

Mul, Y M; van Miltenburg, R T; De Clercq, E; van der Vliet, P C

1989-01-01

The acyclic adenosine analogue (S)-9-(3-hydroxy-2-phosphonylmethoxypropyl)adenine [S]-HPMPA) is a potent and selective inhibitor of adenovirus (Ad) replication in cell culture. We studied the mechanism of inhibition using a reconstituted in vitro DNA replication system. The diphosphoryl derivative (S)-HPMPApp, but not (S)-HPMPA, inhibited the DNA replication of origin containing fragments strongly. The inhibitory effect was exerted at the level of elongation, while initiation was resistant to the drug. Remarkably, the elongation of short strands was only slightly impaired, while inhibition was maximal upon synthesis of long DNA fragments. (S)-HPMPApp appeared to be competitive with dATP, suggesting that the Ad DNA polymerase is the prime target for the drug. We purified the Ad DNA polymerase in complex to the precursor terminal protein to homogeneity from cells infected with overproducing recombinant vaccinia viruses. Employing gapped DNA or poly(dT).oligo(dA) templates, only a weak inhibition was observed. However, inhibition was strongly enhanced in the presence of the adenovirus DNA binding protein (DBP). We interpret this to mean that the increased processivity of the polymerization reaction in the presence of DBP leads to increased drug sensitivity. Images PMID:2587248

Detection of bacterial DNA by PCR in dogs with stifle pathology.

PubMed

Bhandal, Jitender; Hayashi, Kei; Kim, Sun-Young; Klein, Martha; Wong, Alice; Toupadakis, Chrisoula A; Muir, Peter; Yellowley, Clare E

2013-10-01

To determine presence of bacterial DNA in canine stifles with cranial cruciate ligament rupture (CCLR) and medial patellar luxation (MPL) compared to normal canine stifles (control). Prospective clinical study. Dogs (n = 44). Dogs of varying age, breed, sex, and weight residing in California were assessed for stifle pathology (CCLR, MPL, or normal control). Synovial fluid of all stifles was assessed for the presence of bacterial DNA using broad-ranging 16S rRNA primers and PCR. Bacterial DNA was detected in normal control stifles and those with CCLR and MPL. There were no statistical differences in the copy numbers of bacterial DNA in the stifle synovial fluid among groups (P > .05); however, synovial fluid specimens from dogs with stifle pathology (CCLR and MPL combined) tended to have higher copy numbers of bacterial DNA than those from controls (P = .06). There was no significant difference in the number of bacterial DNA between the CCLR and MPL groups (P = .57). The copy numbers of bacterial DNA had a weak positive significant correlation with the duration of lameness in CCLR group (P < .05). Increased detection of bacterial DNA in the stifle synovial fluid may indicate joint pathology but not be directly linked to a specific joint disease. © Copyright 2013 by The American College of Veterinary Surgeons.

Cerium chloride stimulated controlled conversion of B-to-Z DNA in self-assembled nanostructures

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

Bhanjadeo, Madhabi M.; Academy of Scientific & Innovative Research; Nayak, Ashok K.

DNA adopts different conformation not only because of novel base pairs but also while interacting with inorganic or organic compounds. Self-assembled branched DNA (bDNA) structures or DNA origami that change conformation in response to environmental cues hold great promises in sensing and actuation at the nanoscale. Recently, the B-Z transition in DNA is being explored to design various nanomechanical devices. In this communication we have demonstrated that Cerium chloride binds to the phosphate backbone of self-assembled bDNA structure and induce B-to-Z transition at physiological concentration. The mechanism of controlled conversion from right-handed to left-handed has been assayed by various dyemore » binding studies using CD and fluorescence spectroscopy. Three different bDNA structures have been identified to display B-Z transition. This approach provides a rapid and reversible means to change bDNA conformation, which can be used for dynamic and progressive control at the nanoscale. - Highlights: • Cerium-induced B-to-Z DNA transition in self-assembled nanostructures. • Lower melting temperature of Z-DNA than B-DNA confirmed by CD spectroscopy. • Binding mechanism of cerium chloride is explained using fluorescence spectroscopy. • Right-handed to left-handed DNA conformation is also noticed in modified bDNA structure.« less

Imprints from genetic drift and mutation imply relative divergence times across marine transition zones in a pan-European small pelagic fish (Sprattus sprattus).

PubMed

Limborg, M T; Hanel, R; Debes, P V; Ring, A K; André, C; Tsigenopoulos, C S; Bekkevold, D

2012-08-01

Geographic distributions of most temperate marine fishes are affected by postglacial recolonisation events, which have left complex genetic imprints on populations of marine species. This study investigated population structure and demographic history of European sprat (Sprattus sprattus L.) by combining inference from both mtDNA and microsatellite genetic markers throughout the species' distribution. We compared effects from genetic drift and mutation for both genetic markers in shaping genetic differentiation across four transition zones. Microsatellite markers revealed significant isolation by distance and a complex population structure across the species' distribution (overall θ(ST)=0.038, P<0.01). Across transition zones markers indicated larger effects of genetic drift over mutations in the northern distribution of sprat contrasting a stronger relative impact of mutation in the species' southern distribution in the Mediterranean region. These results were interpreted to reflect more recent divergence times between northern populations in accordance with previous findings. This study demonstrates the usefulness of comparing inference from different markers and estimators of divergence for phylogeographic and population genetic studies in species with weak genetic structure, as is the case in many marine species.

High Molecular Weight Forms of Mammalian Respiratory Chain Complex II

PubMed Central

Nůsková, Hana; Holzerová, Eliška; Vrbacký, Marek; Pecina, Petr; Hejzlarová, Kateřina; Kľučková, Katarína; Rohlena, Jakub; Neuzil, Jiri; Houštěk, Josef

2013-01-01

Mitochondrial respiratory chain is organised into supramolecular structures that can be preserved in mild detergent solubilisates and resolved by native electrophoretic systems. Supercomplexes of respiratory complexes I, III and IV as well as multimeric forms of ATP synthase are well established. However, the involvement of complex II, linking respiratory chain with tricarboxylic acid cycle, in mitochondrial supercomplexes is questionable. Here we show that digitonin-solubilised complex II quantitatively forms high molecular weight structures (CIIhmw) that can be resolved by clear native electrophoresis. CIIhmw structures are enzymatically active and differ in electrophoretic mobility between tissues (500 – over 1000 kDa) and cultured cells (400–670 kDa). While their formation is unaffected by isolated defects in other respiratory chain complexes, they are destabilised in mtDNA-depleted, rho0 cells. Molecular interactions responsible for the assembly of CIIhmw are rather weak with the complexes being more stable in tissues than in cultured cells. While electrophoretic studies and immunoprecipitation experiments of CIIhmw do not indicate specific interactions with the respiratory chain complexes I, III or IV or enzymes of the tricarboxylic acid cycle, they point out to a specific interaction between CII and ATP synthase. PMID:23967256

The Crystal Structure of TAL Effector PthXo1 Bound to Its DNA Target

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

Mak, Amanda Nga-Sze; Bradley, Philip; Cernadas, Raul A.

2012-02-10

DNA recognition by TAL effectors is mediated by tandem repeats, each 33 to 35 residues in length, that specify nucleotides via unique repeat-variable diresidues (RVDs). The crystal structure of PthXo1 bound to its DNA target was determined by high-throughput computational structure prediction and validated by heavy-atom derivatization. Each repeat forms a left-handed, two-helix bundle that presents an RVD-containing loop to the DNA. The repeats self-associate to form a right-handed superhelix wrapped around the DNA major groove. The first RVD residue forms a stabilizing contact with the protein backbone, while the second makes a base-specific contact to the DNA sense strand.more » Two degenerate amino-terminal repeats also interact with the DNA. Containing several RVDs and noncanonical associations, the structure illustrates the basis of TAL effector-DNA recognition.« less

Combining crystallography and EPR: crystal and solution structures of the multidomain cochaperone DnaJ

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

Barends, Thomas R. M., E-mail: thomas.barends@mpimf-heidelberg.mpg.de; Brosi, Richard W. W.; Steinmetz, Andrea

2013-08-01

The crystal structure of the N-terminal part of T. thermophilus DnaJ unexpectedly showed an ordered GF domain and guided the design of a construct enabling the first structure determination of a complete DnaJ cochaperone molecule. By combining the crystal structures with spin-labelling EPR and cross-linking in solution, a dynamic view of this flexible molecule was developed. Hsp70 chaperones assist in a large variety of protein-folding processes in the cell. Crucial for these activities is the regulation of Hsp70 by Hsp40 cochaperones. DnaJ, the bacterial homologue of Hsp40, stimulates ATP hydrolysis by DnaK (Hsp70) and thus mediates capture of substrate protein,more » but is also known to possess chaperone activity of its own. The first structure of a complete functional dimeric DnaJ was determined and the mobility of its individual domains in solution was investigated. Crystal structures of the complete molecular cochaperone DnaJ from Thermus thermophilus comprising the J, GF and C-terminal domains and of the J and GF domains alone showed an ordered GF domain interacting with the J domain. Structure-based EPR spin-labelling studies as well as cross-linking results showed the existence of multiple states of DnaJ in solution with different arrangements of the various domains, which has implications for the function of DnaJ.« less

Structural stability of DNA origami nanostructures in the presence of chaotropic agents

NASA Astrophysics Data System (ADS)

Ramakrishnan, Saminathan; Krainer, Georg; Grundmeier, Guido; Schlierf, Michael; Keller, Adrian

2016-05-01

DNA origami represent powerful platforms for single-molecule investigations of biomolecular processes. The required structural integrity of the DNA origami may, however, pose significant limitations regarding their applicability, for instance in protein folding studies that require strongly denaturing conditions. Here, we therefore report a detailed study on the stability of 2D DNA origami triangles in the presence of the strong chaotropic denaturing agents urea and guanidinium chloride (GdmCl) and its dependence on concentration and temperature. At room temperature, the DNA origami triangles are stable up to at least 24 h in both denaturants at concentrations as high as 6 M. At elevated temperatures, however, structural stability is governed by variations in the melting temperature of the individual staple strands. Therefore, the global melting temperature of the DNA origami does not represent an accurate measure of their structural stability. Although GdmCl has a stronger effect on the global melting temperature, its attack results in less structural damage than observed for urea under equivalent conditions. This enhanced structural stability most likely originates from the ionic nature of GdmCl. By rational design of the arrangement and lengths of the individual staple strands used for the folding of a particular shape, however, the structural stability of DNA origami may be enhanced even further to meet individual experimental requirements. Overall, their high stability renders DNA origami promising platforms for biomolecular studies in the presence of chaotropic agents, including single-molecule protein folding or structural switching.DNA origami represent powerful platforms for single-molecule investigations of biomolecular processes. The required structural integrity of the DNA origami may, however, pose significant limitations regarding their applicability, for instance in protein folding studies that require strongly denaturing conditions. Here, we therefore report a detailed study on the stability of 2D DNA origami triangles in the presence of the strong chaotropic denaturing agents urea and guanidinium chloride (GdmCl) and its dependence on concentration and temperature. At room temperature, the DNA origami triangles are stable up to at least 24 h in both denaturants at concentrations as high as 6 M. At elevated temperatures, however, structural stability is governed by variations in the melting temperature of the individual staple strands. Therefore, the global melting temperature of the DNA origami does not represent an accurate measure of their structural stability. Although GdmCl has a stronger effect on the global melting temperature, its attack results in less structural damage than observed for urea under equivalent conditions. This enhanced structural stability most likely originates from the ionic nature of GdmCl. By rational design of the arrangement and lengths of the individual staple strands used for the folding of a particular shape, however, the structural stability of DNA origami may be enhanced even further to meet individual experimental requirements. Overall, their high stability renders DNA origami promising platforms for biomolecular studies in the presence of chaotropic agents, including single-molecule protein folding or structural switching. Electronic supplementary information (ESI) available: Melting curves without baseline subtraction, AFM images of DNA origami after 24 h incubation, calculated melting temperatures of all staple strands. See DOI: 10.1039/c6nr00835f

The N-Terminal Domain of Human DNA Helicase Rtel1 Contains a Redox Active Iron-Sulfur Cluster

PubMed Central

Landry, Aaron P.

2014-01-01

Human telomere length regulator Rtel1 is a superfamily II DNA helicase and is essential for maintaining proper length of telomeres in chromosomes. Here we report that the N-terminal domain of human Rtel1 (RtelN) expressed in Escherichia coli cells produces a protein that contains a redox active iron-sulfur cluster with the redox midpoint potential of −248 ± 10 mV (pH 8.0). The iron-sulfur cluster in RtelN is sensitive to hydrogen peroxide and nitric oxide, indicating that reactive oxygen/nitrogen species may modulate the DNA helicase activity of Rtel1 via modification of its iron-sulfur cluster. Purified RtelN retains a weak binding affinity for the single-stranded (ss) and double-stranded (ds) DNA in vitro. However, modification of the iron-sulfur cluster by hydrogen peroxide or nitric oxide does not significantly affect the DNA binding activity of RtelN, suggesting that the iron-sulfur cluster is not directly involved in the DNA interaction in the N-terminal domain of Rtel1. PMID:25147792

The effect of conjugation to gold nanoparticles on the ability of low molecular weight chitosan to transfer DNA vaccine.

PubMed

Zhou, Xianfeng; Zhang, Xizhen; Yu, Xianghui; Zha, Xiao; Fu, Qiuan; Liu, Bin; Wang, Xueyun; Chen, Yan; Chen, Yue; Shan, Yaming; Jin, Yinghua; Wu, Yongge; Liu, Junqiu; Kong, Wei; Shen, Jiacong

2008-01-01

Nonviral gene delivery systems based on conventional high molecular weight chitosans are efficient as DNA vaccine delivery system, but have poor physical properties such as aggregated shapes, low solubility at neutral pH, high viscosity at concentrations used for in vivo delivery and a slow onset of action. Furthermore, Chitosan oligomers shorter than 14 monomers units were recently found to form only weak complexes with DNA, resulting in physically unstable polyplexes in vitro and in vivo. Here, low molecular weight chitosans with an average molecular mass of 6kDa (Chito6) have been covalently attached to gold nanoparticles (GNPs), and the potency of the resulting Chito6-GNPs conjugates as vectors for the delivery of plasmid DNA has been investigated in vitro and in vivo. After delivery by intramuscular immunization in BALB/c mice, the Chito6-GNPs conjugates induced an enhanced serum antibody response 10 times more potent than naked DNA vaccine. Additionally, in contrast to naked DNA, the Chito6-GNPs conjugates induced potent cytotoxic T lymphocyte responses at a low dose.

The N-terminal domain of human DNA helicase Rtel1 contains a redox active iron-sulfur cluster.

PubMed

Landry, Aaron P; Ding, Huangen

2014-01-01

Human telomere length regulator Rtel1 is a superfamily II DNA helicase and is essential for maintaining proper length of telomeres in chromosomes. Here we report that the N-terminal domain of human Rtel1 (RtelN) expressed in Escherichia coli cells produces a protein that contains a redox active iron-sulfur cluster with the redox midpoint potential of -248 ± 10 mV (pH 8.0). The iron-sulfur cluster in RtelN is sensitive to hydrogen peroxide and nitric oxide, indicating that reactive oxygen/nitrogen species may modulate the DNA helicase activity of Rtel1 via modification of its iron-sulfur cluster. Purified RtelN retains a weak binding affinity for the single-stranded (ss) and double-stranded (ds) DNA in vitro. However, modification of the iron-sulfur cluster by hydrogen peroxide or nitric oxide does not significantly affect the DNA binding activity of RtelN, suggesting that the iron-sulfur cluster is not directly involved in the DNA interaction in the N-terminal domain of Rtel1.

Current-voltage characteristics of double stranded versus single stranded DNA molecules

NASA Astrophysics Data System (ADS)

Hartzell, B.; Chen, Hong; Heremans, J. J.; McCord, B.; Soghomonian, V.

2004-03-01

Investigation of DNA conductivity has focused on the native, duplex structure, with controversial results. Here, we present the influence of the double-helical structure on charge transport through lambda DNA molecules. The current-voltage (I-V) characteristics of both disulfide-labeled double stranded DNA (dsDNA) and disulfide-labeled single stranded DNA (ssDNA) were measured. The ssDNA was formed from the dsDNA using two different methods for comparison purposes: a thermal/chemical denaturation and enzymatic digestion utilizing lambda exonuclease. Resulting I-V characteristics of both the double stranded and single stranded samples were close-to-linear when measured at room temperature. However, the ssDNA samples consistently gave conductivity values about two orders of magnitude smaller in amplitude. Our results suggest an integral relationship between the native structure of DNA with its stacked base pairs and the molecule's ability to support charge transport.(NSF NIRT 0103034)

Rhombic-Shaped Nanostructures and Mechanical Properties of 2D DNA Origami Constructed with Different Crossover/Nick Designs.

PubMed

Ma, Zhipeng; Huang, Yunfei; Park, Seongsu; Kawai, Kentaro; Kim, Do-Nyun; Hirai, Yoshikazu; Tsuchiya, Toshiyuki; Yamada, Hirofumi; Tabata, Osamu

2018-01-01

DNA origami methods enable the fabrication of various nanostructures and nanodevices, but their effective use depends on an understanding of their structural and mechanical properties and the effects of basic structural features. Frequency-modulation atomic force microscopy is introduced to directly characterize, in aqueous solution, the crossover regions of sets of 2D DNA origami based on different crossover/nick designs. Rhombic-shaped nanostructures formed under the influence of flexible crossovers placed between DNA helices are observed in DNA origami incorporating crossovers every 3, 4, or 6 DNA turns. The bending rigidity of crossovers is determined to be only one-third of that of the DNA helix, based on interhelical electrostatic forces reported elsewhere, and the measured pitches of the 3-turn crossover design rhombic-shaped nanostructures undergoing negligible bending. To evaluate the robustness of their structural integrity, they are intentionally and simultaneously stressed using force-controlled atomic force microscopy. DNA crossovers are verified to have a stabilizing effect on the structural robustness, while the nicks have an opposite effect. The structural and mechanical properties of DNA origami and the effects of crossovers and nicks revealed in this paper can provide information essential for the design of versatile DNA origami structures that exhibit specified and desirable properties. © 2017 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

Minimalist Approach to Complexity: Templating the Assembly of DNA Tile Structures with Sequentially Grown Input Strands.

PubMed

Lau, Kai Lin; Sleiman, Hanadi F

2016-07-26

Given its highly predictable self-assembly properties, DNA has proven to be an excellent template toward the design of functional materials. Prominent examples include the remarkable complexity provided by DNA origami and single-stranded tile (SST) assemblies, which require hundreds of unique component strands. However, in many cases, the majority of the DNA assembly is purely structural, and only a small "working area" needs to be aperiodic. On the other hand, extended lattices formed by DNA tile motifs require only a few strands; but they suffer from lack of size control and limited periodic patterning. To overcome these limitations, we adopt a templation strategy, where an input strand of DNA dictates the size and patterning of resultant DNA tile structures. To prepare these templating input strands, a sequential growth technique developed in our lab is used, whereby extended DNA strands of defined sequence and length may be generated simply by controlling their order of addition. With these, we demonstrate the periodic patterning of size-controlled double-crossover (DX) and triple-crossover (TX) tile structures, as well as intentionally designed aperiodicity of a DX tile structure. As such, we are able to prepare size-controlled DNA structures featuring aperiodicity only where necessary with exceptional economy and efficiency.

MDC1: The art of keeping things in focus.

PubMed

Jungmichel, Stephanie; Stucki, Manuel

2010-08-01

The chromatin structure is important for recognition and repair of DNA damage. Many DNA damage response proteins accumulate in large chromatin domains flanking sites of DNA double-strand breaks. The assembly of these structures-usually termed DNA damage foci-is primarily regulated by MDC1, a large nuclear mediator/adaptor protein that is composed of several distinct structural and functional domains. Here, we are summarizing the latest discoveries about the mechanisms by which MDC1 mediates DNA damage foci formation, and we are reviewing the considerable efforts taken to understand the functional implication of these structures.

Sequence-dependent nucleosome sliding in rotation-coupled and uncoupled modes revealed by molecular simulations

PubMed Central

Tan, Cheng; Takada, Shoji

2017-01-01

While nucleosome positioning on eukaryotic genome play important roles for genetic regulation, molecular mechanisms of nucleosome positioning and sliding along DNA are not well understood. Here we investigated thermally-activated spontaneous nucleosome sliding mechanisms developing and applying a coarse-grained molecular simulation method that incorporates both long-range electrostatic and short-range hydrogen-bond interactions between histone octamer and DNA. The simulations revealed two distinct sliding modes depending on the nucleosomal DNA sequence. A uniform DNA sequence showed frequent sliding with one base pair step in a rotation-coupled manner, akin to screw-like motions. On the contrary, a strong positioning sequence, the so-called 601 sequence, exhibits rare, abrupt transitions of five and ten base pair steps without rotation. Moreover, we evaluated the importance of hydrogen bond interactions on the sliding mode, finding that strong and weak bonds favor respectively the rotation-coupled and -uncoupled sliding movements. PMID:29194442

Salient Features of Endonuclease Platforms for Therapeutic Genome Editing.

PubMed

Certo, Michael T; Morgan, Richard A

2016-03-01

Emerging gene-editing technologies are nearing a revolutionary phase in genetic medicine: precisely modifying or repairing causal genetic defects. This may include any number of DNA sequence manipulations, such as knocking out a deleterious gene, introducing a particular mutation, or directly repairing a defective sequence by site-specific recombination. All of these edits can currently be achieved via programmable rare-cutting endonucleases to create targeted DNA breaks that can engage and exploit endogenous DNA repair pathways to impart site-specific genetic changes. Over the past decade, several distinct technologies for introducing site-specific DNA breaks have been developed, yet the different biological origins of these gene-editing technologies bring along inherent differences in parameters that impact clinical implementation. This review aims to provide an accessible overview of the various endonuclease-based gene-editing platforms, highlighting the strengths and weakness of each with respect to therapeutic applications.

Salient Features of Endonuclease Platforms for Therapeutic Genome Editing

PubMed Central

Certo, Michael T; Morgan, Richard A

2016-01-01

Emerging gene-editing technologies are nearing a revolutionary phase in genetic medicine: precisely modifying or repairing causal genetic defects. This may include any number of DNA sequence manipulations, such as knocking out a deleterious gene, introducing a particular mutation, or directly repairing a defective sequence by site-specific recombination. All of these edits can currently be achieved via programmable rare-cutting endonucleases to create targeted DNA breaks that can engage and exploit endogenous DNA repair pathways to impart site-specific genetic changes. Over the past decade, several distinct technologies for introducing site-specific DNA breaks have been developed, yet the different biological origins of these gene-editing technologies bring along inherent differences in parameters that impact clinical implementation. This review aims to provide an accessible overview of the various endonuclease-based gene-editing platforms, highlighting the strengths and weakness of each with respect to therapeutic applications. PMID:26796671

An ameliorative protocol for the quantification of purine 5',8-cyclo-2'-deoxynucleosides in oxidized DNA

NASA Astrophysics Data System (ADS)

Terzidis, Michael; Chatgilialoglu, Chryssostomos

2015-07-01

5',8-Cyclo-2'-deoxyadenosine (cdA) and 5',8-cyclo-2'-deoxyguanosine (cdG) are lesions resulting from hydroxyl radical (HO•) attack on the 5'H of the nucleoside sugar moiety and exist in both 5'R and 5'S diastereomeric forms. Increased levels of cdA and cdG are linked to Nucleotide Excision Repair mechanism deficiency and mutagenesis. Discrepancies in the damage measurements reported over recent years indicated the weakness of the actual protocols, in particular for ensuring the quantitative release of these lesions from the DNA sample and the appropriate method for their analysis. Herein we report the detailed revision leading to a cost-effective and efficient protocol for the DNA damage measurement, consisting of the nuclease benzonase and nuclease P1 enzymatic combination for DNA digestion followed by liquid chromatography isotope dilution tandem mass spectrometry analysis.

