Genetic instability associated with loop or stem–loop structures within transcription units can be independent of nucleotide excision repair

PubMed Central

Burns, John A; Chowdhury, Moinuddin A; Cartularo, Laura; Berens, Christian; Scicchitano, David A

2018-01-01

Abstract Simple sequence repeats (SSRs) are found throughout the genome, and under some conditions can change in length over time. Germline and somatic expansions of trinucleotide repeats are associated with a series of severely disabling illnesses, including Huntington's disease. The underlying mechanisms that effect SSR expansions and contractions have been experimentally elusive, but models suggesting a role for DNA repair have been proposed, in particular the involvement of transcription-coupled nucleotide excision repair (TCNER) that removes transcription-blocking DNA damage from the transcribed strand of actively expressed genes. If the formation of secondary DNA structures that are associated with SSRs were to block RNA polymerase progression, TCNER could be activated, resulting in the removal of the aberrant structure and a concomitant change in the region's length. To test this, TCNER activity in primary human fibroblasts was assessed on defined DNA substrates containing extrahelical DNA loops that lack discernible internal base pairs or DNA stem–loops that contain base pairs within the stem. The results show that both structures impede transcription elongation, but there is no corresponding evidence that nucleotide excision repair (NER) or TCNER operates to remove them. PMID:29474673

RTEL1 dismantles T loops and counteracts telomeric G4-DNA to maintain telomere integrity.

PubMed

Vannier, Jean-Baptiste; Pavicic-Kaltenbrunner, Visnja; Petalcorin, Mark I R; Ding, Hao; Boulton, Simon J

2012-05-11

T loops and telomeric G-quadruplex (G4) DNA structures pose a potential threat to genome stability and must be dismantled to permit efficient telomere replication. Here we implicate the helicase RTEL1 in the removal of telomeric DNA secondary structures, which is essential for preventing telomere fragility and loss. In the absence of RTEL1, T loops are inappropriately resolved by the SLX4 nuclease complex, resulting in loss of the telomere as a circle. Depleting SLX4 or blocking DNA replication abolished telomere circles (TCs) and rescued telomere loss in RTEL1(-/-) cells but failed to suppress telomere fragility. Conversely, stabilization of telomeric G4-DNA or loss of BLM dramatically enhanced telomere fragility in RTEL1-deficient cells but had no impact on TC formation or telomere loss. We propose that RTEL1 performs two distinct functions at telomeres: it disassembles T loops and also counteracts telomeric G4-DNA structures, which together ensure the dynamics and stability of the telomere. Copyright © 2012 Elsevier Inc. All rights reserved.

Asymmetric structure of five and six membered DNA hairpin loops

NASA Technical Reports Server (NTRS)

Baumann, U.; Chang, S.

1995-01-01

The tertiary structure of nucleic acid hairpins was elucidated by means of the accessibility of the single-strand-specific nuclease from mung bean. This molecular probe has proven especially useful in determining details of the structural arrangement of the nucleotides within a loop. In this study 3'-labeling is introduced to complement previously used 5'-labeling in order to assess and to exclude possible artifacts of the method. Both labeling procedures result in mutually consistent cleavage patterns. Therefore, methodological artifacts can be excluded and the potential of the nuclease as structural probe is increased. DNA hairpins with five and six membered loops reveal an asymmetric loop structure with a sharp bend of the phosphate-ribose backbone between the second and third nucleotide on the 3'-side of a loop. These hairpin structures differ from smaller loops with 3 or 4 members, which reveal this type of bend between the first and second 3' nucleotide, and resemble with respect to the asymmetry anticodon loops of tRNA.

RNA polymerase gate loop guides the nontemplate DNA strand in transcription complexes.

PubMed

NandyMazumdar, Monali; Nedialkov, Yuri; Svetlov, Dmitri; Sevostyanova, Anastasia; Belogurov, Georgiy A; Artsimovitch, Irina

2016-12-27

Upon RNA polymerase (RNAP) binding to a promoter, the σ factor initiates DNA strand separation and captures the melted nontemplate DNA, whereas the core enzyme establishes interactions with the duplex DNA in front of the active site that stabilize initiation complexes and persist throughout elongation. Among many core RNAP elements that participate in these interactions, the β' clamp domain plays the most prominent role. In this work, we investigate the role of the β gate loop, a conserved and essential structural element that lies across the DNA channel from the clamp, in transcription regulation. The gate loop was proposed to control DNA loading during initiation and to interact with NusG-like proteins to lock RNAP in a closed, processive state during elongation. We show that the removal of the gate loop has large effects on promoter complexes, trapping an unstable intermediate in which the RNAP contacts with the nontemplate strand discriminator region and the downstream duplex DNA are not yet fully established. We find that although RNAP lacking the gate loop displays moderate defects in pausing, transcript cleavage, and termination, it is fully responsive to the transcription elongation factor NusG. Together with the structural data, our results support a model in which the gate loop, acting in concert with initiation or elongation factors, guides the nontemplate DNA in transcription complexes, thereby modulating their regulatory properties.

Water-soluble mercury ion sensing based on the thymine-Hg2+-thymine base pair using retroreflective Janus particle as an optical signaling probe.

PubMed

Chun, Hyeong Jin; Kim, Saemi; Han, Yong Duk; Kim, Dong Woo; Kim, Ka Ram; Kim, Hyo-Sop; Kim, Jae-Ho; Yoon, Hyun C

2018-05-01

Herein, we report an optical sensing platform for mercury ions (Hg 2+ ) in water based on the integration of Hg 2+ -mediated thymine-thymine (T-T) stabilization, a biotinylated stem-loop DNA probe, and a streptavidin-modified retroreflective Janus particle (SA-RJP). Two oligonucleotide probes, including a stem-loop DNA probe and an assistant DNA probe, were utilized. In the absence of Hg 2+ , the assistant DNA probe does not hybridize with the stem-loop probe due to their T-T mismatch, so the surface-immobilized stem-loop DNA probe remains a closed hairpin structure. In the presence of Hg 2+ , the DNA forms a double-stranded structure with the loop region via Hg 2+ -mediated T-T stabilization. This DNA hybridization induces stretching of the stem-loop DNA probe, exposing biotin. To translate these Hg 2+ -mediated structural changes in DNA probe into measurable signal, SA-RJP, an optical signaling label, is applied to recognize the exposed biotin. The number of biospecifically bound SA-RJPs is proportional to the concentration of Hg 2+ , so that the concentration of Hg 2+ can be quantitatively analyzed by counting the number of RJPs. Using the system, a highly selective and sensitive measurement of Hg 2+ was accomplished with a limit of detection of 0.027nM. Considering the simplified optical instrumentation required for retroreflection-based RJP counting, RJP-assisted Hg 2+ measurement can be accomplished in a much easier and inexpensive manner. Moreover, the detection of Hg 2+ in real drinking water samples including tap and commercial bottled water was successfully carried out. Copyright © 2018 Elsevier B.V. All rights reserved.

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

Myc-induced anchorage of the rDNA IGS region to nucleolar matrix modulates growth-stimulated changes in higher-order rDNA architecture

PubMed Central

Shiue, Chiou-Nan; Nematollahi-Mahani, Amir; Wright, Anthony P.H.

2014-01-01

Chromatin domain organization and the compartmentalized distribution of chromosomal regions are essential for packaging of deoxyribonucleic acid (DNA) in the eukaryotic nucleus as well as regulated gene expression. Nucleoli are the most prominent morphological structures of cell nuclei and nucleolar organization is coupled to cell growth. It has been shown that nuclear scaffold/matrix attachment regions often define the base of looped chromosomal domains in vivo and that they are thereby critical for correct chromosome architecture and gene expression. Here, we show regulated organization of mammalian ribosomal ribonucleic acid genes into distinct chromatin loops by tethering to nucleolar matrix via the non-transcribed inter-genic spacer region of the ribosomal DNA (rDNA). The rDNA gene loop structures are induced specifically upon growth stimulation and are dependent on the activity of the c-Myc protein. Matrix-attached rDNA genes are hypomethylated at the promoter and are thus available for transcriptional activation. rDNA genes silenced by methylation are not recruited to the matrix. c-Myc, which has been shown to induce rDNA transcription directly, is physically associated with rDNA gene looping structures and the intergenic spacer sequence in growing cells. Such a role of Myc proteins in gene activation has not been reported previously. PMID:24609384

Myc-induced anchorage of the rDNA IGS region to nucleolar matrix modulates growth-stimulated changes in higher-order rDNA architecture.

PubMed

Shiue, Chiou-Nan; Nematollahi-Mahani, Amir; Wright, Anthony P H

2014-05-01

Chromatin domain organization and the compartmentalized distribution of chromosomal regions are essential for packaging of deoxyribonucleic acid (DNA) in the eukaryotic nucleus as well as regulated gene expression. Nucleoli are the most prominent morphological structures of cell nuclei and nucleolar organization is coupled to cell growth. It has been shown that nuclear scaffold/matrix attachment regions often define the base of looped chromosomal domains in vivo and that they are thereby critical for correct chromosome architecture and gene expression. Here, we show regulated organization of mammalian ribosomal ribonucleic acid genes into distinct chromatin loops by tethering to nucleolar matrix via the non-transcribed inter-genic spacer region of the ribosomal DNA (rDNA). The rDNA gene loop structures are induced specifically upon growth stimulation and are dependent on the activity of the c-Myc protein. Matrix-attached rDNA genes are hypomethylated at the promoter and are thus available for transcriptional activation. rDNA genes silenced by methylation are not recruited to the matrix. c-Myc, which has been shown to induce rDNA transcription directly, is physically associated with rDNA gene looping structures and the intergenic spacer sequence in growing cells. Such a role of Myc proteins in gene activation has not been reported previously. © 2014 The Author(s). Published by Oxford University Press [on behalf of Nucleic Acids Research].

Biomolecular and structural analyses of cauliflower-like DNAs by ultraviolet, circular dichroism, and fluorescence spectroscopies in comparison with natural DNA.

PubMed

Gill, Pooria; Ranjbar, Bijan; Saber, Reza; Khajeh, Khosro; Mohammadian, Mehdi

2011-07-01

Cauliflower-like DNAs are stem-loop DNAs that are fabricated periodically in inverted repetitions from deoxyribonucleic acid phosphates (dNTPs) by loop-mediated isothermal amplification (LAMP). Cauliflower-like DNAs have ladder-shape behaviors on gel electrophoresis, and increasing the time of LAMP leads to multiplying the repetitions, stem-loops, and electrophoretic bands. Cauliflower-like DNAs were fabricated via LAMP using two loop primers, two bumper primers, dNTPs, a λ-phage DNA template, and a Bst DNA polymerase in 75- and 90-min periods. These times led to manufacturing two types of cauliflower-like DNAs with different contents of inverted repetitions and stem-loops, which were clearly indicated by two comparable electrophoresis patterns in agarose gel. LAMP-fabricated DNAs and natural dsB-DNA (salmon genomic DNA) were dialyzed in Gomori phosphate buffer (10 mM, pH 7.4) to be isolated from salts, nucleotides, and primers. Dialyzed DNAs were studied using UV spectroscopy, circular dichroism spectropolarimetry, and fluorescence spectrophotometry. Structural analyses indicated reduction of the molecular ellipticity and extinction coefficients in comparison with B-DNA. Also, cauliflower-like DNAs demonstrated less intrinsic and more extrinsic fluorescence in comparison with natural DNA. The overwinding and lengthening of the cauliflower-like configurations of LAMP DNAs led to changes in physical parameters of this type of DNA in comparison with natural DNA. The results obtained introduced new biomolecular characteristics of DNA macromolecules fabricated within a LAMP process and show the effects of more inverted repeats and stem-loops, which are manufactured by lengthening the process.

The Activation Domain of the Bovine Papillomavirus E2 Protein Mediates Association of DNA-Bound Dimers to form DNA Loops

NASA Astrophysics Data System (ADS)

Knight, Jonathan D.; Li, Rong; Botchan, Michael

1991-04-01

The E2 transactivator protein of bovine papillomavirus binds its specific DNA target sequence as a dimer. We have found that E2 dimers, performed in solution independent of DNA, exhibit substantial cooperativity of DNA binding as detected by both nitrocellulose filter retention and footprint analysis techniques. If the binding sites are widely spaced, E2 forms stable DNA loops visible by electron microscopy. When three widely separated binding sites reside on te DNA, E2 condenses the molecule into a bow-tie structure. This implies that each E2 dimer has at least two independent surfaces for multimerization. Two naturally occurring shorter forms of the protein, E2C and D8/E2, which function in vivo as repressors of transcription, do not form such loops. Thus, the looping function of E2 maps to the 161-amino acid activation domain. These results support the looping model of transcription activation by enhancers.

Cohesin organizes chromatin loops at DNA replication factories

PubMed Central

Guillou, Emmanuelle; Ibarra, Arkaitz; Coulon, Vincent; Casado-Vela, Juan; Rico, Daniel; Casal, Ignacio; Schwob, Etienne; Losada, Ana; Méndez, Juan

2010-01-01

Genomic DNA is packed in chromatin fibers organized in higher-order structures within the interphase nucleus. One level of organization involves the formation of chromatin loops that may provide a favorable environment to processes such as DNA replication, transcription, and repair. However, little is known about the mechanistic basis of this structuration. Here we demonstrate that cohesin participates in the spatial organization of DNA replication factories in human cells. Cohesin is enriched at replication origins and interacts with prereplication complex proteins. Down-regulation of cohesin slows down S-phase progression by limiting the number of active origins and increasing the length of chromatin loops that correspond with replicon units. These results give a new dimension to the role of cohesin in the architectural organization of interphase chromatin, by showing its participation in DNA replication. PMID:21159821

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.

diffloop: a computational framework for identifying and analyzing differential DNA loops from sequencing data.

PubMed

Lareau, Caleb A; Aryee, Martin J; Berger, Bonnie

2018-02-15

The 3D architecture of DNA within the nucleus is a key determinant of interactions between genes, regulatory elements, and transcriptional machinery. As a result, differences in DNA looping structure are associated with variation in gene expression and cell state. To systematically assess changes in DNA looping architecture between samples, we introduce diffloop, an R/Bioconductor package that provides a suite of functions for the quality control, statistical testing, annotation, and visualization of DNA loops. We demonstrate this functionality by detecting differences between ENCODE ChIA-PET samples and relate looping to variability in epigenetic state. Diffloop is implemented as an R/Bioconductor package available at https://bioconductor.org/packages/release/bioc/html/diffloop.html. aryee.martin@mgh.harvard.edu. Supplementary data are available at Bioinformatics online. © The Author (2017). Published by Oxford University Press. All rights reserved. For Permissions, please email: journals.permissions@oup.com

XRN2 Links Transcription Termination to DNA Damage and Replication Stress

PubMed Central

Patidar, Praveen L.; Motea, Edward A.; Dang, Tuyen T.; Manley, James L.

2016-01-01

XRN2 is a 5’-3’ exoribonuclease implicated in transcription termination. Here we demonstrate an unexpected role for XRN2 in the DNA damage response involving resolution of R-loop structures and prevention of DNA double-strand breaks (DSBs). We show that XRN2 undergoes DNA damage-inducible nuclear re-localization, co-localizing with 53BP1 and R loops, in a transcription and R-loop-dependent process. XRN2 loss leads to increased R loops, genomic instability, replication stress, DSBs and hypersensitivity of cells to various DNA damaging agents. We demonstrate that the DSBs that arise with XRN2 loss occur at transcriptional pause sites. XRN2-deficient cells also exhibited an R-loop- and transcription-dependent delay in DSB repair after ionizing radiation, suggesting a novel role for XRN2 in R-loop resolution, suppression of replication stress, and maintenance of genomic stability. Our study highlights the importance of regulating transcription-related activities as a critical component in maintaining genetic stability. PMID:27437695

XRN2 Links Transcription Termination to DNA Damage and Replication Stress.

PubMed

Morales, Julio C; Richard, Patricia; Patidar, Praveen L; Motea, Edward A; Dang, Tuyen T; Manley, James L; Boothman, David A

2016-07-01

XRN2 is a 5'-3' exoribonuclease implicated in transcription termination. Here we demonstrate an unexpected role for XRN2 in the DNA damage response involving resolution of R-loop structures and prevention of DNA double-strand breaks (DSBs). We show that XRN2 undergoes DNA damage-inducible nuclear re-localization, co-localizing with 53BP1 and R loops, in a transcription and R-loop-dependent process. XRN2 loss leads to increased R loops, genomic instability, replication stress, DSBs and hypersensitivity of cells to various DNA damaging agents. We demonstrate that the DSBs that arise with XRN2 loss occur at transcriptional pause sites. XRN2-deficient cells also exhibited an R-loop- and transcription-dependent delay in DSB repair after ionizing radiation, suggesting a novel role for XRN2 in R-loop resolution, suppression of replication stress, and maintenance of genomic stability. Our study highlights the importance of regulating transcription-related activities as a critical component in maintaining genetic stability.

DNA condensation and size effects of DNA condensation agent

NASA Astrophysics Data System (ADS)

Liu, Yan-Hui; Jiang, Chong-Ming; Guo, Xin-Miao; Tang, Yan-Lin; Hu, Lin

2013-08-01

Based on the model of the strong correlation of counterions condensed on DNA molecule, by tailoring interaction potential, interduplex spacing and correlation spacing between condensed counterions on DNA molecule and interduplex spacing fluctuation strength, toroidal configuration, rod-like configuration and two-hole configurations are possible. The size effects of counterion structure on the toroidal structure can be detected by this model. The autocorrelation function of the tangent vectors is found as an effective way to detect the structure of toroidal conformations and the generic pathway of the process of DNA condensation. The generic pathway of all of the configurations involves an initial nucleation loop, and the next part of the DNA chain is folded on the top of the initial nucleation loop with different manners, in agreement with the recent experimental results.

Protein-mediated loops in supercoiled DNA create large topological domains

PubMed Central

Yan, Yan; Ding, Yue; Leng, Fenfei; Dunlap, David; Finzi, Laura

2018-01-01

Abstract Supercoiling can alter the form and base pairing of the double helix and directly impact protein binding. More indirectly, changes in protein binding and the stress of supercoiling also influence the thermodynamic stability of regulatory, protein-mediated loops and shift the equilibria of fundamental DNA/chromatin transactions. For example, supercoiling affects the hierarchical organization and function of chromatin in topologically associating domains (TADs) in both eukaryotes and bacteria. On the other hand, a protein-mediated loop in DNA can constrain supercoiling within a plectonemic structure. To characterize the extent of constrained supercoiling, 400 bp, lac repressor-secured loops were formed in extensively over- or under-wound DNA under gentle tension in a magnetic tweezer. The protein-mediated loops constrained variable amounts of supercoiling that often exceeded the maximum writhe expected for a 400 bp plectoneme. Loops with such high levels of supercoiling appear to be entangled with flanking domains. Thus, loop-mediating proteins operating on supercoiled substrates can establish topological domains that may coordinate gene regulation and other DNA transactions across spans in the genome that are larger than the separation between the binding sites. PMID:29538766

Optimization of a reusable, DNA pseudoknot-based electrochemical sensor for sequence-specific DNA detection in blood serum.

PubMed

Cash, Kevin J; Heeger, Alan J; Plaxco, Kevin W; Xiao, Yi

2009-01-15

We describe in detail a new electrochemical DNA (E-DNA) sensing platform based on target-induced conformation changes in an electrode-bound DNA pseudoknot. The pseudoknot, a DNA structure containing two stem-loops in which the first stem's loop forms part of the second stem, is modified with a methylene blue redox tag at its 3' terminus and covalently attached to a gold electrode via the 5' terminus. In the absence of a target, the structure of the pseudoknot probe minimizes collisions between the redox tag and the electrode, thus reducing faradaic current. Target binding disrupts the pseudoknot structure, liberating a flexible, single-stranded element that can strike the electrode and efficiently transfer electrons. In this article we report further characterization and optimization of this new E-DNA architecture. We find that optimal signaling is obtained at an intermediate probe density ( approximately 1.8 x 10(13) molecules/cm(2) apparent density), which presumably represents a balance between steric and electrostatic blocking at high probe densities and increased background currents arising from transfer from the pseudoknot probe at lower densities. We also find that optimal 3' stem length, which appears to be 7 base pairs, represents a balance between pseudoknot structural stability and target affinity. Finally, a 3' loop comprised of poly(A) exhibits better mismatch discrimination than the equivalent poly(T) loop, but at the cost of decreased gain. Optimization over this parameter space significantly improves the signaling of the pseudoknot-based E-DNA architecture, leading to the ability to sensitively and specifically detect DNA targets even when challenged in complex, multicomponent samples such as blood serum.

Optimization of a Reusable, DNA Pseudoknot-Based Electrochemical Sensor for Sequence-Specific DNA Detection in Blood Serum

PubMed Central

Cash, Kevin J.; Heeger, Alan J.; Plaxco, Kevin W.; Xiao, Yi

2010-01-01

We describe in detail a new electrochemical DNA (E-DNA) sensing platform based on target-induced conformation changes in an electrode-bound DNA pseudoknot. The pseudoknot, a DNA structure containing two stem-loops in which the first stem’s loop forms part of the second stem, is modified with a methylene blue redox tag at its 3′ terminus and covalently attached to a gold electrode via the 5′ terminus. In the absence of a target, the structure of the pseudoknot probe minimizes collisions between the redox tag and the electrode, thus reducing faradaic current. Target binding disrupts the pseudoknot structure, liberating a flexible, single-stranded element that can strike the electrode and efficiently transfer electrons. In this article we report further characterization and optimization of this new E-DNA architecture. We find that optimal signaling is obtained at an intermediate probe density (~1.8 × 1013 molecules/cm2 apparent density), which presumably represents a balance between steric and electrostatic blocking at high probe densities and increased background currents arising from transfer from the pseudoknot probe at lower densities. We also find that optimal 3′ stem length, which appears to be 7 base pairs, represents a balance between pseudoknot structural stability and target affinity. Finally, a 3′ loop comprised of poly(A) exhibits better mismatch discrimination than the equivalent poly(T) loop, but at the cost of decreased gain. Optimization over this parameter space significantly improves the signaling of the pseudoknot-based E-DNA architecture, leading to the ability to sensitively and specifically detect DNA targets even when challenged in complex, multicomponent samples such as blood serum. PMID:19093760

Human immunodeficiency virus type 1 LTR TATA and TAR region sequences required for transcriptional regulation.

PubMed Central

Garcia, J A; Harrich, D; Soultanakis, E; Wu, F; Mitsuyasu, R; Gaynor, R B

1989-01-01

The human immunodeficiency virus (HIV) type 1 LTR is regulated at the transcriptional level by both cellular and viral proteins. Using HeLa cell extracts, multiple regions of the HIV LTR were found to serve as binding sites for cellular proteins. An untranslated region binding protein UBP-1 has been purified and fractions containing this protein bind to both the TAR and TATA regions. To investigate the role of cellular proteins binding to both the TATA and TAR regions and their potential interaction with other HIV DNA binding proteins, oligonucleotide-directed mutagenesis of both these regions was performed followed by DNase I footprinting and transient expression assays. In the TATA region, two direct repeats TC/AAGC/AT/AGCTGC surround the TATA sequence. Mutagenesis of both of these direct repeats or of the TATA sequence interrupted binding over the TATA region on the coding strand, but only a mutation of the TATA sequence affected in vivo assays for tat-activation. In addition to TAR serving as the site of binding of cellular proteins, RNA transcribed from TAR is capable of forming a stable stem-loop structure. To determine the relative importance of DNA binding proteins as compared to secondary structure, oligonucleotide-directed mutations in the TAR region were studied. Local mutations that disrupted either the stem or loop structure were defective in gene expression. However, compensatory mutations which restored base pairing in the stem resulted in complete tat-activation. This indicated a significant role for the stem-loop structure in HIV gene expression. To determine the role of TAR binding proteins, mutations were constructed which extensively changed the primary structure of the TAR region, yet left stem base pairing, stem energy and the loop sequence intact. These mutations resulted in decreased protein binding to TAR DNA and defects in tat-activation, and revealed factor binding specifically to the loop DNA sequence. Further mutagenesis which inverted this stem and loop mutation relative to the HIV LTR mRNA start site resulted in even larger decreases in tat-activation. This suggests that multiple determinants, including protein binding, the loop sequence, and RNA or DNA secondary structure, are important in tat-activation and suggests that tat may interact with cellular proteins binding to DNA to increase HIV gene expression. Images PMID:2721501

Volumetric contributions of loop regions of G-quadruplex DNA to the formation of the tertiary structure.

PubMed

Takahashi, Shuntaro; Sugimoto, Naoki

2017-12-01

DNA guanine-quadruplexes (G-quadruplexes) are unique DNA structures formed by guanine-rich sequences. The loop regions of G-quadruplexes play key roles in stability and topology of G-quadruplexes. Here, we investigated volumetric changes induced by pressure in the folding of the G-quadruplex formed by the thrombin binding aptamer (TBA) with mutations within the loop regions. The change of partial molar volume in the transition from coil to G-quadruplex, ∆V tr , of TBA with a mutation from T to A in the 5' most loop (TBA T3A) was 75.5cm 3 mol -1 , which was larger than that of TBA (54.6cm 3 mol -1 ). TBA with a G to T mutation in the central loop (TBA G8T) had thermal stability similar to TBA T3A but a smaller ∆V tr of 41.1cm 3 mol -1 . In the presence of poly(ethylene)glycol 200 (PEG200), ∆V tr values were 14.7cm 3 mol -1 for TBA T3A and 13.2cm 3 mol -1 for TBA G8T. These results suggest that the two mutations destabilize the G-quadruplex structure differently. Thus, volumetric data obtained using pressure-based thermodynamic analyses provides information about the dynamics of the loop regions and the roles of loops in the stabilities and folding of G-quadruplex structures. Copyright © 2017 Elsevier B.V. All rights reserved.

Mechanism of foreign DNA selection in a bacterial adaptive immune system

PubMed Central

Sashital, Dipali G.; Wiedenheft, Blake; Doudna, Jennifer A.

2012-01-01

Summary In bacterial and archaeal CRISPR immune pathways, DNA sequences from invading bacteriophage or plasmids are integrated into CRISPR loci within the host genome, conferring immunity against subsequent infections. The ribonucleoprotein complex Cascade utilizes RNAs generated from these loci to target complementary “non-self” DNA sequences for destruction, while avoiding binding to “self” sequences within the CRISPR locus. Here we show that CasA, the largest protein subunit of Cascade, is required for non-self target recognition and binding. Combining a 2.3 Å crystal structure of CasA with cryo-EM structures of Cascade, we have identified a loop that is required for viral defense. This loop contacts a conserved 3-base pair motif that is required for non-self target selection. Our data suggest a model in which the CasA loop scans DNA for this short motif prior to target destabilization and binding, maximizing the efficiency of DNA surveillance by Cascade. PMID:22521690

Solution structure and DNA-binding properties of the C-terminal domain of UvrC from E.coli

PubMed Central

Singh, S.; Folkers, G.E.; Bonvin, A.M.J.J.; Boelens, R.; Wechselberger, R.; Niztayev, A.; Kaptein, R.