Association of health symptoms with low-level exposure to organophosphates, DNA damage, AChE activity, and occupational knowledge and practice among rice, corn, and double-crop farmers

PubMed Central

Hongsibsong, Surat; Sittitoon, Nalin; Sapbamrer, Ratana

2017-01-01

Objectives: This study aims to determine (1) total dialkylphosphate (ΣDAP) levels, occupational knowledge and practice, DNA damage, AChE activity, and health symptoms in rice, corn, and double-crop farmers; (2) the association of health symptoms with ΣDAP levels, occupational knowledge and practice, DNA damage, and AChE activity in farmers; and (3) the prevalence of health symptoms between farmers and non-farmers. Methods: A cross-sectional study was conducted by interviewing as well as analyzing urine and blood samples during July to August 2014. Results: There were no differences in ΣDAP levels, AChE activity, and occupational knowledge and practice scores among all farmer groups. In terms of health symptoms related to ΣDAP, AChE activity, DNA damage, and occupational knowledge and practice, pesticide-related symptoms were determined, including breathlessness, chest pain, dry throat, numbness, muscle weakness, cramp, headache, dizziness, eye irritation, white/red rash, and white/red pimple, which were classified as respiratory, muscle, nervous, and epithelial symptoms. A remarkable finding was that farmers had a significantly higher prevalence of muscle weakness (odds ratio (OR)=3.79) and numbness (OR=3.45) as compared with non-farmers. Conclusion: Our findings, therefore, suggest that a long-term low-level exposure to organophosphates (OPs) may be associated with an increasing prevalence of muscle symptoms. However, a further cohort study incorporating sensitive health outcomes and measurement of multiple pesticides monitoring on a larger scale is warranted. PMID:28077823

An intercalation-locked parallel-stranded DNA tetraplex

DOE PAGES

Tripathi, S.; Zhang, D.; Paukstelis, P. J.

2015-01-27

DNA has proved to be an excellent material for nanoscale construction because complementary DNA duplexes are programmable and structurally predictable. However, in the absence of Watson–Crick pairings, DNA can be structurally more diverse. Here, we describe the crystal structures of d(ACTCGGATGAT) and the brominated derivative, d(AC BrUCGGA BrUGAT). These oligonucleotides form parallel-stranded duplexes with a crystallographically equivalent strand, resulting in the first examples of DNA crystal structures that contains four different symmetric homo base pairs. Two of the parallel-stranded duplexes are coaxially stacked in opposite directions and locked together to form a tetraplex through intercalation of the 5'-most A–A basemore » pairs between adjacent G–G pairs in the partner duplex. The intercalation region is a new type of DNA tertiary structural motif with similarities to the i-motif. 1H– 1H nuclear magnetic resonance and native gel electrophoresis confirmed the formation of a parallel-stranded duplex in solution. Finally, we modified specific nucleotide positions and added d(GAY) motifs to oligonucleotides and were readily able to obtain similar crystals. This suggests that this parallel-stranded DNA structure may be useful in the rational design of DNA crystals and nanostructures.« less

The structure and intermolecular forces of DNA condensates.

PubMed

Yoo, Jejoong; Aksimentiev, Aleksei

2016-03-18

Spontaneous assembly of DNA molecules into compact structures is ubiquitous in biological systems. Experiment has shown that polycations can turn electrostatic self-repulsion of DNA into attraction, yet the physical mechanism of DNA condensation has remained elusive. Here, we report the results of atomistic molecular dynamics simulations that elucidated the microscopic structure of dense DNA assemblies and the physics of interactions that makes such assemblies possible. Reproducing the setup of the DNA condensation experiments, we measured the internal pressure of DNA arrays as a function of the DNA-DNA distance, showing a quantitative agreement between the results of our simulations and the experimental data. Analysis of the MD trajectories determined the DNA-DNA force in a DNA condensate to be pairwise, the DNA condensation to be driven by electrostatics of polycations and not hydration, and the concentration of bridging cations, not adsorbed cations, to determine the magnitude and the sign of the DNA-DNA force. Finally, our simulations quantitatively characterized the orientational correlations of DNA in DNA arrays as well as diffusive motion of DNA and cations. © The Author(s) 2016. Published by Oxford University Press on behalf of Nucleic Acids Research.

Escherichia coli DNA polymerase I can disrupt G-quadruplex structures during DNA replication.

PubMed

Teng, Fang-Yuan; Hou, Xi-Miao; Fan, San-Hong; Rety, Stephane; Dou, Shuo-Xing; Xi, Xu-Guang

2017-12-01

Non-canonical four-stranded G-quadruplex (G4) DNA structures can form in G-rich sequences that are widely distributed throughout the genome. The presence of G4 structures can impair DNA replication by hindering the progress of replicative polymerases (Pols), and failure to resolve these structures can lead to genetic instability. In the present study, we combined different approaches to address the question of whether and how Escherichia coli Pol I resolves G4 obstacles during DNA replication and/or repair. We found that E. coli Pol I-catalyzed DNA synthesis could be arrested by G4 structures at low protein concentrations and the degree of inhibition was strongly dependent on the stability of the G4 structures. Interestingly, at high protein concentrations, E. coli Pol I was able to overcome some kinds of G4 obstacles without the involvement of other molecules and could achieve complete replication of G4 DNA. Mechanistic studies suggested that multiple Pol I proteins might be implicated in G4 unfolding, and the disruption of G4 structures requires energy derived from dNTP hydrolysis. The present work not only reveals an unrealized function of E. coli Pol I, but also presents a possible mechanism by which G4 structures can be resolved during DNA replication and/or repair in E. coli. © 2017 Federation of European Biochemical Societies.

Genetic structure and demographic history of the endangered and endemic schizothoracine fish Gymnodiptychus pachycheilus in Qinghai-Tibetan Plateau.

PubMed

Su, Junhu; Ji, Weihong; Wei, Yanming; Zhang, Yanping; Gleeson, Dianne M; Lou, Zhongyu; Ren, Jing

2014-08-01

The endangered schizothoracine fish Gymnodiptychus pachycheilus is endemic to the Qinghai-Tibetan Plateau (QTP), but very little genetic information is available for this species. Here, we accessed the current genetic divergence of G. pachycheilus population to evaluate their distributions modulated by contemporary and historical processes. Population structure and demographic history were assessed by analyzing 1811-base pairs of mitochondrial DNA from 61 individuals across a large proportion of its geographic range. Our results revealed low nucleotide diversity, suggesting severe historical bottleneck events. Analyses of molecular variance and the conventional population statistic FST (0.0435, P = 0.0215) confirmed weak genetic structure. The monophyly of G. pachycheilus was statistically well-supported, while two divergent evolutionary clusters were identified by phylogenetic analyses, suggesting a microgeographic population structure. The consistent scenario of recent population expansion of two clusters was identified based on several complementary analyses of demographic history (0.096 Ma and 0.15 Ma). This genetic divergence and evolutionary process are likely to have resulted from a series of drainage arrangements triggered by the historical tectonic events of the region. The results obtained here provide the first insights into the evolutionary history and genetic status of this little-known fish.

Structure-directing weak phosphoryl XH...O=P (X = C, N) hydrogen bonds in cyclic oxazaphospholidines and oxazaphosphinanes.

PubMed

van der Lee, A; Rolland, M; Marat, X; Virieux, D; Volle, J N; Pirat, J L

2008-04-01

The structures of six cyclic oxazaphospholidines and three cyclic oxazaphosphinanes have been determined and their supramolecular structures have been compared. The molecules differ with respect to the functional groups attached to the central five- or six-membered rings, but have one phosphoryl group in common. The predominant feature in the supramolecular structures is the existence of relatively weak intermolecular phosphoryl XH...O=P (X = C, N) hydrogen bonds, creating in nearly all cases linear zigzag or double molecular chains. The molecular chains are in general linked to each other via very weak CH...pi or usual hydrogen-bond interactions. A survey of the Cambridge Structural Database on similar XH...O=P interactions shows a very large flexibility of the XH...O angle, which is in agreement with the DFT calculation reported elsewhere. The strength of the XH...O=P interaction can therefore be considered as relatively weak to moderately strong, and is expected to play at least a role in the formation of secondary substructures.

Thymidine kinase 2 (H126N) knockin mice show the essential role of balanced deoxynucleotide pools for mitochondrial DNA maintenance.

PubMed

Akman, Hasan O; Dorado, Beatriz; López, Luis C; García-Cazorla, Angeles; Vilà, Maya R; Tanabe, Lauren M; Dauer, William T; Bonilla, Eduardo; Tanji, Kurenai; Hirano, Michio

2008-08-15

Mitochondrial DNA (mtDNA) depletion syndrome (MDS), an autosomal recessive condition, is characterized by variable organ involvement with decreased mtDNA copy number and activities of respiratory chain enzymes in affected tissues. MtDNA depletion has been associated with mutations in nine autosomal genes, including thymidine kinase (TK2), which encodes a ubiquitous mitochondrial protein. To study the pathogenesis of TK2-deficiency, we generated mice harboring an H126N Tk2 mutation. Homozygous Tk2 mutant (Tk2(-/-)) mice developed rapidly progressive weakness after age 10 days and died between ages 2 and 3 weeks. Tk2(-/-) animals showed Tk2 deficiency, unbalanced dNTP pools, mtDNA depletion and defects of respiratory chain enzymes containing mtDNA-encoded subunits that were most prominent in the central nervous system. Histopathology revealed an encephalomyelopathy with prominent vacuolar changes in the anterior horn of the spinal cord. The H126N TK2 mouse is the first knock-in animal model of human MDS and demonstrates that the severity of TK2 deficiency in tissues may determine the organ-specific phenotype.

Thymidine kinase 2 (H126N) knockin mice show the essential role of balanced deoxynucleotide pools for mitochondrial DNA maintenance

PubMed Central

Akman, Hasan O.; Dorado, Beatriz; López, Luis C.; García-Cazorla, Ángeles; Vilà, Maya R.; Tanabe, Lauren M.; Dauer, William T.; Bonilla, Eduardo; Tanji, Kurenai; Hirano, Michio

2008-01-01

Mitochondrial DNA (mtDNA) depletion syndrome (MDS), an autosomal recessive condition, is characterized by variable organ involvement with decreased mtDNA copy number and activities of respiratory chain enzymes in affected tissues. MtDNA depletion has been associated with mutations in nine autosomal genes, including thymidine kinase (TK2), which encodes a ubiquitous mitochondrial protein. To study the pathogenesis of TK2-deficiency, we generated mice harboring an H126N Tk2 mutation. Homozygous Tk2 mutant (Tk2−/−) mice developed rapidly progressive weakness after age 10 days and died between ages 2 and 3 weeks. Tk2−/− animals showed Tk2 deficiency, unbalanced dNTP pools, mtDNA depletion and defects of respiratory chain enzymes containing mtDNA-encoded subunits that were most prominent in the central nervous system. Histopathology revealed an encephalomyelopathy with prominent vacuolar changes in the anterior horn of the spinal cord. The H126N TK2 mouse is the first knock-in animal model of human MDS and demonstrates that the severity of TK2 deficiency in tissues may determine the organ-specific phenotype. PMID:18467430

Diagnostic Dilemma for Low Viremia with Significant Fibrosis; Is HBV DNA Threshold Level a Good Indicator for Predicting Liver Damage?

PubMed

Yenilmez, Ercan; Çetinkaya, Rıza Aytaç; Tural, Ersin

2018-05-04

The most important difficulties about management of hepatitis B are still determining the liver damage and the right time to start antiviral therapy. To reveal the role of hepatitis B virus DNA threshold level for prediction of liver fibrosis and inflammation in young-aged hepatitis B e antigen negative chronic hepatitis B patients. Diagnostic accuracy study. A total of 273 hepatitis B e antigen negative young chronic hepatitis B patients with any hepatitis B virus DNA levels between 2008 and 2016, who had liver biopsy after at least 6 months follow up period, enrolled in this retrospective study. We created two groups as case and control, cases with hepatitis B virus DNA levels below 2.000 IU/mL and controls with hepatitis B virus DNA levels over 2.000 IU/mL. Having histological activity index ≥4 or/and fibrosis scores ≥2 were defined as significant histological abnormality. Then, we analyzed the relationship between these groups. We showed that significant fibrosis may occur in one third of young chronic hepatitis B patients with low viremia (30.2%, n=42/139 in cases, %55.2, n=74/134 in controls). Among the 42 cases with low viremia and significant fibrosis, 21.4% had alanine aminotransferase level between 40-59 U/L, 42.8% had alanine aminotransferase level between 60-79 U/L, and 35.7% had alanine aminotransferase level over 80 U/L. There was weak correlation between hepatitis B virus DNA threshold level and fibrosis score (p=0.000, rho=0.253). The optimum serum hepatitis B virus DNA threshold level in our study for predicting significant fibrosis was 1293 IU/mL (p=0.00, AUC: 0.657±0.034). The optimum alanine aminotransferase threshold level for predicting significant histological activity index and fibrosis was 64.5 and 59.5 U/L, respectively. The sensitivity and the specificity of 1293 vs 2000 IU/mL hepatitis B virus DNA threshold with 60 U/L alanine aminotransferase threshold level for predicting F≥2 fibrosis score were similar (sensitivity: 0.43 and 0.38, respectively; specificity: 0.76 and 0.77, respectively). Significant fibrosis may occur even in young cases with low viremia. It is not possible to define a single threshold hepatitis B virus DNA level for differentiating inactive carriers from patients with hepatitis B e antigen-negative chronic hepatitis. Diagnostic accuracy of hepatitis B virus DNA with alanine aminotransferase thresholds for the prediction of significant fibrosis is weak.

Cations Form Sequence Selective Motifs within DNA Grooves via a Combination of Cation-Pi and Ion-Dipole/Hydrogen Bond Interactions

PubMed Central

Stewart, Mikaela; Dunlap, Tori; Dourlain, Elizabeth; Grant, Bryce; McFail-Isom, Lori

2013-01-01

The fine conformational subtleties of DNA structure modulate many fundamental cellular processes including gene activation/repression, cellular division, and DNA repair. Most of these cellular processes rely on the conformational heterogeneity of specific DNA sequences. Factors including those structural characteristics inherent in the particular base sequence as well as those induced through interaction with solvent components combine to produce fine DNA structural variation including helical flexibility and conformation. Cation-pi interactions between solvent cations or their first hydration shell waters and the faces of DNA bases form sequence selectively and contribute to DNA structural heterogeneity. In this paper, we detect and characterize the binding patterns found in cation-pi interactions between solvent cations and DNA bases in a set of high resolution x-ray crystal structures. Specifically, we found that monovalent cations (Tl+) and the polarized first hydration shell waters of divalent cations (Mg2+, Ca2+) form cation-pi interactions with DNA bases stabilizing unstacked conformations. When these cation-pi interactions are combined with electrostatic interactions a pattern of specific binding motifs is formed within the grooves. PMID:23940752

Cations form sequence selective motifs within DNA grooves via a combination of cation-pi and ion-dipole/hydrogen bond interactions.

PubMed

Stewart, Mikaela; Dunlap, Tori; Dourlain, Elizabeth; Grant, Bryce; McFail-Isom, Lori

2013-01-01

The fine conformational subtleties of DNA structure modulate many fundamental cellular processes including gene activation/repression, cellular division, and DNA repair. Most of these cellular processes rely on the conformational heterogeneity of specific DNA sequences. Factors including those structural characteristics inherent in the particular base sequence as well as those induced through interaction with solvent components combine to produce fine DNA structural variation including helical flexibility and conformation. Cation-pi interactions between solvent cations or their first hydration shell waters and the faces of DNA bases form sequence selectively and contribute to DNA structural heterogeneity. In this paper, we detect and characterize the binding patterns found in cation-pi interactions between solvent cations and DNA bases in a set of high resolution x-ray crystal structures. Specifically, we found that monovalent cations (Tl⁺) and the polarized first hydration shell waters of divalent cations (Mg²⁺, Ca²⁺) form cation-pi interactions with DNA bases stabilizing unstacked conformations. When these cation-pi interactions are combined with electrostatic interactions a pattern of specific binding motifs is formed within the grooves.

Crystal structure of MboIIA methyltransferase.

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

Osipiuk, J.; Walsh, M. A.; Joachimiak, A.

2003-09-15

DNA methyltransferases (MTases) are sequence-specific enzymes which transfer a methyl group from S-adenosyl-L-methionine (AdoMet) to the amino group of either cytosine or adenine within a recognized DNA sequence. Methylation of a base in a specific DNA sequence protects DNA from nucleolytic cleavage by restriction enzymes recognizing the same DNA sequence. We have determined at 1.74 {angstrom} resolution the crystal structure of a {beta}-class DNA MTase MboIIA (M {center_dot} MboIIA) from the bacterium Moraxella bovis, the smallest DNA MTase determined to date. M {center_dot} MboIIA methylates the 3' adenine of the pentanucleotide sequence 5'-GAAGA-3'. The protein crystallizes with two molecules inmore » the asymmetric unit which we propose to resemble the dimer when M {center_dot} MboIIA is not bound to DNA. The overall structure of the enzyme closely resembles that of M {center_dot} RsrI. However, the cofactor-binding pocket in M {center_dot} MboIIA forms a closed structure which is in contrast to the open-form structures of other known MTases.« less

NMR and computational methods applied to the 3- dimensional structure determination of DNA and ligand-DNA complexes in solution

NASA Astrophysics Data System (ADS)

Smith, Jarrod Anson

2D homonuclear 1H NMR methods and restrained molecular dynamics (rMD) calculations have been applied to determining the three-dimensional structures of DNA and minor groove-binding ligand-DNA complexes in solution. The structure of the DNA decamer sequence d(GCGTTAACGC)2 has been solved both with a distance-based rMD protocol and an NOE relaxation matrix backcalculation-based protocol in order to probe the relative merits of the different refinement methods. In addition, three minor groove binding ligand-DNA complexes have been examined. The solution structure of the oligosaccharide moiety of the antitumor DNA scission agent calicheamicin γ1I has been determined in complex with a decamer duplex containing its high affinity 5'-TCCT- 3' binding sequence. The structure of the complex reinforces the belief that the oligosaccharide moiety is responsible for the sequence selective minor-groove binding activity of the agent, and critical intermolecular contacts are revealed. The solution structures of both the (+) and (-) enantiomers of the minor groove binding DNA alkylating agent duocarmycin SA have been determined in covalent complex with the undecamer DNA duplex d(GACTAATTGTC).d(GAC AATTAGTC). The results support the proposal that the alkylation activity of the duocarmycin antitumor antibiotics is catalyzed by a binding-induced conformational change in the ligand which activates the cyclopropyl group for reaction with the DNA. Comparisons between the structures of the two enantiomers covalently bound to the same DNA sequence at the same 5'-AATTA-3 ' site have provided insight into the binding orientation and site selectivity, as well as the relative rates of reactivity of these two agents.

Mito-nuclear discord in six congeneric lineages of Holarctic ducks (genus Anas).

PubMed

Peters, Jeffrey L; Winker, Kevin; Millam, Kendra C; Lavretsky, Philip; Kulikova, Irina; Wilson, Robert E; Zhuravlev, Yuri N; McCracken, Kevin G

2014-06-01

Many species have Holarctic distributions that extend across Europe, Asia and North America. Most genetics research on these species has examined only mitochondrial (mt) DNA, which has revealed wide variance in divergence between Old World (OW) and New World (NW) populations, ranging from shallow, unstructured genealogies to deeply divergent lineages. In this study, we sequenced 20 nuclear introns to test for concordant patterns of OW-NW differentiation between mtDNA and nuclear (nu) DNA for six lineages of Holarctic ducks (genus Anas). Genetic differentiation for both marker types varied widely among these lineages (idiosyncratic population histories), but mtDNA and nuDNA divergence within lineages was not significantly correlated. Moreover, compared with the association between mtDNA and nuDNA divergence observed among different species, OW-NW nuDNA differentiation was generally lower than mtDNA divergence, at least for lineages with deeply divergent mtDNA. Furthermore, coalescent estimates indicated significantly higher rates of gene flow for nuDNA than mtDNA for four of the six lineages. Thus, Holarctic ducks show prominent mito-nuclear discord between OW and NW populations, and we reject differences in sorting rates as the sole cause of the within-species discord. Male-mediated intercontinental gene flow is likely a leading contributor to this discord, although selection could also cause increased mtDNA divergence relative to weak nuDNA differentiation. The population genetics of these ducks contribute to growing evidence that mtDNA can be an unreliable indicator of stage of speciation and that more holistic approaches are needed for species delimitation. © 2014 John Wiley & Sons Ltd.

The crystal structure of Neisseria gonorrhoeae PriB reveals mechanistic differences among bacterial DNA replication restart pathways

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

Dong, Jinlan; George, Nicholas P.; Duckett, Katrina L.

2010-05-25

Reactivation of repaired DNA replication forks is essential for complete duplication of bacterial genomes. However, not all bacteria encode homologs of the well-studied Escherichia coli DNA replication restart primosome proteins, suggesting that there might be distinct mechanistic differences among DNA replication restart pathways in diverse bacteria. Since reactivation of repaired DNA replication forks requires coordinated DNA and protein binding by DNA replication restart primosome proteins, we determined the crystal structure of Neisseria gonorrhoeae PriB at 2.7 {angstrom} resolution and investigated its ability to physically interact with DNA and PriA helicase. Comparison of the crystal structures of PriB from N. gonorrhoeaemore » and E. coli reveals a well-conserved homodimeric structure consisting of two oligosaccharide/oligonucleotide-binding (OB) folds. In spite of their overall structural similarity, there is significant species variation in the type and distribution of surface amino acid residues. This correlates with striking differences in the affinity with which each PriB homolog binds single-stranded DNA and PriA helicase. These results provide evidence that mechanisms of DNA replication restart are not identical across diverse species and that these pathways have likely become specialized to meet the needs of individual organisms.« less

Behavioral variability of choices versus structural inconsistency of preferences.

PubMed

Regenwetter, Michel; Davis-Stober, Clintin P

2012-04-01

Theories of rational choice often make the structural consistency assumption that every decision maker's binary strict preference among choice alternatives forms a strict weak order. Likewise, the very concept of a utility function over lotteries in normative, prescriptive, and descriptive theory is mathematically equivalent to strict weak order preferences over those lotteries, while intransitive heuristic models violate such weak orders. Using new quantitative interdisciplinary methodologies, we dissociate the variability of choices from the structural inconsistency of preferences. We show that laboratory choice behavior among stimuli of a classical "intransitivity" paradigm is, in fact, consistent with variable strict weak order preferences. We find that decision makers act in accordance with a restrictive mathematical model that, for the behavioral sciences, is extraordinarily parsimonious. Our findings suggest that the best place to invest future behavioral decision research is not in the development of new intransitive decision models but rather in the specification of parsimonious models consistent with strict weak order(s), as well as heuristics and other process models that explain why preferences appear to be weakly ordered.

Hmi1p from Saccharomyces cerevisiae mitochondria is a structure-specific DNA helicase.

PubMed

Kuusk, Silja; Sedman, Tiina; Jõers, Priit; Sedman, Juhan

2005-07-01

Hmi1p is a Saccharomyces cerevisiae mitochondrial DNA helicase that is essential for the maintenance of functional mitochondrial DNA. Hmi1p belongs to the superfamily 1 of helicases and is a close homologue of bacterial PcrA and Rep helicases. We have overexpressed and purified recombinant Hmi1p from Escherichia coli and describe here the biochemical characteristics of its DNA helicase activities. Among nucleotide cofactors, the DNA unwinding by Hmi1p was found to occur efficiently only in the presence of ATP and dATP. Hmi1p could unwind only the DNA substrates with a 3'-single-stranded overhang. The length of the 3'-overhang needed for efficient targeting of the helicase to the substrate depended on the substrate structure. For substrates consisting of duplex DNA with a 3'-single-stranded DNA overhang, at least a 19-nt 3'-overhang was needed. In the case of forked substrates with both 3'- and 5'-overhangs, a 9-nt 3'-overhang was sufficient provided that the 5'-overhang was also 9 nt in length. In flap-structured substrates mimicking the chain displacement structures in DNA recombination process, only a 5-nt 3'-single-stranded DNA tail was required for efficient unwinding by Hmi1p. These data indicate that Hmi1p may be targeted to a specific 3'-flap structure, suggesting its possible role in DNA recombination.

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

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

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.

Atomic Force Microscopy Analysis of the Role of Major DNA-Binding Proteins in Organization of the Nucleoid in Escherichia coli

PubMed Central

Ohniwa, Ryosuke L.; Muchaku, Hiroki; Saito, Shinji; Wada, Chieko; Morikawa, Kazuya

2013-01-01

Bacterial genomic DNA is packed within the nucleoid of the cell along with various proteins and RNAs. We previously showed that the nucleoid in log phase cells consist of fibrous structures with diameters ranging from 30 to 80 nm, and that these structures, upon RNase A treatment, are converted into homogeneous thinner fibers with diameter of 10 nm. In this study, we investigated the role of major DNA-binding proteins in nucleoid organization by analyzing the nucleoid of mutant Escherichia coli strains lacking HU, IHF, H–NS, StpA, Fis, or Hfq using atomic force microscopy. Deletion of particular DNA-binding protein genes altered the nucleoid structure in different ways, but did not release the naked DNA even after the treatment with RNase A. This suggests that major DNA-binding proteins are involved in the formation of higher order structure once 10-nm fiber structure is built up from naked DNA. PMID:23951337

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.

Electrochemical label-free and sensitive nanobiosensing of DNA hybridization by graphene oxide modified pencil graphite electrode.

PubMed

Ahour, F; Shamsi, A

2017-09-01

Based on the strong interaction between single-stranded DNA (ss-DNA) and graphene material, we have constructed a novel label-free electrochemical biosensor for rapid and facile detection of short sequences ss-DNA molecules related to hepatitis C virus 1a using graphene oxide modified pencil graphite electrode. The sensing mechanism is based on the superior adsorption of single-stranded DNA to GO over double stranded DNA (ds-DNA). The intrinsic guanine oxidation signal measured by differential pulse voltammetry (DPV) has been used for duplex DNA formation detection. The probe ss-DNA adsorbs onto the surface of GO via the π- π* stacking interactions leading to a strong background guanine oxidation signal. In the presence of complementary target, formation of helix which has weak binding ability to GO induced ds-DNA to release from the electrode surface and significant variation in differential pulse voltammetric response of guanine bases. The results indicated that the oxidation peak current was proportional to the concentration of complementary strand in the range of 0.1 nM-0.5 μM with a detection limit of 4.3 × 10 -11  M. The simple fabricated electrochemical biosensor has high sensitivity, good selectivity, and could be applied as a new platform for a range of target molecules in future. Copyright © 2017 Elsevier Inc. All rights reserved.

Cr(3+) Binding to DNA Backbone Phosphate and Bases: Slow Ligand Exchange Rates and Metal Hydrolysis.

PubMed

Zhou, Wenhu; Yu, Tianmeng; Vazin, Mahsa; Ding, Jinsong; Liu, Juewen

2016-08-15

The interaction between chromium ions and DNA is of great interest in inorganic chemistry, toxicology, and analytical chemistry. Most previous studies focused on in situ reduction of Cr(VI), producing Cr(3+) for DNA binding. Recently, Cr(3+) was reported to activate the Ce13d DNAzyme for RNA cleavage. Herein, the Ce13d is used to study two types of Cr(3+) and DNA interactions. First, Cr(3+) binds to the DNA phosphate backbone weakly through reversible electrostatic interactions, which is weakened by adding competing inorganic phosphate. However, Cr(3+) coordinates with DNA nucleobases forming stable cross-links that can survive denaturing gel electrophoresis condition. The binding of Cr(3+) to different nucleobases was further studied in terms of binding kinetics and affinity by exploiting carboxyfluorescein-labeled DNA homopolymers. Once binding takes place, the stable Cr(3+)/DNA complex cannot be dissociated by EDTA, attributable to the ultraslow ligand exchange rate of Cr(3+). The binding rate follows the order of G > C > T ≈ A. Finally, Cr(3+) gradually loses its DNA binding ability after being stored at neutral or high pH, attributable to hydrolysis. This hydrolysis can be reversed by lowering the pH. This work provides a deeper insight into the bioinorganic chemistry of Cr(3+) coordination with DNA, clarifies some inconsistency in the previous literature, and offers practically useful information for generating reproducible results.

Charge transport and ac response under light illumination in gate-modulated DNA molecular junctions.

PubMed

Zhang, Yan; Zhu, Wen-Huan; Ding, Guo-Hui; Dong, Bing; Wang, Xue-Feng

2015-05-22

Using a two-strand tight-binding model and within nonequilibrium Green's function approach, we study charge transport through DNA sequences (GC)NGC and (GC)1(TA)NTA (GC)3 sandwiched between two Pt electrodes. We show that at low temperature DNA sequence (GC)NGC exhibits coherent charge carrier transport at very small bias, since the highest occupied molecular orbital in the GC base pair can be aligned with the Fermi energy of the metallic electrodes by a gate voltage. A weak distance dependent conductance is found in DNA sequence (GC)1(TA)NTA (GC)3 with large NTA. Different from the mechanism of thermally induced hopping of charges proposed by the previous experiments, we find that this phenomenon is dominated by quantum tunnelling through discrete quantum well states in the TA base pairs. In addition, ac response of this DNA junction under light illumination is also investigated. The suppression of ac conductances of the left and right lead of DNA sequences at some particular frequencies is attributed to the excitation of electrons in the DNA to the lead Fermi surface by ac potential, or the excitation of electrons in deep DNA energy levels to partially occupied energy levels in the transport window. Therefore, measuring ac response of DNA junctions can reveal a wealth of information about the intrinsic dynamics of DNA molecules.

Block Copolymer Micellization as a Protection Strategy for DNA Origami.

PubMed

Agarwal, Nayan P; Matthies, Michael; Gür, Fatih N; Osada, Kensuke; Schmidt, Thorsten L

2017-05-08

DNA nanotechnology enables the synthesis of nanometer-sized objects that can be site-specifically functionalized with a large variety of materials. For these reasons, DNA-based devices such as DNA origami are being considered for applications in molecular biology and nanomedicine. However, many DNA structures need a higher ionic strength than that of common cell culture buffers or bodily fluids to maintain their integrity and can be degraded quickly by nucleases. To overcome these deficiencies, we coated several different DNA origami structures with a cationic poly(ethylene glycol)-polylysine block copolymer, which electrostatically covered the DNA nanostructures to form DNA origami polyplex micelles (DOPMs). This straightforward, cost-effective, and robust route to protect DNA-based structures could therefore enable applications in biology and nanomedicine where unprotected DNA origami would be degraded. © 2017 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim.

Two high-mobility group box domains act together to underwind and kink DNA

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

Sánchez-Giraldo, R.; Acosta-Reyes, F. J.; Malarkey, C. S.

The crystal structure of HMGB1 box A bound to an unmodified AT-rich DNA fragment is reported at a resolution of 2 Å. A new mode of DNA recognition for HMG box proteins is found in which two box A domains bind in an unusual configuration generating a highly kinked DNA structure. High-mobility group protein 1 (HMGB1) is an essential and ubiquitous DNA architectural factor that influences a myriad of cellular processes. HMGB1 contains two DNA-binding domains, box A and box B, which have little sequence specificity but have remarkable abilities to underwind and bend DNA. Although HMGB1 box A ismore » thought to be responsible for the majority of HMGB1–DNA interactions with pre-bent or kinked DNA, little is known about how it recognizes unmodified DNA. Here, the crystal structure of HMGB1 box A bound to an AT-rich DNA fragment is reported at a resolution of 2 Å. Two box A domains of HMGB1 collaborate in an unusual configuration in which the Phe37 residues of both domains stack together and intercalate the same CG base pair, generating highly kinked DNA. This represents a novel mode of DNA recognition for HMGB proteins and reveals a mechanism by which structure-specific HMG boxes kink linear DNA.« less

Structural diversity of supercoiled DNA

PubMed Central

Irobalieva, Rossitza N.; Fogg, Jonathan M.; Catanese, Daniel J.; Sutthibutpong, Thana; Chen, Muyuan; Barker, Anna K.; Ludtke, Steven J.; Harris, Sarah A.; Schmid, Michael F.; Chiu, Wah; Zechiedrich, Lynn

2015-01-01

By regulating access to the genetic code, DNA supercoiling strongly affects DNA metabolism. Despite its importance, however, much about supercoiled DNA (positively supercoiled DNA, in particular) remains unknown. Here we use electron cryo-tomography together with biochemical analyses to investigate structures of individual purified DNA minicircle topoisomers with defined degrees of supercoiling. Our results reveal that each topoisomer, negative or positive, adopts a unique and surprisingly wide distribution of three-dimensional conformations. Moreover, we uncover striking differences in how the topoisomers handle torsional stress. As negative supercoiling increases, bases are increasingly exposed. Beyond a sharp supercoiling threshold, we also detect exposed bases in positively supercoiled DNA. Molecular dynamics simulations independently confirm the conformational heterogeneity and provide atomistic insight into the flexibility of supercoiled DNA. Our integrated approach reveals the three-dimensional structures of DNA that are essential for its function. PMID:26455586

Structural diversity of supercoiled DNA

NASA Astrophysics Data System (ADS)

Irobalieva, Rossitza N.; Fogg, Jonathan M.; Catanese, Daniel J.; Sutthibutpong, Thana; Chen, Muyuan; Barker, Anna K.; Ludtke, Steven J.; Harris, Sarah A.; Schmid, Michael F.; Chiu, Wah; Zechiedrich, Lynn

2015-10-01

By regulating access to the genetic code, DNA supercoiling strongly affects DNA metabolism. Despite its importance, however, much about supercoiled DNA (positively supercoiled DNA, in particular) remains unknown. Here we use electron cryo-tomography together with biochemical analyses to investigate structures of individual purified DNA minicircle topoisomers with defined degrees of supercoiling. Our results reveal that each topoisomer, negative or positive, adopts a unique and surprisingly wide distribution of three-dimensional conformations. Moreover, we uncover striking differences in how the topoisomers handle torsional stress. As negative supercoiling increases, bases are increasingly exposed. Beyond a sharp supercoiling threshold, we also detect exposed bases in positively supercoiled DNA. Molecular dynamics simulations independently confirm the conformational heterogeneity and provide atomistic insight into the flexibility of supercoiled DNA. Our integrated approach reveals the three-dimensional structures of DNA that are essential for its function.

SINE_scan: an efficient tool to discover short interspersed nuclear elements (SINEs) in large-scale genomic datasets.

PubMed

Mao, Hongliang; Wang, Hao

2017-03-01

Short Interspersed Nuclear Elements (SINEs) are transposable elements (TEs) that amplify through a copy-and-paste mode via RNA intermediates. The computational identification of new SINEs are challenging because of their weak structural signals and rapid diversification in sequences. Here we report SINE_Scan, a highly efficient program to predict SINE elements in genomic DNA sequences. SINE_Scan integrates hallmark of SINE transposition, copy number and structural signals to identify a SINE element. SINE_Scan outperforms the previously published de novo SINE discovery program. It shows high sensitivity and specificity in 19 plant and animal genome assemblies, of which sizes vary from 120 Mb to 3.5 Gb. It identifies numerous new families and substantially increases the estimation of the abundance of SINEs in these genomes. The code of SINE_Scan is freely available at http://github.com/maohlzj/SINE_Scan , implemented in PERL and supported on Linux. wangh8@fudan.edu.cn. Supplementary data are available at Bioinformatics online. © The Author 2016. Published by Oxford University Press.

SINE_scan: an efficient tool to discover short interspersed nuclear elements (SINEs) in large-scale genomic datasets

PubMed Central

Mao, Hongliang

2017-01-01

Abstract Motivation: Short Interspersed Nuclear Elements (SINEs) are transposable elements (TEs) that amplify through a copy-and-paste mode via RNA intermediates. The computational identification of new SINEs are challenging because of their weak structural signals and rapid diversification in sequences. Results: Here we report SINE_Scan, a highly efficient program to predict SINE elements in genomic DNA sequences. SINE_Scan integrates hallmark of SINE transposition, copy number and structural signals to identify a SINE element. SINE_Scan outperforms the previously published de novo SINE discovery program. It shows high sensitivity and specificity in 19 plant and animal genome assemblies, of which sizes vary from 120 Mb to 3.5 Gb. It identifies numerous new families and substantially increases the estimation of the abundance of SINEs in these genomes. Availability and Implementation: The code of SINE_Scan is freely available at http://github.com/maohlzj/SINE_Scan, implemented in PERL and supported on Linux. Contact: wangh8@fudan.edu.cn Supplementary information: Supplementary data are available at Bioinformatics online. PMID:28062442

Phylogeography of colonially nesting seabirds, with special reference to global matrilineal patterns in the sooty tern (Sterna fuscata).

PubMed

Avise, J C; Nelson, W S; Bowen, B W; Walker, D

2000-11-01

Sooty tern (Sterna fuscata) rookeries are scattered throughout the tropical oceans. When not nesting, individuals wander great distances across open seas, but, like many other seabirds, they tend to be site-faithful to nesting locales in successive years. Here we examine the matrilineal history of sooty terns on a global scale. Assayed colonies within an ocean are poorly differentiated in mitochondrial DNA sequence, a result indicating tight historical ties. However, a shallow genealogical partition distinguishes Atlantic from Indo-Pacific rookeries. Phylogeographic patterns in the sooty tern are compared to those in other colonially nesting seabirds, as well as in the green turtle (Chelonia mydas), an analogue of tropical seabirds in some salient aspects of natural history. Phylogeographic structure within an ocean is normally weak in seabirds, unlike the pronounced matrilineal structure in green turtles. However, the phylogeographic partition between Atlantic and Indo-Pacific rookeries in sooty terns mirrors, albeit in shallower evolutionary time, the major matrilineal subdivision in green turtles. Thus, global geology has apparently influenced historical gene movements in these two circumtropical species.

Altered expression of MUC2, MUC4, and MUC5 mucin genes in pancreas tissues and cancer cell lines.

PubMed

Balagué, C; Gambús, G; Carrato, C; Porchet, N; Aubert, J P; Kim, Y S; Real, F X

1994-04-01

Neoplastic transformation of epithelial cells is commonly associated with altered synthesis and structure of mucin glycoproteins. The aim of the study was to determine if altered mucin gene expression takes place in pancreas cancer. To examine mucin gene expression in normal pancreas and pancreas cancer, antibodies detecting the MUC1, MUC2, MUC5B, and MUC5C apomucins were used in immunohistochemical techniques and complementary DNA probes specific for the MUC1-MUC5 genes were used in Northern blots. MUC1 is the major apomucin expressed in normal pancreas, whereas MUC2-MUC5 are weakly expressed or undetectable. In pancreas cancer tissues and cell lines, increased expression of MUC2, MUC4, and MUC5C is shown. The cytoplasmic expression of MUC2 and MUC5C in tumor cells suggests that these apomucins are underglycosylated and abnormally compartmentalized. Enhanced expression of MUC2, MUC4, and MUC5C genes is a frequent event in pancreas cancer and may contribute to the alterations in the biochemical structure of pancreas cancer mucins.

Hands on Group Work Paper Model for Teaching DNA Structure, Central Dogma and Recombinant DNA

ERIC Educational Resources Information Center

Altiparmak, Melek; Nakiboglu Tezer, Mahmure

2009-01-01

Understanding life on a molecular level is greatly enhanced when students are given the opportunity to visualize the molecules. Especially understanding DNA structure and function is essential for understanding key concepts of molecular biology such as DNA, central dogma and the manipulation of DNA. Researches have shown that undergraduate…

The Knowledge of DNA and DNA Technologies among Pre-Service Science Teachers

ERIC Educational Resources Information Center

Cardak, Osman; Dikmenli, Musa

2008-01-01

The purpose of this study is to determine the alternative conceptions of elementary school pre-service science teachers regarding DNA and DNA technologies. The questions asked in the study related to subjects including the structure and role of DNA molecule, structure of genes, some genetic technologies, Genetically Modified Organism (GMO) plants,…

Agarose Gel Electrophoresis Reveals Structural Fluidity of a Phage T3 DNA Packaging Intermediate

PubMed Central

Serwer, Philip; Wright, Elena T.

2012-01-01

We find a new aspect of DNA packaging-associated structural fluidity for phage T3 capsids. The procedure is (1) glutaraldehyde cross-linking of in vivo DNA packaging intermediates for stabilization of structure and then (2) determining of effective radius by two-dimensional agarose gel electrophoresis (2d-AGE). The intermediates are capsids with incompletely packaged DNA (ipDNA) and without an external DNA segment; these intermediates are called ipDNA-capsids. We initially increase production of ipDNA-capsids by raising NaCl concentration during in vivo DNA packaging. By 2d-AGE, we find a new state of contracted shell for some particles of one previously identified ipDNA-capsid. The contracted shell-state is found when ipDNA length/mature DNA length (F) is above 0.17, but not at lower F. Some contracted-shell ipDNA-capsids have the phage tail; others do not. The contracted-shell ipDNA-capsids are explained by premature DNA maturation cleavage that makes accessible a contracted-shell intermediate of a cycle of the T3 DNA packaging motor. The analysis of ipDNA-capsids, rather than intermediates with uncleaved DNA, provides a simplifying strategy for a complete biochemical analysis of in vivo DNA packaging. PMID:22222979

Insights into eukaryotic DNA priming from the structure and functional interactions of the 4Fe-4S cluster domain of human DNA primase

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

Vaithiyalingam, Sivaraja; Warren, Eric M.; Eichman, Brandt F.

2010-10-19

DNA replication requires priming of DNA templates by enzymes known as primases. Although DNA primase structures are available from archaea and bacteria, the mechanism of DNA priming in higher eukaryotes remains poorly understood in large part due to the absence of the structure of the unique, highly conserved C-terminal regulatory domain of the large subunit (p58C). Here, we present the structure of this domain determined to 1.7-{angstrom} resolution by X-ray crystallography. The p58C structure reveals a novel arrangement of an evolutionarily conserved 4Fe-4S cluster buried deeply within the protein core and is not similar to any known protein structure. Analysismore » of the binding of DNA to p58C by fluorescence anisotropy measurements revealed a strong preference for ss/dsDNA junction substrates. This approach was combined with site-directed mutagenesis to confirm that the binding of DNA occurs to a distinctively basic surface on p58C. A specific interaction of p58C with the C-terminal domain of the intermediate subunit of replication protein A (RPA32C) was identified and characterized by isothermal titration calorimetry and NMR. Restraints from NMR experiments were used to drive computational docking of the two domains and generate a model of the p58C-RPA32C complex. Together, our results explain functional defects in human DNA primase mutants and provide insights into primosome loading on RPA-coated ssDNA and regulation of primase activity.« less

Exploring the Interactions of the Dietary Plant Flavonoids Fisetin and Naringenin with G-Quadruplex and Duplex DNA, Showing Contrasting Binding Behavior: Spectroscopic and Molecular Modeling Approaches.

PubMed

Bhattacharjee, Snehasish; Chakraborty, Sandipan; Sengupta, Pradeep K; Bhowmik, Sudipta

2016-09-01

Guanine-rich sequences have the propensity to fold into a four-stranded DNA structure known as a G-quadruplex (G4). G4 forming sequences are abundant in the promoter region of several oncogenes and become a key target for anticancer drug binding. Here we have studied the interactions of two structurally similar dietary plant flavonoids fisetin and naringenin with G4 as well as double stranded (duplex) DNA by using different spectroscopic and modeling techniques. Our study demonstrates the differential binding ability of the two flavonoids with G4 and duplex DNA. Fisetin more strongly interacts with parallel G4 structure than duplex DNA, whereas naringenin shows stronger binding affinity to duplex rather than G4 DNA. Molecular docking results also corroborate our spectroscopic results, and it was found that both of the ligands are stacked externally in the G4 DNA structure. C-ring planarity of the flavonoid structure appears to be a crucial factor for preferential G4 DNA recognition of flavonoids. The goal of this study is to explore the critical effects of small differences in the structure of closely similar chemical classes of such small molecules (flavonoids) which lead to the contrasting binding properties with the two different forms of DNA. The resulting insights may be expected to facilitate the designing of the highly selective G4 DNA binders based on flavonoid scaffolds.