2002-01-01

The C-terminal domain of the UvrC protein (UvrC CTD) is essential for 5′ incision in the prokaryotic nucleotide excision repair process. We have determined the three-dimensional structure of the UvrC CTD using heteronuclear NMR techniques. The structure shows two helix–hairpin–helix (HhH) motifs connected by a small connector helix. The UvrC CTD is shown to mediate structure-specific DNA binding. The domain binds to a single-stranded–double-stranded junction DNA, with a strong specificity towards looped duplex DNA that contains at least six unpaired bases per loop (‘bubble DNA’). Using chemical shift perturbation experiments, the DNA-binding surface is mapped to the first hairpin region encompassing the conserved glycine–valine–glycine residues followed by lysine–arginine–arginine, a positively charged surface patch and the second hairpin region consisting of glycine–isoleucine–serine. A model for the protein– DNA complex is proposed that accounts for this specificity. PMID:12426397

DNA Topoisomerase 1 Prevents R-loop Accumulation to Modulate Auxin-Regulated Root Development in Rice.

PubMed

Shafiq, Sarfraz; Chen, Chunli; Yang, Jing; Cheng, Lingling; Ma, Fei; Widemann, Emilie; Sun, Qianwen

2017-06-05

R-loop structures (RNA:DNA hybrids) have important functions in many biological processes, including transcriptional regulation and genome instability among diverse organisms. DNA topoisomerase 1 (TOP1), an essential manipulator of DNA topology during RNA transcription and DNA replication processes, can prevent R-loop accumulation by removing the positive and negative DNA supercoiling that is made by RNA polymerases during transcription. TOP1 is required for plant development, but little is known about its function in preventing co-transcriptional R-loop accumulation in various biological processes in plants. Here we show that knockdown of OsTOP1 strongly affects rice development, causing defects in root architecture and gravitropism, which are the consequences of misregulation of auxin signaling and transporter genes. We found that R-loops are naturally formed at rice auxin-related gene loci, and overaccumulate when OsTOP1 is knocked down or OsTOP1 protein activity is inhibited. OsTOP1 therefore sets the accurate expression levels of auxin-related genes by preventing the overaccumulation of inherent R-loops. Our data reveal R-loops as important factors in polar auxin transport and plant root development, and highlight that OsTOP1 functions as a key to link transcriptional R-loops with plant hormone signaling, provide new insights into transcriptional regulation of hormone signaling in plants. Copyright © 2017 The Author. Published by Elsevier Inc. All rights reserved.

Dynamic nucleoplasmic and nucleolar localization of mammalian RNase H1 in response to RNAP I transcriptional R-loops

PubMed Central

Sun, Hong; De Hoyos, Cheryl L.; Bailey, Jeffrey K.; Liang, Xue-hai; Crooke, Stanley T.

2017-01-01

Abstract An R-loop is a DNA:RNA hybrid formed during transcription when a DNA duplex is invaded by a nascent RNA transcript. R-loops accumulate in nucleoli during RNA polymerase I (RNAP I) transcription. Here, we report that mammalian RNase H1 enriches in nucleoli and co-localizes with R-loops in cultured human cells. Co-migration of RNase H1 and R-loops from nucleoli to perinucleolar ring structures was observed upon inhibition of RNAP I transcription. Treatment with camptothecin which transiently stabilized nucleolar R-loops recruited RNase H1 to the nucleoli. It has been reported that the absence of Topoisomerase and RNase H activity in Escherichia coli or Saccharomyces cerevisiae caused R-loop accumulation along rDNA. We found that the distribution of RNase H1 and Top1 along rDNA coincided at sites where R-loops accumulated in mammalian cells. Loss of either RNase H1 or Top1 caused R-loop accumulation, and the accumulation of R-loops was exacerbated when both proteins were depleted. Importantly, we observed that protein levels of Top1 were negatively correlated with the abundance of RNase H1. We conclude that Top1 and RNase H1 are partially functionally redundant in mammalian cells to suppress RNAP I transcription-associate R-loops. PMID:28977560

Structure of a preternary complex involving a prokaryotic NHEJ DNA polymerase.

PubMed

Brissett, Nigel C; Martin, Maria J; Pitcher, Robert S; Bianchi, Julie; Juarez, Raquel; Green, Andrew J; Fox, Gavin C; Blanco, Luis; Doherty, Aidan J

2011-01-21

In many prokaryotes, a specific DNA primase/polymerase (PolDom) is required for nonhomologous end joining (NHEJ) repair of DNA double-strand breaks (DSBs). Here, we report the crystal structure of a catalytically active conformation of Mycobacterium tuberculosis PolDom, consisting of a polymerase bound to a DNA end with a 3' overhang, two metal ions, and an incoming nucleotide but, significantly, lacking a primer strand. This structure represents a polymerase:DNA complex in a preternary intermediate state. This polymerase complex occurs in solution, stabilizing the enzyme on DNA ends and promoting nucleotide extension of short incoming termini. We also demonstrate that the invariant Arg(220), contained in a conserved loop (loop 2), plays an essential role in catalysis by regulating binding of a second metal ion in the active site. We propose that this NHEJ intermediate facilitates extension reactions involving critically short or noncomplementary DNA ends, thus promoting break repair and minimizing sequence loss during DSB repair. Copyright © 2011 Elsevier Inc. All rights reserved.

Impact of low-frequency hotspot mutation R282Q on the structure of p53 DNA-binding domain as revealed by crystallography at 1.54 Å resolution

PubMed Central

Tu, Chao; Tan, Yu-Hong; Shaw, Gary; Zhou, Zheng; Bai, Yawen; Luo, Ray; Ji, Xinhua

2008-01-01

Tumor suppressor p53 is a sequence-specific DNA-binding protein and its central DNA-binding domain (DBD) harbors six hotspots (Arg175, Gly245, Arg248, Arg249, Arg273 and Arg282) for human cancers. Here, the crystal structure of a low-frequency hotspot mutant, p53DBD(R282Q), is reported at 1.54 Å resolution together with the results of molecular-dynamics simulations on the basis of the structure. In addition to eliminating a salt bridge, the R282Q mutation has a significant impact on the properties of two DNA-binding loops (L1 and L3). The L1 loop is flexible in the wild type, but it is not flexible in the mutant. The L3 loop of the wild type is not flexible, whereas it assumes two conformations in the mutant. Molecular-dynamics simulations indicated that both conformations of the L3 loop are accessible under biological conditions. It is predicted that the elimination of the salt bridge and the inversion of the flexibility of L1 and L3 are directly or indirectly responsible for deactivating the tumor suppressor p53. PMID:18453682

Searching for DNA Lesions: Structural Evidence for Lower- and Higher-Affinity DNA Binding Conformations of Human Alkyladenine DNA Glycosylase

PubMed Central

2011-01-01

To efficiently repair DNA, human alkyladenine DNA glycosylase (AAG) must search the million-fold excess of unmodified DNA bases to find a handful of DNA lesions. Such a search can be facilitated by the ability of glycosylases, like AAG, to interact with DNA using two affinities: a lower-affinity interaction in a searching process and a higher-affinity interaction for catalytic repair. Here, we present crystal structures of AAG trapped in two DNA-bound states. The lower-affinity depiction allows us to investigate, for the first time, the conformation of this protein in the absence of a tightly bound DNA adduct. We find that active site residues of AAG involved in binding lesion bases are in a disordered state. Furthermore, two loops that contribute significantly to the positive electrostatic surface of AAG are disordered. Additionally, a higher-affinity state of AAG captured here provides a fortuitous snapshot of how this enzyme interacts with a DNA adduct that resembles a one-base loop. PMID:22148158

Favorable 2'-substitution in the loop region of a thrombin-binding DNA aptamer.

PubMed

Awachat, Ragini; Wagh, Atish A; Aher, Manisha; Fernandes, Moneesha; Kumar, Vaijayanti A

2018-06-01

Simple 2'-OMe-chemical modification in the loop region of the 15mer G-rich DNA sequence GGTTGGTGTGGTTGG is reported. The G-quadruplex structure of this thrombin-binding aptamer (TBA), is stabilized by single modifications (T → 2'-OMe-U), depending on the position of the modification. The structural stability also renders significantly increased inhibition of thrombin-induced fibrin polymerization, a process closely associated with blood-clotting. Copyright © 2018 Elsevier Ltd. All rights reserved.

Microbial Abundances in Salt Marsh Soils: A Molecular Approach for Small Spatial Scales

NASA Astrophysics Data System (ADS)

Granse, Dirk; Mueller, Peter; Weingartner, Magdalena; Hoth, Stefan; Jensen, Kai

2016-04-01

The rate of biological decomposition greatly determines the carbon sequestration capacity of salt marshes. Microorganisms are involved in the decomposition of biomass and the rate of decomposition is supposed to be related to microbial abundance. Recent studies quantified microbial abundance by means of quantitative polymerase chain reaction (QPCR), a method that also allows determining the microbial community structure by applying specific primers. The main microbial community structure can be determined by using primers specific for 16S rRNA (Bacteria) and 18S rRNA (Fungi) of the microbial DNA. However, the investigation of microbial abundance pattern at small spatial scales, such as locally varying abiotic conditions within a salt-marsh system, requires high accuracy in DNA extraction and QPCR methods. Furthermore, there is evidence that a single extraction may not be sufficient to reliably quantify rRNA gene copies. The aim of this study was to establish a suitable DNA extraction method and stable QPCR conditions for the measurement of microbial abundances in semi-terrestrial environments. DNA was extracted from two soil samples (top WE{5}{cm}) by using the PowerSoil DNA Extraction Kit (Mo Bio Laboratories, Inc., Carlsbad, CA) and applying a modified extraction protocol. The DNA extraction was conducted in four consecutive DNA extraction loops from three biological replicates per soil sample by reusing the PowerSoil bead tube. The number of Fungi and Bacteria rRNA gene copies of each DNA extraction loop and a pooled DNA solution (extraction loop 1 - 4) was measured by using the QPCR method with taxa specific primer pairs (Bacteria: B341F, B805R; Fungi: FR1, FF390). The DNA yield of the replicates varied at DNA extraction loop 1 between WE{25 and 85}{ng

Structure Basis for Directional R-loop Formation and Substrate Handover Mechanisms in Type I CRISPR-Cas System.

PubMed

Xiao, Yibei; Luo, Min; Hayes, Robert P; Kim, Jonathan; Ng, Sherwin; Ding, Fang; Liao, Maofu; Ke, Ailong

2017-06-29

Type I CRISPR systems feature a sequential dsDNA target searching and degradation process, by crRNA-displaying Cascade and nuclease-helicase fusion enzyme Cas3, respectively. Here we present two cryo-EM snapshots of the Thermobifida fusca type I-E Cascade: (1) unwinding 11 bp of dsDNA at the seed-sequence region to scout for sequence complementarity, and (2) further unwinding of the entire protospacer to form a full R-loop. These structures provide the much-needed temporal and spatial resolution to resolve key mechanistic steps leading to Cas3 recruitment. In the early steps, PAM recognition causes severe DNA bending, leading to spontaneous DNA unwinding to form a seed-bubble. The full R-loop formation triggers conformational changes in Cascade, licensing Cas3 to bind. The same process also generates a bulge in the non-target DNA strand, enabling its handover to Cas3 for cleavage. The combination of both negative and positive checkpoints ensures stringent yet efficient target degradation in type I CRISPR-Cas systems. Copyright © 2017 Elsevier Inc. All rights reserved.

The solution structure of the prototype foamy virus RNase H domain indicates an important role of the basic loop in substrate binding.

PubMed

Leo, Berit; Schweimer, Kristian; Rösch, Paul; Hartl, Maximilian J; Wöhrl, Birgitta M

2012-09-10

The ribonuclease H (RNase H) domains of retroviral reverse transcriptases play an essential role in the replication cycle of retroviruses. During reverse transcription of the viral genomic RNA, an RNA/DNA hybrid is created whose RNA strand needs to be hydrolyzed by the RNase H to enable synthesis of the second DNA strand by the DNA polymerase function of the reverse transcriptase. Here, we report the solution structure of the separately purified RNase H domain from prototype foamy virus (PFV) revealing the so-called C-helix and the adjacent basic loop, which both were suggested to be important in substrate binding and activity. The solution structure of PFV RNase H shows that it contains a mixed five-stranded β-sheet, which is sandwiched by four α-helices (A-D), including the C-helix, on one side and one α-helix (helix E) on the opposite side. NMR titration experiments demonstrate that upon substrate addition signal changes can be detected predominantly in the basic loop as well as in the C-helix. All these regions are oriented towards the bound substrate. In addition, signal intensities corresponding to residues in the B-helix and the active site decrease, while only minor or no changes of the overall structure of the RNase H are detectable upon substrate binding. Dynamic studies confirm the monomeric state of the RNase H domain. Structure comparisons with HIV-1 RNase H, which lacks the basic protrusion, indicate that the basic loop is relevant for substrate interaction, while the C-helix appears to fulfill mainly structural functions, i.e. positioning the basic loop in the correct orientation for substrate binding. The structural data of PFV RNase H demonstrate the importance of the basic loop, which contains four positively charged lysines, in substrate binding and the function of the C-helix in positioning of the loop. In the dimeric full length HIV-1 RT, the function of the basic loop is carried out by a different loop, which also harbors basic residues, derived from the connection domain of the p66 subunit. Our results suggest that RNases H which are also active as separate domains might need a functional basic loop for proper substrate binding.

The Size of the Internal Loop in DNA Hairpins Influences Their Targeting with Partially Complementary Strands

PubMed Central

2015-01-01

Targeting of noncanonical DNA structures, such as hairpin loops, may have significant diagnostic and therapeutic potential. Oligonucleotides can be used for binding to mRNA, forming a DNA/RNA hybrid duplex that inhibits translation. This kind of modulation of gene expression is called the antisense approach. In order to determine the best strategy to target a common structural motif in mRNA, we have designed a set of stem-loop DNA molecules with sequence: d(GCGCTnGTAAT5GTTACTnGCGC), where n = 1, 3, or 5, “T5” is an end loop of five thymines. We used a combination of calorimetric and spectroscopy techniques to determine the thermodynamics for the reaction of a set of hairpins containing internal loops with their respective partially complementary strands. Our aim was to determine if internal- and end-loops are promising regions for targeting with their corresponding complementary strands. Indeed, all targeting reactions were accompanied by negative changes in free energy, indicating that reactions proceed spontaneously. Further investigation showed that these negative free energy terms result from a net balance of unfavorable entropy and favorable enthalpy contributions. In particular, unfolding of hairpins and duplexes is accompanied by positive changes in heat capacity, which may be a result of exposure of hydrophobic groups to the solvent. This study provides a new method for the targeting of mRNA in order to control gene expression. PMID:25486129

Is the kinetoplast DNA a percolating network of linked rings at its critical point?

NASA Astrophysics Data System (ADS)

Michieletto, Davide; Marenduzzo, Davide; Orlandini, Enzo

2015-05-01

In this work we present a computational study of the kinetoplast genome, modelled as a large number of semiflexible unknotted loops, which are allowed to link with each other. As the DNA density increases, the systems shows a percolation transition between a gas of unlinked rings and a network of linked loops which spans the whole system. Close to the percolation transition, we find that the mean valency of the network, i.e. the average number of loops which are linked to any one loop, is around three, as found experimentally for the kinetoplast DNA (kDNA). Even more importantly, by simulating the digestion of the network by a restriction enzyme, we show that the distribution of oligomers, i.e. structures formed by a few loops which remain linked after digestion, quantitatively matches experimental data obtained from gel electrophoresis, provided that the density is, once again, close to the percolation transition. With respect to previous work, our analysis builds on a reduced number of assumptions, yet can still fully explain the experimental data. Our findings suggest that the kDNA can be viewed as a network of linked loops positioned very close to the percolation transition, and we discuss the possible biological implications of this remarkable fact.

Mammalian DNA enriched for replication origins is enriched for snap-back sequences.

PubMed

Zannis-Hadjopoulos, M; Kaufmann, G; Martin, R G

1984-11-15

Using the instability of replication loops as a method for the isolation of double-stranded nascent DNA, extruded DNA enriched for replication origins was obtained and denatured. Snap-back DNA, single-stranded DNA with inverted repeats (palindromic sequences), reassociates rapidly into stem-loop structures with zero-order kinetics when conditions are changed from denaturing to renaturing, and can be assayed by chromatography on hydroxyapatite. Origin-enriched nascent DNA strands from mouse, rat and monkey cells growing either synchronously or asynchronously were purified and assayed for the presence of snap-back sequences. The results show that origin-enriched DNA is also enriched for snap-back sequences, implying that some origins for mammalian DNA replication contain or lie near palindromic sequences.

High-LET Patterns of DSBs in DNA Loops, the HPRT Gene and Phosphorylation Foci

NASA Technical Reports Server (NTRS)

Ponomarev, Artem L.; Huff, Janice L.; Cucinotta, Francis A.

2007-01-01

We present new results obtained with our model based on the track structure and chromatin geometry that predicts the DSB spatial and genomic distributions in a cell nucleus with the full genome represented. The model generates stochastic patterns of DSBs in the physical space of the nucleus filled with the realistic configuration of human chromosomes. The model was re-used to find the distribution of DSBs in a physical volume corresponding to a visible phosphorylation focus believed to be associated with a DSB. The data shows whether there must more than one DSB per foci due to finite size of the visible focus, even if a single DSB is radiochemically responsible for the phosphorylation of DNA in its vicinity. The same model can predict patterns of closely located DSBs in a given gene, or in a DNA loop, one of the large-scale chromatin structures. We demonstrated for the example of the HPRT gene, how different sorts of radiation lead to proximity effect in DSB locations, which is important for modeling gene deletions. The spectrum of intron deletions and total gene deletions was simulated for the HPRT gene. The same proximity effect of DSBs in a loop can hinder DSB restitutions, as parts of the loop between DSBs is deleted with a higher likelihood. The distributions of DSBs and deletions of DNA in a loop are presented.

Active Site Sharing and Subterminal Hairpin Recognition in a New Class of DNA Transposases

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

Ronning, Donald R.; Guynet, Catherine; Ton-Hoang, Bao

2010-07-20

Many bacteria harbor simple transposable elements termed insertion sequences (IS). In Helicobacter pylori, the chimeric IS605 family elements are particularly interesting due to their proximity to genes encoding gastric epithelial invasion factors. Protein sequences of IS605 transposases do not bear the hallmarks of other well-characterized transposases. We have solved the crystal structure of full-length transposase (TnpA) of a representative member, ISHp608. Structurally, TnpA does not resemble any characterized transposase; rather, it is related to rolling circle replication (RCR) proteins. Consistent with RCR, Mg{sup 2+} and a conserved tyrosine, Tyr127, are essential for DNA nicking and the formation of a covalentmore » intermediate between TnpA and DNA. TnpA is dimeric, contains two shared active sites, and binds two DNA stem loops representing the conserved inverted repeats near each end of ISHp608. The cocrystal structure with stem-loop DNA illustrates how this family of transposases specifically recognizes and pairs ends, necessary steps during transposition.« less

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.

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

PubMed

Mukherjee, Goutam; Pal, Arumay; Levy, Yaakov

2017-11-21

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

Structural Characterization of H-1 Parvovirus: Comparison of Infectious Virions to Empty Capsids

PubMed Central

Halder, Sujata; Nam, Hyun-Joo; Govindasamy, Lakshmanan; Vogel, Michèle; Dinsart, Christiane; Salomé, Nathalie; McKenna, Robert

2013-01-01

The structure of single-stranded DNA (ssDNA) packaging H-1 parvovirus (H-1PV), which is being developed as an antitumor gene delivery vector, has been determined for wild-type (wt) virions and noninfectious (empty) capsids to 2.7- and 3.2-Å resolution, respectively, using X-ray crystallography. The capsid viral protein (VP) structure consists of an α-helix and an eight-stranded anti-parallel β-barrel with large loop regions between the strands. The β-barrel and loops form the capsid core and surface, respectively. In the wt structure, 600 nucleotides are ordered in an interior DNA binding pocket of the capsid. This accounts for ∼12% of the H-1PV genome. The wt structure is identical to the empty capsid structure, except for side chain conformation variations at the nucleotide binding pocket. Comparison of the H-1PV nucleotides to those observed in canine parvovirus and minute virus of mice, two members of the genus Parvovirus, showed both similarity in structure and analogous interactions. This observation suggests a functional role, such as in capsid stability and/or ssDNA genome recognition for encapsulation. The VP structure differs from those of other parvoviruses in surface loop regions that control receptor binding, tissue tropism, pathogenicity, and antibody recognition, including VP sequences reported to determine tumor cell tropism for oncotropic rodent parvoviruses. These structures of H-1PV provide insight into structural features that dictate capsid stabilization following genome packaging and three-dimensional information applicable for rational design of tumor-targeted recombinant gene delivery vectors. PMID:23449783

Structural Basis for Guide RNA Processing and Seed-Dependent DNA Targeting by CRISPR-Cas12a.

PubMed

Swarts, Daan C; van der Oost, John; Jinek, Martin

2017-04-20

The CRISPR-associated protein Cas12a (Cpf1), which has been repurposed for genome editing, possesses two distinct nuclease activities: endoribonuclease activity for processing its own guide RNAs and RNA-guided DNase activity for target DNA cleavage. To elucidate the molecular basis of both activities, we determined crystal structures of Francisella novicida Cas12a bound to guide RNA and in complex with an R-loop formed by a non-cleavable guide RNA precursor and a full-length target DNA. Corroborated by biochemical experiments, these structures reveal the mechanisms of guide RNA processing and pre-ordering of the seed sequence in the guide RNA that primes Cas12a for target DNA binding. Furthermore, the R-loop complex structure reveals the strand displacement mechanism that facilitates guide-target hybridization and suggests a mechanism for double-stranded DNA cleavage involving a single active site. Together, these insights advance our mechanistic understanding of Cas12a enzymes and may contribute to further development of genome editing technologies. Copyright © 2017 Elsevier Inc. All rights reserved.

Studies of Xenopus laevis mitochondrial DNA: D-loop mapping and characterization of DNA-binding proteins

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

Cairns, S.S.

1987-01-01

In X. laevis oocytes, mitochondrial DNA accumulates to 10/sup 5/ times the somatic cell complement, and is characterized by a high frequency of a triple-stranded displacement hoop structure at the origin of replication. To map the termini of the single strands, it was necessary to correct the nucleotide sequence of the D-loop region. The revised sequence of 2458 nucleotides contains 54 discrepancies in comparison to a previously published sequence. Radiolabeling of the nascent strands of the D-loop structure either at the 5' end or at the 3' end identifies a major species with a length of 1670 nucleotides. Cleavage ofmore » the 5' labeled strands reveals two families of ends located near several matches to an element, designated CSB-1, that is conserved in this location in several vertebrate genomes. Cleavage of 3' labeled strands produced one fragment. The unique 3' end maps to about 15 nucleotides preceding the tRNA/sup Pro/ gene. A search for proteins which may bind to mtDNA in this region to regulate nucleic acid synthesis has identified three activities in lysates of X. laevis mitochondria. The DNA-binding proteins were assayed by monitoring their ability to retard the migration of labeled double- or single-stranded DNA fragments in polyacrylamide gels. The DNA binding preference was determined by competition with an excess of either ds- or ssDNA.« less

Reactivation of mutant p53: Constraints on mechanism highlighted by principal component analysis of the DNA binding domain.

PubMed

Ouaray, Zahra; ElSawy, Karim M; Lane, David P; Essex, Jonathan W; Verma, Chandra

2016-10-01

Most p53 mutations associated with cancer are located in its DNA binding domain (DBD). Many structures (X-ray and NMR) of this domain are available in the protein data bank (PDB) and a vast conformational heterogeneity characterizes the various free and complexed states. The major difference between the apo and the holo-complexed states appears to lie in the L1 loop. In particular, the conformations of this loop appear to depend intimately on the sequence of DNA to which it binds. This conclusion builds upon recent observations that implicate the tetramerization and the C-terminal domains (respectively TD and Cter) in DNA binding specificity. Detailed PCA analysis of the most recent collection of DBD structures from the PDB have been carried out. In contrast to recommendations that small molecules/drugs stabilize the flexible L1 loop to rescue mutant p53, our study highlights a need to retain the flexibility of the p53 DNA binding surface (DBS). It is the adaptability of this region that enables p53 to engage in the diverse interactions responsible for its functionality. Proteins 2016; 84:1443-1461. © 2016 Wiley Periodicals, Inc. © 2016 Wiley Periodicals, Inc.

Atomistic Free Energy Model for Nucleic Acids: Simulations of Single-Stranded DNA and the Entropy Landscape of RNA Stem-Loop Structures.

PubMed

Mak, Chi H

2015-11-25

While single-stranded (ss) segments of DNAs and RNAs are ubiquitous in biology, details about their structures have only recently begun to emerge. To study ssDNA and RNAs, we have developed a new Monte Carlo (MC) simulation using a free energy model for nucleic acids that has the atomisitic accuracy to capture fine molecular details of the sugar-phosphate backbone. Formulated on the basis of a first-principle calculation of the conformational entropy of the nucleic acid chain, this free energy model correctly reproduced both the long and short length-scale structural properties of ssDNA and RNAs in a rigorous comparison against recent data from fluorescence resonance energy transfer, small-angle X-ray scattering, force spectroscopy and fluorescence correlation transport measurements on sequences up to ∼100 nucleotides long. With this new MC algorithm, we conducted a comprehensive investigation of the entropy landscape of small RNA stem-loop structures. From a simulated ensemble of ∼10(6) equilibrium conformations, the entropy for the initiation of different size RNA hairpin loops was computed and compared against thermodynamic measurements. Starting from seeded hairpin loops, constrained MC simulations were then used to estimate the entropic costs associated with propagation of the stem. The numerical results provide new direct molecular insights into thermodynaimc measurement from macroscopic calorimetry and melting experiments.

Quantitative analysis and prediction of G-quadruplex forming sequences in double-stranded DNA

PubMed Central

Kim, Minji; Kreig, Alex; Lee, Chun-Ying; Rube, H. Tomas; Calvert, Jacob; Song, Jun S.; Myong, Sua

2016-01-01

Abstract G-quadruplex (GQ) is a four-stranded DNA structure that can be formed in guanine-rich sequences. GQ structures have been proposed to regulate diverse biological processes including transcription, replication, translation and telomere maintenance. Recent studies have demonstrated the existence of GQ DNA in live mammalian cells and a significant number of potential GQ forming sequences in the human genome. We present a systematic and quantitative analysis of GQ folding propensity on a large set of 438 GQ forming sequences in double-stranded DNA by integrating fluorescence measurement, single-molecule imaging and computational modeling. We find that short minimum loop length and the thymine base are two main factors that lead to high GQ folding propensity. Linear and Gaussian process regression models further validate that the GQ folding potential can be predicted with high accuracy based on the loop length distribution and the nucleotide content of the loop sequences. Our study provides important new parameters that can inform the evaluation and classification of putative GQ sequences in the human genome. PMID:27095201

Visualization of chromatin domains created by the gypsy insulator of Drosophila.