G-quadruplex and G-rich sequence stimulate Pif1p-catalyzed downstream duplex DNA unwinding through reducing waiting time at ss/dsDNA junction

PubMed Central

Zhang, Bo; Wu, Wen-Qiang; Liu, Na-Nv; Duan, Xiao-Lei; Li, Ming; Dou, Shuo-Xing; Hou, Xi-Miao; Xi, Xu-Guang

2016-01-01

Alternative DNA structures that deviate from B-form double-stranded DNA such as G-quadruplex (G4) DNA can be formed by G-rich sequences that are widely distributed throughout the human genome. We have previously shown that Pif1p not only unfolds G4, but also unwinds the downstream duplex DNA in a G4-stimulated manner. In the present study, we further characterized the G4-stimulated duplex DNA unwinding phenomenon by means of single-molecule fluorescence resonance energy transfer. It was found that Pif1p did not unwind the partial duplex DNA immediately after unfolding the upstream G4 structure, but rather, it would dwell at the ss/dsDNA junction with a ‘waiting time’. Further studies revealed that the waiting time was in fact related to a protein dimerization process that was sensitive to ssDNA sequence and would become rapid if the sequence is G-rich. Furthermore, we identified that the G-rich sequence, as the G4 structure, equally stimulates duplex DNA unwinding. The present work sheds new light on the molecular mechanism by which G4-unwinding helicase Pif1p resolves physiological G4/duplex DNA structures in cells. PMID:27471032

Presence of extracellular DNA in the Candida albicans biofilm matrix and its contribution to biofilms.

PubMed

Martins, Margarida; Uppuluri, Priya; Thomas, Derek P; Cleary, Ian A; Henriques, Mariana; Lopez-Ribot, José L; Oliveira, Rosário

2010-05-01

DNA has been described as a structural component of the extracellular matrix (ECM) in bacterial biofilms. In Candida albicans, there is a scarce knowledge concerning the contribution of extracellular DNA (eDNA) to biofilm matrix and overall structure. This work examined the presence and quantified the amount of eDNA in C. albicans biofilm ECM and the effect of DNase treatment and the addition of exogenous DNA on C. albicans biofilm development as indicators of a role for eDNA in biofilm development. We were able to detect the accumulation of eDNA in biofilm ECM extracted from C. albicans biofilms formed under conditions of flow, although the quantity of eDNA detected differed according to growth conditions, in particular with regards to the medium used to grow the biofilms. Experiments with C. albicans biofilms formed statically using a microtiter plate model indicated that the addition of exogenous DNA (>160 ng/ml) increases biofilm biomass and, conversely, DNase treatment (>0.03 mg/ml) decreases biofilm biomass at later time points of biofilm development. We present evidence for the role of eDNA in C. albicans biofilm structure and formation, consistent with eDNA being a key element of the ECM in mature C. albicans biofilms and playing a predominant role in biofilm structural integrity and maintenance.

The unusual and dynamic character of PX-DNA

DOE PAGES

Niu, Dong; Jiang, Hualin; Sha, Ruojie; ...

2015-07-15

PX-DNA is a four-stranded DNA structure that has been implicated in the recognition of homology, either continuously, or in an every-other-half-turn fashion. Some of the structural features of the molecule have been noted previously, but the structure requires further characterization. Here, we report atomic force microscopic characterization of PX molecules that contain periodically placed biotin groups, enabling the molecule to be labeled by streptavidin molecules at these sites. In comparison with conventional double stranded DNA and with antiparallel DNA double crossover molecules, it is clear that PX-DNA is a more dynamic structure. Moreover, the spacing between the nucleotide pairs alongmore » the helix axis is shorter, suggesting a mixed B/A structure. Circular dichroism spectroscopy indicates unusual features in the PX molecule that are absent in both the molecules to which it is compared.« less

Intracellular Delivery of a Planar DNA Origami Structure by the Transferrin-Receptor Internalization Pathway.

PubMed

Schaffert, David H; Okholm, Anders H; Sørensen, Rasmus S; Nielsen, Jesper S; Tørring, Thomas; Rosen, Christian B; Kodal, Anne Louise B; Mortensen, Michael R; Gothelf, Kurt V; Kjems, Jørgen

2016-05-01

DNA origami provides rapid access to easily functionalized, nanometer-sized structures making it an intriguing platform for the development of defined drug delivery and sensor systems. Low cellular uptake of DNA nanostructures is a major obstacle in the development of DNA-based delivery platforms. Herein, significant strong increase in cellular uptake in an established cancer cell line by modifying a planar DNA origami structure with the iron transport protein transferrin (Tf) is demonstrated. A variable number of Tf molecules are coupled to the origami structure using a DNA-directed, site-selective labeling technique to retain ligand functionality. A combination of confocal fluorescence microscopy and quantitative (qPCR) techniques shows up to 22-fold increased cytoplasmic uptake compared to unmodified structures and with an efficiency that correlates to the number of transferrin molecules on the origami surface. © 2016 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

Large-Scale Mitochondrial DNA Analysis of the Domestic Goat Reveals Six Haplogroups with High Diversity

PubMed Central

Naderi, Saeid; Rezaei, Hamid-Reza; Taberlet, Pierre; Zundel, Stéphanie; Rafat, Seyed-Abbas; Naghash, Hamid-Reza; El-Barody, Mohamed A. A.; Ertugrul, Okan; Pompanon, François

2007-01-01

Background From the beginning of domestication, the transportation of domestic animals resulted in genetic and demographic processes that explain their present distribution and genetic structure. Thus studying the present genetic diversity helps to better understand the history of domestic species. Methodology/Principal Findings The genetic diversity of domestic goats has been characterized with 2430 individuals from all over the old world, including 946 new individuals from regions poorly studied until now (mainly the Fertile Crescent). These individuals represented 1540 haplotypes for the HVI segment of the mitochondrial DNA (mtDNA) control region. This large-scale study allowed the establishment of a clear nomenclature of the goat maternal haplogroups. Only five of the six previously defined groups of haplotypes were divergent enough to be considered as different haplogroups. Moreover a new mitochondrial group has been localized around the Fertile Crescent. All groups showed very high haplotype diversity. Most of this diversity was distributed among groups and within geographic regions. The weak geographic structure may result from the worldwide distribution of the dominant A haplogroup (more than 90% of the individuals). The large-scale distribution of other haplogroups (except one), may be related to human migration. The recent fragmentation of local goat populations into discrete breeds is not detectable with mitochondrial markers. The estimation of demographic parameters from mismatch analyses showed that all groups had a recent demographic expansion corresponding roughly to the period when domestication took place. But even with a large data set it remains difficult to give relative dates of expansion for different haplogroups because of large confidence intervals. Conclusions/Significance We propose standard criteria for the definition of the different haplogroups based on the result of mismatch analysis and on the use of sequences of reference. Such a method could be also applied for clarifying the nomenclature of mitochondrial haplogroups in other domestic species. PMID:17925860

From the Tunnels into the Treetops: New Lineages of Black Yeasts from Biofilm in the Stockholm Metro System and Their Relatives among Ant-Associated Fungi in the Chaetothyriales

PubMed Central

Hubka, Vit; Thureborn, Olle; Lundberg, Johannes; Sallstedt, Therese; Wedin, Mats; Ivarsson, Magnus

2016-01-01

Rock-inhabiting fungi harbour species-rich, poorly differentiated, extremophilic taxa of polyphyletic origin. Their closest relatives are often well-known species from various biotopes with significant pathogenic potential. Speleothems represent a unique rock-dwelling habitat, whose mycobiota are largely unexplored. Isolation of fungi from speleothem biofilm covering bare granite walls in the Kungsträdgården metro station in Stockholm yielded axenic cultures of two distinct black yeast morphotypes. Phylogenetic analyses of DNA sequences from six nuclear loci, ITS, nuc18S and nuc28S rDNA, rpb1, rpb2 and β-tubulin, support their placement in the Chaetothyriales (Ascomycota). They are described as a new genus Bacillicladium with the type species B. lobatum, and a new species Bradymyces graniticola. Bacillicladium is distantly related to the known five chaetothyrialean families and is unique in the Chaetothyriales by variable morphology showing hyphal, meristematic and yeast-like growth in vitro. The nearest relatives of Bacillicladium are recruited among fungi isolated from cardboard-like construction material produced by arboricolous non-attine ants. Their sister relationship is weakly supported by the Maximum likelihood analysis, but strongly supported by Bayesian inference. The genus Bradymyces is placed amidst members of the Trichomeriaceae and is ecologically undefined; it includes an opportunistic animal pathogen while two other species inhabit rock surfaces. ITS rDNA sequences of three species accepted in Bradymyces and other undescribed species and environmental samples were subjected to phylogenetic analysis and in-depth comparative analysis of ITS1 and ITS2 secondary structures in order to study their intraspecific variability. Compensatory base change criterion in the ITS2 secondary structure supported delimitation of species in Bradymyces, which manifest a limited number of phenotypic features useful for species recognition. The role of fungi in the speleothem biofilm and relationships of Bacillicladium and Bradymyces with other members of the Chaetothyriales are discussed. PMID:27732675

From the Tunnels into the Treetops: New Lineages of Black Yeasts from Biofilm in the Stockholm Metro System and Their Relatives among Ant-Associated Fungi in the Chaetothyriales.

PubMed

Réblová, Martina; Hubka, Vit; Thureborn, Olle; Lundberg, Johannes; Sallstedt, Therese; Wedin, Mats; Ivarsson, Magnus

2016-01-01

Rock-inhabiting fungi harbour species-rich, poorly differentiated, extremophilic taxa of polyphyletic origin. Their closest relatives are often well-known species from various biotopes with significant pathogenic potential. Speleothems represent a unique rock-dwelling habitat, whose mycobiota are largely unexplored. Isolation of fungi from speleothem biofilm covering bare granite walls in the Kungsträdgården metro station in Stockholm yielded axenic cultures of two distinct black yeast morphotypes. Phylogenetic analyses of DNA sequences from six nuclear loci, ITS, nuc18S and nuc28S rDNA, rpb1, rpb2 and β-tubulin, support their placement in the Chaetothyriales (Ascomycota). They are described as a new genus Bacillicladium with the type species B. lobatum, and a new species Bradymyces graniticola. Bacillicladium is distantly related to the known five chaetothyrialean families and is unique in the Chaetothyriales by variable morphology showing hyphal, meristematic and yeast-like growth in vitro. The nearest relatives of Bacillicladium are recruited among fungi isolated from cardboard-like construction material produced by arboricolous non-attine ants. Their sister relationship is weakly supported by the Maximum likelihood analysis, but strongly supported by Bayesian inference. The genus Bradymyces is placed amidst members of the Trichomeriaceae and is ecologically undefined; it includes an opportunistic animal pathogen while two other species inhabit rock surfaces. ITS rDNA sequences of three species accepted in Bradymyces and other undescribed species and environmental samples were subjected to phylogenetic analysis and in-depth comparative analysis of ITS1 and ITS2 secondary structures in order to study their intraspecific variability. Compensatory base change criterion in the ITS2 secondary structure supported delimitation of species in Bradymyces, which manifest a limited number of phenotypic features useful for species recognition. The role of fungi in the speleothem biofilm and relationships of Bacillicladium and Bradymyces with other members of the Chaetothyriales are discussed.

Purification and functional characterization of p16, the ATPase of the bacteriophage Φ29 packaging machinery

PubMed Central

Ibarra, Borja; Valpuesta, José María; Carrascosa, José L.

2001-01-01

Bacteriophage Φ29 codes for a protein (p16) that is required for viral DNA packaging both in vivo and in vitro. Co-expression of p16 with the chaperonins GroEL and GroES has allowed its purification in a soluble form. Purified p16 shows a weak ATPase activity that is stimulated by either DNA or RNA, irrespective of the presence of any other viral component. The stimulation of ATPase activity of p16, although induced under packaging conditions, is not dependent of the actual DNA packaging and in this respect the Φ29 enzyme is similar to other viral terminases. Protein p16 competes with DNA and RNA in the interaction with the viral prohead, which occurs through the N-terminal region of the connector protein (p10). In fact, p16 interacts in a nucleotide-dependent fashion with the viral Φ29-encoded RNA (pRNA) involved in DNA packaging, and this binding can be competed with DNA. Our results are consistent with a model for DNA translocation in which p16, bound and organized around the connector, acts as a power stroke to pump the DNA into the prohead, using the hydrolysis of ATP as an energy source. PMID:11691914

Genetic control of enhanced mutability of mitochondrial DNA and gamma-ray sensitivity in Saccharomyces cerevisiae.

PubMed Central

Foury, F; Goffeau, A

1979-01-01

Five nuclear mutants enhancing the spontaneous mutation rate of mtDNA have been isolated in Saccharomyces cerevisiae. These mutators fall into five complementation groups and are located at five genetic loci different from rad50 to rad57 loci. Three mutants (gam1, gam2, and gam4), insensitive or weakly sensitive to gamma-rays, exhibit increased frequency of spontaneous production of mutants with large deletions of the mtDNA (p-) and of all tested mitochondrial drug-resistant mutants. Two other mutants (gam3 and gam5), highly sensitive to gamma-rays, increase only the mutation rate of particular alleles of the mtDNA. The mutant gam5 enhances only the production of p- and erythromycin-resistant clones. The mutant gam3 exhibits an enhanced rate of oligomycin-resistant clones as well as a collateral increase of nuclear mutability. The existence of gam3 and gam5 mutants indicates that at least two common steps control both nuclear DNA repair and the mutability of particular alleles of the mtDNA. However, the general spontaneous mutability of the mtDNA includes at least three steps not involved in the repair of nuclear DNA, as revealed by the gam1, gam2, and gam4 mutations. PMID:392521

Abasic and oxidized ribonucleotides embedded in DNA are processed by human APE1 and not by RNase H2.

PubMed

Malfatti, Matilde Clarissa; Balachander, Sathya; Antoniali, Giulia; Koh, Kyung Duk; Saint-Pierre, Christine; Gasparutto, Didier; Chon, Hyongi; Crouch, Robert J; Storici, Francesca; Tell, Gianluca

2017-11-02

Ribonucleoside 5'-monophosphates (rNMPs) are the most common non-standard nucleotides found in DNA of eukaryotic cells, with over 100 million rNMPs transiently incorporated in the mammalian genome per cell cycle. Human ribonuclease (RNase) H2 is the principal enzyme able to cleave rNMPs in DNA. Whether RNase H2 may process abasic or oxidized rNMPs incorporated in DNA is unknown. The base excision repair (BER) pathway is mainly responsible for repairing oxidized and abasic sites into DNA. Here we show that human RNase H2 is unable to process an abasic rNMP (rAP site) or a ribose 8oxoG (r8oxoG) site embedded in DNA. On the contrary, we found that recombinant purified human apurinic/apyrimidinic endonuclease-1 (APE1) and APE1 from human cell extracts efficiently process an rAP site in DNA and have weak endoribonuclease and 3'-exonuclease activities on r8oxoG substrate. Using biochemical assays, our results provide evidence of a human enzyme able to recognize and process abasic and oxidized ribonucleotides embedded in DNA. © The Author(s) 2017. Published by Oxford University Press on behalf of Nucleic Acids Research.

A small-molecule-linked DNA-graphene oxide-based fluorescence-sensing system for detection of biotin.

PubMed

Zhang, Hao; Li, Yan; Su, Xingguang

2013-11-15

In this paper, we establish a novel fluorescence-sensing system for the detection of biotin based on the interaction between DNA and graphene oxide and on protection of the terminal of the biotinylated single-stranded DNA fluorescent probe by streptavidin. In this system, streptavidin binds to the biotinylated DNA, which protects the DNA from hydrolysis by exonuclease I. The streptavidin-DNA conjugate is then adsorbed to the graphene oxide resulting in the fluorescence being quenched. Upon the addition of free biotin, it competes with the labeled biotin for the binding sites of streptavidin and then the exonuclease I digests the unbound DNA probe from the 3' to the 5' terminal, releasing the fluorophore from the DNA. Because of the weak affinity between the fluorophore and graphene oxide, the fluorescence is recovered. Under optimal conditions, the fluorescence intensity is proportional to the concentration of biotin in the concentration range of 0.5-20nmol/L. The detection limit for biotin is 0.44nmol/L. The proposed fluorescence-sensing system was applied to the determination of biotin in some real samples with satisfactory reproducibility and accuracy. This work could provide a common platform for detecting small biomolecules based on protein-small molecule ligand binding. Copyright © 2013 Elsevier Inc. All rights reserved.

CROSS-DISCIPLINARY PHYSICS AND RELATED AREAS OF SCIENCE AND TECHNOLOGY: Characteristics of alternating current hopping conductivity in DNA sequences

NASA Astrophysics Data System (ADS)

Ma, Song-Shan; Xu, Hui; Wang, Huan-You; Guo, Rui

2009-08-01

This paper presents a model to describe alternating current (AC) conductivity of DNA sequences, in which DNA is considered as a one-dimensional (1D) disordered system, and electrons transport via hopping between localized states. It finds that AC conductivity in DNA sequences increases as the frequency of the external electric field rises, and it takes the form of øac(ω) ~ ω2 ln2(1/ω). Also AC conductivity of DNA sequences increases with the increase of temperature, this phenomenon presents characteristics of weak temperature-dependence. Meanwhile, the AC conductivity in an off-diagonally correlated case is much larger than that in the uncorrelated case of the Anderson limit in low temperatures, which indicates that the off-diagonal correlations in DNA sequences have a great effect on the AC conductivity, while at high temperature the off-diagonal correlations no longer play a vital role in electric transport. In addition, the proportion of nucleotide pairs p also plays an important role in AC electron transport of DNA sequences. For p < 0.5, the conductivity of DNA sequence decreases with the increase of p, while for p >= 0.5, the conductivity increases with the increase of p.

Purification and functional characterization of p16, the ATPase of the bacteriophage Phi29 packaging machinery.

PubMed

Ibarra, B; Valpuesta, J M; Carrascosa, J L

2001-11-01

Bacteriophage Phi29 codes for a protein (p16) that is required for viral DNA packaging both in vivo and in vitro. Co-expression of p16 with the chaperonins GroEL and GroES has allowed its purification in a soluble form. Purified p16 shows a weak ATPase activity that is stimulated by either DNA or RNA, irrespective of the presence of any other viral component. The stimulation of ATPase activity of p16, although induced under packaging conditions, is not dependent of the actual DNA packaging and in this respect the Phi29 enzyme is similar to other viral terminases. Protein p16 competes with DNA and RNA in the interaction with the viral prohead, which occurs through the N-terminal region of the connector protein (p10). In fact, p16 interacts in a nucleotide-dependent fashion with the viral Phi29-encoded RNA (pRNA) involved in DNA packaging, and this binding can be competed with DNA. Our results are consistent with a model for DNA translocation in which p16, bound and organized around the connector, acts as a power stroke to pump the DNA into the prohead, using the hydrolysis of ATP as an energy source.

Potentiation of anthrax vaccines using protective antigen-expressing viral replicon vectors.

PubMed

Wang, Hai-Chao; An, Huai-Jie; Yu, Yun-Zhou; Xu, Qing

2015-02-01

DNA vaccines require improvement for human use because they are generally weak stimulators of the immune system in humans. The efficacy of DNA vaccines can be improved using a viral replicon as vector to administer antigen of pathogen. In this study, we comprehensively evaluated the conventional non-viral DNA, viral replicon DNA or viral replicon particles (VRP) vaccines encoding different forms of anthrax protective antigen (PA) for specific immunity and protective potency against anthrax. Our current results clearly suggested that these viral replicon DNA or VRP vaccines derived from Semliki Forest virus (SFV) induced stronger PA-specific immune responses than the conventional non-viral DNA vaccines when encoding the same antigen forms, which resulted in potent protection against challenge with the Bacillus anthracis strain A16R. Additionally, the naked PA-expressing SFV replicon DNA or VRP vaccines without the need for high doses or demanding particular delivery regimens elicited robust immune responses and afforded completely protective potencies, which indicated the potential of the SFV replicon as vector of anthrax vaccines for use in clinical application. Therefore, our results suggest that these PA-expressing SFV replicon DNA or VRP vaccines may be suitable as candidate vaccines against anthrax. Copyright © 2015 Elsevier B.V. All rights reserved.

A Paper Model of DNA Structure and Replication.

ERIC Educational Resources Information Center

Sigismondi, Linda A.

1989-01-01

A paper model which is designed to give students a hands-on experience during lecture and blackboard instruction on DNA structure is provided. A list of materials, paper patterns, and procedures for using the models to teach DNA structure and replication are given. (CW)

Structural insights into 5‧ flap DNA unwinding and incision by the human FAN1 dimer

NASA Astrophysics Data System (ADS)

Zhao, Qi; Xue, Xiaoyu; Longerich, Simonne; Sung, Patrick; Xiong, Yong

2014-12-01

Human FANCD2-associated nuclease 1 (FAN1) is a DNA structure-specific nuclease involved in the processing of DNA interstrand crosslinks (ICLs). FAN1 maintains genomic stability and prevents tissue decline in multiple organs, yet it confers ICL-induced anti-cancer drug resistance in several cancer subtypes. Here we report three crystal structures of human FAN1 in complex with a 5‧ flap DNA substrate, showing that two FAN1 molecules form a head-to-tail dimer to locate the lesion, orient the DNA and unwind a 5‧ flap for subsequent incision. Biochemical experiments further validate our model for FAN1 action, as structure-informed mutations that disrupt protein dimerization, substrate orientation or flap unwinding impair the structure-specific nuclease activity. Our work elucidates essential aspects of FAN1-DNA lesion recognition and a unique mechanism of incision. These structural insights shed light on the cellular mechanisms underlying organ degeneration protection and cancer drug resistance mediated by FAN1.

The complex between a four-way DNA junction and T7 endonuclease I

PubMed Central

Déclais, Anne-Cécile; Fogg, Jonathan M.; Freeman, Alasdair D.J.; Coste, Franck; Hadden, Jonathan M.; Phillips, Simon E.V.; Lilley, David M.J.

2003-01-01

The junction-resolving enzyme endonuclease I is selective for the structure of the DNA four-way (Holliday) junction. The enzyme binds to a four-way junction in two possible orientations, with a 4:1 ratio, opening the DNA structure at the centre and changing the global structure into a 90° cross of approximately coaxial helices. The nuclease cleaves the continuous strands of the junction in each orientation. Binding leads to pronounced regions of protection of the DNA against hydroxyl radical attack. Using all this information together with the known structure of the enzyme and the structure of the BglI–DNA complex, we have constructed a model of the complex of endonuclease I and a DNA junction. This shows how the enzyme is selective for the structure of a four-way junction, such that both continuous strands can be accommodated into the two active sites so that a productive resolution event is possible. PMID:12628932

The Human DNA glycosylases NEIL1 and NEIL3 Excise Psoralen-Induced DNA-DNA Cross-Links in a Four-Stranded DNA Structure.

PubMed

Martin, Peter R; Couvé, Sophie; Zutterling, Caroline; Albelazi, Mustafa S; Groisman, Regina; Matkarimov, Bakhyt T; Parsons, Jason L; Elder, Rhoderick H; Saparbaev, Murat K

2017-12-12

Interstrand cross-links (ICLs) are highly cytotoxic DNA lesions that block DNA replication and transcription by preventing strand separation. Previously, we demonstrated that the bacterial and human DNA glycosylases Nei and NEIL1 excise unhooked psoralen-derived ICLs in three-stranded DNA via hydrolysis of the glycosidic bond between the crosslinked base and deoxyribose sugar. Furthermore, NEIL3 from Xenopus laevis has been shown to cleave psoralen- and abasic site-induced ICLs in Xenopus egg extracts. Here we report that human NEIL3 cleaves psoralen-induced DNA-DNA cross-links in three-stranded and four-stranded DNA substrates to generate unhooked DNA fragments containing either an abasic site or a psoralen-thymine monoadduct. Furthermore, while Nei and NEIL1 also cleave a psoralen-induced four-stranded DNA substrate to generate two unhooked DNA duplexes with a nick, NEIL3 targets both DNA strands in the ICL without generating single-strand breaks. The DNA substrate specificities of these Nei-like enzymes imply the occurrence of long uninterrupted three- and four-stranded crosslinked DNA-DNA structures that may originate in vivo from DNA replication fork bypass of an ICL. In conclusion, the Nei-like DNA glycosylases unhook psoralen-derived ICLs in various DNA structures via a genuine repair mechanism in which complex DNA lesions can be removed without generation of highly toxic double-strand breaks.

Structural basis of DNA target recognition by the B3 domain of Arabidopsis epigenome reader VAL1

PubMed Central

Sasnauskas, Giedrius; Kauneckaitė, Kotryna; Siksnys, Virginijus

2018-01-01

Abstract Arabidopsis thaliana requires a prolonged period of cold exposure during winter to initiate flowering in a process termed vernalization. Exposure to cold induces epigenetic silencing of the FLOWERING LOCUS C (FLC) gene by Polycomb group (PcG) proteins. A key role in this epigenetic switch is played by transcriptional repressors VAL1 and VAL2, which specifically recognize Sph/RY DNA sequences within FLC via B3 DNA binding domains, and mediate recruitment of PcG silencing machinery. To understand the structural mechanism of site-specific DNA recognition by VAL1, we have solved the crystal structure of VAL1 B3 domain (VAL1-B3) bound to a 12 bp oligoduplex containing the canonical Sph/RY DNA sequence 5′-CATGCA-3′/5′-TGCATG-3′. We find that VAL1-B3 makes H-bonds and van der Waals contacts to DNA bases of all six positions of the canonical Sph/RY element. In agreement with the structure, in vitro DNA binding studies show that VAL1-B3 does not tolerate substitutions at any position of the 5′-TGCATG-3′ sequence. The VAL1-B3–DNA structure presented here provides a structural model for understanding the specificity of plant B3 domains interacting with the Sph/RY and other DNA sequences. PMID:29660015

Destabilization of the MutSα's protein-protein interface due to binding to the DNA adduct induced by anticancer agent carboplatin via molecular dynamics simulations.

PubMed

Negureanu, Lacramioara; Salsbury, Freddie R

2013-11-01

DNA mismatch repair (MMR) proteins maintain genetic integrity in all organisms by recognizing and repairing DNA errors. Such alteration of hereditary information can lead to various diseases, including cancer. Besides their role in DNA repair, MMR proteins detect and initiate cellular responses to certain type of DNA damage. Its response to the damaged DNA has made the human MMR pathway a useful target for anticancer agents such as carboplatin. This study indicates that strong, specific interactions at the interface of MutSα in response to the mismatched DNA recognition are replaced by weak, non-specific interactions in response to the damaged DNA recognition. Data suggest a severe impairment of the dimerization of MutSα in response to the damaged DNA recognition. While the core of MutSα is preserved in response to the damaged DNA recognition, the loss of contact surface and the rearrangement of contacts at the protein interface suggest a different packing in response to the damaged DNA recognition. Coupled in response to the mismatched DNA recognition, interaction energies, hydrogen bonds, salt bridges, and solvent accessible surface areas at the interface of MutSα and within the subunits are uncoupled or asynchronously coupled in response to the damaged DNA recognition. These pieces of evidence suggest that the loss of a synchronous mode of response in the MutSα's surveillance for DNA errors would possibly be one of the mechanism(s) of signaling the MMR-dependent programed cell death much wanted in anticancer therapies. The analysis was drawn from dynamics simulations.

Structure and free energy of cholesteric DNA droplets

NASA Astrophysics Data System (ADS)

Strey, Helmut; Hong, Helen; Easwar, Nalini

2000-03-01

Liquid crystals of DNA are the simplest model systems for DNA packing in cell nuclei or in phage heads. With increasing concentration DNA solutions exhibit the following phases: hexagonal, line hexatic, cholesteric, blue phases. We will present measurements of defect structure and pitch of cholesteric spherulites of short fragment DNA (146 base pairs). DNA concentration as well as salt concentrations are controlled by bathing the spherulites in poly (ethylene glycol) (MW 35,000u) solutions of known osmotic pressure. Combining polarizing microscopy and x-ray scattering with the osmotic stress method allows us to monitor the cholesteric structure and pitch as a function of interaxial distance between DNA molecules as well as salt concentration and type. In particular, we present data on how the DNA cholesteric pitch unwinds when the line hexatic phase is approached.

Raman spectroscopic study of plasma-treated salmon DNA

NASA Astrophysics Data System (ADS)

Joon Lee, Geon; Kwon, Young-Wan; Hee Kim, Yong; Ha Choi, Eun

2013-01-01

In this research, we studied the effect of plasma treatment on the optical/structural properties of the deoxyribonucleic acid (DNA) extracted from salmon sperm. DNA-cetyltrimethylammonium (CTMA) films were obtained by complexation of DNA with CTMA. Circular dichroism (CD) and Raman spectra indicated that DNA retained its double helical structure in the solid film. The Raman spectra exhibited several vibration modes corresponding to the nuclear bases and the deoxyribose-phosphate backbones of the DNA, as well as the alkylchains of CTMA. Dielectric-barrier-discharge (DBD) plasma treatment induced structural modification and damage to the DNA, as observed by changes in the ultraviolet-visible absorption, CD, and Raman spectra. The optical emission spectra of the DBD plasma confirmed that DNA modification was induced by plasma ions such as reactive oxygen species and reactive nitrogen species.

Structural polymorphism at LCR and its role in beta-globin gene regulation.

PubMed

Kukreti, Shrikant; Kaur, Harpreet; Kaushik, Mahima; Bansal, Aparna; Saxena, Sarika; Kaushik, Shikha; Kukreti, Ritushree

2010-09-01

Information on the secondary structures and conformational manifestations of eukaryotic DNA and their biological significance with reference to gene regulation and expression is limited. The human beta-globin gene Locus Control Region (LCR), a dominant regulator of globin gene expression, is a contiguous piece of DNA with five tissue-specific DNase I-hypersensitive sites (HSs). Since these HSs have a high density of transcription factor binding sites, structural interdependencies between HSs and different promoters may directly or indirectly regulate LCR functions. Mutations and SNPs may stabilize or destabilize the local secondary structures, affecting the gene expression by changes in the protein-DNA recognition patterns. Various palindromic or quasi-palindromic segments within LCR, could cause structural polymorphism and geometrical switching of DNA. This emphasizes the importance of understanding of the sequence-dependent variations of the DNA structure. Such structural motifs might act as regulatory elements. The local conformational variability of a DNA segment or action of a DNA specific protein is key to create and maintain active chromatin domains and affect transcription of various tissue specific beta-globin genes. We, summarize here the current status of beta-globin LCR structure and function. Further structural studies at molecular level and functional genomics might solve the regulatory puzzles that control the beta-globin gene locus. Copyright (c) 2010 Elsevier Masson SAS. All rights reserved.

Hybrid Methods Reveal Multiple Flexibly Linked DNA Polymerases within the Bacteriophage T7 Replisome

DOE PAGES

Wallen, Jamie R.; Zhang, Hao; Weis, Caroline; ...

2017-01-03

The physical organization of DNA enzymes at a replication fork enables efficient copying of two antiparallel DNA strands, yet dynamic protein interactions within the replication complex complicate replisome structural studies. We employed a combination of crystallographic, native mass spectrometry and small-angle X-ray scattering experiments to capture alternative structures of a model replication system encoded by bacteriophage T7. then, the two molecules of DNA polymerase bind the ring-shaped primase-helicase in a conserved orientation and provide structural insight into how the acidic C-terminal tail of the primase-helicase contacts the DNA polymerase to facilitate loading of the polymerase onto DNA. A third DNA polymerasemore » binds the ring in an offset manner that may enable polymerase exchange during replication. Alternative polymerase binding modes are also detected by small-angle X-ray scattering with DNA substrates present. The collective results unveil complex motions within T7 replisome higher-order structures that are underpinned by multivalent protein-protein interactions with functional implications.« less

Hybrid Methods Reveal Multiple Flexibly Linked DNA Polymerases within the Bacteriophage T7 Replisome

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

Wallen, Jamie R.; Zhang, Hao; Weis, Caroline

The physical organization of DNA enzymes at a replication fork enables efficient copying of two antiparallel DNA strands, yet dynamic protein interactions within the replication complex complicate replisome structural studies. We employed a combination of crystallographic, native mass spectrometry and small-angle X-ray scattering experiments to capture alternative structures of a model replication system encoded by bacteriophage T7. then, the two molecules of DNA polymerase bind the ring-shaped primase-helicase in a conserved orientation and provide structural insight into how the acidic C-terminal tail of the primase-helicase contacts the DNA polymerase to facilitate loading of the polymerase onto DNA. A third DNA polymerasemore » binds the ring in an offset manner that may enable polymerase exchange during replication. Alternative polymerase binding modes are also detected by small-angle X-ray scattering with DNA substrates present. The collective results unveil complex motions within T7 replisome higher-order structures that are underpinned by multivalent protein-protein interactions with functional implications.« less

Computer-aided design of DNA origami structures.

PubMed

Selnihhin, Denis; Andersen, Ebbe Sloth

2015-01-01

The DNA origami method enables the creation of complex nanoscale objects that can be used to organize molecular components and to function as reconfigurable mechanical devices. Of relevance to synthetic biology, DNA origami structures can be delivered to cells where they can perform complicated sense-and-act tasks, and can be used as scaffolds to organize enzymes for enhanced synthesis. The design of DNA origami structures is a complicated matter and is most efficiently done using dedicated software packages. This chapter describes a procedure for designing DNA origami structures using a combination of state-of-the-art software tools. First, we introduce the basic method for calculating crossover positions between DNA helices and the standard crossover patterns for flat, square, and honeycomb DNA origami lattices. Second, we provide a step-by-step tutorial for the design of a simple DNA origami biosensor device, from schematic idea to blueprint creation and to 3D modeling and animation, and explain how careful modeling can facilitate later experimentation in the laboratory.

Kinetic gating mechanism of DNA damage recognition by Rad4/XPC

NASA Astrophysics Data System (ADS)

Chen, Xuejing; Velmurugu, Yogambigai; Zheng, Guanqun; Park, Beomseok; Shim, Yoonjung; Kim, Youngchang; Liu, Lili; van Houten, Bennett; He, Chuan; Ansari, Anjum; Min, Jung-Hyun

2015-01-01

The xeroderma pigmentosum C (XPC) complex initiates nucleotide excision repair by recognizing DNA lesions before recruiting downstream factors. How XPC detects structurally diverse lesions embedded within normal DNA is unknown. Here we present a crystal structure that captures the yeast XPC orthologue (Rad4) on a single register of undamaged DNA. The structure shows that a disulphide-tethered Rad4 flips out normal nucleotides and adopts a conformation similar to that seen with damaged DNA. Contrary to many DNA repair enzymes that can directly reject non-target sites as structural misfits, our results suggest that Rad4/XPC uses a kinetic gating mechanism whereby lesion selectivity arises from the kinetic competition between DNA opening and the residence time of Rad4/XPC per site. This mechanism is further supported by measurements of Rad4-induced lesion-opening times using temperature-jump perturbation spectroscopy. Kinetic gating may be a general mechanism used by site-specific DNA-binding proteins to minimize time-consuming interrogations of non-target sites.

Three-dimensional structural dynamics and fluctuations of DNA-nanogold conjugates by individual-particle electron tomography

NASA Astrophysics Data System (ADS)

Zhang, Lei; Lei, Dongsheng; Smith, Jessica M.; Zhang, Meng; Tong, Huimin; Zhang, Xing; Lu, Zhuoyang; Liu, Jiankang; Alivisatos, A. Paul; Ren, Gang

2016-03-01

DNA base pairing has been used for many years to direct the arrangement of inorganic nanocrystals into small groupings and arrays with tailored optical and electrical properties. The control of DNA-mediated assembly depends crucially on a better understanding of three-dimensional structure of DNA-nanocrystal-hybridized building blocks. Existing techniques do not allow for structural determination of these flexible and heterogeneous samples. Here we report cryo-electron microscopy and negative-staining electron tomography approaches to image, and three-dimensionally reconstruct a single DNA-nanogold conjugate, an 84-bp double-stranded DNA with two 5-nm nanogold particles for potential substrates in plasmon-coupling experiments. By individual-particle electron tomography reconstruction, we obtain 14 density maps at ~2-nm resolution. Using these maps as constraints, we derive 14 conformations of dsDNA by molecular dynamics simulations. The conformational variation is consistent with that from liquid solution, suggesting that individual-particle electron tomography could be an expected approach to study DNA-assembling and flexible protein structure and dynamics.

Kinetic gating mechanism of DNA damage recognition by Rad4/XPC

DOE PAGES

Chen, Xuejing; Velmurugu, Yogambigai; Zheng, Guanqun; ...

2015-01-06

The xeroderma pigmentosum C (XPC) complex initiates nucleotide excision repair by recognizing DNA lesions before recruiting downstream factors. How XPC detects structurally diverse lesions embedded within normal DNA is unknown. Here we present a crystal structure that captures the yeast XPC orthologue (Rad4) on a single register of undamaged DNA. The structure shows that a disulphide-tethered Rad4 flips out normal nucleotides and adopts a conformation similar to that seen with damaged DNA. Contrary to many DNA repair enzymes that can directly reject non-target sites as structural misfits, our results suggest that Rad4/XPC uses a kinetic gating mechanism whereby lesion selectivitymore » arises from the kinetic competition between DNA opening and the residence time of Rad4/XPC per site. This mechanism is further supported by measurements of Rad4-induced lesion-opening times using temperature-jump perturbation spectroscopy. Lastly, kinetic gating may be a general mechanism used by site-specific DNA-binding proteins to minimize time-consuming interrogations of non-target sites.« less

The secondary structure of the ets domain of human Fli-1 resembles that of the helix-turn-helix DNA-binding motif of the Escherichia coli catabolite gene activator protein.

PubMed Central

Liang, H; Olejniczak, E T; Mao, X; Nettesheim, D G; Yu, L; Thompson, C B; Fesik, S W

1994-01-01

The ets family of eukaryotic transcription factors is characterized by a conserved DNA-binding domain of approximately 85 amino acids for which the three-dimensional structure is not known. By using multidimensional NMR spectroscopy, we have determined the secondary structure of the ets domain of one member of this gene family, human Fli-1, both in the free form and in a complex with a 16-bp cognate DNA site. The secondary structure of the Fli-1 ets domain consists of three alpha-helices and a short four-stranded antiparallel beta-sheet. This secondary structure arrangement resembles that of the DNA-binding domain of the catabolite gene activator protein of Escherichia coli, as well as those of several eukaryotic DNA-binding proteins including histone H5, HNF-3/fork head, and the heat shock transcription factor. Differences in chemical shifts of backbone resonances and amide exchange rates between the DNA-bound and free forms of the Fli-1 ets domain suggest that the third helix is the DNA recognition helix, as in the catabolite gene activator protein and other structurally related proteins. These results suggest that the ets domain is structurally similar to the catabolite gene activator protein family of helix-turn-helix DNA-binding proteins. Images PMID:7972119

An enhanced chemiluminescence resonance energy transfer system based on target recycling G-guadruplexes/hemin DNAzyme catalysis and its application in ultrasensitive detection of DNA.

PubMed

Chen, Jia; Huang, Yong; Vdovenko, Marina; Sakharov, Ivan Yu; Su, Guifa; Zhao, Shulin

2015-06-01

An enhanced chemiluminescence resonance energy transfer (CRET) system based on target recycling G-guadruplexes/hemin DNAzyme catalysis was developed for ultrasensitive detection of DNA. CRET system consists of luminol as chemiluminescent donor, and fluorescein isothiocyanate (FITC) as acceptor. The sensitive detection was achieved by using the system consisted of G-riched DNA, blocker DNA, and the Nb.BbvCI biocatalyst. Upon addition of target DNA to the system, target DNA hybridizes with the quasi-circular DNA structure, and forms a DNA duplex. The formation of DNA duplex triggers selective enzymatic cleavage of quasi-circular DNA by Nb.BbvCI, resulting in the release of target DNA and two G-riched DNAzyme segments. Released target DNA then hybridizes with another quasi-circular DNA structure to initiate the cleavage of the quasi-circular DNA structure. Eventually, each target DNA can go through many cycles, resulting in the digestion of many quasi-circular DNA structures, generating many G-riched DNAzyme segments. G-riched DNAzyme segment products assemble with hemin to form stable hemin/G-quadruplexes that exhibit peroxidase-like activity which can catalyze the oxidation of luminol by H2O2 to produce CL signals. In the presence of FITC, CL of luminol can excite FITC molecules, and thus produced CRET between the luminol and FITC. This unique analysis strategy gives a detection limit down to 80 fM, which is at least four orders of magnitude lower than that of unamplified DNA detection methods. Copyright © 2015 Elsevier B.V. All rights reserved.

Repetitive DNA loci and their modulation by the non-canonical nucleic acid structures R-loops and G-quadruplexes

PubMed Central

Hall, Amanda C.; Ostrowski, Lauren A.; Mekhail, Karim

2017-01-01

ABSTRACT Cells have evolved intricate mechanisms to maintain genome stability despite allowing mutational changes to drive evolutionary adaptation. Repetitive DNA sequences, which represent the bulk of most genomes, are a major threat to genome stability often driving chromosome rearrangements and disease. The major source of repetitive DNA sequences and thus the most vulnerable constituents of the genome are the rDNA (rDNA) repeats, telomeres, and transposable elements. Maintaining the stability of these loci is critical to overall cellular fitness and lifespan. Therefore, cells have evolved mechanisms to regulate rDNA copy number, telomere length and transposon activity, as well as DNA repair at these loci. In addition, non-canonical structure-forming DNA motifs can also modulate the function of these repetitive DNA loci by impacting their transcription, replication, and stability. Here, we discuss key mechanisms that maintain rDNA repeats, telomeres, and transposons in yeast and human before highlighting emerging roles for non-canonical DNA structures at these repetitive loci. PMID:28406751

In the Absence of Writhe, DNA Relieves Torsional Stress with Localized, Sequence-Dependent Structural Failure to Preserve B-form

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

Randall, Graham L.; Zechiedrich, E. L.; Pettitt, Bernard M.

2009-09-01

To understand how underwinding and overwinding the DNA helix affects its structure, we simulated 19 independent DNA systems with fixed degrees of twist using molecular dynamics in a system that does not allow writhe. Underwinding DNA induced spontaneous, sequence-dependent base flipping and local denaturation, while overwinding DNA induced the formation of Pauling-like DNA (P-DNA). The winding resulted in a bimodal state simultaneously including local structural failure and B-form DNA for both underwinding and extreme overwinding. Our simulations suggest that base flipping and local denaturation may provide a landscape influencing protein recognition of DNA sequence to affect, for examples, replication, transcriptionmore » and recombination. Additionally, our findings help explain results from singlemolecule experiments and demonstrate that elastic rod models are strictly valid on average only for unstressed or overwound DNA up to P-DNA formation. Finally, our data support a model in which base flipping can result from torsional stress.« less

Structural stability of DNA origami nanostructures in the presence of chaotropic agents.

PubMed

Ramakrishnan, Saminathan; Krainer, Georg; Grundmeier, Guido; Schlierf, Michael; Keller, Adrian

2016-05-21

DNA origami represent powerful platforms for single-molecule investigations of biomolecular processes. The required structural integrity of the DNA origami may, however, pose significant limitations regarding their applicability, for instance in protein folding studies that require strongly denaturing conditions. Here, we therefore report a detailed study on the stability of 2D DNA origami triangles in the presence of the strong chaotropic denaturing agents urea and guanidinium chloride (GdmCl) and its dependence on concentration and temperature. At room temperature, the DNA origami triangles are stable up to at least 24 h in both denaturants at concentrations as high as 6 M. At elevated temperatures, however, structural stability is governed by variations in the melting temperature of the individual staple strands. Therefore, the global melting temperature of the DNA origami does not represent an accurate measure of their structural stability. Although GdmCl has a stronger effect on the global melting temperature, its attack results in less structural damage than observed for urea under equivalent conditions. This enhanced structural stability most likely originates from the ionic nature of GdmCl. By rational design of the arrangement and lengths of the individual staple strands used for the folding of a particular shape, however, the structural stability of DNA origami may be enhanced even further to meet individual experimental requirements. Overall, their high stability renders DNA origami promising platforms for biomolecular studies in the presence of chaotropic agents, including single-molecule protein folding or structural switching.

Structural Mechanism of Replication Stalling on a Bulky Amino-Polycyclic Aromatic Hydrocarbon DNA Adduct by a Y Family DNA Polymerase

PubMed Central

Kirouac, Kevin N.; Basu, Ashis K.; Ling, Hong

2013-01-01

Polycyclic aromatic hydrocarbons and their nitro derivatives are culprits of the detrimental health effects of environmental pollution. These hydrophobic compounds metabolize to reactive species and attach to DNA producing bulky lesions, such as N-[deoxyguanosine-8-yl]-1-aminopyrene (APG), in genomic DNA. The bulky adducts block DNA replication by high-fidelity polymerases and compromise replication fidelities and efficiencies by specialized lesion bypass polymerases. Here we present three crystal structures of the DNA polymerase Dpo4, a model translesion DNA polymerase of the Y family, in complex with APG-lesion-containing DNA in pre-insertion and extension stages. APG is captured in two conformations in the pre-insertion complex; one is highly exposed to the solvent, whereas the other is harbored in a shallow cleft between the finger and unique Y family little finger domain. In contrast, APG is in a single conformation at the extension stage, in which the pyrene ring is sandwiched between the little finger domain and a base from the turning back single-stranded template strand. Strikingly, a nucleotide intercalates the DNA helix to form a quaternary complex with Dpo4, DNA, and an incoming nucleotide, which stabilizes the distorted DNA structure at the extension stage. The unique APG DNA conformations in Dpo4 inhibit DNA translocation through the polymerase active site for APG bypass. We also modeled an insertion complex that illustrates a solvent-exposed pyrene ring contributing to an unstable insertion state. The structural work combined with our lesion replication assays provides a novel structural mechanism on bypass of DNA adducts containing polycyclic aromatic hydrocarbon moieties. PMID:23876706

Structural mechanism of replication stalling on a bulky amino-polycyclic aromatic hydrocarbon DNA adduct by a y family DNA polymerase.