PubMed

Byrd, Keith; Corces, Victor G

2003-08-18

Insulators might regulate gene expression by establishing and maintaining the organization of the chromatin fiber within the nucleus. Biochemical fractionation and in situ high salt extraction of lysed cells show that two known protein components of the gypsy insulator are present in the nuclear matrix. Using FISH with DNA probes located between two endogenous Su(Hw) binding sites, we show that the intervening DNA is arranged in a loop, with the two insulators located at the base. Mutations in insulator proteins, subjecting the cells to a brief heat shock, or destruction of the nuclear matrix lead to disruption of the loop. Insertion of an additional gypsy insulator in the center of the loop results in the formation of paired loops through the attachment of the inserted sequences to the nuclear matrix. These results suggest that the gypsy insulator might establish higher-order domains of chromatin structure and regulate nuclear organization by tethering the DNA to the nuclear matrix and creating chromatin loops.

Mechanisms underlying radiosensitivity : iIvestigations in xrs-5, an X-ray sensitive hamster cell line

NASA Astrophysics Data System (ADS)

Johnston, Peter James

The damage caused to cells by ionising radiation is believed to center on damage to the DNA. In particular, the induction of DNA double strand breaks (DSB) have been implicated in biological end-points such as cell killing and the formation of chromosomal aberrations. The xrs-5 cell line is a mutant Chinese hamster ovary fibroblast (CHO-K1) mutant which exhibits sensitivity to ionising radiation and a number of other DNA damaging agents. This mutation, postulated to involve the hamster homologue of the human XRCC5 gene, is believed to be involved in the repair of the DSB. In addition, there are constitutive differences between the wild type and xrs cells involving the structure and function of the nucleus and higher order chromatin structures. The aims of this thesis were to study further the xrs-5 cell line and its response to DNA damage and to investigate the possible link between chromatin structure and DSB repair. By the examination of the response of xrs-5 cells to a number of DNA damaging agents and potential modulators of this response using the cytokinesis block micronucleus assay [Fenech and Morley, 1985] a possible cell cycle defect was identified in addition to elevated levels of chromosomal damage. Xrs-5 cells appeared to be partially defective in the cell cycle checkpoints involving the passage from G2 phase to mitosis. By the use of a modified neutral filter elution procedure variations in the repair of DSB were observed between xrs-5 and CHO. Conventional neutral filter elution requires harsh lysis conditions to remove higher order chromatin structures which interfere with the elution of DNA containing DSB. By lysing cells with non-ionic detergent in the presence of 2 M NaC1, histone depleted structures which retain the higher order nuclear matrix organisation, including chromatin loops, can be produced. Elution from these structures will only occur if two or more DSB lie within a single looped domain delineated by points of attachment to the nuclear matrix. Repair experiments indicate that in CHO cells repair of DSB in loops containing multiple DSB are repaired with "slow" kinetics (t1/2 = 5 hrs) whilst DSB occurring in loops containing single DSB are repaired with "fast" kinetics (t1/2 " 10 min). Xrs- 5 cells are incapable of repairing these multiply damaged loops. This work indicates that the spatial orientation of DSB in higher order structures of chromatin are a possible factor in the repair of these lesions. By construction of a mathematical model of the process of elution from chromatin loops it was possible to postulate the size of the loops to approximate to 2.5-3 Mbp. Further evidence of a potential structural defect in the chromatin of xrs-5 cells was provided by examination of the polypeptide composition and DNA binding activity of nuclear extracts. The affinity of extracted proteins for double-stranded calf-thymus DNA was measured in nuclear extracts of xrs-5 and CHO cells. There was an alteration in the DNA binding activity of salt extractable proteins from xrs-5 as measured by a filter binding assay. By the use of SDS-PAGE and the technique of South-Western blotting, it was possible to identify the approximate molecular weights of these DNA binding proteins. Differences were found in DNA binding between proteins from CHO and xrs-5 extracts of both non-irradiated and irradiated cells. Two proteins with apparent molecular weights of 32.2 and 31.8 kDa exhibited a lower DNA binding activity in xrs-5 than proteins of similar extracts from CHO. The amount of the 32.2 kDa protein was less in the xrs-5 extracts than in CHO extracts, as measured by Coomassie blue staining. The two proteins have not yet been identified but comprise a major DNA binding activity in CHO extracts obtained by detergent-free extraction procedures. This work provides circumstantial evidence that suggests these two polypeptides may form part of the histone H1 family.

Single-molecule manipulation reveals supercoiling-dependent modulation of lac repressor-mediated DNA looping

PubMed Central

Normanno, Davide; Vanzi, Francesco; Pavone, Francesco Saverio

2008-01-01

Gene expression regulation is a fundamental biological process which deploys specific sets of genomic information depending on physiological or environmental conditions. Several transcription factors (including lac repressor, LacI) are present in the cell at very low copy number and increase their local concentration by binding to multiple sites on DNA and looping the intervening sequence. In this work, we employ single-molecule manipulation to experimentally address the role of DNA supercoiling in the dynamics and stability of LacI-mediated DNA looping. We performed measurements over a range of degrees of supercoiling between −0.026 and +0.026, in the absence of axial stretching forces. A supercoiling-dependent modulation of the lifetimes of both the looped and unlooped states was observed. Our experiments also provide evidence for multiple structural conformations of the LacI–DNA complex, depending on torsional constraints. The supercoiling-dependent modulation demonstrated here adds an important element to the model of the lac operon. In fact, the complex network of proteins acting on the DNA in a living cell constantly modifies its topological and mechanical properties: our observations demonstrate the possibility of establishing a signaling pathway from factors affecting DNA supercoiling to transcription factors responsible for the regulation of specific sets of genes. PMID:18310101

Loop L1 governs the DNA-binding specificity and order for RecA-catalyzed reactions in homologous recombination and DNA repair

PubMed Central

Shinohara, Takeshi; Ikawa, Shukuko; Iwasaki, Wakana; Hiraki, Toshiki; Hikima, Takaaki; Mikawa, Tsutomu; Arai, Naoto; Kamiya, Nobuo; Shibata, Takehiko

2015-01-01

In all organisms, RecA-family recombinases catalyze homologous joint formation in homologous genetic recombination, which is essential for genome stability and diversification. In homologous joint formation, ATP-bound RecA/Rad51-recombinases first bind single-stranded DNA at its primary site and then interact with double-stranded DNA at another site. The underlying reason and the regulatory mechanism for this conserved binding order remain unknown. A comparison of the loop L1 structures in a DNA-free RecA crystal that we originally determined and in the reported DNA-bound active RecA crystals suggested that the aspartate at position 161 in loop L1 in DNA-free RecA prevented double-stranded, but not single-stranded, DNA-binding to the primary site. This was confirmed by the effects of the Ala-replacement of Asp-161 (D161A), analyzed directly by gel-mobility shift assays and indirectly by DNA-dependent ATPase activity and SOS repressor cleavage. When RecA/Rad51-recombinases interact with double-stranded DNA before single-stranded DNA, homologous joint-formation is suppressed, likely by forming a dead-end product. We found that the D161A-replacement reduced this suppression, probably by allowing double-stranded DNA to bind preferentially and reversibly to the primary site. Thus, Asp-161 in the flexible loop L1 of wild-type RecA determines the preference for single-stranded DNA-binding to the primary site and regulates the DNA-binding order in RecA-catalyzed recombinase reactions. PMID:25561575

Defining the location of promoter-associated R-loops at near-nucleotide resolution using bisDRIP-seq

PubMed Central

Dumelie, Jason G

2017-01-01

R-loops are features of chromatin consisting of a strand of DNA hybridized to RNA, as well as the expelled complementary DNA strand. R-loops are enriched at promoters where they have recently been shown to have important roles in modifying gene expression. However, the location of promoter-associated R-loops and the genomic domains they perturb to modify gene expression remain unclear. To resolve this issue, we developed a bisulfite-based approach, bisDRIP-seq, to map R-loops across the genome at near-nucleotide resolution in MCF-7 cells. We found the location of promoter-associated R-loops is dependent on the presence of introns. In intron-containing genes, R-loops are bounded between the transcription start site and the first exon-intron junction. In intronless genes, the 3' boundary displays gene-specific heterogeneity. Moreover, intronless genes are often associated with promoter-associated R-loop formation. Together, these studies provide a high-resolution map of R-loops and identify gene structure as a critical determinant of R-loop formation. PMID:29072160

The Crystal Structure of PF-8, the DNA Polymerase Accessory Subunit from Kaposi's Sarcoma-Associated Herpesvirus

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

Baltz, Jennifer L.; Filman, David J.; Ciustea, Mihai

2009-12-01

Kaposi's sarcoma-associated herpesvirus is an emerging pathogen whose mechanism of replication is poorly understood. PF-8, the presumed processivity factor of Kaposi's sarcoma-associated herpesvirus DNA polymerase, acts in combination with the catalytic subunit, Pol-8, to synthesize viral DNA. We have solved the crystal structure of residues 1 to 304 of PF-8 at a resolution of 2.8 {angstrom}. This structure reveals that each monomer of PF-8 shares a fold common to processivity factors. Like human cytomegalovirus UL44, PF-8 forms a head-to-head dimer in the form of a C clamp, with its concave face containing a number of basic residues that are predictedmore » to be important for DNA binding. However, there are several differences with related proteins, especially in loops that extend from each monomer into the center of the C clamp and in the loops that connect the two subdomains of each protein, which may be important for determining PF-8's mode of binding to DNA and to Pol-8. Using the crystal structures of PF-8, the herpes simplex virus catalytic subunit, and RB69 bacteriophage DNA polymerase in complex with DNA and initial experiments testing the effects of inhibition of PF-8-stimulated DNA synthesis by peptides derived from Pol-8, we suggest a model for how PF-8 might form a ternary complex with Pol-8 and DNA. The structure and the model suggest interesting similarities and differences in how PF-8 functions relative to structurally similar proteins.« less

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

The rolling-circle melting-pot model for porcine circovirus DNA replication

USDA-ARS?s Scientific Manuscript database

A stem-loop structure, formed by a pair of inverted repeats during DNA replication, is a conserved feature at the origin of DNA replication (Ori) among plant and animal viruses, bacteriophages and plasmids that replicate their genomes via the rolling-circle replication (RCR) mechanism. Porcine circo...

Understanding the Elementary Steps in DNA Tile-Based Self-Assembly.

PubMed

Jiang, Shuoxing; Hong, Fan; Hu, Huiyu; Yan, Hao; Liu, Yan

2017-09-26

Although many models have been developed to guide the design and implementation of DNA tile-based self-assembly systems with increasing complexity, the fundamental assumptions of the models have not been thoroughly tested. To expand the quantitative understanding of DNA tile-based self-assembly and to test the fundamental assumptions of self-assembly models, we investigated DNA tile attachment to preformed "multi-tile" arrays in real time and obtained the thermodynamic and kinetic parameters of single tile attachment in various sticky end association scenarios. With more sticky ends, tile attachment becomes more thermostable with an approximately linear decrease in the free energy change (more negative). The total binding free energy of sticky ends is partially compromised by a sequence-independent energy penalty when tile attachment forms a constrained configuration: "loop". The minimal loop is a 2 × 2 tetramer (Loop4). The energy penalty of loops of 4, 6, and 8 tiles was analyzed with the independent loop model assuming no interloop tension, which is generalizable to arbitrary tile configurations. More sticky ends also contribute to a faster on-rate under isothermal conditions when nucleation is the rate-limiting step. Incorrect sticky end contributes to neither the thermostability nor the kinetics. The thermodynamic and kinetic parameters of DNA tile attachment elucidated here will contribute to the future improvement and optimization of tile assembly modeling, precise control of experimental conditions, and structural design for error-free self-assembly.

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.

Phi29 Connector-DNA Interactions Govern DNA Crunching and Rotation, Supporting the Check-Valve Model

PubMed Central

Kumar, Rajendra; Grubmüller, Helmut

2016-01-01

During replication of the ϕ29 bacteriophage inside a bacterial host cell, a DNA packaging motor transports the viral DNA into the procapsid against a pressure difference of up to 40 ± 20 atm. Several models have been proposed for the underlying molecular mechanism. Here we have used molecular dynamics simulations to examine the role of the connector part of the motor, and specifically the one-way revolution and the push-roll model. We have focused at the structure and intermolecular interactions between the DNA and the connector, for which a near-complete structure is available. The connector is found to induce considerable DNA deformations with respect to its canonical B-form. We further assessed by force-probe simulations to which extent the connector is able to prevent DNA leakage and found that the connector can act as a partial one-way valve by a check-valve mechanism via its mobile loops. Analysis of the geometry, flexibility, and energetics of channel lysine residues suggested that this arrangement of residues is incompatible with the observed DNA packaging step-size of ∼2.5 bp, such that the step-size is probably determined by the other components of the motor. Previously proposed DNA revolution and rolling motions inside the connector channel are both found implausible due to structural entanglement between the DNA and connector loops that have not been resolved in the crystal structure. Rather, in the simulations, the connector facilitates minor DNA rotation during the packaging process compatible with recent optical-tweezers experiments. Combined with the available experimental data, our simulation results suggest that the connector acts as a check-valve that prevents DNA leakage and induces DNA compression and rotation during DNA packaging. PMID:26789768

Sequence Effect on the Formation of DNA Minidumbbells.

PubMed

Liu, Yuan; Lam, Sik Lok

2017-11-16

The DNA minidumbbell (MDB) is a recently identified non-B structure. The reported MDBs contain two TTTA, CCTG, or CTTG type II loops. At present, the knowledge and understanding of the sequence criteria for MDB formation are still limited. In this study, we performed a systematic high-resolution nuclear magnetic resonance (NMR) and native gel study to investigate the effect of sequence variations in tandem repeats on the formation of MDBs. Our NMR results reveal the importance of hydrogen bonds, base-base stacking, and hydrophobic interactions from each of the participating residues. We conclude that in the MDBs formed by tandem repeats, C-G loop-closing base pairs are more stabilizing than T-A loop-closing base pairs, and thymine residues in both the second and third loop positions are more stabilizing than cytosine residues. The results from this study enrich our knowledge on the sequence criteria for the formation of MDBs, paving a path for better exploring their potential roles in biological systems and DNA nanotechnology.

NF-κB– and AP-1–Mediated DNA Looping Regulates Osteopontin Transcription in Endotoxin-Stimulated Murine Macrophages

PubMed Central

Zhao, Wei; Wang, Lijuan; Zhang, Meng; Wang, Peng; Zhang, Lei; Yuan, Chao; Qi, Jianni; Qiao, Yu; Kuo, Paul C.; Gao, Chengjiang

2013-01-01

Osteopontin (OPN) is expressed by various immune cells and modulates both innate and adaptive immune responses. However, the molecular mechanisms that control opn gene expression, especially at the chromatin level, remain largely unknown. We have previously demonstrated many specific cis- and trans-regulatory elements that determine the extent of endotoxin (LPS)-mediated induction of OPN synthesis in murine macrophages. In the present study, we confirm that NF-κB also plays an important role in the setting of LPS-stimulated OPN expression through binding to a distal regulatory element. Importantly, we demonstrate that LPS stimulates chromosomal loops in the OPN promoter between NF-κB binding site and AP-1 binding site using chromosome conformation capture technology. The crucial role of NF-κB and AP-1 in LPS-stimulated DNA looping was confirmed, as small interfering RNA knock-down of NF-κB p65 and AP-1 c-Jun exhibited decreased levels of DNA looping. Furthermore, we demonstrate that p300 can form a complex with NF-κB and AP-1 and is involved in DNA looping and LPS-induced OPN expression. Therefore, we have identified an essential mechanism to remodel the local chromatin structures and spatial conformations to regulate LPS-induced OPN expression. PMID:21257959

DNA looping by FokI: the impact of twisting and bending rigidity on protein-induced looping dynamics

PubMed Central

Laurens, Niels; Rusling, David A.; Pernstich, Christian; Brouwer, Ineke; Halford, Stephen E.; Wuite, Gijs J. L.

2012-01-01

Protein-induced DNA looping is crucial for many genetic processes such as transcription, gene regulation and DNA replication. Here, we use tethered-particle motion to examine the impact of DNA bending and twisting rigidity on loop capture and release, using the restriction endonuclease FokI as a test system. To cleave DNA efficiently, FokI bridges two copies of an asymmetric sequence, invariably aligning the sites in parallel. On account of the fixed alignment, the topology of the DNA loop is set by the orientation of the sites along the DNA. We show that both the separation of the FokI sites and their orientation, altering, respectively, the twisting and the bending of the DNA needed to juxtapose the sites, have profound effects on the dynamics of the looping interaction. Surprisingly, the presence of a nick within the loop does not affect the observed rigidity of the DNA. In contrast, the introduction of a 4-nt gap fully relaxes all of the torque present in the system but does not necessarily enhance loop stability. FokI therefore employs torque to stabilise its DNA-looping interaction by acting as a ‘torsional’ catch bond. PMID:22373924

Structural and sequencing analysis of local target DNA recognition by MLV integrase.

PubMed

Aiyer, Sriram; Rossi, Paolo; Malani, Nirav; Schneider, William M; Chandar, Ashwin; Bushman, Frederic D; Montelione, Gaetano T; Roth, Monica J

2015-06-23

Target-site selection by retroviral integrase (IN) proteins profoundly affects viral pathogenesis. We describe the solution nuclear magnetic resonance structure of the Moloney murine leukemia virus IN (M-MLV) C-terminal domain (CTD) and a structural homology model of the catalytic core domain (CCD). In solution, the isolated MLV IN CTD adopts an SH3 domain fold flanked by a C-terminal unstructured tail. We generated a concordant MLV IN CCD structural model using SWISS-MODEL, MMM-tree and I-TASSER. Using the X-ray crystal structure of the prototype foamy virus IN target capture complex together with our MLV domain structures, residues within the CCD α2 helical region and the CTD β1-β2 loop were predicted to bind target DNA. The role of these residues was analyzed in vivo through point mutants and motif interchanges. Viable viruses with substitutions at the IN CCD α2 helical region and the CTD β1-β2 loop were tested for effects on integration target site selection. Next-generation sequencing and analysis of integration target sequences indicate that the CCD α2 helical region, in particular P187, interacts with the sequences distal to the scissile bonds whereas the CTD β1-β2 loop binds to residues proximal to it. These findings validate our structural model and disclose IN-DNA interactions relevant to target site selection. © The Author(s) 2015. Published by Oxford University Press on behalf of Nucleic Acids Research.

Structure-function analysis of the DNA translocating portal of the bacteriophage T4 packaging machine.

PubMed

Padilla-Sanchez, Victor; Gao, Song; Kim, Hyung Rae; Kihara, Daisuke; Sun, Lei; Rossmann, Michael G; Rao, Venigalla B

2014-03-06

Tailed bacteriophages and herpesviruses consist of a structurally well conserved dodecameric portal at a special 5-fold vertex of the capsid. The portal plays critical roles in head assembly, genome packaging, neck/tail attachment, and genome ejection. Although the structures of portals from phages φ29, SPP1, and P22 have been determined, their mechanistic roles have not been well understood. Structural analysis of phage T4 portal (gp20) has been hampered because of its unusual interaction with the Escherichia coli inner membrane. Here, we predict atomic models for the T4 portal monomer and dodecamer, and we fit the dodecamer into the cryo-electron microscopy density of the phage portal vertex. The core structure, like that from other phages, is cone shaped with the wider end containing the "wing" and "crown" domains inside the phage head. A long "stem" encloses a central channel, and a narrow "stalk" protrudes outside the capsid. A biochemical approach was developed to analyze portal function by incorporating plasmid-expressed portal protein into phage heads and determining the effect of mutations on head assembly, DNA translocation, and virion production. We found that the protruding loops of the stalk domain are involved in assembling the DNA packaging motor. A loop that connects the stalk to the channel might be required for communication between the motor and the portal. The "tunnel" loops that project into the channel are essential for sealing the packaged head. These studies established that the portal is required throughout the DNA packaging process, with different domains participating at different stages of genome packaging. © 2013.

Selectivity in ligand recognition of G-quadruplex loops.

PubMed

Campbell, Nancy H; Patel, Manisha; Tofa, Amina B; Ghosh, Ragina; Parkinson, Gary N; Neidle, Stephen

2009-03-03

A series of disubstituted acridine ligands have been cocrystallized with a bimolecular DNA G-quadruplex. The ligands have a range of cyclic amino end groups of varying size. The crystal structures show that the diagonal loop in this quadruplex results in a large cavity for these groups, in contrast to the steric constraints imposed by propeller loops in human telomeric quadruplexes. We conclude that the nature of the loop has a significant influence on ligand selectivity for particular quadruplex folds.

Ca2+ improves organization of single-stranded DNA bases in human Rad51 filament, explaining stimulatory effect on gene recombination.

PubMed

Fornander, Louise H; Frykholm, Karolin; Reymer, Anna; Renodon-Cornière, Axelle; Takahashi, Masayuki; Nordén, Bengt

2012-06-01

Human RAD51 protein (HsRad51) catalyses the DNA strand exchange reaction for homologous recombination. To clarify the molecular mechanism of the reaction in vitro being more effective in the presence of Ca(2+) than of Mg(2+), we have investigated the effect of these ions on the structure of HsRad51 filament complexes with single- and double-stranded DNA, the reaction intermediates. Flow linear dichroism spectroscopy shows that the two ionic conditions induce significantly different structures in the HsRad51/single-stranded DNA complex, while the HsRad51/double-stranded DNA complex does not demonstrate this ionic dependence. In the HsRad51/single-stranded DNA filament, the primary intermediate of the strand exchange reaction, ATP/Ca(2+) induces an ordered conformation of DNA, with preferentially perpendicular orientation of nucleobases relative to the filament axis, while the presence of ATP/Mg(2+), ADP/Mg(2+) or ADP/Ca(2+) does not. A high strand exchange activity is observed for the filament formed with ATP/Ca(2+), whereas the other filaments exhibit lower activity. Molecular modelling suggests that the structural variation is caused by the divalent cation interfering with the L2 loop close to the DNA-binding site. It is proposed that the larger Ca(2+) stabilizes the loop conformation and thereby the protein-DNA interaction. A tight binding of DNA, with bases perpendicularly oriented, could facilitate strand exchange.

A small protein inhibits proliferating cell nuclear antigen by breaking the DNA clamp

DOE PAGES

Altieri, Amanda S.; Ladner, Jane E.; Li, Zhuo; ...

2016-05-03

Here, proliferating cell nuclear antigen (PCNA) forms a trimeric ring that encircles duplex DNA and acts as an anchor for a number of proteins involved in DNA metabolic processes. PCNA has two structurally similar domains (I and II) linked by a long loop (inter-domain connector loop, IDCL) on the outside of each monomer of the trimeric structure that makes up the DNA clamp. All proteins that bind to PCNA do so via a PCNA-interacting peptide (PIP) motif that binds near the IDCL. A small protein, called TIP, binds to PCNA and inhibits PCNA-dependent activities although it does not contain amore » canonical PIP motif. The X-ray crystal structure of TIP bound to PCNA reveals that TIP binds to the canonical PIP interaction site, but also extends beyond it through a helix that relocates the IDCL. TIP alters the relationship between domains I and II within the PCNA monomer such that the trimeric ring structure is broken, while the individual domains largely retain their native structure. Small angle X-ray scattering (SAXS) confirms the disruption of the PCNA trimer upon addition of the TIP protein in solution and together with the X-ray crystal data, provides a structural basis for the mechanism of PCNA inhibition by TIP.« less

Protein-mediated looping of DNA under tension requires supercoiling

PubMed Central

Yan, Yan; Leng, Fenfei; Finzi, Laura; Dunlap, David

2018-01-01

Abstract Protein-mediated DNA looping is ubiquitous in chromatin organization and gene regulation, but to what extent supercoiling or nucleoid associated proteins promote looping is poorly understood. Using the lac repressor (LacI), a paradigmatic loop-mediating protein, we measured LacI-induced looping as a function of either supercoiling or the concentration of the HU protein, an abundant nucleoid protein in Escherichia coli. Negative supercoiling to physiological levels with magnetic tweezers easily drove the looping probability from 0 to 100% in single DNA molecules under slight tension that likely exists in vivo. In contrast, even saturating (micromolar) concentrations of HU could not raise the looping probability above 30% in similarly stretched DNA or 80% in DNA without tension. Negative supercoiling is required to induce significant looping of DNA under any appreciable tension. PMID:29365152

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

Evaluating the role of coherent delocalized phonon-like modes in DNA cyclization

DOE PAGES

Alexandrov, Ludmil B.; Rasmussen, Kim Ø.; Bishop, Alan R.; ...

2017-08-29

The innate flexibility of a DNA sequence is quantified by the Jacobson-Stockmayer’s J-factor, which measures the propensity for DNA loop formation. Recent studies of ultra-short DNA sequences revealed a discrepancy of up to six orders of magnitude between experimentally measured and theoretically predicted J-factors. These large differences suggest that, in addition to the elastic moduli of the double helix, other factors contribute to loop formation. We develop a new theoretical model that explores how coherent delocalized phonon-like modes in DNA provide single-stranded ”flexible hinges” to assist in loop formation. We also combine the Czapla-Swigon-Olson structural model of DNA with ourmore » extended Peyrard-Bishop-Dauxois model and, without changing any of the parameters of the two models, apply this new computational framework to 86 experimentally characterized DNA sequences. Our results demonstrate that the new computational framework can predict J-factors within an order of magnitude of experimental measurements for most ultra-short DNA sequences, while continuing to accurately describe the J-factors of longer sequences. Furthermore, we demonstrate that our computational framework can be used to describe the cyclization of DNA sequences that contain a base pair mismatch. Overall, our results support the conclusion that coherent delocalized phonon-like modes play an important role in DNA cyclization.« less

Evaluating the role of coherent delocalized phonon-like modes in DNA cyclization

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

Alexandrov, Ludmil B.; Rasmussen, Kim Ø.; Bishop, Alan R.

The innate flexibility of a DNA sequence is quantified by the Jacobson-Stockmayer’s J-factor, which measures the propensity for DNA loop formation. Recent studies of ultra-short DNA sequences revealed a discrepancy of up to six orders of magnitude between experimentally measured and theoretically predicted J-factors. These large differences suggest that, in addition to the elastic moduli of the double helix, other factors contribute to loop formation. We develop a new theoretical model that explores how coherent delocalized phonon-like modes in DNA provide single-stranded ”flexible hinges” to assist in loop formation. We also combine the Czapla-Swigon-Olson structural model of DNA with ourmore » extended Peyrard-Bishop-Dauxois model and, without changing any of the parameters of the two models, apply this new computational framework to 86 experimentally characterized DNA sequences. Our results demonstrate that the new computational framework can predict J-factors within an order of magnitude of experimental measurements for most ultra-short DNA sequences, while continuing to accurately describe the J-factors of longer sequences. Furthermore, we demonstrate that our computational framework can be used to describe the cyclization of DNA sequences that contain a base pair mismatch. Overall, our results support the conclusion that coherent delocalized phonon-like modes play an important role in DNA cyclization.« less

Double stranded nucleic acid biochips

DOEpatents

Chernov, Boris; Golova, Julia

2006-05-23

This invention describes a new method of constructing double-stranded DNA (dsDNA) microarrays based on the use of pre-synthesized or natural DNA duplexes without a stem-loop structure. The complementary oligonucleotide chains are bonded together by a novel connector that includes a linker for immobilization on a matrix. A non-enzymatic method for synthesizing double-stranded nucleic acids with this novel connector enables the construction of inexpensive and robust dsDNA/dsRNA microarrays. DNA-DNA and DNA-protein interactions are investigated using the microarrays.