PubMed

Kirouac, Kevin N; Basu, Ashis K; Ling, Hong

2013-11-15

Polycyclic aromatic hydrocarbons and their nitro derivatives are culprits of the detrimental health effects of environmental pollution. These hydrophobic compounds metabolize to reactive species and attach to DNA producing bulky lesions, such as N-[deoxyguanosine-8-yl]-1-aminopyrene (APG), in genomic DNA. The bulky adducts block DNA replication by high-fidelity polymerases and compromise replication fidelities and efficiencies by specialized lesion bypass polymerases. Here we present three crystal structures of the DNA polymerase Dpo4, a model translesion DNA polymerase of the Y family, in complex with APG-lesion-containing DNA in pre-insertion and extension stages. APG is captured in two conformations in the pre-insertion complex; one is highly exposed to the solvent, whereas the other is harbored in a shallow cleft between the finger and unique Y family little finger domain. In contrast, APG is in a single conformation at the extension stage, in which the pyrene ring is sandwiched between the little finger domain and a base from the turning back single-stranded template strand. Strikingly, a nucleotide intercalates the DNA helix to form a quaternary complex with Dpo4, DNA, and an incoming nucleotide, which stabilizes the distorted DNA structure at the extension stage. The unique APG DNA conformations in Dpo4 inhibit DNA translocation through the polymerase active site for APG bypass. We also modeled an insertion complex that illustrates a solvent-exposed pyrene ring contributing to an unstable insertion state. The structural work combined with our lesion replication assays provides a novel structural mechanism on bypass of DNA adducts containing polycyclic aromatic hydrocarbon moieties. © 2013.

Distinct Mechanisms of Nuclease-Directed DNA-Structure-Induced Genetic Instability in Cancer Genomes.

PubMed

Zhao, Junhua; Wang, Guliang; Del Mundo, Imee M; McKinney, Jennifer A; Lu, Xiuli; Bacolla, Albino; Boulware, Stephen B; Zhang, Changsheng; Zhang, Haihua; Ren, Pengyu; Freudenreich, Catherine H; Vasquez, Karen M

2018-01-30

Sequences with the capacity to adopt alternative DNA structures have been implicated in cancer etiology; however, the mechanisms are unclear. For example, H-DNA-forming sequences within oncogenes have been shown to stimulate genetic instability in mammals. Here, we report that H-DNA-forming sequences are enriched at translocation breakpoints in human cancer genomes, further implicating them in cancer etiology. H-DNA-induced mutations were suppressed in human cells deficient in the nucleotide excision repair nucleases, ERCC1-XPF and XPG, but were stimulated in cells deficient in FEN1, a replication-related endonuclease. Further, we found that these nucleases cleaved H-DNA conformations, and the interactions of modeled H-DNA with ERCC1-XPF, XPG, and FEN1 proteins were explored at the sub-molecular level. The results suggest mechanisms of genetic instability triggered by H-DNA through distinct structure-specific, cleavage-based replication-independent and replication-dependent pathways, providing critical evidence for a role of the DNA structure itself in the etiology of cancer and other human diseases. Copyright © 2018 The Authors. Published by Elsevier Inc. All rights reserved.

Dielectrophoresis of gold nanoparticles conjugated to DNA origami structures

PubMed Central

Wiens, Matthew; Lakatos, Mathias; Heerwig, Andreas; Ostermaier, Frieder; Haufe, Nora

2016-01-01

Summary DNA nanostructures are promising construction materials to bridge the gap between self-assembly of functional molecules and conventional top-down fabrication methods in nanotechnology. Their positioning onto specific locations of a microstructured substrate is an important task towards this aim. Here we study manipulation and positioning of pristine and of gold nanoparticle-conjugated tubular DNA origami structures using ac dielectrophoresis. The dielectrophoretic behavior was investigated employing fluorescence microscopy. For the pristine origami, a significant dielectrophoretic response was found to take place in the megahertz range, whereas, due to the higher polarizability of the metallic nanoparticles, the nanoparticle/DNA hybrid structures required a lower electrical field strength and frequency for a comparable trapping at the edges of the electrode structure. The nanoparticle conjugation additionally resulted in a remarkable alteration of the DNA structure arrangement. The growth of linear, chain-like structures in between electrodes at applied frequencies in the megahertz range was observed. The long-range chain formation is caused by a local, gold nanoparticle-induced field concentration along the DNA nanostructures, which in turn, creates dielectrophoretic forces that enable the observed self-alignment of the hybrid structures. PMID:27547612

Crystal structures of 3-methyladenine DNA glycosylase MagIII and the recognition of alkylated bases

PubMed Central

Eichman, Brandt F.; O’Rourke, Eyleen J.; Radicella, J.Pablo; Ellenberger, Tom

2003-01-01

DNA glycosylases catalyze the excision of chemically modified bases from DNA. Although most glycosylases are specific to a particular base, the 3-methyladenine (m3A) DNA glycosylases include both highly specific enzymes acting on a single modified base, and enzymes with broader specificity for alkylation-damaged DNA. Our structural understanding of these different enzymatic specificities is currently limited to crystal and NMR structures of the unliganded enzymes and complexes with abasic DNA inhibitors. Presented here are high-resolution crystal structures of the m3A DNA glycosylase from Helicobacter pylori (MagIII) in the unliganded form and bound to alkylated bases 3,9-dimethyladenine and 1,N6-ethenoadenine. These are the first structures of a nucleobase bound in the active site of a m3A glycosylase belonging to the helix–hairpin–helix superfamily. MagIII achieves its specificity for positively-charged m3A not by direct interactions with purine or methyl substituent atoms, but rather by stacking the base between two aromatic side chains in a pocket that excludes 7-methylguanine. We report base excision and DNA binding activities of MagIII active site mutants, together with a structural comparison of the HhH glycosylases. PMID:14517230

StructAlign, a Program for Alignment of Structures of DNA-Protein Complexes.

PubMed

Popov, Ya V; Galitsyna, A A; Alexeevski, A V; Karyagina, A S; Spirin, S A

2015-11-01

Comparative analysis of structures of complexes of homologous proteins with DNA is important in the analysis of DNA-protein recognition. Alignment is a necessary stage of the analysis. An alignment is a matching of amino acid residues and nucleotides of one complex to residues and nucleotides of the other. Currently, there are no programs available for aligning structures of DNA-protein complexes. We present the program StructAlign, which should fill this gap. The program inputs a pair of complexes of DNA double helix with proteins and outputs an alignment of DNA chains corresponding to the best spatial fit of the protein chains.

Hybrid DNA i-motif: Aminoethylprolyl-PNA (pC5) enhance the stability of DNA (dC5) i-motif structure.

PubMed

Gade, Chandrasekhar Reddy; Sharma, Nagendra K

2017-12-15

This report describes the synthesis of C-rich sequence, cytosine pentamer, of aep-PNA and its biophysical studies for the formation of hybrid DNA:aep-PNAi-motif structure with DNA cytosine pentamer (dC 5 ) under acidic pH conditions. Herein, the CD/UV/NMR/ESI-Mass studies strongly support the formation of stable hybrid DNA i-motif structure with aep-PNA even near acidic conditions. Hence aep-PNA C-rich sequence cytosine could be considered as potential DNA i-motif stabilizing agents in vivo conditions. Copyright © 2017 Elsevier Ltd. All rights reserved.

[The role of remodeling complexes CHD1 and ISWI in spontaneous and UV-induced mutagenesis control in yeast Saccharomyces cerevisiae].

PubMed

Evstiukhina, T A; Alekseeva, E A; Fedorov, D V; Peshekhonov, V T; Korolev, V G

2017-02-01

Chromatin remodulators are special multiprotein machines capable of transforming the structure, constitution, and positioning of nucleosomes on DNA. Biochemical activities of remodeling complexes CHD1 and ISWI from the SWI2/SNF2 family are well established. They ensure correct positioning of nucleosomes along the genome, which is probably critical for genome stability, in particular, after action of polymerases, repair enzymes, and transcription. In this paper, we show that single mutations in genes ISW1, ISW2, and CHD1 weakly affect repair and mutagenic processes in yeast cells. At the same time, there are differences in the effect of these mutations on spontaneous mutation levels, which indicates certain specificity of action of protein complexes ISW1, ISW2, and CHD1 on expression of different genes that control repair and mutation processes in yeast.

Multilocus Phylogeography and Species Delimitation in the Cumberland Plateau Salamander, Plethodon kentucki: Incongruence among Data Sets and Methods

PubMed Central

Kuchta, Shawn R.; Brown, Ashley D.; Converse, Paul E.; Highton, Richard

2016-01-01

Species are a fundamental unit of biodiversity, yet can be challenging to delimit objectively. This is particularly true of species complexes characterized by high levels of population genetic structure, hybridization between genetic groups, isolation by distance, and limited phenotypic variation. Previous work on the Cumberland Plateau Salamander, Plethodon kentucki, suggested that it might constitute a species complex despite occupying a relatively small geographic range. To examine this hypothesis, we sampled 135 individuals from 43 populations, and used four mitochondrial loci and five nuclear loci (5693 base pairs) to quantify phylogeographic structure and probe for cryptic species diversity. Rates of evolution for each locus were inferred using the multidistribute package, and time calibrated gene trees and species trees were inferred using BEAST 2 and *BEAST 2, respectively. Because the parameter space relevant for species delimitation is large and complex, and all methods make simplifying assumptions that may lead them to fail, we conducted an array of analyses. Our assumption was that strongly supported species would be congruent across methods. Putative species were first delimited using a Bayesian implementation of the GMYC model (bGMYC), Geneland, and Brownie. We then validated these species using the genealogical sorting index and BPP. We found substantial phylogeographic diversity using mtDNA, including four divergent clades and an inferred common ancestor at 14.9 myr (95% HPD: 10.8–19.7 myr). By contrast, this diversity was not corroborated by nuclear sequence data, which exhibited low levels of variation and weak phylogeographic structure. Species trees estimated a far younger root than did the mtDNA data, closer to 1.0 myr old. Mutually exclusive putative species were identified by the different approaches. Possible causes of data set discordance, and the problem of species delimitation in complexes with high levels of population structure and introgressive hybridization, are discussed. PMID:26974148

Measurement of changes in impedance of DNA nanowires due to radiation induced structural damage. A novel approach for a DNA-based radiosensitive device

NASA Astrophysics Data System (ADS)

Heimbach, Florian; Arndt, Alexander; Nettelbeck, Heidi; Langner, Frank; Giesen, Ulrich; Rabus, Hans; Sellner, Stefan; Toppari, Jussi; Shen, Boxuan; Baek, Woon Yong

2017-08-01

The ability of DNA to conduct electric current has been the topic of numerous investigations over the past few decades. Those investigations indicate that this ability is dependent on the molecular structure of the DNA. Radiation-induced damages, which lead to an alteration of the molecular structure, should therefore change the electrical impedance of a DNA molecule. In this paper, the damage due to ionising radiation is shown to have a direct effect on the electrical transport properties of DNA. Impedance measurements of DNA samples were carried out by an AC impedance spectrometer before, during and after irradiation. The samples comprised of DNA segments, which were immobilized between gold electrodes with a gap of 12 μm. The impedance of all DNA samples exhibited rising capacitive behaviour with increasing absorbed dose.

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

Relative information content of polymorphic microsatellites and mitochondrial DNA for inferring dispersal and population genetic structure in the olive sea snake, Aipysurus laevis.

PubMed

Lukoschek, V; Waycott, M; Keogh, J S

2008-07-01

Polymorphic microsatellites are widely considered more powerful for resolving population structure than mitochondrial DNA (mtDNA) markers, particularly for recently diverged lineages or geographically proximate populations. Weaker population subdivision for biparentally inherited nuclear markers than maternally inherited mtDNA may signal male-biased dispersal but can also be attributed to marker-specific evolutionary characteristics and sampling properties. We discriminated between these competing explanations with a population genetic study on olive sea snakes, Aipysurus laevis. A previous mtDNA study revealed strong regional population structure for A. laevis around northern Australia, where Pleistocene sea-level fluctuations have influenced the genetic signatures of shallow-water marine species. Divergences among phylogroups dated to the Late Pleistocene, suggesting recent range expansions by previously isolated matrilines. Fine-scale population structure within regions was, however, poorly resolved for mtDNA. In order to improve estimates of fine-scale genetic divergence and to compare population structure between nuclear and mtDNA, 354 olive sea snakes (previously sequenced for mtDNA) were genotyped for five microsatellite loci. F statistics and Bayesian multilocus genotype clustering analyses found similar regional population structure as mtDNA and, after standardizing microsatellite F statistics for high heterozygosities, regional divergence estimates were quantitatively congruent between marker classes. Over small spatial scales, however, microsatellites recovered almost no genetic structure and standardized F statistics were orders of magnitude smaller than for mtDNA. Three tests for male-biased dispersal were not significant, suggesting that recent demographic expansions to the typically large population sizes of A. laevis have prevented microsatellites from reaching mutation-drift equilibrium and local populations may still be diverging.

Structural DNA Nanotechnology: State of the Art and Future Perspective

PubMed Central

2015-01-01

Over the past three decades DNA has emerged as an exceptional molecular building block for nanoconstruction due to its predictable conformation and programmable intra- and intermolecular Watson–Crick base-pairing interactions. A variety of convenient design rules and reliable assembly methods have been developed to engineer DNA nanostructures of increasing complexity. The ability to create designer DNA architectures with accurate spatial control has allowed researchers to explore novel applications in many directions, such as directed material assembly, structural biology, biocatalysis, DNA computing, nanorobotics, disease diagnosis, and drug delivery. This Perspective discusses the state of the art in the field of structural DNA nanotechnology and presents some of the challenges and opportunities that exist in DNA-based molecular design and programming. PMID:25029570

OligArch: A software tool to allow artificially expanded genetic information systems (AEGIS) to guide the autonomous self-assembly of long DNA constructs from multiple DNA single strands.

PubMed

Bradley, Kevin M; Benner, Steven A

2014-01-01

Synthetic biologists wishing to self-assemble large DNA (L-DNA) constructs from small DNA fragments made by automated synthesis need fragments that hybridize predictably. Such predictability is difficult to obtain with nucleotides built from just the four standard nucleotides. Natural DNA's peculiar combination of strong and weak G:C and A:T pairs, the context-dependence of the strengths of those pairs, unimolecular strand folding that competes with desired interstrand hybridization, and non-Watson-Crick interactions available to standard DNA, all contribute to this unpredictability. In principle, adding extra nucleotides to the genetic alphabet can improve the predictability and reliability of autonomous DNA self-assembly, simply by increasing the information density of oligonucleotide sequences. These extra nucleotides are now available as parts of artificially expanded genetic information systems (AEGIS), and tools are now available to generate entirely standard DNA from AEGIS DNA during PCR amplification. Here, we describe the OligArch (for "oligonucleotide architecting") software, an application that permits synthetic biologists to engineer optimally self-assembling DNA constructs from both six- and eight-letter AEGIS alphabets. This software has been used to design oligonucleotides that self-assemble to form complete genes from 20 or more single-stranded synthetic oligonucleotides. OligArch is therefore a key element of a scalable and integrated infrastructure for the rapid and designed engineering of biology.

On the origin of mongrels: evolutionary history of free-breeding dogs in Eurasia

PubMed Central

Pilot, Małgorzata; Malewski, Tadeusz; Moura, Andre E.; Grzybowski, Tomasz; Oleński, Kamil; Ruść, Anna; Kamiński, Stanisław; Ruiz Fadel, Fernanda; Mills, Daniel S.; Alagaili, Abdulaziz N.; Mohammed, Osama B.; Kłys, Grzegorz; Okhlopkov, Innokentiy M.; Suchecka, Ewa; Bogdanowicz, Wiesław

2015-01-01

Although a large part of the global domestic dog population is free-ranging and free-breeding, knowledge of genetic diversity in these free-breeding dogs (FBDs) and their ancestry relations to pure-breed dogs is limited, and the indigenous status of FBDs in Asia is still uncertain. We analyse genome-wide SNP variability of FBDs across Eurasia, and show that they display weak genetic structure and are genetically distinct from pure-breed dogs rather than constituting an admixture of breeds. Our results suggest that modern European breeds originated locally from European FBDs. East Asian and Arctic breeds show closest affinity to East Asian FBDs, and they both represent the earliest branching lineages in the phylogeny of extant Eurasian dogs. Our biogeographic reconstruction of ancestral distributions indicates a gradual westward expansion of East Asian indigenous dogs to the Middle East and Europe through Central and West Asia, providing evidence for a major expansion that shaped the patterns of genetic differentiation in modern dogs. This expansion was probably secondary and could have led to the replacement of earlier resident populations in Western Eurasia. This could explain why earlier studies based on modern DNA suggest East Asia as the region of dog origin, while ancient DNA and archaeological data point to Western Eurasia. PMID:26631564

On the origin of mongrels: evolutionary history of free-breeding dogs in Eurasia.

PubMed

Pilot, Małgorzata; Malewski, Tadeusz; Moura, Andre E; Grzybowski, Tomasz; Oleński, Kamil; Ruść, Anna; Kamiński, Stanisław; Ruiz Fadel, Fernanda; Mills, Daniel S; Alagaili, Abdulaziz N; Mohammed, Osama B; Kłys, Grzegorz; Okhlopkov, Innokentiy M; Suchecka, Ewa; Bogdanowicz, Wiesław

2015-12-07

Although a large part of the global domestic dog population is free-ranging and free-breeding, knowledge of genetic diversity in these free-breeding dogs (FBDs) and their ancestry relations to pure-breed dogs is limited, and the indigenous status of FBDs in Asia is still uncertain. We analyse genome-wide SNP variability of FBDs across Eurasia, and show that they display weak genetic structure and are genetically distinct from pure-breed dogs rather than constituting an admixture of breeds. Our results suggest that modern European breeds originated locally from European FBDs. East Asian and Arctic breeds show closest affinity to East Asian FBDs, and they both represent the earliest branching lineages in the phylogeny of extant Eurasian dogs. Our biogeographic reconstruction of ancestral distributions indicates a gradual westward expansion of East Asian indigenous dogs to the Middle East and Europe through Central and West Asia, providing evidence for a major expansion that shaped the patterns of genetic differentiation in modern dogs. This expansion was probably secondary and could have led to the replacement of earlier resident populations in Western Eurasia. This could explain why earlier studies based on modern DNA suggest East Asia as the region of dog origin, while ancient DNA and archaeological data point to Western Eurasia. © 2015 The Author(s).

Coelenterazine-binding protein of Renilla muelleri: cDNA cloning, overexpression, and characterization as a substrate of luciferase.

PubMed

Titushin, Maxim S; Markova, Svetlana V; Frank, Ludmila A; Malikova, Natalia P; Stepanyuk, Galina A; Lee, John; Vysotski, Eugene S

2008-02-01

The Renilla bioluminescent system in vivo is comprised of three proteins--the luciferase, green-fluorescent protein, and coelenterazine-binding protein (CBP), previously called luciferin-binding protein (LBP). This work reports the cloning of the full-size cDNA encoding CBP from soft coral Renilla muelleri, its overexpression and properties of the recombinant protein. The apo-CBP was quantitatively converted to CBP by simple incubation with coelenterazine. The physicochemical properties of this recombinant CBP are determined to be practically the same as those reported for the CBP (LBP) of R. reniformis. CBP is a member of the four-EF-hand Ca(2+)-binding superfamily of proteins with only three of the EF-hand loops having the Ca(2+)-binding consensus sequences. There is weak sequence homology with the Ca(2+)-regulated photoproteins but only as a result of the necessary Ca(2+)-binding loop structure. In combination with Renilla luciferase, addition of only one Ca(2+) is sufficient to release the coelenterazine as a substrate for the luciferase for bioluminescence. This combination of the two proteins generates bioluminescence with higher reaction efficiency than using free coelenterazine alone as the substrate for luciferase. This increased quantum yield, a difference of bioluminescence spectra, and markedly different kinetics, implicate that a CBP-luciferase complex might be involved.

Structural basis for recognition and remodeling of the TBP:DNA:NC2 complex by Mot1

PubMed Central

Butryn, Agata; Schuller, Jan M; Stoehr, Gabriele; Runge-Wollmann, Petra; Förster, Friedrich; Auble, David T; Hopfner, Karl-Peter

2015-01-01

Swi2/Snf2 ATPases remodel substrates such as nucleosomes and transcription complexes to control a wide range of DNA-associated processes, but detailed structural information on the ATP-dependent remodeling reactions is largely absent. The single subunit remodeler Mot1 (modifier of transcription 1) dissociates TATA box-binding protein (TBP):DNA complexes, offering a useful system to address the structural mechanisms of Swi2/Snf2 ATPases. Here, we report the crystal structure of the N-terminal domain of Mot1 in complex with TBP, DNA, and the transcription regulator negative cofactor 2 (NC2). Our data show that Mot1 reduces DNA:NC2 interactions and unbends DNA as compared to the TBP:DNA:NC2 state, suggesting that Mot1 primes TBP:NC2 displacement in an ATP-independent manner. Electron microscopy and cross-linking data suggest that the Swi2/Snf2 domain of Mot1 associates with the upstream DNA and the histone fold of NC2, thereby revealing parallels to some nucleosome remodelers. This study provides a structural framework for how a Swi2/Snf2 ATPase interacts with its substrate DNA:protein complex. DOI: http://dx.doi.org/10.7554/eLife.07432.001 PMID:26258880

Effect of point substitutions within the minimal DNA-binding domain of xeroderma pigmentosum group A protein on interaction with DNA intermediates of nucleotide excision repair.

PubMed

Maltseva, E A; Krasikova, Y S; Naegeli, H; Lavrik, O I; Rechkunova, N I

2014-06-01

Xeroderma pigmentosum factor A (XPA) is one of the key proteins in the nucleotide excision repair (NER) process. The effects of point substitutions in the DNA-binding domain of XPA (positively charged lysine residues replaced by negatively charged glutamate residues: XPA K204E, K179E, K141E, and tandem mutant K141E/K179E) on the interaction of the protein with DNA structures modeling intermediates of the damage recognition and pre-incision stages in NER were analyzed. All these mutations decreased the affinity of the protein to DNA, the effect depending on the substitution and the DNA structure. The mutant as well as wild-type proteins bind with highest efficiency partly open damaged DNA duplex, and the affinity of the mutants to this DNA is reduced in the order: K204E > K179E > K141E = K141/179E. For all the mutants, decrease in DNA binding efficiency was more pronounced in the case of full duplex and single-stranded DNA than with bubble-DNA structure, the difference between protein affinities to different DNA structures increasing as DNA binding activity of the mutant decreased. No effect of the studied XPA mutations on the location of the protein on the partially open DNA duplex was observed using photoinduced crosslinking with 5-I-dUMP in different positions of the damaged DNA strand. These results combined with earlier published data suggest no direct correlation between DNA binding and activity in NER for these XPA mutants.

DNA origami metallized site specifically to form electrically conductive nanowires.

PubMed

Pearson, Anthony C; Liu, Jianfei; Pound, Elisabeth; Uprety, Bibek; Woolley, Adam T; Davis, Robert C; Harb, John N

2012-09-06

DNA origami is a promising tool for use as a template in the design and fabrication of nanoscale structures. The ability to engineer selected staple strands on a DNA origami structure provides a high density of addressable locations across the structure. Here we report a method using site-specific attachment of gold nanoparticles to modified staple strands and subsequent metallization to fabricate conductive wires from DNA origami templates. We have modified DNA origami structures by lengthening each staple strand in select regions with a 10-base nucleotide sequence and have attached DNA-modified gold nanoparticles to the lengthened staple strands via complementary base-pairing. The high density of extended staple strands allowed the gold nanoparticles to pack tightly in the modified regions of the DNA origami, where the measured median gap size between neighboring particles was 4.1 nm. Gold metallization processes were optimized so that the attached gold nanoparticles grew until gaps between particles were filled and uniform continuous nanowires were formed. Finally, electron beam lithography was used to pattern electrodes in order to measure the electrical conductivity of metallized DNA origami, which showed an average resistance of 2.4 kΩ per metallized structure.

Crystal structures of the structure-selective nuclease Mus81-Eme1 bound to flap DNA substrates

PubMed Central

Gwon, Gwang Hyeon; Jo, Aera; Baek, Kyuwon; Jin, Kyeong Sik; Fu, Yaoyao; Lee, Jong-Bong; Kim, YoungChang; Cho, Yunje

2014-01-01

The Mus81-Eme1 complex is a structure-selective endonuclease with a critical role in the resolution of recombination intermediates during DNA repair after interstrand cross-links, replication fork collapse, or double-strand breaks. To explain the molecular basis of 3′ flap substrate recognition and cleavage mechanism by Mus81-Eme1, we determined crystal structures of human Mus81-Eme1 bound to various flap DNA substrates. Mus81-Eme1 undergoes gross substrate-induced conformational changes that reveal two key features: (i) a hydrophobic wedge of Mus81 that separates pre- and post-nick duplex DNA and (ii) a “5′ end binding pocket” that hosts the 5′ nicked end of post-nick DNA. These features are crucial for comprehensive protein-DNA interaction, sharp bending of the 3′ flap DNA substrate, and incision strand placement at the active site. While Mus81-Eme1 unexpectedly shares several common features with members of the 5′ flap nuclease family, the combined structural, biochemical, and biophysical analyses explain why Mus81-Eme1 preferentially cleaves 3′ flap DNA substrates with 5′ nicked ends. PMID:24733841

DeoR repression at-a-distance only weakly responds to changes in interoperator separation and DNA topology.

PubMed Central

Dandanell, G

1992-01-01

The interoperator distance between a synthetic operator Os and the deoP2O2-galK fusion was varied between 46 and 176 bp. The repression of the deoP2 directed galK expression as a function of the interoperator distance (center-to-center) was measured in vivo in a single-copy system. The results show that the DeoR repressor efficiently can repress transcription at all the interoperator distances tested. The degree of repression depends very little on the spacing between the operators, however, a weak periodic dependency of 8-11 bp may exist. PMID:1437558

Extracting DNA from FFPE Tissue Biospecimens Using User-Friendly Automated Technology: Is There an Impact on Yield or Quality?

PubMed

Mathieson, William; Guljar, Nafia; Sanchez, Ignacio; Sroya, Manveer; Thomas, Gerry A

2018-05-03

DNA extracted from formalin-fixed, paraffin-embedded (FFPE) tissue blocks is amenable to analytical techniques, including sequencing. DNA extraction protocols are typically long and complex, often involving an overnight proteinase K digest. Automated platforms that shorten and simplify the process are therefore an attractive proposition for users wanting a faster turn-around or to process large numbers of biospecimens. It is, however, unclear whether automated extraction systems return poorer DNA yields or quality than manual extractions performed by experienced technicians. We extracted DNA from 42 FFPE clinical tissue biospecimens using the QiaCube (Qiagen) and ExScale (ExScale Biospecimen Solutions) automated platforms, comparing DNA yields and integrities with those from manual extractions. The QIAamp DNA FFPE Spin Column Kit was used for manual and QiaCube DNA extractions and the ExScale extractions were performed using two of the manufacturer's magnetic bead kits: one extracting DNA only and the other simultaneously extracting DNA and RNA. In all automated extraction methods, DNA yields and integrities (assayed using DNA Integrity Numbers from a 4200 TapeStation and the qPCR-based Illumina FFPE QC Assay) were poorer than in the manual method, with the QiaCube system performing better than the ExScale system. However, ExScale was fastest, offered the highest reproducibility when extracting DNA only, and required the least intervention or technician experience. Thus, the extraction methods have different strengths and weaknesses, would appeal to different users with different requirements, and therefore, we cannot recommend one method over another.

Complex DNA Brick Assembly.

PubMed

Ong, Luvena L; Ke, Yonggang

2017-01-01

DNA nanostructures are a useful technology for precisely organizing and manipulating nanomaterials. The DNA bricks method is a modular and versatile platform for applications requiring discrete or periodic structures with complex three-dimensional features. Here, we describe how structures are designed from the fundamental strand architecture through assembly and characterization of the formed structures.

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

Tripathi, S.; Zhang, D.; Paukstelis, P. J.

DNA has proved to be an excellent material for nanoscale construction because complementary DNA duplexes are programmable and structurally predictable. However, in the absence of Watson–Crick pairings, DNA can be structurally more diverse. Here, we describe the crystal structures of d(ACTCGGATGAT) and the brominated derivative, d(AC BrUCGGA BrUGAT). These oligonucleotides form parallel-stranded duplexes with a crystallographically equivalent strand, resulting in the first examples of DNA crystal structures that contains four different symmetric homo base pairs. Two of the parallel-stranded duplexes are coaxially stacked in opposite directions and locked together to form a tetraplex through intercalation of the 5'-most A–A basemore » pairs between adjacent G–G pairs in the partner duplex. The intercalation region is a new type of DNA tertiary structural motif with similarities to the i-motif. 1H– 1H nuclear magnetic resonance and native gel electrophoresis confirmed the formation of a parallel-stranded duplex in solution. Finally, we modified specific nucleotide positions and added d(GAY) motifs to oligonucleotides and were readily able to obtain similar crystals. This suggests that this parallel-stranded DNA structure may be useful in the rational design of DNA crystals and nanostructures.« less

High-Throughput Analysis of T-DNA Location and Structure Using Sequence Capture.

PubMed

Inagaki, Soichi; Henry, Isabelle M; Lieberman, Meric C; Comai, Luca

2015-01-01

Agrobacterium-mediated transformation of plants with T-DNA is used both to introduce transgenes and for mutagenesis. Conventional approaches used to identify the genomic location and the structure of the inserted T-DNA are laborious and high-throughput methods using next-generation sequencing are being developed to address these problems. Here, we present a cost-effective approach that uses sequence capture targeted to the T-DNA borders to select genomic DNA fragments containing T-DNA-genome junctions, followed by Illumina sequencing to determine the location and junction structure of T-DNA insertions. Multiple probes can be mixed so that transgenic lines transformed with different T-DNA types can be processed simultaneously, using a simple, index-based pooling approach. We also developed a simple bioinformatic tool to find sequence read pairs that span the junction between the genome and T-DNA or any foreign DNA. We analyzed 29 transgenic lines of Arabidopsis thaliana, each containing inserts from 4 different T-DNA vectors. We determined the location of T-DNA insertions in 22 lines, 4 of which carried multiple insertion sites. Additionally, our analysis uncovered a high frequency of unconventional and complex T-DNA insertions, highlighting the needs for high-throughput methods for T-DNA localization and structural characterization. Transgene insertion events have to be fully characterized prior to use as commercial products. Our method greatly facilitates the first step of this characterization of transgenic plants by providing an efficient screen for the selection of promising lines.

Global DNA methylation loss associated with mercury contamination and aging in the American alligator (Alligator mississippiensis).

PubMed

Nilsen, Frances M; Parrott, Benjamin B; Bowden, John A; Kassim, Brittany L; Somerville, Stephen E; Bryan, Teresa A; Bryan, Colleen E; Lange, Ted R; Delaney, J Patrick; Brunell, Arnold M; Long, Stephen E; Guillette, Louis J

2016-03-01

Mercury is a widespread environmental contaminant with exposures eliciting a well-documented catalog of adverse effects. Yet, knowledge regarding the underlying mechanisms by which mercury exposures are translated into biological effects remains incomplete. DNA methylation is an epigenetic modification that is sensitive to environmental cues, and alterations in DNA methylation at the global level are associated with a variety of diseases. Using a liquid chromatography tandem mass spectrometry-based (LC-MS/MS) approach, global DNA methylation levels were measured in red blood cells of 144 wild American alligators (Alligator mississippiensis) from 6 sites with variable levels of mercury contamination across Florida's north-south axis. Variation in mercury concentrations measured in whole blood was highly associated with location, allowing the comparison of global DNA methylation levels across different "treatments" of mercury. Global DNA methylation in alligators across all locations was weakly associated with increased mercury exposure. However, a much more robust relationship was observed in those animals sampled from locations more highly contaminated with mercury. Also, similar to other vertebrates, global DNA methylation appears to decline with age in alligators. The relationship between age-associated loss of global DNA methylation and varying mercury exposures was examined to reveal a potential interaction. These findings demonstrate that global DNA methylation levels are associated with mercury exposure, and give insights into interactions between contaminants, aging, and epigenetics. Published by Elsevier B.V.

Rosalind Franklin: Unsung Hero of the DNA Revolution

ERIC Educational Resources Information Center

Rapoport, Sarah

2002-01-01

On April 25, 1953, three papers were published in "Nature," the prestigious scientific journal, which exposed the "fundamentally beautiful" structure of DNA to the public, and sounded the starting gun of the DNA Revolution. The authors of these papers revealed the now-famous double-helix structure of DNA, thereby unlocking the…

Two distinct overstretched DNA structures revealed by single-molecule thermodynamics measurements

PubMed Central

Zhang, Xinghua; Chen, Hu; Fu, Hongxia; Doyle, Patrick S.; Yan, Jie

2012-01-01

Double-stranded DNA is a dynamic molecule whose structure can change depending on conditions. While there is consensus in the literature about many structures DNA can have, the state of highly-stretched DNA is still not clear. Several groups have shown that DNA in the torsion-unconstrained B-form undergoes an “overstretching” transition at a stretching force of around 65 pN, which leads to approximately 1.7-fold elongation of the DNA contour length. Recent experiments have revealed that two distinct structural transitions are involved in the overstretching process: (i) a hysteretic “peeling” off one strand from its complementary strand, and (ii) a nonhysteretic transition that leads to an undetermined DNA structure. We report the first simultaneous determination of the entropy (ΔS) and enthalpy changes (ΔH) pertaining to these respective transitions. For the hysteretic peeling transition, we determined ΔS ∼ 20 cal/(K.mol) and ΔH ∼ 7 kcal/mol. In the case of the nonhysteretic transition, ΔS ∼ -3 cal/(K.mol) and ΔH ∼ 1 kcal/mol. Furthermore, the response of the transition force to salt concentration implies that the two DNA strands are spatially separated after the hysteretic peeling transition. In contrast, the corresponding response after the nonhysteretic transition indicated that the strands remained in close proximity. The selection between the two transitions depends on DNA base-pair stability, and it can be illustrated by a multidimensional phase diagram. Our results provide important insights into the thermodynamics of DNA overstretching and conformational structures of overstretched DNA that may play an important role in vivo. PMID:22532662

Innovative approaches to the use of polyamines for DNA nanoparticle preparation for gene therapy.

PubMed

Vijayanathan, Veena; Agostinelli, Enzo; Thomas, Thresia; Thomas, T J

2014-03-01

Advances in genomic technologies, such as next generation sequencing and disease specific gene targeting through anti-sense, anti-gene, siRNA and microRNA approaches require the transport of nucleic acid drugs through the cell membrane. Membrane transport of DNA/RNA drugs is an inefficient process, and the mechanism(s) by which this process occurs is not clear. A pre-requisite for effective transport of DNA and RNA in cells is their condensation to nanoparticles of ~100 nm size. Although viral vectors are effective in gene therapy, the immune response elicited by viral proteins poses a major challenge. Multivalent cations, such as natural polyamines are excellent promoters of DNA/RNA condensation to nanoparticles. During the past 20 years, our laboratory has synthesized and tested several analogs of the natural polyamine, spermine, for their efficacy to provoke DNA condensation to nanoparticles. We determined the thermodynamics of polyamine-mediated DNA condensation, measured the structural specificity effects of polyamine analogs in facilitating the cellular uptake of oligonucleotides, and evaluated the gene silencing activity of DNA nanoparticles in breast cancer cells. Polyamine-complexed oligonucleotides showed a synergistic effect on target gene inhibition at the mRNA level compared to the use of polyamines and oligonucleotides as single agents. Ionic and structural specificity effects were evident in DNA condensation and cellular transportation effects of polyamines. In condensed DNA structures, correlation exists between the attractive and repulsive forces with structurally different polyamines and cobalt hexamine, indicating the existence of a common force in stabilizing the condensed structures. Future studies aimed at defining the mechanism(s) of DNA compaction and structural features of DNA nanoparticles might aid in the development of novel gene delivery vehicles.

Bullied no more:when and how DNA shoves proteins around

PubMed Central

Pettitt, B. Montgomery; Sumners, De Witt L.; Harris, Sarah A.; Zechiedrich, Lynn

2016-01-01

The predominant protein-centric perspective in protein–DNA-binding studies assumes that the protein drives the interaction. Research focuses on protein structural motifs, electrostatic surfaces and contact potentials, while DNA is often ignored as a passive polymer to be manipulated. Recent studies of DNA topology, the supercoiling, knotting, and linking of the helices, have shown that DNA has the capability to be an active participant in its transactions. DNA topology-induced structural and geometric changes can drive, or at least strongly influence, the interactions between protein and DNA. Deformations of the B-form structure arise from both the considerable elastic energy arising from supercoiling and from the electrostatic energy. Here, we discuss how these energies are harnessed for topology-driven, sequence-specific deformations that can allow DNA to direct its own metabolism. PMID:22850561

Programmed folding of DNA origami structures through single-molecule force control.

PubMed

Bae, Wooli; Kim, Kipom; Min, Duyoung; Ryu, Je-Kyung; Hyeon, Changbong; Yoon, Tae-Young

2014-12-03

Despite the recent development in the design of DNA origami, its folding yet relies on thermal or chemical annealing methods. We here demonstrate mechanical folding of the DNA origami structure via a pathway that has not been accessible to thermal annealing. Using magnetic tweezers, we stretch a single scaffold DNA with mechanical tension to remove its secondary structures, followed by base pairing of the stretched DNA with staple strands. When the force is subsequently quenched, folding of the DNA nanostructure is completed through displacement between the bound staple strands. Each process in the mechanical folding is well defined and free from kinetic traps, enabling us to complete folding within 10 min. We also demonstrate parallel folding of DNA nanostructures through multiplexed manipulation of the scaffold DNAs. Our results suggest a path towards programmability of the folding pathway of DNA nanostructures.

Structural insights into NHEJ: building up an integrated picture of the dynamic DSB repair super complex, one component and interaction at a time

PubMed Central

Williams, Gareth J.; Hammel, Michal; Radhakrishnan, Sarvan Kumar; Ramsden, Dale; Lees-Miller, Susan P.; Tainer, John A.

2014-01-01

Non-homologous end joining (NHEJ) is the major pathway for repair of DNA double-strand breaks (DSBs) in human cells. NHEJ is also needed for V(D)J recombination and the development of T and B cells in vertebrate immune systems, and acts in both the generation and prevention of non-homologous chromosomal translocations, a hallmark of genomic instability and many human cancers. X-ray crystal structures, cryo-electron microscopy envelopes, and small angle X-ray scattering (SAXS) solution conformations and assemblies are defining most of the core protein components for NHEJ: Ku70/Ku80 heterodimer; the DNA dependent protein kinase catalytic subunit (DNA-PKcs); the structure-specific endonuclease Artemis along with polynucleotide kinase/phosphatase (PNKP), aprataxin and PNKP related protein (APLF); the scaffolding proteins XRCC4 and XLF (XRCC4-like factor); DNA polymerases, and DNA ligase IV (Lig IV). The dynamic assembly of multi-protein NHEJ complexes at DSBs is regulated in part by protein phosphorylation. The basic steps of NHEJ have been biochemically defined to require: 1) DSB detection by the Ku heterodimer with subsequent DNA-PKcs tethering to form the DNA-PKcs-Ku-DNA complex (termed DNA-PK), 2) lesion processing, and 3) DNA end ligation by Lig IV, which functions in complex with XRCC4 and XLF. The current integration of structures by combined methods is resolving puzzles regarding the mechanisms, coordination and regulation of these three basic steps. Overall, structural results suggest the NHEJ system forms a flexing scaffold with the DNA-PKcs HEAT repeats acting as compressible macromolecular springs suitable to store and release conformational energy to apply forces to regulate NHEJ complexes and the DNA substrate for DNA end protection, processing, and ligation. PMID:24656613

Structural basis for suppression of hypernegative DNA supercoiling by E. coli topoisomerase I

DOE PAGES

Tan, Kemin; Zhou, Qingxuan; Cheng, Bokun; ...

2015-10-20

Escherichia coli topoisomerase I has an essential function in preventing hypernegative supercoiling of DNA. A full length structure of E. coli topoisomerase I reported here shows how the C-terminal domains bind single-stranded DNA (ssDNA) to recognize the accumulation of negative supercoils in duplex DNA. These C-terminal domains of E. coli topoisomerase I are known to interact with RNA polymerase, and two flexible linkers within the C-terminal domains may assist in the movement of the ssDNA for the rapid removal of transcription driven negative supercoils. The structure has also unveiled for the first time how the 4-Cys zinc ribbon domain andmore » zinc ribbon-like domain bind ssDNA with primarily π -stacking interactions. Finally, this novel structure, in combination with new biochemical data, provides important insights into the mechanism of genome regulation by type IA topoisomerases that is essential for life, as well as the structures of homologous type IA TOP3α and TOP3β from higher eukaryotes that also have multiple 4-Cys zinc ribbon domains required for their physiological functions.« less

High-Mobility Group Chromatin Proteins 1 and 2 Functionally Interact with Steroid Hormone Receptors To Enhance Their DNA Binding In Vitro and Transcriptional Activity in Mammalian Cells

PubMed Central

Boonyaratanakornkit, Viroj; Melvin, Vida; Prendergast, Paul; Altmann, Magda; Ronfani, Lorenza; Bianchi, Marco E.; Taraseviciene, Laima; Nordeen, Steven K.; Allegretto, Elizabeth A.; Edwards, Dean P.

1998-01-01

We previously reported that the chromatin high-mobility group protein 1 (HMG-1) enhances the sequence-specific DNA binding activity of progesterone receptor (PR) in vitro, thus providing the first evidence that HMG-1 may have a coregulatory role in steroid receptor-mediated gene transcription. Here we show that HMG-1 and the highly related HMG-2 stimulate DNA binding by other steroid receptors, including estrogen, androgen, and glucocorticoid receptors, but have no effect on DNA binding by several nonsteroid nuclear receptors, including retinoid acid receptor (RAR), retinoic X receptor (RXR), and vitamin D receptor (VDR). As highly purified recombinant full-length proteins, all steroid receptors tested exhibited weak binding affinity for their optimal palindromic hormone response elements (HREs), and the addition of purified HMG-1 or -2 substantially increased their affinity for HREs. Purified RAR, RXR, and VDR also exhibited little to no detectable binding to their cognate direct repeat HREs but, in contrast to results with steroid receptors, the addition of HMG-1 or HMG-2 had no stimulatory effect. Instead, the addition of purified RXR enhanced RAR and VDR DNA binding through a heterodimerization mechanism and HMG-1 or HMG-2 had no further effect on DNA binding by RXR-RAR or RXR-VDR heterodimers. HMG-1 and HMG-2 (HMG-1/-2) themselves do not bind to progesterone response elements, but in the presence of PR they were detected as part of an HMG-PR-DNA ternary complex. HMG-1/-2 can also interact transiently in vitro with PR in the absence of DNA; however, no direct protein interaction was detected with VDR. These results, taken together with the fact that PR can bend its target DNA and that HMG-1/-2 are non-sequence-specific DNA binding proteins that recognize DNA structure, suggest that HMG-1/-2 are recruited to the PR-DNA complex by the combined effect of transient protein interaction and DNA bending. In transient-transfection assays, coexpression of HMG-1 or HMG-2 increased PR-mediated transcription in mammalian cells by as much as 7- to 10-fold without altering the basal promoter activity of target reporter genes. This increase in PR-mediated gene activation by coexpression of HMG-1/-2 was observed in different cell types and with different target promoters, suggesting a generality to the functional interaction between HMG-1/-2 and PR in vivo. Cotransfection of HMG-1 also increased reporter gene activation mediated by other steroid receptors, including glucocorticoid and androgen receptors, but it had a minimal influence on VDR-dependent transcription in vivo. These results support the conclusion that HMG-1/-2 are coregulatory proteins that increase the DNA binding and transcriptional activity of the steroid hormone class of receptors but that do not functionally interact with certain nonsteroid classes of nuclear receptors. PMID:9671457

Identifying DNA-binding proteins using structural motifs and the electrostatic potential

PubMed Central

Shanahan, Hugh P.; Garcia, Mario A.; Jones, Susan; Thornton, Janet M.

2004-01-01

Robust methods to detect DNA-binding proteins from structures of unknown function are important for structural biology. This paper describes a method for identifying such proteins that (i) have a solvent accessible structural motif necessary for DNA-binding and (ii) a positive electrostatic potential in the region of the binding region. We focus on three structural motifs: helix–turn-helix (HTH), helix–hairpin–helix (HhH) and helix–loop–helix (HLH). We find that the combination of these variables detect 78% of proteins with an HTH motif, which is a substantial improvement over previous work based purely on structural templates and is comparable to more complex methods of identifying DNA-binding proteins. Similar true positive fractions are achieved for the HhH and HLH motifs. We see evidence of wide evolutionary diversity for DNA-binding proteins with an HTH motif, and much smaller diversity for those with an HhH or HLH motif. PMID:15356290

Structural basis for DNA binding by replication initiator Mcm10

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

Warren, Eric M.; Vaithiyalingam, Sivaraja; Haworth, Justin

2009-06-30

Mcm10 is an essential eukaryotic DNA replication protein required for assembly and progression of the replication fork. The highly conserved internal domain (Mcm10-ID) has been shown to physically interact with single-stranded (ss) DNA, DNA polymerase alpha, and proliferating cell nuclear antigen (PCNA). The crystal structure of Xenopus laevis Mcm10-ID presented here reveals a DNA binding architecture composed of an oligonucleotide/oligosaccharide-fold followed in tandem by a variant and highly basic zinc finger. NMR chemical shift perturbation and mutational studies of DNA binding activity in vitro reveal how Mcm10 uses this unique surface to engage ssDNA. Corresponding mutations in Saccharomyces cerevisiae resultmore » in increased sensitivity to replication stress, demonstrating the functional importance of DNA binding by this region of Mcm10 to replication. In addition, mapping Mcm10 mutations known to disrupt PCNA, polymerase alpha, and DNA interactions onto the crystal structure provides insight into how Mcm10 might coordinate protein and DNA binding within the replisome.« less

Strand invasion structures in the inverted repeat of Candida albicans mitochondrial DNA reveal a role for homologous recombination in replication.