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

PubMed

Ishida, Hisashi; Matsumoto, Atsushi

2016-09-01

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

Spectroscopic insights into quadruplexes of five-repeat telomere DNA sequences upon G-block damage.

PubMed

Dvořáková, Zuzana; Vorlíčková, Michaela; Renčiuk, Daniel

2017-11-01

The DNA lesions, resulting from oxidative damage, were shown to destabilize human telomere four-repeat quadruplex and to alter its structure. Long telomere DNA, as a repetitive sequence, offers, however, other mechanisms of dealing with the lesion: extrusion of the damaged repeat into loop or shifting the quadruplex position by one repeat. Using circular dichroism and UV absorption spectroscopy and polyacrylamide electrophoresis, we studied consequences of lesions at different positions of the model five-repeat human telomere DNA sequences on the structure and stability of their quadruplexes in sodium and in potassium. The repeats affected by lesion are preferentially positioned as terminal overhangs of the core quadruplex structurally similar to the four-repeat one. Forced affecting of the inner repeats leads to presence of variety of more parallel folds in potassium. In sodium the designed models form mixture of two dominant antiparallel quadruplexes whose population varies with the position of the affected repeat. The shapes of quadruplex CD spectra, namely the height of dominant peaks, significantly correlate with melting temperatures. Lesion in one guanine tract of a more than four repeats long human telomere DNA sequence may cause re-positioning of its quadruplex arrangement associated with a shift of the structure to less common quadruplex conformations. The type of the quadruplex depends on the loop position and external conditions. The telomere DNA quadruplexes are quite resistant to the effect of point mutations due to the telomere DNA repetitive nature, although their structure and, consequently, function might be altered. Copyright © 2017. Published by Elsevier B.V.

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

DNA–DNA kissing complexes as a new tool for the assembly of DNA nanostructures

PubMed Central

Barth, Anna; Kobbe, Daniela; Focke, Manfred

2016-01-01

Kissing-loop annealing of nucleic acids occurs in nature in several viruses and in prokaryotic replication, among other circumstances. Nucleobases of two nucleic acid strands (loops) interact with each other, although the two strands cannot wrap around each other completely because of the adjacent double-stranded regions (stems). In this study, we exploited DNA kissing-loop interaction for nanotechnological application. We functionalized the vertices of DNA tetrahedrons with DNA stem-loop sequences. The complementary loop sequence design allowed the hybridization of different tetrahedrons via kissing-loop interaction, which might be further exploited for nanotechnology applications like cargo transport and logical elements. Importantly, we were able to manipulate the stability of those kissing-loop complexes based on the choice and concentration of cations, the temperature and the number of complementary loops per tetrahedron either at the same or at different vertices. Moreover, variations in loop sequences allowed the characterization of necessary sequences within the loop as well as additional stability control of the kissing complexes. Therefore, the properties of the presented nanostructures make them an important tool for DNA nanotechnology. PMID:26773051

Structure-Function Analysis of the DNA Translocating Portal of the Bacteriophage T4 Packaging Machine

PubMed Central

Padilla-Sanchez, Victor; Gao, Song; Kim, Hyung Rae; Kihara, Daisuke; Sun, Lei; Rossmann, Michael G.; Rao, Venigalla B.

2013-01-01

Tailed bacteriophages and herpesviruses consist of a structurally well conserved dodecameric portal at a special five-fold vertex of the capsid. The portal plays critical roles in head assembly, genome packaging, neck/tail attachment, and genome ejection. Although the structures of portals from phages φ29, SPP1 and P22 have been determined, their mechanistic roles have not been well understood. Structural analysis of phage T4 portal (gp20) has been hampered because of its unusual interaction with the E. coli inner membrane. Here, we predict atomic models for the T4 portal monomer and dodecamer, and fit the dodecamer into the cryoEM density of the phage portal vertex. The core structure, like that from other phages, is cone-shaped with the wider end containing the “wing” and “crown” domains inside the phage head. A long “stem” encloses a central channel, and a narrow “stalk” protrudes outside the capsid. A biochemical approach was developed to analyze portal function by incorporating plasmid-expressed portal protein into phage heads and determining the effect of mutations on head assembly, DNA translocation, and virion production. We found that the protruding loops of the stalk domain are involved in assembling the DNA packaging motor. A loop that connects the stalk to the channel might be required for communication between the motor and portal. The “tunnel” loops that project into the channel are essential for sealing the packaged head. These studies established that the portal is required throughout the DNA packaging process, with different domains participating at different stages of genome packaging. PMID:24126213

Strand displacement synthesis by yeast DNA polymerase ε

PubMed Central

Ganai, Rais A.; Zhang, Xiao-Ping; Heyer, Wolf-Dietrich; Johansson, Erik

2016-01-01

DNA polymerase ε (Pol ε) is a replicative DNA polymerase with an associated 3′–5′ exonuclease activity. Here, we explored the capacity of Pol ε to perform strand displacement synthesis, a process that influences many DNA transactions in vivo. We found that Pol ε is unable to carry out extended strand displacement synthesis unless its 3′–5′ exonuclease activity is removed. However, the wild-type Pol ε holoenzyme efficiently displaced one nucleotide when encountering double-stranded DNA after filling a gap or nicked DNA. A flap, mimicking a D-loop or a hairpin structure, on the 5′ end of the blocking primer inhibited Pol ε from synthesizing DNA up to the fork junction. This inhibition was observed for Pol ε but not with Pol δ, RB69 gp43 or Pol η. Neither was Pol ε able to extend a D-loop in reconstitution experiments. Finally, we show that the observed strand displacement synthesis by exonuclease-deficient Pol ε is distributive. Our results suggest that Pol ε is unable to extend the invading strand in D-loops during homologous recombination or to add more than two nucleotides during long-patch base excision repair. Our results support the hypothesis that Pol ε participates in short-patch base excision repair and ribonucleotide excision repair. PMID:27325747

Mechanism and manipulation of DNA:RNA hybrid G-quadruplex formation in transcription of G-rich DNA.

PubMed

Zhang, Jia-yu; Zheng, Ke-wei; Xiao, Shan; Hao, Yu-hua; Tan, Zheng

2014-01-29

We recently reported that a DNA:RNA hybrid G-quadruplex (HQ) forms during transcription of DNA that bears two or more tandem guanine tracts (G-tract) on the nontemplate strand. Putative HQ-forming sequences are enriched in the nearby 1000 nt region right downstream of transcription start sites in the nontemplate strand of warm-blooded animals, and HQ regulates transcription under both in vitro and in vivo conditions. Therefore, knowledge of the mechanism of HQ formation is important for understanding the biological function of HQ as well as for manipulating gene expression by targeting HQ. In this work, we studied the mechanism of HQ formation using an in vitro T7 transcription model. We show that RNA synthesis initially produces an R-loop, a DNA:RNA heteroduplex formed by a nascent RNA transcript and the template DNA strand. In the following round of transcription, the RNA in the R-loop is displaced, releasing the RNA in single-stranded form (ssRNA). Then the G-tracts in the RNA can jointly form HQ with those in the nontemplate DNA strand. We demonstrate that the structural cascade R-loop → ssRNA → HQ offers opportunities to intercept HQ formation, which may provide a potential method to manipulate gene expression.

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

Altieri, Amanda S.; Ladner, Jane E.; Li, Zhuo

Proliferating cell nuclear antigen (PCNA) forms a trimeric ring that encircles duplex DNA and acts as an anchor for a number of proteins involved in DNA metabolic processes. PCNA has two structurally similar domains (I and II) linked by a long loop (inter-domain connector loop, IDCL) on the outside of each monomer of the trimeric structure that makes up the DNA clamp. All proteins that bind to PCNA do so via a PCNA-interacting peptide (PIP) motif that binds near the IDCL. A small protein, called TIP, binds to PCNA and inhibits PCNA-dependent activities although it does not contain a canonicalmore » PIP motif. The X-ray crystal structure of TIP bound to PCNA reveals that TIP binds to the canonical PIP interaction site, but also extends beyond it through a helix that relocates the IDCL. TIP alters the relationship between domains I and II within the PCNA monomer such that the trimeric ring structure is broken, while the individual domains largely retain their native structure. Small angle X-ray scattering (SAXS) confirms the disruption of the PCNA trimer upon addition of the TIP protein in solution and together with the X-ray crystal data, provides a structural basis for the mechanism of PCNA inhibition by TIP.« less

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

Altieri, Amanda S.; Ladner, Jane E.; Li, Zhuo

Here, proliferating cell nuclear antigen (PCNA) forms a trimeric ring that encircles duplex DNA and acts as an anchor for a number of proteins involved in DNA metabolic processes. PCNA has two structurally similar domains (I and II) linked by a long loop (inter-domain connector loop, IDCL) on the outside of each monomer of the trimeric structure that makes up the DNA clamp. All proteins that bind to PCNA do so via a PCNA-interacting peptide (PIP) motif that binds near the IDCL. A small protein, called TIP, binds to PCNA and inhibits PCNA-dependent activities although it does not contain amore » canonical PIP motif. The X-ray crystal structure of TIP bound to PCNA reveals that TIP binds to the canonical PIP interaction site, but also extends beyond it through a helix that relocates the IDCL. TIP alters the relationship between domains I and II within the PCNA monomer such that the trimeric ring structure is broken, while the individual domains largely retain their native structure. Small angle X-ray scattering (SAXS) confirms the disruption of the PCNA trimer upon addition of the TIP protein in solution and together with the X-ray crystal data, provides a structural basis for the mechanism of PCNA inhibition by TIP.« less

Top3-Rmi1 dissolve Rad51-mediated D loops by a topoisomerase-based mechanism.

PubMed

Fasching, Clare L; Cejka, Petr; Kowalczykowski, Stephen C; Heyer, Wolf-Dietrich

2015-02-19

The displacement loop (D loop) is a DNA strand invasion product formed during homologous recombination. Disruption of nascent D loops prevents recombination, and during synthesis-dependent strand annealing (SDSA), disruption of D loops extended by DNA polymerase ensures a non-crossover outcome. The proteins implicated in D loop disruption are DNA motor proteins/helicases that act by moving DNA junctions. Here we report that D loops can also be disrupted by DNA topoisomerase 3 (Top3), and this disruption depends on Top3's catalytic activity. Yeast Top3 specifically disrupts D loops mediated by yeast Rad51/Rad54; protein-free D loops or D loop mediated by bacterial RecA protein or human RAD51/RAD54 resist dissolution. Also, the human Topoisomerase IIIa-RMI1-RMI2 complex is capable of dissolving D loops. Consistent with genetic data, we suggest that the extreme growth defect and hyper-recombination phenotype of Top3-deficient yeast cells is partially a result of unprocessed D loops. Copyright © 2015 Elsevier Inc. All rights reserved.

Highly sensitive self-complementary DNA nanoswitches triggered by polyelectrolytes

NASA Astrophysics Data System (ADS)

Wu, Jincai; Yu, Feng; Zhang, Zheng; Chen, Yong; Du, Jie; Maruyama, Atsushi

2015-12-01

Dimerization of two homologous strands of genomic DNA/RNA is an essential feature of retroviral replication. Herein we show that a cationic comb-type copolymer (CCC), poly(l-lysine)-graft-dextran, accelerates the dimerization of self-complementary stem-loop DNA, frequently found in functional DNA/RNA molecules, such as aptamers. Furthermore, an anionic polymer poly(sodium vinylsulfonate) (PVS) dissociates CCC from the duplex shortly within a few seconds. Then single stem-loop DNA spontaneously transforms from its dimer. Thus we can easily control the dimer and stem-loop DNA by switching on/off CCC activity. Both polyelectrolytes and DNA concentrations are in the nanomole per liter range. The polyelectrolyte-assisted transconformation and sequences design strategy ensures the reversible state control with rapid response and effective switching under physiologically relevant conditions. A further application of this sensitive assembly is to construct an aptamer-type drug delivery system, bind or release functional molecules responding to its transconformation.Dimerization of two homologous strands of genomic DNA/RNA is an essential feature of retroviral replication. Herein we show that a cationic comb-type copolymer (CCC), poly(l-lysine)-graft-dextran, accelerates the dimerization of self-complementary stem-loop DNA, frequently found in functional DNA/RNA molecules, such as aptamers. Furthermore, an anionic polymer poly(sodium vinylsulfonate) (PVS) dissociates CCC from the duplex shortly within a few seconds. Then single stem-loop DNA spontaneously transforms from its dimer. Thus we can easily control the dimer and stem-loop DNA by switching on/off CCC activity. Both polyelectrolytes and DNA concentrations are in the nanomole per liter range. The polyelectrolyte-assisted transconformation and sequences design strategy ensures the reversible state control with rapid response and effective switching under physiologically relevant conditions. A further application of this sensitive assembly is to construct an aptamer-type drug delivery system, bind or release functional molecules responding to its transconformation. Electronic supplementary information (ESI) available: I. Sequences of DIS25, DIS25-2a and DIS25-3a. II. Structural formula of poly(l-lysine)-graft-dextran (PLL-g-Dex). 1H-NMR spectra of PLL-g-Dex in D2O. III. Gel electrophoretic analysis of dimerization of DIS25 with various N/P ratios. IV. The effect of polyelectrolyte on the fluorescence polarity of TAMRA-labeled duplex. V. UV absorption/Tm profiles of DIS25. VI. Arrhenius plots for spontaneous dissociation of the DIS25 dimer and PLL-g-Dex-assisted dimerization of DIS25.VII. Switching between double stem-loop DIS42 and extended multiplex drived by PLL-g-Dex and PVS. See DOI: 10.1039/c5nr05193b

Functions of Replication Protein A as a Sensor of R Loops and a Regulator of RNaseH1

PubMed Central

Nguyen, Hai Dang; Yadav, Tribhuwan; Giri, Sumanprava; Saez, Borja; Graubert, Timothy A.; Zou, Lee

2017-01-01

R loop, a transcription intermediate containing RNA:DNA hybrids and displaced single-stranded DNA (ssDNA), has emerged as a major source of genomic instability. RNaseH1, which cleaves the RNA in RNA:DNA hybrids, plays an important role in R loop suppression. Here, we show that replication protein A (RPA), a ssDNA-binding protein, interacts with RNaseH1 and colocalizes with both RNaseH1 and R loops in cells. In vitro, purified RPA directly enhances the association of RNaseH1 with RNA:DNA hybrids and stimulates the activity of RNaseH1 on R loops. An RPA binding-defective RNaseH1 mutant is not efficiently stimulated by RPA in vitro, fails to accumulate at R loops in cells, and loses the ability to suppress R loops and associated genomic instability. Thus, in addition to sensing DNA damage and replication stress, RPA is a sensor of R loops and a regulator of RNaseH1, extending the versatile role of RPA in suppression of genomic instability. PMID:28257700

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

PubMed

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

2017-08-10

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

Genetic diversity of Guangxi chicken breeds assessed with microsatellites and the mitochondrial DNA D-loop region.

PubMed

Liao, Yuying; Mo, Guodong; Sun, Junli; Wei, Fengying; Liao, Dezhong Joshua

2016-05-01

The domestic chicken (Gallus gallus domesticus) is an excellent model for genetic studies of phenotypic diversity. The Guangxi Region of China possesses several native chicken breeds displaying a broad range of phenotypes well adapted to the extreme hot-and-wet environments in the region. We thus evaluated the genetic diversity and relationships among six native chicken populations of the Guangxi region and also evaluated two commercial breeds (Arbor Acres and Roman chickens). We analyzed the sequences of the D-loop region of the mitochondrial DNA (mtDNA) and 18 microsatellite loci of 280 blood samples from six Guangxi native chicken breeds and from Arbor Acres and Roman chickens, and used the neighbor-joining method to construct the phylogenetic tree of these eight breeds. Our results showed that the genetic diversity of Guangxi native breeds was relatively rich. The phylogenetic tree using the unweighed pair-group method with arithmetic means (UPGAM) on microsatellite marks revealed two main clusters. Arbor Acres chicken and Roman chicken were in one cluster, while the Guangxi breeds were in the other cluster. Moreover, the UPGAM tree of Guangxi native breeds based on microsatellite loci was more consistent with the genesis, breeding history, differentiation and location than the mtDNA D-loop region. STRUCTURE analysis further confirmed the genetic structure of Guangxi native breeds in the Neighbor-Net dendrogram. The nomenclature of mtDNA sequence polymorphisms suggests that the Guangxi native chickens are distributed across four clades, but most of them are clustered in two main clades (B and E), with the other haplotypes within the clades A and C. The Guangxi native breeds revealed abundant genetic diversity not only on microsatellite loci but also on mtDNA D-loop region, and contained multiple maternal lineages, including one from China and another from Europe or the Middle East.

Replication, checkpoint suppression and structure of centromeric DNA

PubMed Central

Romeo, Francesco; Costanzo, Vincenzo

2016-01-01

ABSTRACT Human centromeres contain large amounts of repetitive DNA sequences known as α satellite DNA, which can be difficult to replicate and whose functional role is unclear. Recently, we have characterized protein composition, structural organization and checkpoint response to stalled replication forks of centromeric chromatin reconstituted in Xenopus laevis egg extract. We showed that centromeric DNA has high affinity for SMC2-4 subunits of condensins and for CENP-A, it is enriched for DNA repair factors and suppresses the ATR checkpoint to ensure its efficient replication. We also showed that centromeric chromatin forms condensins enriched and topologically constrained DNA loops, which likely contribute to the overall structure of the centromere. These findings have important implications on how chromosomes are organized and genome stability is maintained in mammalian cells. PMID:27893298

Multiprotein DNA Looping

NASA Astrophysics Data System (ADS)

Vilar, Jose M. G.; Saiz, Leonor

2006-06-01

DNA looping plays a fundamental role in a wide variety of biological processes, providing the backbone for long range interactions on DNA. Here we develop the first model for DNA looping by an arbitrarily large number of proteins and solve it analytically in the case of identical binding. We uncover a switchlike transition between looped and unlooped phases and identify the key parameters that control this transition. Our results establish the basis for the quantitative understanding of fundamental cellular processes like DNA recombination, gene silencing, and telomere maintenance.

Biopolymer Chain Elasticity: a novel concept and a least deformation energy principle predicts backbone and overall folding of DNA TTT hairpins in agreement with NMR distances

PubMed Central

Pakleza, Christophe; Cognet, Jean A. H.

2003-01-01

A new molecular modelling methodology is presented and shown to apply to all published solution structures of DNA hairpins with TTT in the loop. It is based on the theory of elasticity of thin rods and on the assumption that single-stranded B-DNA behaves as a continuous, unshearable, unstretchable and flexible thin rod. It requires four construction steps: (i) computation of the tri-dimensional trajectory of the elastic line, (ii) global deformation of single-stranded helical DNA onto the elastic line, (iii) optimisation of the nucleoside rotations about the elastic line, (iv) energy minimisation to restore backbone bond lengths and bond angles. This theoretical approach called ‘Biopolymer Chain Elasticity’ (BCE) is capable of reproducing the tri-dimensional course of the sugar–phosphate chain and, using NMR-derived distances, of reproducing models close to published solution structures. This is shown by computing three different types of distance criteria. The natural description provided by the elastic line and by the new parameter, Ω, which corresponds to the rotation angles of nucleosides about the elastic line, offers a considerable simplification of molecular modelling of hairpin loops. They can be varied independently from each other, since the global shape of the hairpin loop is preserved in all cases. PMID:12560506

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

Strand displacement synthesis by yeast DNA polymerase ε.

PubMed

Ganai, Rais A; Zhang, Xiao-Ping; Heyer, Wolf-Dietrich; Johansson, Erik

2016-09-30

DNA polymerase ε (Pol ε) is a replicative DNA polymerase with an associated 3'-5' exonuclease activity. Here, we explored the capacity of Pol ε to perform strand displacement synthesis, a process that influences many DNA transactions in vivo We found that Pol ε is unable to carry out extended strand displacement synthesis unless its 3'-5' exonuclease activity is removed. However, the wild-type Pol ε holoenzyme efficiently displaced one nucleotide when encountering double-stranded DNA after filling a gap or nicked DNA. A flap, mimicking a D-loop or a hairpin structure, on the 5' end of the blocking primer inhibited Pol ε from synthesizing DNA up to the fork junction. This inhibition was observed for Pol ε but not with Pol δ, RB69 gp43 or Pol η. Neither was Pol ε able to extend a D-loop in reconstitution experiments. Finally, we show that the observed strand displacement synthesis by exonuclease-deficient Pol ε is distributive. Our results suggest that Pol ε is unable to extend the invading strand in D-loops during homologous recombination or to add more than two nucleotides during long-patch base excision repair. Our results support the hypothesis that Pol ε participates in short-patch base excision repair and ribonucleotide excision repair. © The Author(s) 2016. Published by Oxford University Press on behalf of Nucleic Acids Research.

Mechanism of mismatch recognition revealed by human MutSβ bound to unpaired DNA loops

PubMed Central

Gupta, Shikha; Gellert, Martin; Yang, Wei

2011-01-01

DNA mismatch repair corrects replication errors, thus reducing mutation rates and microsatellite instability. Genetic defects in this pathway cause Lynch Syndrome and various cancers in humans. Binding of a mispaired or unpaired base by bacterial MutS and eukaryotic MutSα is well characterized. We report here crystal structures of human MutSβ complexed with DNA containing insertion-deletion loops (IDL) of 2, 3, 4, or 6 unpaired nucleotides. In contrast to eukaryotic MutSα and bacterial MutS, which bind the base of a mismatched nucleotide, MutSβ binds three phosphates in an IDL. DNA is severely bent at the IDL; unpaired bases are flipped out into the major groove and partially exposed to solvent. A normal downstream basepair can become unpaired; thereby a single unpaired base can be converted to an IDL of 2 nucleotides and recognized by MutSβ. The C-terminal dimerization domains form an integral part of the MutS structure and coordinate asymmetrical ATP hydrolysis by Msh2 and Msh3 with mismatch binding to signal for repair. PMID:22179786

Structural mechanism of Smad4 recognition by the nuclear oncoprotein Ski: insights on Ski-mediated repression of TGF-beta signaling.

PubMed

Wu, Jia Wei; Krawitz, Ariel R; Chai, Jijie; Li, Wenyu; Zhang, Fangjiu; Luo, Kunxin; Shi, Yigong

2002-11-01

The Ski family of nuclear oncoproteins represses TGF-beta signaling through interactions with the Smad proteins. The crystal structure of the Smad4 binding domain of human c-Ski in complex with the MH2 domain of Smad4 reveals specific recognition of the Smad4 L3 loop region by a highly conserved interaction loop (I loop) from Ski. The Ski binding surface on Smad4 significantly overlaps with that required for binding of the R-Smads. Indeed, Ski disrupts the formation of a functional complex between the Co- and R-Smads, explaining how it could lead to repression of TGF-beta, activin, and BMP responses. Intriguingly, the structure of the Ski fragment, stabilized by a bound zinc atom, resembles the SAND domain, in which the corresponding I loop is responsible for DNA binding.

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.

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

Mining protein loops using a structural alphabet and statistical exceptionality

PubMed Central

2010-01-01

Background Protein loops encompass 50% of protein residues in available three-dimensional structures. These regions are often involved in protein functions, e.g. binding site, catalytic pocket... However, the description of protein loops with conventional tools is an uneasy task. Regular secondary structures, helices and strands, have been widely studied whereas loops, because they are highly variable in terms of sequence and structure, are difficult to analyze. Due to data sparsity, long loops have rarely been systematically studied. Results We developed a simple and accurate method that allows the description and analysis of the structures of short and long loops using structural motifs without restriction on loop length. This method is based on the structural alphabet HMM-SA. HMM-SA allows the simplification of a three-dimensional protein structure into a one-dimensional string of states, where each state is a four-residue prototype fragment, called structural letter. The difficult task of the structural grouping of huge data sets is thus easily accomplished by handling structural letter strings as in conventional protein sequence analysis. We systematically extracted all seven-residue fragments in a bank of 93000 protein loops and grouped them according to the structural-letter sequence, named structural word. This approach permits a systematic analysis of loops of all sizes since we consider the structural motifs of seven residues rather than complete loops. We focused the analysis on highly recurrent words of loops (observed more than 30 times). Our study reveals that 73% of loop-lengths are covered by only 3310 highly recurrent structural words out of 28274 observed words). These structural words have low structural variability (mean RMSd of 0.85 Å). As expected, half of these motifs display a flanking-region preference but interestingly, two thirds are shared by short (less than 12 residues) and long loops. Moreover, half of recurrent motifs exhibit a significant level of amino-acid conservation with at least four significant positions and 87% of long loops contain at least one such word. We complement our analysis with the detection of statistically over-represented patterns of structural letters as in conventional DNA sequence analysis. About 30% (930) of structural words are over-represented, and cover about 40% of loop lengths. Interestingly, these words exhibit lower structural variability and higher sequential specificity, suggesting structural or functional constraints. Conclusions We developed a method to systematically decompose and study protein loops using recurrent structural motifs. This method is based on the structural alphabet HMM-SA and not on structural alignment and geometrical parameters. We extracted meaningful structural motifs that are found in both short and long loops. To our knowledge, it is the first time that pattern mining helps to increase the signal-to-noise ratio in protein loops. This finding helps to better describe protein loops and might permit to decrease the complexity of long-loop analysis. Detailed results are available at http://www.mti.univ-paris-diderot.fr/publication/supplementary/2009/ACCLoop/. PMID:20132552

Mining protein loops using a structural alphabet and statistical exceptionality.

PubMed

Regad, Leslie; Martin, Juliette; Nuel, Gregory; Camproux, Anne-Claude

2010-02-04

Protein loops encompass 50% of protein residues in available three-dimensional structures. These regions are often involved in protein functions, e.g. binding site, catalytic pocket... However, the description of protein loops with conventional tools is an uneasy task. Regular secondary structures, helices and strands, have been widely studied whereas loops, because they are highly variable in terms of sequence and structure, are difficult to analyze. Due to data sparsity, long loops have rarely been systematically studied. We developed a simple and accurate method that allows the description and analysis of the structures of short and long loops using structural motifs without restriction on loop length. This method is based on the structural alphabet HMM-SA. HMM-SA allows the simplification of a three-dimensional protein structure into a one-dimensional string of states, where each state is a four-residue prototype fragment, called structural letter. The difficult task of the structural grouping of huge data sets is thus easily accomplished by handling structural letter strings as in conventional protein sequence analysis. We systematically extracted all seven-residue fragments in a bank of 93000 protein loops and grouped them according to the structural-letter sequence, named structural word. This approach permits a systematic analysis of loops of all sizes since we consider the structural motifs of seven residues rather than complete loops. We focused the analysis on highly recurrent words of loops (observed more than 30 times). Our study reveals that 73% of loop-lengths are covered by only 3310 highly recurrent structural words out of 28274 observed words). These structural words have low structural variability (mean RMSd of 0.85 A). As expected, half of these motifs display a flanking-region preference but interestingly, two thirds are shared by short (less than 12 residues) and long loops. Moreover, half of recurrent motifs exhibit a significant level of amino-acid conservation with at least four significant positions and 87% of long loops contain at least one such word. We complement our analysis with the detection of statistically over-represented patterns of structural letters as in conventional DNA sequence analysis. About 30% (930) of structural words are over-represented, and cover about 40% of loop lengths. Interestingly, these words exhibit lower structural variability and higher sequential specificity, suggesting structural or functional constraints. We developed a method to systematically decompose and study protein loops using recurrent structural motifs. This method is based on the structural alphabet HMM-SA and not on structural alignment and geometrical parameters. We extracted meaningful structural motifs that are found in both short and long loops. To our knowledge, it is the first time that pattern mining helps to increase the signal-to-noise ratio in protein loops. This finding helps to better describe protein loops and might permit to decrease the complexity of long-loop analysis. Detailed results are available at http://www.mti.univ-paris-diderot.fr/publication/supplementary/2009/ACCLoop/.

Mutations in yeast proliferating cell nuclear antigen define distinct sites for interaction with DNA polymerase delta and DNA polymerase epsilon.

PubMed Central

Eissenberg, J C; Ayyagari, R; Gomes, X V; Burgers, P M

1997-01-01

The importance of the interdomain connector loop and of the carboxy-terminal domain of Saccharomyces cerevisiae proliferating cell nuclear antigen (PCNA) for functional interaction with DNA polymerases delta (Poldelta) and epsilon (Pol epsilon) was investigated by site-directed mutagenesis. Two alleles, pol30-79 (IL126,128AA) in the interdomain connector loop and pol30-90 (PK252,253AA) near the carboxy terminus, caused growth defects and elevated sensitivity to DNA-damaging agents. These two mutants also had elevated rates of spontaneous mutations. The mutator phenotype of pol30-90 was due to partially defective mismatch repair in the mutant. In vitro, the mutant PCNAs showed defects in DNA synthesis. Interestingly, the pol30-79 mutant PCNA (pcna-79) was most defective in replication with Poldelta, whereas pcna-90 was defective in replication with Pol epsilon. Protein-protein interaction studies showed that pcna-79 and pcna-90 failed to interact with Pol delta and Pol epsilon, respectively. In addition, pcna-90 was defective in interaction with the FEN-1 endo-exonuclease (RTH1 product). A loss of interaction between pcna-79 and the smallest subunit of Poldelta, the POL32 gene product, implicates this interaction in the observed defect with the polymerase. Neither PCNA mutant showed a defect in the interaction with replication factor C or in loading by this complex. Processivity of DNA synthesis by the mutant holoenzyme containing pcna-79 was unaffected on poly(dA) x oligo(dT) but was dramatically reduced on a natural template with secondary structure. A stem-loop structure with a 20-bp stem formed a virtually complete block for the holoenzyme containing pcna-79 but posed only a minor pause site for wild-type holoenzyme, indicating a function of the POL32 gene product in allowing replication past structural blocks. PMID:9343398

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 determination of uracil-DNA N-glycosylase from Deinococcus radiodurans in complex with DNA.

PubMed

Pedersen, Hege Lynum; Johnson, Kenneth A; McVey, Colin E; Leiros, Ingar; Moe, Elin

2015-10-01

Uracil-DNA N-glycosylase (UNG) is a DNA-repair enzyme in the base-excision repair (BER) pathway which removes uracil from DNA. Here, the crystal structure of UNG from the extremophilic bacterium Deinococcus radiodurans (DrUNG) in complex with DNA is reported at a resolution of 1.35 Å. Prior to the crystallization experiments, the affinity between DrUNG and different DNA oligonucleotides was tested by electrophoretic mobility shift assays (EMSAs). As a result of this analysis, two 16 nt double-stranded DNAs were chosen for the co-crystallization experiments, one of which (16 nt AU) resulted in well diffracting crystals. The DNA in the co-crystal structure contained an abasic site (substrate product) flipped into the active site of the enzyme, with no uracil in the active-site pocket. Despite the high resolution, it was not possible to fit all of the terminal nucleotides of the DNA complex into electron density owing to disorder caused by a lack of stabilizing interactions. However, the DNA which was in contact with the enzyme, close to the active site, was well ordered and allowed detailed analysis of the enzyme-DNA interaction. The complex revealed that the interaction between DrUNG and DNA is similar to that in the previously determined crystal structure of human UNG (hUNG) in complex with DNA [Slupphaug et al. (1996). Nature (London), 384, 87-92]. Substitutions in a (here defined) variable part of the leucine loop result in a shorter loop (eight residues instead of nine) in DrUNG compared with hUNG; regardless of this, it seems to fulfil its role and generate a stabilizing force with the minor groove upon flipping out of the damaged base into the active site. The structure also provides a rationale for the previously observed high catalytic efficiency of DrUNG caused by high substrate affinity by demonstrating an increased number of long-range electrostatic interactions between the enzyme and the DNA. Interestingly, specific interactions between residues in the N-terminus of a symmetry-related molecule and the complementary DNA strand facing away from the active site were also observed which seem to stabilize the enzyme-DNA complex. However, the significance of this observation remains to be investigated. The results provide new insights into the current knowledge about DNA damage recognition and repair by uracil-DNA glycosylases.

Complete Sequence of the mitochondrial genome of the tapeworm Hymenolepis diminuta: Gene arrangements indicate that platyhelminths are eutrochozoans

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

von Nickisch-Rosenegk, Markus; Brown, Wesley M.; Boore, Jeffrey L.

2001-01-01

Using ''long-PCR'' we have amplified in overlapping fragments the complete mitochondrial genome of the tapeworm Hymenolepis diminuta (Platyhelminthes: Cestoda) and determined its 13,900 nucleotide sequence. The gene content is the same as that typically found for animal mitochondrial DNA (mtDNA) except that atp8 appears to be lacking, a condition found previously for several other animals. Despite the small size of this mtDNA, there are two large non-coding regions, one of which contains 13 repeats of a 31 nucleotide sequence and a potential stem-loop structure of 25 base pairs with an 11-member loop. Large potential secondary structures are identified also formore » the non-coding regions of two other cestode mtDNAs. Comparison of the mitochondrial gene arrangement of H. diminuta with those previously published supports a phylogenetic position of flatworms as members of the Eutrochozoa, rather than being basal to either a clade of protostomes or a clade of coelomates.« less

Binding of DNA hairpins to an assembler-strand as part of a primordial translation device

NASA Astrophysics Data System (ADS)

Baumann, Ulrich

1987-09-01

A crucial event in the process leading to the origin of life is the emergence of a simple translation device. To approach experimental realization of this device the binding ability of short DNA hairpins to complementary oligonucleotides fixed on a solid support was investigated. The binding is achieved by base pairing between the loop nucleotides of the hairpins containing different numbers of adenosine residues and oligothymidylates covalently linked to cellulose. The loop has to consist of at least five nucleotides to achieve binding. The exact number of established base pairs was determined in two ways. First, the elution temperatures of hairpins and those of oligoadenylates which had the length of the loop were compared. Secondly, the architecture of the loop was analyzed by means of the single-strand-specific nuclease from mung bean acting as structural probe. Onlyn-2 of n loop nucleotides of a hairpin are able to form base pairs. Therefore, a strong evidence for the formation of a triplet of base pairs between primeval tRNA and mRNA sufficient to stabilize the complex enzyme-free is given.

Structural features of the DNA hairpin d(ATCCTA-GTTA-TAGGAT): formation of a G-A base pair in the loop.

PubMed Central

van Dongen, M J; Mooren, M M; Willems, E F; van der Marel, G A; van Boom, J H; Wijmenga, S S; Hilbers, C W

1997-01-01

The three-dimensional structure of the hairpin formed by d(ATCCTA-GTTA-TAGGAT) has been determined by means of two-dimensional NMR studies, distance geometry and molecular dynamics calculations. The first and the last residues of the tetraloop of this hairpin form a sheared G-A base pair on top of the six Watson-Crick base pairs in the stem. The glycosidic torsion angles of the guanine and adenine residues in the G-A base pair reside in the anti and high- anti domain ( approximately -60 degrees ) respectively. Several dihedral angles in the loop adopt non-standard values to accommodate this base pair. The first and second residue in the loop are stacked in a more or less normal helical fashion; the fourth loop residue also stacks upon the stem, while the third residue is directed away from the loop region. The loop structure can be classified as a so-called type-I loop, in which the bases at the 5'-end of the loop stack in a continuous fashion. In this situation, loop stability is unlikely to depend heavily on the nature of the unpaired bases in the loop. Moreover, the present study indicates that the influence of the polarity of a closing A.T pair is much less significant than that of a closing C.G base pair. PMID:9092659

Stable loop in the crystal structure of the intercalated four-stranded cytosine-rich metazoan telomere

NASA Technical Reports Server (NTRS)

Kang, C.; Berger, I.; Lockshin, C.; Ratliff, R.; Moyzis, R.; Rich, A.

1995-01-01

In most metazoans, the telomeric cytosine-rich strand repeating sequence is d(TAACCC). The crystal structure of this sequence was solved to 1.9-A resolution. Four strands associate via the cytosine-containing parts to form a four-stranded intercalated structure held together by C.C+ hydrogen bonds. The base-paired strands are parallel to each other, and the two duplexes are intercalated into each other in opposite orientations. One TAA end forms a highly stabilized loop with the 5' thymine Hoogsteen-base-paired to the third adenine. The 5' end of this loop is in close proximity to the 3' end of one of the other intercalated cytosine strands. Instead of being entirely in a DNA duplex, this structure suggests the possibility of an alternative conformation for the cytosine-rich telomere strands.

Hydration Changes upon DNA Folding Studied by Osmotic Stress Experiments

PubMed Central

Nakano, Shu-ichi; Yamaguchi, Daisuke; Tateishi-Karimata, Hisae; Miyoshi, Daisuke; Sugimoto, Naoki

2012-01-01

The thermal stability of nucleic acid structures is perturbed under the conditions that mimic the intracellular environment, typically rich in inert components and under osmotic stress. We now describe the thermodynamic stability of DNA oligonucleotide structures in the presence of high background concentrations of neutral cosolutes. Small cosolutes destabilize the basepair structures, and the DNA structures consisting of the same nearest-neighbor composition show similar thermodynamic parameters in the presence of various types of cosolutes. The osmotic stress experiments reveal that water binding to flexible loops, unstable mismatches, and an abasic site upon DNA folding are almost negligible, whereas the binding to stable mismatch pairs is significant. The studies using the basepair-mimic nucleosides and the peptide nucleic acid suggest that the sugar-phosphate backbone and the integrity of the basepair conformation make important contributions to the binding of water molecules to the DNA bases and helical grooves. The study of the DNA hydration provides the basis for understanding and predicting nucleic acid structures in nonaqueous solvent systems. PMID:22735531

Quadruplexes in 'Dicty': crystal structure of a four-quartet G-quadruplex formed by G-rich motif found in the Dictyostelium discoideum genome.

PubMed

Guédin, Aurore; Lin, Linda Yingqi; Armane, Samir; Lacroix, Laurent; Mergny, Jean-Louis; Thore, Stéphane; Yatsunyk, Liliya A

2018-06-01

Guanine-rich DNA has the potential to fold into non-canonical G-quadruplex (G4) structures. Analysis of the genome of the social amoeba Dictyostelium discoideum indicates a low number of sequences with G4-forming potential (249-1055). Therefore, D. discoideum is a perfect model organism to investigate the relationship between the presence of G4s and their biological functions. As a first step in this investigation, we crystallized the dGGGGGAGGGGTACAGGGGTACAGGGG sequence from the putative promoter region of two divergent genes in D. discoideum. According to the crystal structure, this sequence folds into a four-quartet intramolecular antiparallel G4 with two lateral and one diagonal loops. The G-quadruplex core is further stabilized by a G-C Watson-Crick base pair and a A-T-A triad and displays high thermal stability (Tm > 90°C at 100 mM KCl). Biophysical characterization of the native sequence and loop mutants suggests that the DNA adopts the same structure in solution and in crystalline form, and that loop interactions are important for the G4 stability but not for its folding. Four-tetrad G4 structures are sparse. Thus, our work advances understanding of the structural diversity of G-quadruplexes and yields coordinates for in silico drug screening programs and G4 predictive tools.

A conserved loop-wedge motif moderates reaction site search and recognition by FEN1.

PubMed

Thompson, Mark J; Gotham, Victoria J B; Ciani, Barbara; Grasby, Jane A

2018-06-07

DNA replication and repair frequently involve intermediate two-way junction structures with overhangs, or flaps, that must be promptly removed; a task performed by the essential enzyme flap endonuclease 1 (FEN1). We demonstrate a functional relationship between two intrinsically disordered regions of the FEN1 protein, which recognize opposing sides of the junction and order in response to the requisite substrate. Our results inform a model in which short-range translocation of FEN1 on DNA facilitates search for the annealed 3'-terminus of a primer strand, which is recognized by breaking the terminal base pair to generate a substrate with a single nucleotide 3'-flap. This recognition event allosterically signals hydrolytic removal of the 5'-flap through reaction in the opposing junction duplex, by controlling access of the scissile phosphate diester to the active site. The recognition process relies on a highly-conserved 'wedge' residue located on a mobile loop that orders to bind the newly-unpaired base. The unanticipated 'loop-wedge' mechanism exerts control over substrate selection, rate of reaction and reaction site precision, and shares features with other enzymes that recognize irregular DNA structures. These new findings reveal how FEN1 precisely couples 3'-flap verification to function.

A cyclometalated iridium(III) complex used as a conductor for the electrochemical sensing of IFN-γ

NASA Astrophysics Data System (ADS)

Miao, Xiangmin; Ko, Chung-Nga; Vellaisamy, Kasipandi; Li, Zongbing; Yang, Guanjun; Leung, Chung-Hang; Ma, Dik-Lung

2017-02-01

A novel iridium(III) complex was prepared and used as a conductor for sensitive and enzyme-free electrochemical detection of interferon gamma (IFN-γ). This assay is based on a dual signal amplification mechanism involving positively charged gold nanoparticles ((+)AuNPs) and hybridization chain reaction (HCR). To construct the sensor, nafion (Nf) and (+)AuNPs composite membrane was first immobilized onto the electrode surface. Subsequently, a loop-stem structured capture probe (CP) containing a special IFN-γ interact strand was modified onto the (+)AuNP surface via the formation of Au-S bonds. Upon addition of IFN-γ, the loop-stem structure of CP was opened, and the newly exposed “sticky” region of CP then hybridized with DNA hairpin-1 (H1), which in turn opened its hairpin structure for hybridizing with DNA hairpin-2 (H2). Happen of HCR between H1 and H2 thus generated a polymeric duplex DNA (dsDNA) chain. Meanwhile, the iridium(III) complex could interact with the grooves of the dsDNA polymer, producing a strong current signal that was proportional to IFN-γ concentration. Thus, sensitive detection of IFN-γ could be realized with a detection limit down to 16.3 fM. Moreover, satisfied results were achieved by using this method for the detection of IFN-γ in human serum samples.

Structural and thermodynamic analysis of modified nucleosides in self-assembled DNA cross-tiles.

PubMed

Hakker, Lauren; Marchi, Alexandria N; Harris, Kimberly A; LaBean, Thomas H; Agris, Paul F

2014-01-01

DNA Holliday junctions are important natural strand-exchange structures that form during homologous recombination. Immobile four-arm junctions, analogs to Holliday junctions, have been designed to self-assemble into cross-tile structures by maximizing Watson-Crick base pairing and fixed crossover points. The cross-tiles, self-assembled from base pair recognition between designed single-stranded DNAs, form higher order lattice structures through cohesion of self-associating sticky ends. These cross-tiles have 16 unpaired nucleosides in the central loop at the junction of the four duplex stems. The importance of the centralized unpaired nucleosides to the structure's thermodynamic stability and self-assembly is unknown. Cross-tile DNA nanostructures were designed and constructed from nine single-stranded DNAs with four shell strands, four arms, and a central loop containing 16 unpaired bases. The 16 unpaired bases were either 2'-deoxyribothymidines, 2'-O-methylribouridines, or abasic 1',2'-dideoxyribonucleosides. Thermodynamic profiles and structural base-stacking contributions were assessed using UV absorption spectroscopy during thermal denaturation and circular dichroism spectroscopy, respectively, and the resulting structures were observed by atomic force microscopy. There were surprisingly significant changes in the thermodynamic and structural properties of lattice formation as a result of altering only the 16 unpaired, centralized nucleosides. The 16 unpaired 2'-O-methyluridines were stabilizing and produced uniform tubular structures. In contrast, the abasic nucleosides were destabilizing producing a mixture of structures. These results strongly indicate the importance of a small number of centrally located unpaired nucleosides within the structures. Since minor modifications lead to palpable changes in lattice formation, DNA cross-tiles present an easily manipulated structure convenient for applications in biomedical and biosensing devices.

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

NASA Technical Reports Server (NTRS)

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

Cell-cycle dynamics of chromosomal organisation at single-cell resolution

PubMed Central

Nagano, Takashi; Lubling, Yaniv; Várnai, Csilla; Dudley, Carmel; Leung, Wing; Baran, Yael; Mendelson-Cohen, Netta; Wingett, Steven; Fraser, Peter; Tanay, Amos

2017-01-01

Summary Chromosomes in proliferating metazoan cells undergo dramatic structural metamorphoses every cell cycle, alternating between highly condensed mitotic structures facilitating chromosome segregation, and decondensed interphase structures accommodating transcription, gene silencing and DNA replication. Here we use single-cell Hi-C to study chromosome conformations in thousands of individual cells, and discover a continuum of cis-interaction profiles that finely position individual cells along the cell cycle. We show that chromosomal compartments, topological associated domains (TADs), contact insulation and long-range loops, all defined by bulk Hi-C maps, are governed by distinct cell-cycle dynamics. In particular, DNA replication correlates with build-up of compartments and reduction in TAD insulation, while loops are generally stable from G1 through S and G2. Whole-genome 3D structural models reveal a radial architecture of chromosomal compartments with distinct epigenomic signatures. Our single-cell data thereby allow for re-interpretation of chromosome conformation maps through the prism of the cell cycle. PMID:28682332

Top3-Rmi1 dissolve Rad51-mediated D-loops by a topoisomerase-based mechanism

PubMed Central

Fasching, Clare L.; Cejka, Petr; Kowalczykowski, Stephen C.; Heyer, Wolf-Dietrich

2015-01-01

Summary The displacement loop (D-loop) is the DNA strand invasion product formed during homologous recombination. Disruption of nascent D-loops represents a mechanism of anti-recombination. During Synthesis-Dependent Strand Annealing D-loop disruption after extension of the invading strand is an integral step of the pathway and ensures a non-crossover outcome. The proteins implicated in D-loop disruption are DNA motor proteins/helicases acting by migrating DNA junctions. Here we report an unanticipated mechanism of D-loop dissolution mediated by DNA topoisomerase 3 (Top3) and dependent on its catalytic activity. D-loop dissolution catalyzed by yeast Top3 is highly specific for yeast Rad51/Rad54-mediated D-loops, whereas protein-free D-loops or D-loop mediated by bacterial RecA protein or human RAD51/RAD54 resist dissolution. Also the human Topoisomerase IIIα-RMI1–RMI2 complex is capable of dissolving D-loops. Consistent with genetic data, we suggest that the extreme growth defect and hyper-recombination phenotype of Top3-deficient yeast cells is in part a result of unprocessed D-loops. PMID:25699708

Molecular dynamics simulations of electrostatics and hydration distributions around RNA and DNA motifs

NASA Astrophysics Data System (ADS)

Marlowe, Ashley E.; Singh, Abhishek; Semichaevsky, Andrey V.; Yingling, Yaroslava G.

2009-03-01

Nucleic acid nanoparticles can self-assembly through the formation of complementary loop-loop interactions or stem-stem interactions. Presence and concentration of ions can significantly affect the self-assembly process and the stability of the nanostructure. In this presentation we use explicit molecular dynamics simulations to examine the variations in cationic distributions and hydration environment around DNA and RNA helices and loop-loop interactions. Our simulations show that the potassium and sodium ionic distributions are different around RNA and DNA motifs which could be indicative of ion mediated relative stability of loop-loop complexes. Moreover in RNA loop-loop motifs ions are consistently present and exchanged through a distinct electronegative channel. We will also show how we used the specific RNA loop-loop motif to design a RNA hexagonal nanoparticle.

Detection of HbsAg and hATIII genetically modified goats (Caprahircus) by loop-mediated isothermal amplification.

PubMed

Tao, Chenyu; Zhang, Qingde; Zhai, Shanli; Liu, Bang

2013-11-01

In this study, sensitive and rapid detection systems were designed using a loop-mediated isothermal amplification (LAMP) method to detect the genetically modified goats. A set of 4 primers were designed for each exogenous nucleic acids HBsAg and hATIII. The DNA samples were first amplified with the outer and inner primers and released a single-stranded DNA,of which both ends were stem-loop structure. Then one inner primer hybridized with the loop, and initiated displacement synthesis in less than 1 h. The result could be visualized by both agarose gel electrophoresis and unaided eyes directly after adding SYBR GREEN 1. The detection limit of LAMP was ten copies of target molecules, indicating that LAMP was tenfold more sensitive than the classical PCR. Furthermore, all the samples of genetically modified goats were tested positively by LAMP, and the results demonstrated that the LAMP was a rapid and sensitive method for detecting the genetically modified organism.

A double chain reversal loop and two diagonal loops define the architecture of a unimolecular DNA quadruplex containing a pair of stacked G(syn)-G(syn)-G(anti)-G(anti) tetrads flanked by a G-(T-T) Triad and a T-T-T triple.

PubMed

Kuryavyi, V; Majumdar, A; Shallop, A; Chernichenko, N; Skripkin, E; Jones, R; Patel, D J

2001-06-29

The architecture of G-G-G-G tetrad-aligned DNA quadruplexes in monovalent cation solution is dependent on the directionality of the four strands, which in turn are defined by loop connectivities and the guanine syn/anti distribution along individual strands and within individual G-G-G-G tetrads. The smallest unimolecular G-quadruplex belongs to the d(G2NnG2NnG2NnG2) family, which has the potential to form two stacked G-tetrads linked by Nn loop connectivities. Previous studies have focused on the thrombin-binding DNA aptamer d(G2T2G2TGTG2T2G2), where Nn was T2 for the first and third connecting loops and TGT for the middle connecting loop. This DNA aptamer in K(+) cation solution forms a unimolecular G-quadruplex stabilized by two stacked G(syn)-G(anti)-G(syn)-G(anti) tetrads, adjacent strands which are antiparallel to each other and edge-wise connecting T2, TGT and T2 loops. We now report on the NMR-based solution structure of the d(G2T4G2CAG2GT4G2T) sequence, which differs from the thrombin-binding DNA aptamer sequence in having longer first (T4) and third (GT4) loops and a shorter (CA) middle loop. This d(G2T4G2CAG2GT4G2T) sequence in Na(+) cation solution forms a unimolecular G-quadruplex stabilized by two stacked G(syn)-G(syn)-G(anti)-G(anti) tetrads, adjacent strands which have one parallel and one antiparallel neighbors and distinct non-edge-wise loop connectivities. Specifically, the longer first (T4) and third (GT4) loops are of the diagonal type while the shorter middle loop is of the double chain reversal type. In addition, the pair of stacked G-G-G-G tetrads are flanked on one side by a G-(T-T) triad and on the other side by a T-T-T triple. The distinct differences in strand directionalities, loop connectivities and syn/anti distribution within G-G-G-G tetrads between the thrombin-binding DNA aptamer d(G2T2G2TGTG2T2G2) quadruplex reported previously, and the d(G2T4G2CAG2GT4G2T) quadruplex reported here, reinforces the polymorphic nature of higher-order DNA architectures. Further, these two small unimolecular G-quadruplexes, which are distinct from each other and from parallel-stranded G-quadruplexes, provide novel targets for ligand recognition. Our results demonstrate that the double chain reversal loop connectivity identified previously by our laboratory within the Tetrahymena telomere d(T2G4)4 quadruplex, is a robust folding topology, since it has now also been observed within the d(G2T4G2CAG2GT4G2T) quadruplex. The identification of a G-(T-T) triad and a T-T-T triple, expands on the available recognition alignments for base triads and triples. Copyright 2001 Academic Press.

Human Y-Family DNA Polymerase κ Is More Tolerant to Changes in Its Active Site Loop than Its Ortholog Escherichia coli DinB.

PubMed

Antczak, Nicole M; Packer, Morgan R; Lu, Xueguang; Zhang, Ke; Beuning, Penny J

2017-11-20

DNA damage is a constant threat and can be bypassed in a process called translesion synthesis, which is typically carried out by Y-family DNA polymerases. Y-family DNA polymerases are conserved in all domains of life and tend to have specificity for certain types of DNA damage. Escherichia coli DinB and its human ortholog pol κ can bypass specific minor groove deoxyguanine adducts efficiently and are inhibited by major groove adducts, as Y-family DNA polymerases make contacts with the minor groove side of the DNA substrate and lack contacts with the major groove at the nascent base pair. DinB is inhibited by major groove adducts more than pol κ, and they each have active site loops of different lengths, with four additional amino acids in the DinB loop. We previously showed that the R35A active site loop mutation in DinB allows for bypass of the major groove adduct N 6 -furfuryl-dA. These observations led us to investigate the different active site loops by creating loop swap chimeras of DinB with a pol κ loop and vice versa by changing the loop residues in a stepwise fashion. We then determined their activity with undamaged DNA or DNA containing N 2 -furfuryl-dG or N 6 -furfuryl-dA. The DinB proteins with the pol kappa loop have low activity on all templates but have decreased misincorporation compared to either wild-type protein. The kappa proteins with the DinB loop retain activity on all templates and have decreased misincorporation compared to either wild-type protein. We assessed the thermal stability of the proteins and observed an increase in stability in the presence of all DNA templates and additional increases generally only in the presence of the undamaged and N 2 -furfuryl-dG adduct and dCTP, which correlates with activity. Overall we find that pol κ is more tolerant to changes in the active site loop than DinB.

Thermodynamics-hydration relationships within loops that affect G-quadruplexes under molecular crowding conditions.

PubMed

Fujimoto, Takeshi; Nakano, Shu-ichi; Sugimoto, Naoki; Miyoshi, Daisuke

2013-01-31

We systematically investigated the effects of loop length on the conformation, thermodynamic stability, and hydration of DNA G-quadruplexes under dilute and molecular crowding conditions in the presence of Na(+). Structural analysis showed that molecular crowding induced conformational switches of oligonucleotides with the longer guanine stretch and the shorter thymine loop. Thermodynamic parameters further demonstrated that the thermodynamic stability of G-quadruplexes increased by increasing the loop length from two to four, whereas it decreased by increasing the loop length from four to six. Interestingly, we found by osmotic pressure analysis that the number of water molecules released from the G-quadruplex decreased with increasing thermodynamic stability. We assumed that base-stacking interactions within the loops not only stabilized the whole G-quadruplex structure but also created hydration sites by accumulating nucleotide functional groups. The molecular crowding effects on the stability of G-quadruplexes composed of abasic sites, which reduce the stacking interactions at the loops, further demonstrated that G-quadruplexes with fewer stacking interactions within the loops released a larger number of water molecules upon folding. These results showed that the stacking interactions within the loops determined the thermodynamic stability and hydration of the whole G-quadruplex.