PubMed

Gerhold, Joachim M; Aun, Anu; Sedman, Tiina; Jõers, Priit; Sedman, Juhan

2010-09-24

Molecular recombination and transcription are proposed mechanisms to initiate mitochondrial DNA (mtDNA) replication in yeast. We conducted a comprehensive analysis of mtDNA from the yeast Candida albicans. Two-dimensional agarose gel electrophoresis of mtDNA intermediates reveals no bubble structures diagnostic of specific replication origins, but rather supports recombination-driven replication initiation of mtDNA in yeast. Specific species of Y structures together with DNA copy number analyses of a C. albicans mutant strain provide evidence that a region in a mainly noncoding inverted repeat is predominantly involved in replication initiation via homologous recombination. Our further findings show that the C. albicans mtDNA forms a complex branched network that does not contain detectable amounts of circular molecules. We provide topological evidence for recombination-driven mtDNA replication initiation and introduce C. albicans as a suitable model organism to study wild-type mtDNA maintenance in yeast. Copyright © 2010 Elsevier Inc. All rights reserved.

Molecular Toxicology of Chromatin

DTIC Science & Technology

1992-01-01

towards the DNA analogs used as coenzymes suggests that the maximal activation by spermine , that depends on coDNA, may involve DNA structures which...evidence for the participation of spermine in an ADPRT-mediated regulatory system that can modify DNA structures , it seems plausible to assume tnat ADPRT may...DNA-dependent manner. The binding properties of spermine -, polylysine- and p olyarginine-Sepharose 4B affinity matrices were also determined. The

Preparation and self-folding of amphiphilic DNA origami.

PubMed

Zhou, Chao; Wang, Dianming; Dong, Yuanchen; Xin, Ling; Sun, Yawei; Yang, Zhongqiang; Liu, Dongsheng

2015-03-01

Amphiphilic DNA origami is prepared by dressing multiple hydrophobic molecules on a rectangular single layer DNA origami, which is then folded or coupled in sandwich-like structures with two outer DNA origami layer and one inner hydrophobic molecules layer. The preference to form different kinds of structures could be tailored by rational design of DNA origami. © 2014 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

DNA Architectures for Templated Material Growth

DTIC Science & Technology

2011-09-01

Solubilization of the DNA in non-aqueous solvents is achieved by replacing charge stabilizing salts with surfactants. Retention of DNA hierarchical structure...solvents is achieved by replacing charge stabilizing salts with surfactants. Retention of DNA hierarchical structure under both conditions will be...studied and explained, and is only being reproduced in this experiment.12-14 Both CTAB and CTAC, were reacted with the reconstituted DNA (salmon sperm

Minireview: DNA Replication in Plant Mitochondria

PubMed Central

Cupp, John D.; Nielsen, Brent L.

2014-01-01

Higher plant mitochondrial genomes exhibit much greater structural complexity as compared to most other organisms. Unlike well-characterized metazoan mitochondrial DNA (mtDNA) replication, an understanding of the mechanism(s) and proteins involved in plant mtDNA replication remains unclear. Several plant mtDNA replication proteins, including DNA polymerases, DNA primase/helicase, and accessory proteins have been identified. Mitochondrial dynamics, genome structure, and the complexity of dual-targeted and dual-function proteins that provide at least partial redundancy suggest that plants have a unique model for maintaining and replicating mtDNA when compared to the replication mechanism utilized by most metazoan organisms. PMID:24681310

An exonic missense mutation c.28G>A is associated with weak B blood group by affecting RNA splicing of the ABO gene.

PubMed

Cai, Xiaohong; Qian, Chengrui; Wu, Wenman; Lei, Hang; Ding, Qiulan; Zou, Wei; Xiang, Dong; Wang, Xuefeng

2017-09-01

The amino acid substitutions caused by ABO gene mutations are usually predicted to impact glycosyltransferase's function or its biosynthesis. Here we report an ABO exonic missense mutation that affects B-antigen expression by decreasing the mRNA level of the ABO gene rather than the amino acid change. Serologic studies including plasma total GTB transfer capacity were performed. The exon sequences of the ABO gene were analyzed by Sanger sequencing. B 310 cDNA with c.28G>A (p.G10R) mutation was expressed in HeLa cells and total GTB transfer capacity in cell supernatant was measured. Flow cytometry was performed on these HeLa cells after transfection, and agglutination of Hela-B weak cells was also examined. The mRNA of the ABO gene was analyzed by direct sequencing and real-time reverse transcriptase-polymerase chain reaction. A minigene construct was prepared to evaluate the potential of splicing. While plasma total GTB transfer capacity was undetectable in this B 3 -like individual, the relative percentage of antigen-expressing cells and mean fluorescence index of the B weak red blood cells (RBCs) were 19 and 14% of normal B RBCs, respectively. There was no significant difference of total GTB transfer capacity in cell supernatant and B-antigen expression on cell surfaces between HeLa cells transfected with B 310 cDNA and B cDNA. The mRNA expression level of B 310 in peripheral whole blood was significantly reduced. The amount of splicing is significantly lower in c.28G>A construct compared to that in wild-type construct after transfection in K562 cells. ABO c.28G>A mutation may cause B 3 -like subgroup by affecting RNA splicing of the ABO gene. © 2017 AABB.

Solution structure of CEH-37 homeodomain of the nematode Caenorhabditis elegans

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

Moon, Sunjin; Lee, Yong Woo; Kim, Woo Taek

Highlights: •We have determined solution structures of CEH-37 homedomain. •CEH-37 HD has a compact α-helical structure with HTH DNA binding motif. •Solution structure of CEH-37 HD shares its molecular topology with that of the homeodomain proteins. •Residues in the N-terminal region and HTH motif are important in binding to Caenorhabditis elegans telomeric DNA. •CEH-37 could play an important role in telomere function via DNA binding. -- Abstract: The nematode Caenorhabditis elegans protein CEH-37 belongs to the paired OTD/OTX family of homeobox-containing homeodomain proteins. CEH-37 shares sequence similarity with homeodomain proteins, although it specifically binds to double-stranded C. elegans telomeric DNA,more » which is unusual to homeodomain proteins. Here, we report the solution structure of CEH-37 homeodomain and molecular interaction with double-stranded C. elegans telomeric DNA using nuclear magnetic resonance (NMR) spectroscopy. NMR structure shows that CEH-37 homeodomain is composed of a flexible N-terminal region and three α-helices with a helix-turn-helix (HTH) DNA binding motif. Data from size-exclusion chromatography and fluorescence spectroscopy reveal that CEH-37 homeodomain interacts strongly with double-stranded C. elegans telomeric DNA. NMR titration experiments identified residues responsible for specific binding to nematode double-stranded telomeric DNA. These results suggest that C. elegans homeodomain protein, CEH-37 could play an important role in telomere function via DNA binding.« less

Study on the Weak Stress in Flexural MEMS Cantilever

NASA Astrophysics Data System (ADS)

Ge, Yuetao; Ren, Yan

2018-03-01

In order to design a better piezoresistive MEMS cantilever beam, especially for cantilever beams that will detect weak forces or will be subjected to weak forces, this paper uses study on the weak stress in flexural MEMS cantilever. The sensor design structure, divided into protective layer, piezoresistive layer, support layer. The protective layer is responsible for protecting the piezoresistive layer so that the varistor is insulated from the outside; the piezoresistive layer is used to make the varistor; the support layer forms the main part of the cantilever beam, the majority of the cantilever beam. This paper has some value for cantilever multilayer structure design and cantilever beam size design.

Altering DNA-Programmable Colloidal Crystallization Paths by Modulating Particle Repulsion

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

Wang, Mary X.; Brodin, Jeffrey D.; Millan, Jaime A.

Colloidal crystal engineering with DNA can be used to realize precise control over nanoparticle (NP) arrangement. Here, we investigate a case of DNA-based assembly where the properties of DNA as a polyelectrolyte brush are employed to alter a hybridization-driven NP crystallization pathway. Using the co-assembly of DNA-conjugated proteins and spherical gold 2 nanoparticles (AuNPs) as a model system, we explore how steric repulsion between non-complementary, neighboring DNA-NPs due to overlapping DNA shells can influence their ligand-directed behavior. Specifically, our experimental data coupled with coarse-grained molecular dynamics (MD) simulations reveal that by changing factors related to NP repulsion, two structurally distinctmore » outcomes can be achieved. When steric repulsion between DNA-AuNPs is significantly greater than that between DNA-proteins, a lower packing density crystal lattice is favored over the structure that is predicted by design rules based on DNA-hybridization considerations alone. This is enabled by the large difference in DNA density on AuNPs versus proteins and can be tuned by modulating the flexibility, and thus conformational entropy, of the DNA on the constituent particles. At intermediate ligand flexibility, the crystallization pathways are energetically similar and the structural outcome can be adjusted using the density of DNA duplexes on DNA-AuNPs and by screening the Coulomb potential between them. Such lattices are shown to undergo dynamic reorganization upon changing salt concentration. These data help elucidate the structural considerations necessary for understanding repulsive forces in DNA-assembly and lay the groundwork for using them to increase architectural diversity in engineering colloidal crystals.« less

Prediction of weak topological insulators in layered semiconductors.

PubMed

Yan, Binghai; Müchler, Lukas; Felser, Claudia

2012-09-14

We report the discovery of weak topological insulators by ab initio calculations in a honeycomb lattice. We propose a structure with an odd number of layers in the primitive unit cell as a prerequisite for forming weak topological insulators. Here, the single-layered KHgSb is the most suitable candidate for its large bulk energy gap of 0.24 eV. Its side surface hosts metallic surface states, forming two anisotropic Dirac cones. Although the stacking of even-layered structures leads to trivial insulators, the structures can host a quantum spin Hall layer with a large bulk gap, if an additional single layer exists as a stacking fault in the crystal. The reported honeycomb compounds can serve as prototypes to aid in the finding of new weak topological insulators in layered small-gap semiconductors.

Moving beyond Watson-Crick models of coarse grained DNA dynamics.

PubMed

Linak, Margaret C; Tourdot, Richard; Dorfman, Kevin D

2011-11-28

DNA produces a wide range of structures in addition to the canonical B-form of double-stranded DNA. Some of these structures are stabilized by Hoogsteen bonds. We developed an experimentally parameterized, coarse-grained model that incorporates such bonds. The model reproduces many of the microscopic features of double-stranded DNA and captures the experimental melting curves for a number of short DNA hairpins, even when the open state forms complicated secondary structures. We demonstrate the utility of the model by simulating the folding of a thrombin aptamer, which contains G-quartets, and strand invasion during triplex formation. Our results highlight the importance of including Hoogsteen bonding in coarse-grained models of DNA.

Lack of sex-biased dispersal promotes fine-scale genetic structure in alpine ungulates

USGS Publications Warehouse

Roffler, Gretchen H.; Talbot, Sandra L.; Luikart, Gordon; Sage, George K.; Pilgrim, Kristy L.; Adams, Layne G.; Schwartz, Michael K.

2014-01-01

Identifying patterns of fine-scale genetic structure in natural populations can advance understanding of critical ecological processes such as dispersal and gene flow across heterogeneous landscapes. Alpine ungulates generally exhibit high levels of genetic structure due to female philopatry and patchy configuration of mountain habitats. We assessed the spatial scale of genetic structure and the amount of gene flow in 301 Dall’s sheep (Ovis dalli dalli) at the landscape level using 15 nuclear microsatellites and 473 base pairs of the mitochondrial (mtDNA) control region. Dall’s sheep exhibited significant genetic structure within contiguous mountain ranges, but mtDNA structure occurred at a broader geographic scale than nuclear DNA within the study area, and mtDNA structure for other North American mountain sheep populations. No evidence of male-mediated gene flow or greater philopatry of females was observed; there was little difference between markers with different modes of inheritance (pairwise nuclear DNA F ST = 0.004–0.325; mtDNA F ST = 0.009–0.544), and males were no more likely than females to be recent immigrants. Historical patterns based on mtDNA indicate separate northern and southern lineages and a pattern of expansion following regional glacial retreat. Boundaries of genetic clusters aligned geographically with prominent mountain ranges, icefields, and major river valleys based on Bayesian and hierarchical modeling of microsatellite and mtDNA data. Our results suggest that fine-scale genetic structure in Dall’s sheep is influenced by limited dispersal, and structure may be weaker in populations occurring near ancestral levels of density and distribution in continuous habitats compared to other alpine ungulates that have experienced declines and marked habitat fragmentation.

T7 RNA polymerase non-specifically transcribes and induces disassembly of DNA nanostructures

PubMed Central

Schaffter, Samuel W; Green, Leopold N; Schneider, Joanna; Subramanian, Hari K K; Schulman, Rebecca

2018-01-01

Abstract The use of proteins that bind and catalyze reactions with DNA alongside DNA nanostructures has broadened the functionality of DNA devices. DNA binding proteins have been used to specifically pattern and tune structural properties of DNA nanostructures and polymerases have been employed to directly and indirectly drive structural changes in DNA structures and devices. Despite these advances, undesired and poorly understood interactions between DNA nanostructures and proteins that bind DNA continue to negatively affect the performance and stability of DNA devices used in conjunction with enzymes. A better understanding of these undesired interactions will enable the construction of robust DNA nanostructure-enzyme hybrid systems. Here, we investigate the undesired disassembly of DNA nanotubes in the presence of viral RNA polymerases (RNAPs) under conditions used for in vitro transcription. We show that nanotubes and individual nanotube monomers (tiles) are non-specifically transcribed by T7 RNAP, and that RNA transcripts produced during non-specific transcription disassemble the nanotubes. Disassembly requires a single-stranded overhang on the nanotube tiles where transcripts can bind and initiate disassembly through strand displacement, suggesting that single-stranded domains on other DNA nanostructures could cause unexpected interactions in the presence of viral RNA polymerases. PMID:29718412

Human DNA ligase III recognizes DNA ends by dynamic switching between two DNA-bound states.

PubMed

Cotner-Gohara, Elizabeth; Kim, In-Kwon; Hammel, Michal; Tainer, John A; Tomkinson, Alan E; Ellenberger, Tom

2010-07-27

Human DNA ligase III has essential functions in nuclear and mitochondrial DNA replication and repair and contains a PARP-like zinc finger (ZnF) that increases the extent of DNA nick joining and intermolecular DNA ligation, yet the bases for ligase III specificity and structural variation among human ligases are not understood. Here combined crystal structure and small-angle X-ray scattering results reveal dynamic switching between two nick-binding components of ligase III: the ZnF-DNA binding domain (DBD) forms a crescent-shaped surface used for DNA end recognition which switches to a ring formed by the nucleotidyl transferase (NTase) and OB-fold (OBD) domains for catalysis. Structural and mutational analyses indicate that high flexibility and distinct DNA binding domain features in ligase III assist both nick sensing and the transition from nick sensing by the ZnF to nick joining by the catalytic core. The collective results support a "jackknife model" in which the ZnF loads ligase III onto nicked DNA and conformational changes deliver DNA into the active site. This work has implications for the biological specificity of DNA ligases and functions of PARP-like zinc fingers.

T7 RNA polymerase non-specifically transcribes and induces disassembly of DNA nanostructures.

PubMed

Schaffter, Samuel W; Green, Leopold N; Schneider, Joanna; Subramanian, Hari K K; Schulman, Rebecca; Franco, Elisa

2018-06-01

The use of proteins that bind and catalyze reactions with DNA alongside DNA nanostructures has broadened the functionality of DNA devices. DNA binding proteins have been used to specifically pattern and tune structural properties of DNA nanostructures and polymerases have been employed to directly and indirectly drive structural changes in DNA structures and devices. Despite these advances, undesired and poorly understood interactions between DNA nanostructures and proteins that bind DNA continue to negatively affect the performance and stability of DNA devices used in conjunction with enzymes. A better understanding of these undesired interactions will enable the construction of robust DNA nanostructure-enzyme hybrid systems. Here, we investigate the undesired disassembly of DNA nanotubes in the presence of viral RNA polymerases (RNAPs) under conditions used for in vitro transcription. We show that nanotubes and individual nanotube monomers (tiles) are non-specifically transcribed by T7 RNAP, and that RNA transcripts produced during non-specific transcription disassemble the nanotubes. Disassembly requires a single-stranded overhang on the nanotube tiles where transcripts can bind and initiate disassembly through strand displacement, suggesting that single-stranded domains on other DNA nanostructures could cause unexpected interactions in the presence of viral RNA polymerases.

Weak silica nanomaterial-induced genotoxicity can be explained by indirect DNA damage as shown by the OGG1-modified comet assay and genomic analysis.

PubMed

Pfuhler, Stefan; Downs, Thomas R; Allemang, Ashley J; Shan, Yuching; Crosby, Meredith E

2017-01-01

In a previous study, 15-nm silica nanoparticles (NPs) caused small increases in DNA damage in liver as measured in the in vivo comet and micronucleus assays after intravenous administration to rats at their maximum tolerated dose, a worst-case exposure scenario. Histopathological examination supported a particle-induced, tissue damage-mediated inflammatory response. This study used a targeted approach to provide insight into the mode of action (MoA) by examining transcriptional regulation of genes in liver in a time and dose-dependent manner at 1, 2, 4, 8 and 24 h after intravenous administration of 15-nm silica NPs. DNA damage was assessed using the standard comet assay and hOGG1 glycosylase-modified comet assay that also measures oxidative DNA damage. Potassium bromate, an IARC Class 2B carcinogen that specifically operates via an oxidative stress MoA, was used as a positive control for the hOGG1 comet assay and gave a strong signal in its main target organ, the kidney, while showing less activity in liver. Treatment of rats with silica NPs at 50 mg/kg body weight (bw) caused small, statistically insignificant increases in DNA damage in liver measured by the standard comet assay, while a statistically significant increase was observed at 4 h with the hOGG1 comet assay, consistent with a MoA involving reactive oxygen species. Histopathology showed liver damage and neutrophil involvement while genomic analysis and response pattern of key genes involved in inflammation and oxidative stress supported a tissue damage-mediated inflammatory response involving the complement system for removing/phagocytising damaged cells. No changes were observed for histopathology or gene array for the low-dose (5 mg/kg bw) silica NPs. The results of this study confirm our hypothesis that the weak DNA damage observed by silica NPs occurs secondary to inflammation/immune response, indicating that a threshold can be applied in the risk assessment of these materials. © The Author 2016. Published by Oxford University Press on behalf of the UK Environmental Mutagen Society. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.

The extent of the uptake of plasmid into the skin determines the immune responses induced by a DNA vaccine applied topically onto the skin

PubMed Central

Yu, Zhen; Chung, Woon-Gye; Sloat, Brian R.; Löhr, Christiane V.; Weiss, Richard; Rodriguez, B. Leticia; Li, Xinran; Cui, Zhengrong

2011-01-01

Objectives Non-invasive immunization by applying plasmid DNA topically onto the skin is an attractive immunization approach. However, the immune responses induced are generally weak. Previously, we showed that the antibody responses induced by topical DNA vaccine were significantly enhanced when hair follicles in the application area were induced into anagen (growth) stage by hair plucking. In the present study, we further investigated the mechanism of immune enhancement. Methods Three different methods, hair plucking or treatment with retinoic acid (RA) or O- tetradecanoylphorbol-13-acetate (TPA), were used to induce hair follicles into anagen stage before mice were dosed with a β-galactosidase-encoding plasmid, and the specific antibody responses induced were evaluated. Key findings The hair plucking method was more effective at enhancing the resultant antibody responses. Treatment with RA or TPA caused more damages to the skin and induced more severe local inflammations than hair plucking. However, hair plucking was most effective at enhancing the uptake or retention of the DNA in the application area. Conclusions The uptake of plasmid DNA in the application area correlated with the antibody responses induced by a topically applied DNA. PMID:21235583

Evolutionary relationships in Panicoid grasses based on plastome phylogenomics (Panicoideae; Poaceae).

PubMed

Burke, Sean V; Wysocki, William P; Zuloaga, Fernando O; Craine, Joseph M; Pires, J Chris; Edger, Patrick P; Mayfield-Jones, Dustin; Clark, Lynn G; Kelchner, Scot A; Duvall, Melvin R

2016-06-18

Panicoideae are the second largest subfamily in Poaceae (grass family), with 212 genera and approximately 3316 species. Previous studies have begun to reveal relationships within the subfamily, but largely lack resolution and/or robust support for certain tribal and subtribal groups. This study aims to resolve these relationships, as well as characterize a putative mitochondrial insert in one linage. 35 newly sequenced Panicoideae plastomes were combined in a phylogenomic study with 37 other species: 15 Panicoideae and 22 from outgroups. A robust Panicoideae topology largely congruent with previous studies was obtained, but with some incongruences with previously reported subtribal relationships. A mitochondrial DNA (mtDNA) to plastid DNA (ptDNA) transfer was discovered in the Paspalum lineage. The phylogenomic analysis returned a topology that largely supports previous studies. Five previously recognized subtribes appear on the topology to be non-monophyletic. Additionally, evidence for mtDNA to ptDNA transfer was identified in both Paspalum fimbriatum and P. dilatatum, and suggests a single rare event that took place in a common progenitor. Finally, the framework from this study can guide larger whole plastome sampling to discern the relationships in Cyperochloeae, Steyermarkochloeae, Gynerieae, and other incertae sedis taxa that are weakly supported or unresolved.

Mms1 is an assistant for regulating G-quadruplex DNA structures.

PubMed

Schwindt, Eike; Paeschke, Katrin

2018-06-01

The preservation of genome stability is fundamental for every cell. Genomic integrity is constantly challenged. Among those challenges are also non-canonical nucleic acid structures. In recent years, scientists became aware of the impact of G-quadruplex (G4) structures on genome stability. It has been shown that folded G4-DNA structures cause changes in the cell, such as transcriptional up/down-regulation, replication stalling, or enhanced genome instability. Multiple helicases have been identified to regulate G4 structures and by this preserve genome stability. Interestingly, although these helicases are mostly ubiquitous expressed, they show specificity for G4 regulation in certain cellular processes (e.g., DNA replication). To this date, it is not clear how this process and target specificity of helicases are achieved. Recently, Mms1, an ubiquitin ligase complex protein, was identified as a novel G4-DNA-binding protein that supports genome stability by aiding Pif1 helicase binding to these regions. In this perspective review, we discuss the question if G4-DNA interacting proteins are fundamental for helicase function and specificity at G4-DNA structures.

DNA attachment to support structures

DOEpatents

Balhorn, Rodney L.; Barry, Christopher H.

2002-01-01

Microscopic beads or other structures are attached to nucleic acids (DNA) using a terminal transferase. The transferase adds labeled dideoxy nucleotide bases to the ends of linear strands of DNA. The labels, such as the antigens digoxigenin and biotin, bind to the antibody compounds or other appropriate complementary ligands, which are bound to the microscopic beads or other support structures. The method does not require the synthesis of a synthetic oligonucleotide probe. The method can be used to tag or label DNA even when the DNA has an unknown sequence, has blunt ends, or is a very large fragment (e.g., >500 kilobase pairs).