Conformational trapping of mismatch recognition complex MSH2/MSH3 on repair-resistant DNA loops.

PubMed

Lang, Walter H; Coats, Julie E; Majka, Jerzy; Hura, Greg L; Lin, Yuyen; Rasnik, Ivan; McMurray, Cynthia T

2011-10-18

Insertion and deletion of small heteroduplex loops are common mutations in DNA, but why some loops are prone to mutation and others are efficiently repaired is unknown. Here we report that the mismatch recognition complex, MSH2/MSH3, discriminates between a repair-competent and a repair-resistant loop by sensing the conformational dynamics of their junctions. MSH2/MSH3 binds, bends, and dissociates from repair-competent loops to signal downstream repair. Repair-resistant Cytosine-Adenine-Guanine (CAG) loops adopt a unique DNA junction that traps nucleotide-bound MSH2/MSH3, and inhibits its dissociation from the DNA. We envision that junction dynamics is an active participant and a conformational regulator of repair signaling, and governs whether a loop is removed by MSH2/MSH3 or escapes to become a precursor for mutation.

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

Zahn, Karl E.; Averill, April M.; Aller, Pierre

DNA polymerase θ protects against genomic instability via an alternative end-joining repair pathway for DNA double-strand breaks. Polymerase θ is overexpressed in breast, lung and oral cancers, and reduction of its activity in mammalian cells increases sensitivity to double-strand break–inducing agents, including ionizing radiation. Reported in this paper are crystal structures of the C-terminal polymerase domain from human polymerase θ, illustrating two potential modes of dimerization. One structure depicts insertion of ddATP opposite an abasic-site analog during translesion DNA synthesis. The second structure describes a cognate ddGTP complex. Polymerase θ uses a specialized thumb subdomain to establish unique upstream contactsmore » to the primer DNA strand, including an interaction with the 3'-terminal phosphate from one of five distinctive insertion loops. Finally, these observations demonstrate how polymerase θ grasps the primer to bypass DNA lesions or extend poorly annealed DNA termini to mediate end-joining.« less

The NMR solution structure of a mutant of the Max b/HLH/LZ free of DNA: insights into the specific and reversible DNA binding mechanism of dimeric transcription factors.

PubMed

Sauvé, Simon; Tremblay, Luc; Lavigne, Pierre

2004-09-17

Basic region-helix1-loop-helix2-leucine zipper (b/H(1)LH(2)/LZ) transcription factors bind specific DNA sequence in their target gene promoters as dimers. Max, a b/H(1)LH(2)/LZ transcription factor, is the obligate heterodimeric partner of the related b/H(1)LH(2)/LZ proteins of the Myc and Mad families. These heterodimers specifically bind E-box DNA sequence (CACGTG) to activate (e.g. c-Myc/Max) and repress (e.g. Mad1/Max) transcription. Max can also homodimerize and bind E-box sequences in c-Myc target gene promoters. While the X-ray structure of the Max b/H(1)LH(2)/LZ/DNA complex and that of others have been reported, the precise sequence of events leading to the reversible and specific binding of these important transcription factors is still largely unknown. In order to provide insights into the DNA binding mechanism, we have solved the NMR solution structure of a covalently homodimerized version of a Max b/H(1)LH(2)/LZ protein with two stabilizing mutations in the LZ, and characterized its backbone dynamics from (15)N spin-relaxation measurements in the absence of DNA. Apart from minor differences in the pitch of the LZ, possibly resulting from the mutations in the construct, we observe that the packing of the helices in the H(1)LH(2) domain is almost identical to that of the two crystal structures, indicating that no important conformational change in these helices occurs upon DNA binding. Conversely to the crystal structures of the DNA complexes, the first 14 residues of the basic region are found to be mostly unfolded while the loop is observed to be flexible. This indicates that these domains undergo conformational changes upon DNA binding. On the other hand, we find the last four residues of the basic region form a persistent helical turn contiguous to H(1). In addition, we provide evidence of the existence of internal motions in the backbone of H(1) that are of larger amplitude and longer time-scale (nanoseconds) than the ones in the H(2) and LZ domain. Most interestingly, we note that conformers in the ensemble of calculated structures have highly conserved basic residues (located in the persistent helical turn of the basic region and in the loop) known to be important for specific binding in a conformation that matches that of the DNA-bound state. These partially prefolded conformers can directly fit into the major groove of DNA and as such are proposed to lie on the pathway leading to the reversible and specific DNA binding. In these conformers, the conserved basic side-chains form a cluster that elevates the local electrostatic potential and could provide the necessary driving force for the generation of the internal motions localized in the H(1) and therefore link structural determinants with the DNA binding function. Overall, our results suggests that the Max homodimeric b/H(1)LH(2)/LZ can rapidly and preferentially bind DNA sequence through transient and partially prefolded states and subsequently, adopt the fully helical bound state in a DNA-assisted mechanism or induced-fit.

The Arabidopsis thaliana homolog of the helicase RTEL1 plays multiple roles in preserving genome stability.

PubMed

Recker, Julia; Knoll, Alexander; Puchta, Holger

2014-12-01

In humans, mutations in the DNA helicase Regulator of Telomere Elongation Helicase1 (RTEL1) lead to Hoyeraal-Hreidarsson syndrome, a severe, multisystem disorder. Here, we demonstrate that the RTEL1 homolog in Arabidopsis thaliana plays multiple roles in preserving genome stability. RTEL1 suppresses homologous recombination in a pathway parallel to that of the DNA translocase FANCM. Cytological analyses of root meristems indicate that RTEL1 is involved in processing DNA replication intermediates independently from FANCM and the nuclease MUS81. Moreover, RTEL1 is involved in interstrand and intrastrand DNA cross-link repair independently from FANCM and (in intrastrand cross-link repair) parallel to MUS81. RTEL1 contributes to telomere homeostasis; the concurrent loss of RTEL1 and the telomerase TERT leads to rapid, severe telomere shortening, which occurs much more rapidly than it does in the single-mutant line tert, resulting in developmental arrest after four generations. The double mutant rtel1-1 recq4A-4 exhibits massive growth defects, indicating that this RecQ family helicase, which is also involved in the suppression of homologous recombination and the repair of DNA lesions, can partially replace RTEL1 in the processing of DNA intermediates. The requirement for RTEL1 in multiple pathways to preserve genome stability in plants can be explained by its putative role in the destabilization of DNA loop structures, such as D-loops and T-loops. © 2014 American Society of Plant Biologists. All rights reserved.

The Arabidopsis thaliana Homolog of the Helicase RTEL1 Plays Multiple Roles in Preserving Genome Stability[C][W

PubMed Central

Recker, Julia; Knoll, Alexander; Puchta, Holger

2014-01-01

In humans, mutations in the DNA helicase Regulator of Telomere Elongation Helicase1 (RTEL1) lead to Hoyeraal-Hreidarsson syndrome, a severe, multisystem disorder. Here, we demonstrate that the RTEL1 homolog in Arabidopsis thaliana plays multiple roles in preserving genome stability. RTEL1 suppresses homologous recombination in a pathway parallel to that of the DNA translocase FANCM. Cytological analyses of root meristems indicate that RTEL1 is involved in processing DNA replication intermediates independently from FANCM and the nuclease MUS81. Moreover, RTEL1 is involved in interstrand and intrastrand DNA cross-link repair independently from FANCM and (in intrastrand cross-link repair) parallel to MUS81. RTEL1 contributes to telomere homeostasis; the concurrent loss of RTEL1 and the telomerase TERT leads to rapid, severe telomere shortening, which occurs much more rapidly than it does in the single-mutant line tert, resulting in developmental arrest after four generations. The double mutant rtel1-1 recq4A-4 exhibits massive growth defects, indicating that this RecQ family helicase, which is also involved in the suppression of homologous recombination and the repair of DNA lesions, can partially replace RTEL1 in the processing of DNA intermediates. The requirement for RTEL1 in multiple pathways to preserve genome stability in plants can be explained by its putative role in the destabilization of DNA loop structures, such as D-loops and T-loops. PMID:25516598

Crystal structures of thrombin in complex with chemically modified thrombin DNA aptamers reveal the origins of enhanced affinity.

PubMed

Dolot, Rafal; Lam, Curtis H; Sierant, Malgorzata; Zhao, Qiang; Liu, Feng-Wu; Nawrot, Barbara; Egli, Martin; Yang, Xianbin

2018-05-18

Thrombin-binding aptamer (TBA) is a DNA 15-mer of sequence 5'-GGT TGG TGT GGT TGG-3' that folds into a G-quadruplex structure linked by two T-T loops located on one side and a T-G-T loop on the other. These loops are critical for post-SELEX modification to improve TBA target affinity. With this goal in mind we synthesized a T analog, 5-(indolyl-3-acetyl-3-amino-1-propenyl)-2'-deoxyuridine (W) to substitute one T or a pair of Ts. Subsequently, the affinity for each analog was determined by biolayer interferometry. An aptamer with W at position 4 exhibited about 3-fold increased binding affinity, and replacing both T4 and T12 with W afforded an almost 10-fold enhancement compared to native TBA. To better understand the role of the substituent's aromatic moiety, an aptamer with 5-(methyl-3-acetyl-3-amino-1-propenyl)-2'-deoxyuridine (K; W without the indole moiety) in place of T4 was also synthesized. This K4 aptamer was found to improve affinity 7-fold relative to native TBA. Crystal structures of aptamers with T4 replaced by either W or K bound to thrombin provide insight into the origins of the increased affinities. Our work demonstrates that facile chemical modification of a simple DNA aptamer can be used to significantly improve its binding affinity for a well-established pharmacological target protein.

Increased immunostimulatory activity of polypod-like structured DNA by ligation of the terminal loop structures.

PubMed

Mohri, Kohta; Takahashi, Natsuki; Nishikawa, Makiya; Kusuki, Eri; Shiomi, Tomoki; Takahashi, Yuki; Takakura, Yoshinobu

2012-11-10

The immunostimulatory activity of phosphodiester DNA containing unmethylated cytosine-guanine (CpG) dinucleotides can be increased by converting it into branched structures. These structures could be stabilized by ligating the 5'- and 3'-ends to form a closed loop with no terminal ends. To further increase the ability of branched DNA assemblies to induce cytokines, a series of tetrapod-like structured DNA, or tetrapodna, were designed using four 48-base oligodeoxynucleotides (ODNs). All these preparations were designed to have the same sequence except for the nick sites, and all the ODNs of one of the tetrapodna preparations were ligated to obtain circular tetrapodna. The nick site significantly influenced the formation of the structure and melting temperature (Tm), but hardly affected the enzymatic stability of the tetrapodna preparations. Circular tetrapodna exhibited a significantly higher Tm and was more stable in mouse serum than its non-ligated counterparts. The amounts of cytokines released from macrophage-like RAW264.7 cells or dendritic DC2.4 cells after addition of circular tetrapodna were not significantly higher than those after addition of other tetrapodna preparations under conditions when no serum was present. However, when serum was present, circular tetrapodna induced the greatest amount of tumor necrosis factor-α, indicating that circular tetrapodna is effective in inducing cytokines under conditions where DNA-degrading enzymes are present. The cellular association of tetrapodna preparations was almost unaffected by ligation of the terminal ends. These results indicate that circular tetrapodna with no terminal ends is more effective than its non-ligated counterparts in the presence of serum. Copyright © 2012 Elsevier B.V. All rights reserved.

Human DNA polymerase θ grasps the primer terminus to mediate DNA repair

DOE PAGES

Zahn, Karl E.; Averill, April M.; Aller, Pierre; ...

2015-03-16

DNA polymerase θ protects against genomic instability via an alternative end-joining repair pathway for DNA double-strand breaks. Polymerase θ is overexpressed in breast, lung and oral cancers, and reduction of its activity in mammalian cells increases sensitivity to double-strand break–inducing agents, including ionizing radiation. Reported in this paper are crystal structures of the C-terminal polymerase domain from human polymerase θ, illustrating two potential modes of dimerization. One structure depicts insertion of ddATP opposite an abasic-site analog during translesion DNA synthesis. The second structure describes a cognate ddGTP complex. Polymerase θ uses a specialized thumb subdomain to establish unique upstream contactsmore » to the primer DNA strand, including an interaction with the 3'-terminal phosphate from one of five distinctive insertion loops. Finally, these observations demonstrate how polymerase θ grasps the primer to bypass DNA lesions or extend poorly annealed DNA termini to mediate end-joining.« less

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

DNA Looping Facilitates Targeting of a Chromatin Remodeling Enzyme

PubMed Central

Yadon, Adam N; Singh, Badri Nath; Hampsey, Michael; Tsukiyama, Toshio

2013-01-01

Summary ATP-dependent chromatin remodeling enzymes are highly abundant and play pivotal roles regulating DNA-dependent processes. The mechanisms by which they are targeted to specific loci have not been well understood on a genome-wide scale. Here we present evidence that a major targeting mechanism for the Isw2 chromatin remodeling enzyme to specific genomic loci is through sequence-specific transcription factor (TF)-dependent recruitment. Unexpectedly, Isw2 is recruited in a TF-dependent fashion to a large number of loci without TF binding sites. Using the 3C assay, we show that Isw2 can be targeted by Ume6- and TFIIB-dependent DNA looping. These results identify DNA looping as a previously unknown mechanism for the recruitment of a chromatin remodeling enzyme and defines a novel function for DNA looping. We also present evidence suggesting that Ume6-dependent DNA looping is involved in chromatin remodeling and transcriptional repression, revealing a mechanism by which the three-dimensional folding of chromatin affects DNA-dependent processes. PMID:23478442

Structure and mechanism of human DNA polymerase [eta

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

Biertümpfel, Christian; Zhao, Ye; Kondo, Yuji

2010-11-03

The variant form of the human syndrome xeroderma pigmentosum (XPV) is caused by a deficiency in DNA polymerase {eta} (Pol{eta}), a DNA polymerase that enables replication through ultraviolet-induced pyrimidine dimers. Here we report high-resolution crystal structures of human Pol{eta} at four consecutive steps during DNA synthesis through cis-syn cyclobutane thymine dimers. Pol{eta} acts like a 'molecular splint' to stabilize damaged DNA in a normal B-form conformation. An enlarged active site accommodates the thymine dimer with excellent stereochemistry for two-metal ion catalysis. Two residues conserved among Pol{eta} orthologues form specific hydrogen bonds with the lesion and the incoming nucleotide to assistmore » translesion synthesis. On the basis of the structures, eight Pol{eta} missense mutations causing XPV can be rationalized as undermining the molecular splint or perturbing the active-site alignment. The structures also provide an insight into the role of Pol{eta} in replicating through D loop and DNA fragile sites.« less

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

RNA Helicase DDX1 Converts RNA G-Quadruplex Structures into R-Loops to Promote IgH Class Switch Recombination.

PubMed

Ribeiro de Almeida, Claudia; Dhir, Somdutta; Dhir, Ashish; Moghaddam, Amin E; Sattentau, Quentin; Meinhart, Anton; Proudfoot, Nicholas J

2018-05-17

Class switch recombination (CSR) at the immunoglobulin heavy-chain (IgH) locus is associated with the formation of R-loop structures over switch (S) regions. While these often occur co-transcriptionally between nascent RNA and template DNA, we now show that they also form as part of a post-transcriptional mechanism targeting AID to IgH S-regions. This depends on the RNA helicase DDX1 that is also required for CSR in vivo. DDX1 binds to G-quadruplex (G4) structures present in intronic switch transcripts and converts them into S-region R-loops. This in turn targets the cytidine deaminase enzyme AID to S-regions so promoting CSR. Notably R-loop levels over S-regions are diminished by chemical stabilization of G4 RNA or by the expression of a DDX1 ATPase-deficient mutant that acts as a dominant-negative protein to reduce CSR efficiency. In effect, we provide evidence for how S-region transcripts interconvert between G4 and R-loop structures to promote CSR in the IgH locus. Copyright © 2018 The Author(s). Published by Elsevier Inc. All rights reserved.

Mechanism of mismatch recognition revealed by human MutS[beta] bound to unpaired DNA loops

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

Gupta, Shikha; Gellert, Martin; Yang, Wei

2012-04-17

DNA mismatch repair corrects replication errors, thus reducing mutation rates and microsatellite instability. Genetic defects in this pathway cause Lynch syndrome and various cancers in humans. Binding of a mispaired or unpaired base by bacterial MutS and eukaryotic MutS{alpha} is well characterized. We report here crystal structures of human MutS{beta} in complex with DNA containing insertion-deletion loops (IDL) of two, three, four or six unpaired nucleotides. In contrast to eukaryotic MutS{alpha} and bacterial MutS, which bind the base of a mismatched nucleotide, MutS{beta} binds three phosphates in an IDL. DNA is severely bent at the IDL; unpaired bases are flippedmore » out into the major groove and partially exposed to solvent. A normal downstream base pair can become unpaired; a single unpaired base can thereby be converted to an IDL of two nucleotides and recognized by MutS{beta}. The C-terminal dimerization domains form an integral part of the MutS structure and coordinate asymmetrical ATP hydrolysis by Msh2 and Msh3 with mismatch binding to signal for repair.« less

Effect of supercoiling on formation of protein-mediated DNA loops

NASA Astrophysics Data System (ADS)

Purohit, P. K.; Nelson, P. C.

2006-12-01

DNA loop formation is one of several mechanisms used by organisms to regulate genes. The free energy of forming a loop is an important factor in determining whether the associated gene is switched on or off. In this paper we use an elastic rod model of DNA to determine the free energy of forming short (50-100 basepair), protein mediated DNA loops. Superhelical stress in the DNA of living cells is a critical factor determining the energetics of loop formation, and we explicitly account for it in our calculations. The repressor protein itself is regarded as a rigid coupler; its geometry enters the problem through the boundary conditions it applies on the DNA. We show that a theory with these ingredients is sufficient to explain certain features observed in modulation of in vivo gene activity as a function of the distance between operator sites for the lac repressor. We also use our theory to make quantitative predictions for the dependence of looping on superhelical stress, which may be testable both in vivo and in single-molecule experiments such as the tethered particle assay and the magnetic bead assay.

The chloroplast tRNALys(UUU) gene from mustard (Sinapis alba) contains a class II intron potentially coding for a maturase-related polypeptide.

PubMed

Neuhaus, H; Link, G

1987-01-01

The trnK gene endocing the tRNALys(UUU) has been located on mustard (Sinapis alba) chloroplast DNA, 263 bp upstream of the psbA gene on the same strand. The nucleotide sequence of the trnK gene and its flanking regions as well as the putative transcription start and termination sites are shown. The 5' end of the transcript lies 121 bp upstream of the 5' tRNA coding region and is preceded by procaryotic-type "-10" and "-35" sequence elements, while the 3' end maps 2.77 kb downstream to a DNA region with possible stemloop secondary structure. The anticodon loop of the tRNALys is interrupted by a 2,574 bp intron containing a long open reading frame, which codes for 524 amino acids. Based on conserved stem and loop structures, this intron has characteristic features of a class II intron. A region near the carboxyl terminus of the derived polypeptide appears structurally related to maturases.

Analysis of the mechanism of nucleosome survival during transcription

PubMed Central

Chang, Han-Wen; Kulaeva, Olga I.; Shaytan, Alexey K.; Kibanov, Mikhail; Kuznedelov, Konstantin; Severinov, Konstantin V.; Kirpichnikov, Mikhail P.; Clark, David J.; Studitsky, Vasily M.

2014-01-01

Maintenance of nucleosomal structure in the cell nuclei is essential for cell viability, regulation of gene expression and normal aging. Our previous data identified a key intermediate (a small intranucleosomal DNA loop, Ø-loop) that is likely required for nucleosome survival during transcription by RNA polymerase II (Pol II) through chromatin, and suggested that strong nucleosomal pausing guarantees efficient nucleosome survival. To evaluate these predictions, we analysed transcription through a nucleosome by different, structurally related RNA polymerases and mutant yeast Pol II having different histone-interacting surfaces that presumably stabilize the Ø-loop. The height of the nucleosomal barrier to transcription and efficiency of nucleosome survival correlate with the net negative charges of the histone-interacting surfaces. Molecular modeling and analysis of Pol II-nucleosome intermediates by DNase I footprinting suggest that efficient Ø-loop formation and nucleosome survival are mediated by electrostatic interactions between the largest subunit of Pol II and core histones. PMID:24234452

APOBEC3G Inhibits HIV-1 RNA Elongation by Inactivating the Viral Trans-Activation Response Element

PubMed Central

Nowarski, Roni; Prabhu, Ponnandy; Kenig, Edan; Smith, Yoav; Britan-Rosich, Elena; Kotler, Moshe

2014-01-01

Deamination of cytidine residues in viral DNA (vDNA) is a major mechanism by which APOBEC3G (A3G) inhibits vif-deficient HIV-1 replication. dC to dU transition following RNase-H activity leads to viral cDNA degradation, production of non-functional proteins, formation of undesired stop codons and decreased viral protein synthesis. Here we demonstrate that A3G provides an additional layer of defence against HIV-1 infection dependent on inhibition of proviral transcription. HIV-1 transcription elongation is regulated by the trans-activation response (TAR) element, a short stem-loop RNA structure required for elongation factors binding. Vif-deficient HIV-1-infected cells accumulate short viral transcripts and produce lower amounts of full-length HIV-1 transcripts due to A3G deamination of the TAR apical loop cytidine, highlighting the requirement for TAR loop integrity in HIV-1 transcription. Finally, we show that free ssDNA termini are not essential for A3G activity and a gap of CCC motif blocked with juxtaposed DNA or RNA on either or 3′+5′ ends is sufficient for A3G deamination, identifying A3G as an efficient mutator, and that deamination of (−)SSDNA results in an early block of HIV-1 transcription. PMID:24859335

APOBEC3G inhibits HIV-1 RNA elongation by inactivating the viral trans-activation response element.

PubMed

Nowarski, Roni; Prabhu, Ponnandy; Kenig, Edan; Smith, Yoav; Britan-Rosich, Elena; Kotler, Moshe

2014-07-29

Deamination of cytidine residues in viral DNA is a major mechanism by which APOBEC3G (A3G) inhibits vif-deficient human immunodeficiency virus type 1 (HIV-1) replication. dC-to-dU transition following RNase-H activity leads to viral cDNA degradation, production of non-functional proteins, formation of undesired stop codons and decreased viral protein synthesis. Here, we demonstrate that A3G provides an additional layer of defense against HIV-1 infection dependent on inhibition of proviral transcription. HIV-1 transcription elongation is regulated by the trans-activation response (TAR) element, a short stem-loop RNA structure required for elongation factors binding. Vif-deficient HIV-1-infected cells accumulate short viral transcripts and produce lower amounts of full-length HIV-1 transcripts due to A3G deamination of the TAR apical loop cytidine, highlighting the requirement for TAR loop integrity in HIV-1 transcription. We further show that free single-stranded DNA (ssDNA) termini are not essential for A3G activity and a gap of CCC motif blocked with juxtaposed DNA or RNA on either or 3'+5' ends is sufficient for A3G deamination. These results identify A3G as an efficient mutator and that deamination of (-)SSDNA results in an early block of HIV-1 transcription. Copyright © 2014 Elsevier Ltd. All rights reserved.

DNA glycosylases search for and remove oxidized DNA bases.

PubMed

Wallace, Susan S

2013-12-01

This review article presents, an overview of the DNA glycosylases that recognize oxidized DNA bases using the Fpg/Nei family of DNA glycosylases as models for how structure can inform function. For example, even though human NEIL1 and the plant and fungal orthologs lack the zinc finger shown to be required for binding, DNA crystal structures revealed a "zincless finger" with the same properties. Moreover, the "lesion recognition loop" is not involved in lesion recognition, rather, it stabilizes 8-oxoG in the active site pocket. Unlike the other Fpg/Nei family members, Neil3 lacks two of the three void-filling residues that stabilize the DNA duplex and interact with the opposite strand to the damage which may account for its preference for lesions in single-stranded DNA. Also single-molecule approaches show that DNA glycosylases search for their substrates in a sea of undamaged DNA by using a wedge residue that is inserted into the DNA helix to probe for the presence of damage. Copyright © 2013 Wiley Periodicals, Inc.

Mutations in the C-terminal fragment of DnaK affecting peptide binding.

PubMed Central

Burkholder, W F; Zhao, X; Zhu, X; Hendrickson, W A; Gragerov, A; Gottesman, M E

1996-01-01

Escherichia coli DnaK acts as a molecular chaperone through its ATP-regulated binding and release of polypeptide substrates. Overexpressing a C-terminal fragment (CTF) of DnaK (Gly-384 to Lys-638) containing the polypeptide substrate binding domain is lethal in wild-type E. coli. This dominant-negative phenotype may result from the nonproductive binding of CTF to cellular polypeptide targets of DnaK. Mutations affecting DnaK substrate binding were identified by selecting noncytotoxic CTF mutants followed by in vitro screening. The clustering of such mutations in the three-dimensional structure of CTF suggests the model that loops L1,2 and L4,5 form a rigid core structure critical for interactions with substrate. Images Fig. 1 Fig. 2 Fig. 3 PMID:8855230

Solution structure of the N-terminal domain of a replication restart primosome factor, PriC, in Escherichia coli

PubMed Central

Aramaki, Takahiko; Abe, Yoshito; Katayama, Tsutomu; Ueda, Tadashi

2013-01-01

In eubacterial organisms, the oriC-independent primosome plays an essential role in replication restart after the dissociation of the replication DNA-protein complex by DNA damage. PriC is a key protein component in the replication restart primosome. Our recent study suggested that PriC is divided into two domains: an N-terminal and a C-terminal domain. In the present study, we determined the solution structure of the N-terminal domain, whose structure and function have remained unknown until now. The revealed structure was composed of three helices and one extended loop. We also observed chemical shift changes in the heteronuclear NMR spectrum and oligomerization in the presence of ssDNA. These abilities may contribute to the PriC-ssDNA complex, which is important for the replication restart primosome. PMID:23868391

The basic helix-loop-helix region of the transcriptional repressor hairy and enhancer of split 1 is preorganized to bind DNA.

PubMed

Popovic, Matija; Wienk, Hans; Coglievina, Maristella; Boelens, Rolf; Pongor, Sándor; Pintar, Alessandro

2014-04-01

Hairy and enhancer of split 1, one of the main downstream effectors in Notch signaling, is a transcriptional repressor of the basic helix-loop-helix (bHLH) family. Using nuclear magnetic resonance methods, we have determined the structure and dynamics of a recombinant protein, H1H, which includes an N-terminal segment, b1, containing functionally important phosphorylation sites, the basic region b2, required for binding to DNA, and the HLH domain. We show that a proline residue in the sequence divides the protein in two parts, a flexible and disordered N-terminal region including b1 and a structured, mainly helical region comprising b2 and the HLH domain. Binding of H1H to a double strand DNA oligonucleotide was monitored through the chemical shift perturbation of backbone amide resonances, and showed that the interaction surface involves not only the b2 segment but also several residues in the b1 and HLH regions. Copyright © 2014 Wiley Periodicals, Inc.

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

Crystal structure of a transcribing RNA Polymerase II complex reveals a complete transcription bubble

PubMed Central

Barnes, Christopher O.; Calero, Monica; Malik, Indranil; Graham, Brian W.; Spahr, Henrik; Lin, Guowu; Cohen, Aina; Brown, Ian S.; Zhang, Qiangmin; Pullara, Filippo; Trakselis, Michael A.; Kaplan, Craig D.; Calero, Guillermo

2015-01-01

Summary Notwithstanding numerous published structures of RNA Polymerase II (Pol II), structural details of Pol II engaging a complete nucleic acid scaffold have been lacking. Here, we report the structures of TFIIF stabilized transcribing Pol II complexes, revealing the upstream duplex and full transcription bubble. The upstream duplex lies over a wedge-shaped loop from Rpb2 that engages its minor groove, providing part of the structural framework for DNA tracking during elongation. At the upstream transcription bubble fork, rudder and fork loop-1 residues spatially coordinate strand annealing and the nascent RNA transcript. At the downstream fork, a network of Pol II interactions with the non-template strand forms a rigid domain with the Trigger Loop (TL), allowing visualization of its open state. Overall, our observations suggest that “open/closed” conformational transitions of the TL may be linked to interactions with the non-template strand, possibly in a synchronized ratcheting manner conducive to polymerase translocation. PMID:26186291

RECQ-like helicases Sgs1 and BLM regulate R-loop-associated genome instability.

PubMed

Chang, Emily Yun-Chia; Novoa, Carolina A; Aristizabal, Maria J; Coulombe, Yan; Segovia, Romulo; Chaturvedi, Richa; Shen, Yaoqing; Keong, Christelle; Tam, Annie S; Jones, Steven J M; Masson, Jean-Yves; Kobor, Michael S; Stirling, Peter C

2017-12-04

Sgs1, the orthologue of human Bloom's syndrome helicase BLM, is a yeast DNA helicase functioning in DNA replication and repair. We show that SGS1 loss increases R-loop accumulation and sensitizes cells to transcription-replication collisions. Yeast lacking SGS1 accumulate R-loops and γ-H2A at sites of Sgs1 binding, replication pausing regions, and long genes. The mutation signature of sgs1 Δ reveals copy number changes flanked by repetitive regions with high R-loop-forming potential. Analysis of BLM in Bloom's syndrome fibroblasts or by depletion of BLM from human cancer cells confirms a role for Sgs1/BLM in suppressing R-loop-associated genome instability across species. In support of a potential direct effect, BLM is found physically proximal to DNA:RNA hybrids in human cells, and can efficiently unwind R-loops in vitro. Together, our data describe a conserved role for Sgs1/BLM in R-loop suppression and support an increasingly broad view of DNA repair and replication fork stabilizing proteins as modulators of R-loop-mediated genome instability. © 2017 Chang et al.

The free-energy cost of interaction between DNA loops.

PubMed

Huang, Lifang; Liu, Peijiang; Yuan, Zhanjiang; Zhou, Tianshou; Yu, Jianshe

2017-10-03

From the viewpoint of thermodynamics, the formation of DNA loops and the interaction between them, which are all non-equilibrium processes, result in the change of free energy, affecting gene expression and further cell-to-cell variability as observed experimentally. However, how these processes dissipate free energy remains largely unclear. Here, by analyzing a mechanic model that maps three fundamental topologies of two interacting DNA loops into a 4-state model of gene transcription, we first show that a longer DNA loop needs more mean free energy consumption. Then, independent of the type of interacting two DNA loops (nested, side-by-side or alternating), the promotion between them always consumes less mean free energy whereas the suppression dissipates more mean free energy. More interestingly, we find that in contrast to the mechanism of direct looping between promoter and enhancer, the facilitated-tracking mechanism dissipates less mean free energy but enhances the mean mRNA expression, justifying the facilitated-tracking hypothesis, a long-standing debate in biology. Based on minimal energy principle, we thus speculate that organisms would utilize the mechanisms of loop-loop promotion and facilitated tracking to survive in complex environments. Our studies provide insights into the understanding of gene expression regulation mechanism from the view of energy consumption.

An ATR-dependent function for the Ddx19 RNA helicase in nuclear R-loop metabolism.

PubMed

Hodroj, Dana; Recolin, Bénédicte; Serhal, Kamar; Martinez, Susan; Tsanov, Nikolay; Abou Merhi, Raghida; Maiorano, Domenico

2017-05-02

Coordination between transcription and replication is crucial in the maintenance of genome integrity. Disturbance of these processes leads to accumulation of aberrant DNA:RNA hybrids (R-loops) that, if unresolved, generate DNA damage and genomic instability. Here we report a novel, unexpected role for the nucleopore-associated mRNA export factor Ddx19 in removing nuclear R-loops formed upon replication stress or DNA damage. We show, in live cells, that Ddx19 transiently relocalizes from the nucleopore to the nucleus upon DNA damage, in an ATR/Chk1-dependent manner, and that Ddx19 nuclear relocalization is required to clear R-loops. Ddx19 depletion induces R-loop accumulation, proliferation-dependent DNA damage and defects in replication fork progression. Further, we show that Ddx19 resolves R-loops in vitro via its helicase activity. Furthermore, mutation of a residue phosphorylated by Chk1 in Ddx19 disrupts its interaction with Nup214 and allows its nuclear relocalization. Finally, we show that Ddx19 operates in resolving R-loops independently of the RNA helicase senataxin. Altogether these observations put forward a novel, ATR-dependent function for Ddx19 in R-loop metabolism to preserve genome integrity in mammalian cells. © 2017 The Authors.

Processing of double-R-loops in (CAG)·(CTG) and C9orf72 (GGGGCC)·(GGCCCC) repeats causes instability

PubMed Central

Reddy, Kaalak; Schmidt, Monika H.M.; Geist, Jaimie M.; Thakkar, Neha P.; Panigrahi, Gagan B.; Wang, Yuh-Hwa; Pearson, Christopher E.

2014-01-01

R-loops, transcriptionally-induced RNA:DNA hybrids, occurring at repeat tracts (CTG)n, (CAG)n, (CGG)n, (CCG)n and (GAA)n, are associated with diseases including myotonic dystrophy, Huntington's disease, fragile X and Friedreich's ataxia. Many of these repeats are bidirectionally transcribed, allowing for single- and double-R-loop configurations, where either or both DNA strands may be RNA-bound. R-loops can trigger repeat instability at (CTG)·(CAG) repeats, but the mechanism of this is unclear. We demonstrate R-loop-mediated instability through processing of R-loops by HeLa and human neuron-like cell extracts. Double-R-loops induced greater instability than single-R-loops. Pre-treatment with RNase H only partially suppressed instability, supporting a model in which R-loops directly generate instability by aberrant processing, or via slipped-DNA formation upon RNA removal and its subsequent aberrant processing. Slipped-DNAs were observed to form following removal of the RNA from R-loops. Since transcriptionally-induced R-loops can occur in the absence of DNA replication, R-loop processing may be a source of repeat instability in the brain. Double-R-loop formation and processing to instability was extended to the expanded C9orf72 (GGGGCC)·(GGCCCC) repeats, known to cause amyotrophic lateral sclerosis and frontotemporal dementia, providing the first suggestion through which these repeats may become unstable. These findings provide a mechanistic basis for R-loop-mediated instability at disease-associated repeats. PMID:25147206

Human ribonuclease H1 resolves R-loops and thereby enables progression of the DNA replication fork.

PubMed

Parajuli, Shankar; Teasley, Daniel C; Murali, Bhavna; Jackson, Jessica; Vindigni, Alessandro; Stewart, Sheila A

2017-09-15

Faithful DNA replication is essential for genome stability. To ensure accurate replication, numerous complex and redundant replication and repair mechanisms function in tandem with the core replication proteins to ensure DNA replication continues even when replication challenges are present that could impede progression of the replication fork. A unique topological challenge to the replication machinery is posed by RNA-DNA hybrids, commonly referred to as R-loops. Although R-loops play important roles in gene expression and recombination at immunoglobulin sites, their persistence is thought to interfere with DNA replication by slowing or impeding replication fork progression. Therefore, it is of interest to identify DNA-associated enzymes that help resolve replication-impeding R-loops. Here, using DNA fiber analysis, we demonstrate that human ribonuclease H1 (RNH1) plays an important role in replication fork movement in the mammalian nucleus by resolving R-loops. We found that RNH1 depletion results in accumulation of RNA-DNA hybrids, slowing of replication forks, and increased DNA damage. Our data uncovered a role for RNH1 in global DNA replication in the mammalian nucleus. Because accumulation of RNA-DNA hybrids is linked to various human cancers and neurodegenerative disorders, our study raises the possibility that replication fork progression might be impeded, adding to increased genomic instability and contributing to disease. © 2017 by The American Society for Biochemistry and Molecular Biology, Inc.

Different mutation patterns of mitochondrial DNA displacement-loop in hepatocellular carcinomas induced by N-nitrosodiethylamine and a choline-deficient l-amino acid-defined diet in rats.

PubMed

Onishi, Mariko; Sokuza, Yui; Nishikawa, Tomoki; Mori, Chiharu; Uwataki, Kimiko; Honoki, Kanya; Tsujiuchi, Toshifumi

2007-10-12

Mutations of the mitochondria DNA (mtDNA) displacement loop (D-loop) were investigated to clarify different changes of exogenous and endogenous liver carcinogenesis in rats. We induced hepatocellular carcinomas (HCCs) in rats with N-nitrosodiethylamine (DEN) and a choline-deficient l-amino acid-defined (CDAA) diet. DNAs were extracted from 10 HCCs induced by DEN and 10 HCCs induced by the CDAA diet. To identify mutations in mtDNA D-loop, polymerase chain reaction (PCR)-single strand conformation polymorphism (SSCP) analysis, followed by nucleotide sequencing, was performed. Mutations were detected in 5 out of 10 HCCs (50%) induced by DEN. Four out of 5 mutations were G/C to A/T transitions at positions 15707, 15717, 15930, and 16087, and one T/A to C/G transition at position 15559. By contrast, no mutations were found in 10 HCCs induced by the CDAA diet. These results demonstrated that mutations in mtDNA D-loop occur in rat HCCs induced by DEN but not by the CDAA diet, suggesting that mtDNA D-loop is a target of exogenous liver carcinogenesis in rats.

DNA looping by FokI: the impact of synapse geometry on loop topology at varied site orientations

PubMed Central

Rusling, David A.; Laurens, Niels; Pernstich, Christian; Wuite, Gijs J. L.; Halford, Stephen E.

2012-01-01

Most restriction endonucleases, including FokI, interact with two copies of their recognition sequence before cutting DNA. On DNA with two sites they act in cis looping out the intervening DNA. While many restriction enzymes operate symmetrically at palindromic sites, FokI acts asymmetrically at a non-palindromic site. The directionality of its sequence means that two FokI sites can be bridged in either parallel or anti-parallel alignments. Here we show by biochemical and single-molecule biophysical methods that FokI aligns two recognition sites on separate DNA molecules in parallel and that the parallel arrangement holds for sites in the same DNA regardless of whether they are in inverted or repeated orientations. The parallel arrangement dictates the topology of the loop trapped between sites in cis: the loop from inverted sites has a simple 180° bend, while that with repeated sites has a convoluted 360° turn. The ability of FokI to act at asymmetric sites thus enabled us to identify the synapse geometry for sites in trans and in cis, which in turn revealed the relationship between synapse geometry and loop topology. PMID:22362745

Probing the dynamics of restriction endonuclease NgoMIV-DNA interaction by single-molecule FRET.

PubMed

Tutkus, Marijonas; Sasnauskas, Giedrius; Rutkauskas, Danielis

2017-12-01

Many type II restriction endonucleases require two copies of their recognition sequence for optimal activity. Concomitant binding of two DNA sites by such an enzyme produces a DNA loop. Here we exploit single-molecule Förster resonance energy transfer (smFRET) of surface-immobilized DNA fragments to study the dynamics of DNA looping induced by tetrameric endonuclease NgoMIV. We have employed a DNA fragment with two NgoMIV recognition sites and a FRET dye pair such that upon protein-induced DNA looping the dyes are brought to close proximity resulting in a FRET signal. The dynamics of DNA-NgoMIV interactions proved to be heterogeneous, with individual smFRET trajectories exhibiting broadly different average looped state durations. Distinct types of the dynamics were attributed to different types of DNA-protein complexes, mediated either by one NgoMIV tetramer simultaneously bound to two specific sites ("slow" trajectories) or by semi-specific interactions of two DNA-bound NgoMIV tetramers ("fast" trajectories), as well as to conformational heterogeneity of individual NgoMIV molecules. © 2017 Wiley Periodicals, Inc.

Super-resolution optical microscopy study of telomere structure.

PubMed

Phipps, Mary Lisa; Goodwin, Peter M; Martinez, Jennifer S; Goodwin, Edwin H

2016-09-01

Chromosome ends are shielded from exonucleolytic attack and inappropriate end-joining by terminal structures called telomeres; these structures are potential targets for anticancer drugs. Telomeres are composed of a simple DNA sequence (5?-TTAGGG-3? in humans) repeated more than a thousand times, a short 3? single-stranded overhang, and numerous proteins. Electron microscopy has shown that the 3? overhang pairs with the complementary strand at an internal site creating a small displacement loop and a large double-stranded “t-loop.” Our goal is to determine whether all telomeres adopt the t-loop configuration, or whether there are two or more distinct configurations. Progress in optimizing super-resolution (SR) microscopy for this ongoing investigation is reported here. Results suggest that under certain conditions sample preparation procedures may disrupt chromatin by causing loss of nucleosomes. This finding may limit the use of SR microscopy in telomere studies.

Super-resolution optical microscopy study of telomere structure

NASA Astrophysics Data System (ADS)

Phipps, Mary Lisa; Goodwin, Peter M.; Martinez, Jennifer S.; Goodwin, Edwin H.

2016-09-01

Chromosome ends are shielded from exonucleolytic attack and inappropriate end-joining by terminal structures called telomeres; these structures are potential targets for anticancer drugs. Telomeres are composed of a simple DNA sequence (5‧-TTAGGG-3‧ in humans) repeated more than a thousand times, a short 3‧ single-stranded overhang, and numerous proteins. Electron microscopy has shown that the 3‧ overhang pairs with the complementary strand at an internal site creating a small displacement loop and a large double-stranded "t-loop." Our goal is to determine whether all telomeres adopt the t-loop configuration, or whether there are two or more distinct configurations. Progress in optimizing super-resolution (SR) microscopy for this ongoing investigation is reported here. Results suggest that under certain conditions sample preparation procedures may disrupt chromatin by causing loss of nucleosomes. This finding may limit the use of SR microscopy in telomere studies.

Identification, cloning and characterization of a new DNA-binding protein from the hyperthermophilic methanogen Methanopyrus kandleri

PubMed Central

Pavlov, Nikolai A.; Cherny, Dmitry I.; Nazimov, Igor V.; Slesarev, Alexei I.; Subramaniam, Vinod

2002-01-01

Three novel DNA-binding proteins with apparent molecular masses of 7, 10 and 30 kDa have been isolated from the hyperthermophilic methanogen Methanopyrus kandleri. The proteins were identified using a blot overlay assay that was modified to emulate the high ionic strength intracellular environment of M.kandleri proteins. A 7 kDa protein, named 7kMk, was cloned and expressed in Escherichia coli. As indicated by CD spectroscopy and computer-assisted structure prediction methods, 7kMk is a substantially α-helical protein possibly containing a short N-terminal β-strand. According to analytical gel filtration chromatography and chemical crosslinking, 7kMk exists as a stable dimer, susceptible to further oligomerization. Electron microscopy showed that 7kMk bends DNA and also leads to the formation of loop-like structures of ∼43.5 ± 3.5 nm (136 ± 11 bp for B-form DNA) circumference. A topoisomerase relaxation assay demonstrated that looped DNA is negatively supercoiled under physiologically relevant conditions (high salt and temperature). A BLAST search did not yield 7kMk homologs at the amino acid sequence level, but based on a multiple alignment with ribbon–helix–helix (RHH) transcriptional regulators, fold features and self-association properties of 7kMk we hypothesize that it could be related to RHH proteins. PMID:11809880

Determinants for DNA target structure selectivity of the human LINE-1 retrotransposon endonuclease.

PubMed

Repanas, Kostas; Zingler, Nora; Layer, Liliana E; Schumann, Gerald G; Perrakis, Anastassis; Weichenrieder, Oliver

2007-01-01

The human LINE-1 endonuclease (L1-EN) is the targeting endonuclease encoded by the human LINE-1 (L1) retrotransposon. L1-EN guides the genomic integration of new L1 and Alu elements that presently account for approximately 28% of the human genome. L1-EN bears considerable technological interest, because its target selectivity may ultimately be engineered to allow the site-specific integration of DNA into defined genomic locations. Based on the crystal structure, we generated L1-EN mutants to analyze and manipulate DNA target site recognition. Crystal structures and their dynamic and functional analysis show entire loop grafts to be feasible, resulting in altered specificity, while individual point mutations do not change the nicking pattern of L1-EN. Structural parameters of the DNA target seem more important for recognition than the nucleotide sequence, and nicking profiles on DNA oligonucleotides in vitro are less well defined than the respective integration site consensus in vivo. This suggests that additional factors other than the DNA nicking specificity of L1-EN contribute to the targeted integration of non-LTR retrotransposons.

CRISPR-Cas9 Structures and Mechanisms.

PubMed

Jiang, Fuguo; Doudna, Jennifer A

2017-05-22

Many bacterial clustered regularly interspaced short palindromic repeats (CRISPR)-CRISPR-associated (Cas) systems employ the dual RNA-guided DNA endonuclease Cas9 to defend against invading phages and conjugative plasmids by introducing site-specific double-stranded breaks in target DNA. Target recognition strictly requires the presence of a short protospacer adjacent motif (PAM) flanking the target site, and subsequent R-loop formation and strand scission are driven by complementary base pairing between the guide RNA and target DNA, Cas9-DNA interactions, and associated conformational changes. The use of CRISPR-Cas9 as an RNA-programmable DNA targeting and editing platform is simplified by a synthetic single-guide RNA (sgRNA) mimicking the natural dual trans-activating CRISPR RNA (tracrRNA)-CRISPR RNA (crRNA) structure. This review aims to provide an in-depth mechanistic and structural understanding of Cas9-mediated RNA-guided DNA targeting and cleavage. Molecular insights from biochemical and structural studies provide a framework for rational engineering aimed at altering catalytic function, guide RNA specificity, and PAM requirements and reducing off-target activity for the development of Cas9-based therapies against genetic diseases.

Impact of Somatic Mutations in the D-Loop of Mitochondrial DNA on the Survival of Oral Squamous Cell Carcinoma Patients

PubMed Central

Lin, Jin-Ching; Wang, Chen-Chi; Jiang, Rong-San; Wang, Wen-Yi; Liu, Shih-An

2015-01-01

Objectives The aim of this study was to investigate somatic mutations in the D-loop of mitochondrial DNA (mtDNA) and their impact on survival in oral squamous cell carcinoma patients. Materials and Methods Surgical specimen confirmed by pathological examination and corresponding non-cancerous tissues were collected from 120 oral squamous cell carcinoma patients. The sequence in the D-loop of mtDNA from non-cancerous tissues was compared with that from paired cancer samples and any sequence differences were recognized as somatic mutations. Results Somatic mutations in the D-loop of mtDNA were identified in 75 (62.5%) oral squamous cell carcinoma patients and most of them occurred in the poly-C tract. Although there were no significant differences in demographic and tumor-related features between participants with and without somatic mutation, the mutation group had a better survival rate (5 year disease-specific survival rate: 64.0% vs. 43.0%, P = 0.0266). Conclusion Somatic mutation in D-loop of mtDNA was associated with a better survival in oral squamous cell carcinoma patients. PMID:25906372

Programmable and Multifunctional DNA-Based Materials for Biomedical Applications.

PubMed

Zhang, Yuezhou; Tu, Jing; Wang, Dongqing; Zhu, Haitao; Maity, Sajal Kumar; Qu, Xiangmeng; Bogaert, Bram; Pei, Hao; Zhang, Hongbo

2018-06-01

DNA encodes the genetic information; recently, it has also become a key player in material science. Given the specific Watson-Crick base-pairing interactions between only four types of nucleotides, well-designed DNA self-assembly can be programmable and predictable. Stem-loops, sticky ends, Holliday junctions, DNA tiles, and lattices are typical motifs for forming DNA-based structures. The oligonucleotides experience thermal annealing in a near-neutral buffer containing a divalent cation (usually Mg 2+ ) to produce a variety of DNA nanostructures. These structures not only show beautiful landscape, but can also be endowed with multifaceted functionalities. This Review begins with the fundamental characterization and evolutionary trajectory of DNA-based artificial structures, but concentrates on their biomedical applications. The coverage spans from controlled drug delivery to high therapeutic profile and accurate diagnosis. A variety of DNA-based materials, including aptamers, hydrogels, origamis, and tetrahedrons, are widely utilized in different biomedical fields. In addition, to achieve better performance and functionality, material hybridization is widely witnessed, and DNA nanostructure modification is also discussed. Although there are impressive advances and high expectations, the development of DNA-based structures/technologies is still hindered by several commonly recognized challenges, such as nuclease instability, lack of pharmacokinetics data, and relatively high synthesis cost. © 2018 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

Multiple maternal origins of Indonesian crowing chickens revealed by mitochondrial DNA analysis.

PubMed

Ulfah, Maria; Perwitasari, Dyah; Jakaria, Jakaria; Muladno, Muhammad; Farajallah, Achmad

2017-03-01

The utilization of Indonesian crowing chickens is increasing; as such, assessing their genetic structures is important to support the conservation of their genetic resources. This study analyzes the matrilineal evolution of Indonesian crowing chickens based on the mtDNA displacement loop D-loop region to clarify their phylogenetic relationships, possible maternal origin, and possible routes of chicken dispersal. The neighbor-joining tree reveals that the majority of Indonesian crowing chickens belong to haplogroups B, D, and E, but haplogroup D harbored most of them. The Bayesian analysis also reveals that Indonesian crowing chickens derive from Bekisar chicken, a hybrid of the green junglefowl, suggesting the possible contribution of green junglefowl to chicken domestication. There appear at least three maternal lineages of Indonesian chicken origins indicated by the median network profile of mtDNA D-loop haplotypes, namely (1) Chinese; (2) Chinese, Indian, and other Southeast Asian chickens; and (3) Indian, Chinese, Southeast Asian, Japanese, and European chickens. Chicken domestication might be centered in China, India, Indonesia, and other Southeast Asian countries, supporting multiple maternal origins of Indonesian crowing chickens. A systematic breeding program of indigenous chickens will be very important to retain the genetic diversity for future use and conservation.

Detection of somatic mutations in the mitochondrial DNA control region D-loop in brain tumors: The first report in Malaysian patients.

PubMed

Mohamed Yusoff, Abdul Aziz; Mohd Nasir, Khairol Naaim; Haris, Khalilah; Mohd Khair, Siti Zulaikha Nashwa; Abdul Ghani, Abdul Rahman Izaini; Idris, Zamzuri; Abdullah, Jafri Malin

2017-11-01

Although the role of nuclear-encoded gene alterations has been well documented in brain tumor development, the involvement of the mitochondrial genome in brain tumorigenesis has not yet been fully elucidated and remains controversial. The present study aimed to identify mutations in the mitochondrial DNA (mtDNA) control region D-loop in patients with brain tumors in Malaysia. A mutation analysis was performed in which DNA was extracted from paired tumor tissue and blood samples obtained from 49 patients with brain tumors. The D-loop region DNA was amplified using the PCR technique, and genetic data from DNA sequencing analyses were compared with the published revised Cambridge sequence to identify somatic mutations. Among the 49 brain tumor tissue samples evaluated, 25 cases (51%) had somatic mutations of the mtDNA D-loop, with a total of 48 mutations. Novel mutations that had not previously been identified in the D-loop region (176 A-deletion, 476 C>A, 566 C>A and 16405 A-deletion) were also classified. No significant associations between the D-loop mutation status and the clinicopathological parameters were observed. To the best of our knowledge, the current study presents the first evidence of alterations in the mtDNA D-loop regions in the brain tumors of Malaysian patients. These results may provide an overview and data regarding the incidence of mitochondrial genome alterations in Malaysian patients with brain tumors. In addition to nuclear genome aberrations, these specific mitochondrial genome alterations may also be considered as potential cancer biomarkers for the diagnosis and staging of brain cancers.

Productive mRNA stem loop-mediated transcriptional slippage: Crucial features in common with intrinsic terminators.

PubMed

Penno, Christophe; Sharma, Virag; Coakley, Arthur; O'Connell Motherway, Mary; van Sinderen, Douwe; Lubkowska, Lucyna; Kireeva, Maria L; Kashlev, Mikhail; Baranov, Pavel V; Atkins, John F

2015-04-21

Escherichia coli and yeast DNA-dependent RNA polymerases are shown to mediate efficient nascent transcript stem loop formation-dependent RNA-DNA hybrid realignment. The realignment was discovered on the heteropolymeric sequence T5C5 and yields transcripts lacking a C residue within a corresponding U5C4. The sequence studied is derived from a Roseiflexus insertion sequence (IS) element where the resulting transcriptional slippage is required for transposase synthesis. The stability of the RNA structure, the proximity of the stem loop to the slippage site, the length and composition of the slippage site motif, and the identity of its 3' adjacent nucleotides (nt) are crucial for transcripts lacking a single C. In many respects, the RNA structure requirements for this slippage resemble those for hairpin-dependent transcription termination. In a purified in vitro system, the slippage efficiency ranges from 5% to 75% depending on the concentration ratios of the nucleotides specified by the slippage sequence and the 3' nt context. The only previous proposal of stem loop mediated slippage, which was in Ebola virus expression, was based on incorrect data interpretation. We propose a mechanical slippage model involving the RNAP translocation state as the main motor in slippage directionality and efficiency. It is distinct from previously described models, including the one proposed for paramyxovirus, where following random movement efficiency is mainly dependent on the stability of the new realigned hybrid. In broadening the scope for utilization of transcription slippage for gene expression, the stimulatory structure provides parallels with programmed ribosomal frameshifting at the translation level.

Label-free fluorescence strategy for sensitive detection of adenosine triphosphate using a loop DNA probe with low background noise.

PubMed

Lin, Chunshui; Cai, Zhixiong; Wang, Yiru; Zhu, Zhi; Yang, Chaoyong James; Chen, Xi

2014-07-15

A simple, rapid, label-free, and ultrasensitive fluorescence strategy for adenosine triphosphate (ATP) detection was developed using a loop DNA probe with low background noise. In this strategy, a loop DNA probe, which is the substrate for both ligation and digestion enzyme reaction, was designed. SYBR green I (SG I), a double-stranded specific dye, was applied for the readout fluorescence signal. Exonuclease I (Exo I) and exonuclease III (Exo III), sequence-independent nucleases, were selected to digest the loop DNA probe in order to minimize the background fluorescence signal. As a result, in the absence of ATP, the loop DNA was completely digested by Exo I and Exo III, leading to low background fluorescence owing to the weak electrostatic interaction between SG I and mononucleotides. On the other hand, ATP induced the ligation of the nicking site, and the sealed loop DNA resisted the digestion of Exo I and ExoIII, resulting in a remarkable increase of fluorescence response. Upon background noise reduction, the sensitivity of the ATP determination was improved significantly, and the detection limitation was found to be 1.2 pM, which is much lower than that in almost all the previously reported methods. This strategy has promise for wide application in the determination of ATP.

Structure of transcribed chromatin is a sensor of DNA damage

PubMed Central

Pestov, Nikolay A.; Gerasimova, Nadezhda S.; Kulaeva, Olga I.; Studitsky, Vasily M.

2015-01-01

Early detection and repair of damaged DNA is essential for cell functioning and survival. Although multiple cellular systems are involved in the repair of single-strand DNA breaks (SSBs), it remains unknown how SSBs present in the nontemplate strand (NT-SSBs) of DNA organized in chromatin are detected. The effect of NT-SSBs on transcription through chromatin by RNA polymerase II was studied. NT-SSBs localized in the promoter-proximal region of nucleosomal DNA and hidden in the nucleosome structure can induce a nearly quantitative arrest of RNA polymerase downstream of the break, whereas more promoter-distal SSBs moderately facilitate transcription. The location of the arrest sites on nucleosomal DNA suggests that formation of small intranucleosomal DNA loops causes the arrest. This mechanism likely involves relief of unconstrained DNA supercoiling accumulated during transcription through chromatin by NT-SSBs. These data suggest the existence of a novel chromatin-specific mechanism that allows the detection of NT-SSBs by the transcribing enzyme. PMID:26601207

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

C9orf72 nucleotide repeat structures initiate molecular cascades of disease.

PubMed

Haeusler, Aaron R; Donnelly, Christopher J; Periz, Goran; Simko, Eric A J; Shaw, Patrick G; Kim, Min-Sik; Maragakis, Nicholas J; Troncoso, Juan C; Pandey, Akhilesh; Sattler, Rita; Rothstein, Jeffrey D; Wang, Jiou

2014-03-13

A hexanucleotide repeat expansion (HRE), (GGGGCC)n, in C9orf72 is the most common genetic cause of the neurodegenerative diseases amyotrophic lateral sclerosis (ALS) and frontotemporal dementia (FTD). Here we identify a molecular mechanism by which structural polymorphism of the HRE leads to ALS/FTD pathology and defects. The HRE forms DNA and RNA G-quadruplexes with distinct structures and promotes RNA•DNA hybrids (R-loops). The structural polymorphism causes a repeat-length-dependent accumulation of transcripts aborted in the HRE region. These transcribed repeats bind to ribonucleoproteins in a conformation-dependent manner. Specifically, nucleolin, an essential nucleolar protein, preferentially binds the HRE G-quadruplex, and patient cells show evidence of nucleolar stress. Our results demonstrate that distinct C9orf72 HRE structural polymorphism at both DNA and RNA levels initiates molecular cascades leading to ALS/FTD pathologies, and provide the basis for a mechanistic model for repeat-associated neurodegenerative diseases.

Low mitochondrial DNA diversity of Japanese Polled and Kuchinoshima feral cattle.

PubMed

Mannen, Hideyuki; Yonesaka, Riku; Noda, Aoi; Shimogiri, Takeshi; Oshima, Ichiro; Katahira, Kiyomi; Kanemaki, Misao; Kunieda, Tetsuo; Inayoshi, Yousuke; Mukai, Fumio; Sasazaki, Shinji

2017-05-01

This study aims to estimate the mitochondrial genetic diversity and structure of Japanese Polled and Kuchinoshima feral cattle, which are maintained in small populations. We determined the mitochondrial DMA (mtDNA) displacement loop (D-loop) sequences for both cattle populations and analyzed these in conjunction with previously published data from Northeast Asian cattle populations. Our findings showed that Japanese native cattle have a predominant, Asian-specific mtDNA haplogroup T4 with high frequencies (0.43-0.81). This excluded Kuchinoshima cattle (32 animals), which had only one mtDNA haplotype belonging to the haplogroup T3. Japanese Polled showed relatively lower mtDNA diversity in the average sequence divergence (0.0020) than other Wagyu breeds (0.0036-0.0047). Japanese Polled have been maintained in a limited area of Yamaguchi, and the population size is now less than 200. Therefore, low mtDNA diversity in the Japanese Polled could be explained by the decreasing population size in the last three decades. We found low mtDNA diversity in both Japanese Polled and Kuchinoshima cattle. The genetic information obtained in this study will be useful for maintaining these populations and for understanding the origin of Japanese native cattle. © 2016 Japanese Society of Animal Science.

Historical explanation of genetic variation in the Mediterranean horseshoe bat Rhinolophus euryale (Chiroptera: Rhinolophidae) inferred from mitochondrial cytochrome-b and D-loop genes in Iran.

PubMed

Najafi, Nargess; Akmali, Vahid; Sharifi, Mozafar

2018-04-26

Molecular phylogeography and species distribution modelling (SDM) suggest that late Quaternary glacial cycles have portrayed a significant role in structuring current population genetic structure and diversity. Based on phylogenetic relationships using Bayesian inference and maximum likelihood of 535 bp mtDNA (D-loop) and 745 bp mtDNA (Cytb) in 62 individuals of the Mediterranean Horseshoe Bat, Rhinolophus euryale, from 13 different localities in Iran we identified two subspecific populations with differing population genetic structure distributed in southern Zagros Mts. and northern Elburz Mts. Analysis of molecular variance (AMOVA) obtained from D-loop sequences indicates that 21.18% of sequence variation is distributed among populations and 10.84% within them. Moreover, a degree of genetic subdivision, mainly attributable to the existence of significant variance among the two regions is shown (θCT = 0.68, p = .005). The positive and significant correlation between geographic and genetic distances (R 2  = 0.28, r = 0.529, p = .000) is obtained following controlling for environmental distance. Spatial distribution of haplotypes indicates that marginal population of the species in southern part of the species range have occupied this section as a glacial refugia. However, this genetic variation, in conjunction with results of the SDM shows a massive postglacial range expansion for R. euryale towards higher latitudes in Iran.

Structural basis for the recognition of guide RNA and target DNA heteroduplex by Argonaute

PubMed Central

Miyoshi, Tomohiro; Ito, Kosuke; Murakami, Ryo; Uchiumi, Toshio

2016-01-01

Argonaute proteins are key players in the gene silencing mechanisms mediated by small nucleic acids in all domains of life from bacteria to eukaryotes. However, little is known about the Argonaute protein that recognizes guide RNA/target DNA. Here, we determine the 2 Å crystal structure of Rhodobacter sphaeroides Argonaute (RsAgo) in a complex with 18-nucleotide guide RNA and its complementary target DNA. The heteroduplex maintains Watson–Crick base-pairing even in the 3′-region of the guide RNA between the N-terminal and PIWI domains, suggesting a recognition mode by RsAgo for stable interaction with the target strand. In addition, the MID/PIWI interface of RsAgo has a system that specifically recognizes the 5′ base-U of the guide RNA, and the duplex-recognition loop of the PAZ domain is important for the DNA silencing activity. Furthermore, we show that Argonaute discriminates the nucleic acid type (RNA/DNA) by recognition of the duplex structure of the seed region. PMID:27325485

Structural basis for the recognition of guide RNA and target DNA heteroduplex by Argonaute.

PubMed

Miyoshi, Tomohiro; Ito, Kosuke; Murakami, Ryo; Uchiumi, Toshio

2016-06-21

Argonaute proteins are key players in the gene silencing mechanisms mediated by small nucleic acids in all domains of life from bacteria to eukaryotes. However, little is known about the Argonaute protein that recognizes guide RNA/target DNA. Here, we determine the 2 Å crystal structure of Rhodobacter sphaeroides Argonaute (RsAgo) in a complex with 18-nucleotide guide RNA and its complementary target DNA. The heteroduplex maintains Watson-Crick base-pairing even in the 3'-region of the guide RNA between the N-terminal and PIWI domains, suggesting a recognition mode by RsAgo for stable interaction with the target strand. In addition, the MID/PIWI interface of RsAgo has a system that specifically recognizes the 5' base-U of the guide RNA, and the duplex-recognition loop of the PAZ domain is important for the DNA silencing activity. Furthermore, we show that Argonaute discriminates the nucleic acid type (RNA/DNA) by recognition of the duplex structure of the seed region.

The Structure and Specificity of the Type III Secretion System Effector NleC Suggest a DNA Mimicry Mechanism of Substrate Recognition

PubMed Central

2015-01-01

Many pathogenic bacteria utilize the type III secretion system (T3SS) to translocate effector proteins directly into host cells, facilitating colonization. In enterohemmorhagic Escherichia coli (EHEC), a subset of T3SS effectors is essential for suppression of the inflammatory response in hosts, including humans. Identified as a zinc protease that cleaves NF-κB transcription factors, NleC is one such effector. Here, we investigate NleC substrate specificity, showing that four residues around the cleavage site in the DNA-binding loop of the NF-κB subunit RelA strongly influence the cleavage rate. Class I NF-κB subunit p50 is cleaved at a reduced rate consistent with conservation of only three of these four residues. However, peptides containing 10 residues on each side of the scissile bond were not efficiently cleaved by NleC, indicating that elements distal from the cleavage site are also important for substrate recognition. We present the crystal structure of NleC and show that it mimics DNA structurally and electrostatically. Consistent with this model, mutation of phosphate-mimicking residues in NleC reduces the level of RelA cleavage. We propose that global recognition of NF-κB subunits by DNA mimicry combined with a high sequence selectivity for the cleavage site results in exquisite NleC substrate specificity. The structure also shows that despite undetectable similarity of its sequence to those of other Zn2+ proteases beyond its conserved HExxH Zn2+-binding motif, NleC is a member of the Zincin protease superfamily, albeit divergent from its structural homologues. In particular, NleC displays a modified Ψ-loop motif that may be important for folding and refolding requirements implicit in T3SS translocation. PMID:25040221

TFBSshape: a motif database for DNA shape features of transcription factor binding sites.

PubMed

Yang, Lin; Zhou, Tianyin; Dror, Iris; Mathelier, Anthony; Wasserman, Wyeth W; Gordân, Raluca; Rohs, Remo

2014-01-01

Transcription factor binding sites (TFBSs) are most commonly characterized by the nucleotide preferences at each position of the DNA target. Whereas these sequence motifs are quite accurate descriptions of DNA binding specificities of transcription factors (TFs), proteins recognize DNA as a three-dimensional object. DNA structural features refine the description of TF binding specificities and provide mechanistic insights into protein-DNA recognition. Existing motif databases contain extensive nucleotide sequences identified in binding experiments based on their selection by a TF. To utilize DNA shape information when analysing the DNA binding specificities of TFs, we developed a new tool, the TFBSshape database (available at http://rohslab.cmb.usc.edu/TFBSshape/), for calculating DNA structural features from nucleotide sequences provided by motif databases. The TFBSshape database can be used to generate heat maps and quantitative data for DNA structural features (i.e., minor groove width, roll, propeller twist and helix twist) for 739 TF datasets from 23 different species derived from the motif databases JASPAR and UniPROBE. As demonstrated for the basic helix-loop-helix and homeodomain TF families, our TFBSshape database can be used to compare, qualitatively and quantitatively, the DNA binding specificities of closely related TFs and, thus, uncover differential DNA binding specificities that are not apparent from nucleotide sequence alone.

TFBSshape: a motif database for DNA shape features of transcription factor binding sites

PubMed Central

Yang, Lin; Zhou, Tianyin; Dror, Iris; Mathelier, Anthony; Wasserman, Wyeth W.; Gordân, Raluca; Rohs, Remo

2014-01-01

Transcription factor binding sites (TFBSs) are most commonly characterized by the nucleotide preferences at each position of the DNA target. Whereas these sequence motifs are quite accurate descriptions of DNA binding specificities of transcription factors (TFs), proteins recognize DNA as a three-dimensional object. DNA structural features refine the description of TF binding specificities and provide mechanistic insights into protein–DNA recognition. Existing motif databases contain extensive nucleotide sequences identified in binding experiments based on their selection by a TF. To utilize DNA shape information when analysing the DNA binding specificities of TFs, we developed a new tool, the TFBSshape database (available at http://rohslab.cmb.usc.edu/TFBSshape/), for calculating DNA structural features from nucleotide sequences provided by motif databases. The TFBSshape database can be used to generate heat maps and quantitative data for DNA structural features (i.e., minor groove width, roll, propeller twist and helix twist) for 739 TF datasets from 23 different species derived from the motif databases JASPAR and UniPROBE. As demonstrated for the basic helix-loop-helix and homeodomain TF families, our TFBSshape database can be used to compare, qualitatively and quantitatively, the DNA binding specificities of closely related TFs and, thus, uncover differential DNA binding specificities that are not apparent from nucleotide sequence alone. PMID:24214955

A surface-confined DNA assembly amplification strategy on DNA nanostructural scaffold for electrochemiluminescence biosensing.

PubMed

Feng, Qiu-Mei; Guo, Yue-Hua; Xu, Jing-Juan; Chen, Hong-Yuan

2018-02-15

A critical challenge in surface-based DNA assembly amplification is the reduced accessibility of DNA strands arranged on a heterogeneous surface compared to that in homogeneous solution. Here, a novel in situ surface-confined DNA assembly amplification electrochemiluminescence (ECL) biosensor based on DNA nanostructural scaffold was presented. In this design, a stem-loop structural DNA segment (Hairpin 1) was constructed on the vertex of DNA nanostructural scaffold as recognition probe. In the present of target DNA, the hairpin structure changed to rod-like through complementary hybridization with target DNA, resulting in the formation of Hairpin 1:target DNA. When the obtained Hairpin 1:target DNA met Hairpin 2 labeled with glucose oxidase (GOD), the DNA cyclic amplification was activated, releasing target DNA into homogeneous solution for the next recycling. Thus, the ECL signal of Ru(bpy) 3 2+ -TPrA system was quenched by H 2 O 2 , the product of GOD catalyzing glucose. As a result, this proposed method achieved a linear range response from 50 aM to 10 pM with lower detection limit of 20 aM. Copyright © 2017 Elsevier B.V. All rights reserved.

Indirect DNA Readout by an H-NS Related Protein: Structure of the DNA Complex of the C-Terminal Domain of Ler

PubMed Central

Cordeiro, Tiago N.; Schmidt, Holger; Madrid, Cristina; Juárez, Antonio; Bernadó, Pau; Griesinger, Christian; García, Jesús; Pons, Miquel

2011-01-01

Ler, a member of the H-NS protein family, is the master regulator of the LEE pathogenicity island in virulent Escherichia coli strains. Here, we determined the structure of a complex between the DNA-binding domain of Ler (CT-Ler) and a 15-mer DNA duplex. CT-Ler recognizes a preexisting structural pattern in the DNA minor groove formed by two consecutive regions which are narrower and wider, respectively, compared with standard B-DNA. The compressed region, associated with an AT-tract, is sensed by the side chain of Arg90, whose mutation abolishes the capacity of Ler to bind DNA. The expanded groove allows the approach of the loop in which Arg90 is located. This is the first report of an experimental structure of a DNA complex that includes a protein belonging to the H-NS family. The indirect readout mechanism not only explains the capacity of H-NS and other H-NS family members to modulate the expression of a large number of genes but also the origin of the specificity displayed by Ler. Our results point to a general mechanism by which horizontally acquired genes may be specifically recognized by members of the H-NS family. PMID:22114557

Indirect DNA readout by an H-NS related protein: structure of the DNA complex of the C-terminal domain of Ler.

PubMed

Cordeiro, Tiago N; Schmidt, Holger; Madrid, Cristina; Juárez, Antonio; Bernadó, Pau; Griesinger, Christian; García, Jesús; Pons, Miquel

2011-11-01

Ler, a member of the H-NS protein family, is the master regulator of the LEE pathogenicity island in virulent Escherichia coli strains. Here, we determined the structure of a complex between the DNA-binding domain of Ler (CT-Ler) and a 15-mer DNA duplex. CT-Ler recognizes a preexisting structural pattern in the DNA minor groove formed by two consecutive regions which are narrower and wider, respectively, compared with standard B-DNA. The compressed region, associated with an AT-tract, is sensed by the side chain of Arg90, whose mutation abolishes the capacity of Ler to bind DNA. The expanded groove allows the approach of the loop in which Arg90 is located. This is the first report of an experimental structure of a DNA complex that includes a protein belonging to the H-NS family. The indirect readout mechanism not only explains the capacity of H-NS and other H-NS family members to modulate the expression of a large number of genes but also the origin of the specificity displayed by Ler. Our results point to a general mechanism by which horizontally acquired genes may be specifically recognized by members of the H-NS family.

A strand graph semantics for DNA-based computation

PubMed Central

Petersen, Rasmus L.; Lakin, Matthew R.; Phillips, Andrew

2015-01-01

DNA nanotechnology is a promising approach for engineering computation at the nanoscale, with potential applications in biofabrication and intelligent nanomedicine. DNA strand displacement is a general strategy for implementing a broad range of nanoscale computations, including any computation that can be expressed as a chemical reaction network. Modelling and analysis of DNA strand displacement systems is an important part of the design process, prior to experimental realisation. As experimental techniques improve, it is important for modelling languages to keep pace with the complexity of structures that can be realised experimentally. In this paper we present a process calculus for modelling DNA strand displacement computations involving rich secondary structures, including DNA branches and loops. We prove that our calculus is also sufficiently expressive to model previous work on non-branching structures, and propose a mapping from our calculus to a canonical strand graph representation, in which vertices represent DNA strands, ordered sites represent domains, and edges between sites represent bonds between domains. We define interactions between strands by means of strand graph rewriting, and prove the correspondence between the process calculus and strand graph behaviours. Finally, we propose a mapping from strand graphs to an efficient implementation, which we use to perform modelling and simulation of DNA strand displacement systems with rich secondary structure. PMID:27293306

Topological Interaction by Entanglement of DNA

NASA Astrophysics Data System (ADS)

Feng, Lang; Sha, Ruojie; Seeman, Nadrian; Chaikin, Paul

2012-02-01

We find and study a new type of interaction between colloids, Topological Interaction by Entanglement of DNA (TIED), due to concatenation of loops formed by palindromic DNA. Consider a particle coated with palindromic DNA of sequence ``P1.'' Below the DNA hybridization temperature (Tm), loops of the self-complementary DNA form on the particle surface. Direct hybridization with similar particle covered with a different sequence P2 do not occur. However when particles are held together at T > Tm, then cooled to T < Tm, some of the loops entangle and link, similar to a Olympic Gel. We quantitatively observe and measure this topological interaction between colloids in a ˜5^o C temperature window, ˜6^o C lower than direct binding of complementary DNA with similar strength and introduce the concept of entanglement binding free energy. To prove our interaction to be topological, we unknot the purely entangled binding sites between colloids by adding Topoisomerase I which unconcatenates our loops. This research suggests novel history dependent ways of binding particles and serves as a new design tool in colloidal self-assembly.

RNA-Mediated cis Regulation in Acinetobacter baumannii Modulates Stress-Induced Phenotypic Variation

PubMed Central

Ching, Carly; Gozzi, Kevin; Heinemann, Björn; Chai, Yunrong

2017-01-01

ABSTRACT In the nosocomial opportunistic pathogen Acinetobacter baumannii, RecA-dependent mutagenesis, which causes antibiotic resistance acquisition, is linked to the DNA damage response (DDR). Notably, unlike the Escherichia coli paradigm, recA and DDR gene expression in A. baumannii is bimodal. Namely, there is phenotypic variation upon DNA damage, which may provide a bet-hedging strategy for survival. Thus, understanding recA gene regulation is key to elucidate the yet unknown DDR regulation in A. baumannii. Here, we identify a structured 5′ untranslated region (UTR) in the recA transcript which serves as a cis-regulatory element. We show that a predicted stem-loop structure in this 5′ UTR affects mRNA half-life and underlies bimodal gene expression and thus phenotypic variation in response to ciprofloxacin treatment. We furthermore show that the stem-loop structure of the recA 5′ UTR influences intracellular RecA protein levels and, in vivo, impairing the formation of the stem-loop structure of the recA 5′ UTR lowers cell survival of UV treatment and decreases rifampin resistance acquisition from DNA damage-induced mutagenesis. We hypothesize that the 5′ UTR allows for stable recA transcripts during stress, including antibiotic treatment, enabling cells to maintain suitable RecA levels for survival. This innovative strategy to regulate the DDR in A. baumannii may contribute to its success as a pathogen. IMPORTANCE Acinetobacter baumannii is an opportunistic pathogen quickly gaining antibiotic resistances. Mutagenesis and antibiotic resistance acquisition are linked to the DNA damage response (DDR). However, how the DDR is regulated in A. baumannii remains unknown, since unlike most bacteria, A. baumannii does not follow the regulation of the Escherichia coli paradigm. In this study, we have started to uncover the mechanisms regulating the novel A. baumannii DDR. We have found that a cis-acting 5′ UTR regulates recA transcript stability, RecA protein levels, and DNA damage-induced phenotypic variation. Though 5′ UTRs are known to provide stability to transcripts in bacteria, this is the first example in which it regulates a bimodal DDR response through recA transcript stabilization, potentially enabling cells to have enough RecA for survival and genetic variability. PMID:28320880

Stabilised DNA secondary structures with increasing transcription localise hypermutable bases for somatic hypermutation in IGHV3-23.

PubMed

Duvvuri, Bhargavi; Duvvuri, Venkata R; Wu, Jianhong; Wu, Gillian E

2012-07-01

Somatic hypermutation (SHM) mediated by activation-induced cytidine deaminase (AID) is a transcription-coupled mechanism most responsible for generating high affinity antibodies. An issue remaining enigmatic in SHM is how AID is preferentially targeted during transcription to hypermutable bases in its substrates (WRC motifs) on both DNA strands. AID targets only single stranded DNA. By modelling the dynamical behaviour of IGHV3-23 DNA, a commonly used human variable gene segment, we observed that hypermutable bases on the non-transcribed strand are paired whereas those on transcribed strand are mostly unpaired. Hypermutable bases (both paired and unpaired) are made accessible to AID in stabilised secondary structures formed with increasing transcription levels. This observation provides a rationale for the hypermutable bases on both the strands of DNA being targeted to a similar extent despite having differences in unpairedness. We propose that increasing transcription and RNAP II stalling resulting in the formation and stabilisation of stem-loop structures with AID hotspots in negatively supercoiled region can localise the hypermutable bases of both strands of DNA, to AID-mediated SHM.

Transcription of telomeric DNA leads to high levels of homologous recombination and t-loops.

PubMed

Kar, Anirban; Willcox, Smaranda; Griffith, Jack D

2016-11-02

The formation of DNA loops at chromosome ends (t-loops) and the transcription of telomeres producing G-rich RNA (TERRA) represent two central features of telomeres. To explore a possible link between them we employed artificial human telomeres containing long arrays of TTAGGG repeats flanked by the T7 or T3 promoters. Transcription of these DNAs generates a high frequency of t-loops within individual molecules and homologous recombination events between different DNAs at their telomeric sequences. T-loop formation does not require a single strand overhang, arguing that both terminal strands insert into the preceding duplex. The loops are very stable and some RNase H resistant TERRA remains at the t-loop, likely adding to their stability. Transcription of DNAs containing TTAGTG or TGAGTG repeats showed greatly reduced loop formation. While in the cell multiple pathways may lead to t-loop formation, the pathway revealed here does not depend on the shelterins but rather on the unique character of telomeric DNA when it is opened for transcription. Hence, telomeric sequences may have evolved to facilitate their ability to loop back on themselves. © The Author(s) 2016. Published by Oxford University Press on behalf of Nucleic Acids Research.

Thermal stability of DNA quadruplex-duplex hybrids.

PubMed

Lim, Kah Wai; Khong, Zi Jian; Phan, Anh Tuân

2014-01-14

DNA has the capacity to adopt several distinct structural forms, such as duplex and quadruplex helices, which have been implicated in cellular processes and shown to exhibit important functional properties. Quadruplex-duplex hybrids, generated from the juxtaposition of these two structural elements, could find applications in therapeutics and nanotechnology. Here we used NMR and CD spectroscopy to investigate the thermal stability of two classes of quadruplex-duplex hybrids comprising fundamentally distinct modes of duplex and quadruplex connectivity: Construct I involves the coaxial orientation of the duplex and quadruplex helices with continual base stacking across the two components; Construct II involves the orthogonal orientation of the duplex and quadruplex helices with no base stacking between the two components. We have found that for both constructs, the stability of the quadruplex generally increases with the length of the stem-loop incorporated, with respect to quadruplexes comprising nonstructured loops of the same length, which showed a continuous drop in stability with increasing loop length. The stability of these complexes, particularly Construct I, can be substantially influenced by the base-pair steps proximal to the quadruplex-duplex junction. Bulges at the junction are largely detrimental to the adoption of the desired G-quadruplex topology for Construct I but not for Construct II. These findings should facilitate future design and prediction of quadruplex-duplex hybrids.

Characterizing DNA Star-Tile-Based Nanostructures Using a Coarse-Grained Model.

PubMed

Schreck, John S; Romano, Flavio; Zimmer, Matthew H; Louis, Ard A; Doye, Jonathan P K

2016-04-26

We use oxDNA, a coarse-grained model of DNA at the nucleotide level, to simulate large nanoprisms that are composed of multi-arm star tiles, in which the size of bulge loops that have been incorporated into the tile design is used to control the flexibility of the tiles. The oxDNA model predicts equilibrium structures for several different nanoprism designs that are in excellent agreement with the experimental structures as measured by cryoTEM. In particular we reproduce the chiral twisting of the top and bottom faces of the nanoprisms, as the bulge sizes in these structures are varied due to the greater flexibility of larger bulges. We are also able to follow how the properties of the star tiles evolve as the prisms are assembled. Individual star tiles are very flexible, but their structures become increasingly well-defined and rigid as they are incorporated into larger assemblies. oxDNA also finds that the experimentally observed prisms are more stable than their inverted counterparts, but interestingly this preference for the arms of the tiles to bend in a given direction only emerges after they are part of larger assemblies. These results show the potential for oxDNA to provide detailed structural insight as well as to predict the properties of DNA nanostructures and hence to aid rational design in DNA nanotechnology.

Phylogeography and Sex-Biased Dispersal across Riverine Manatee Populations (Trichechus inunguis and Trichechus manatus) in South America

PubMed Central

Satizábal, Paula; Mignucci-Giannoni, Antonio A.; Duchêne, Sebastián; Caicedo-Herrera, Dalila; Perea-Sicchar, Carlos M.; García-Dávila, Carmen R.; Trujillo, Fernando; Caballero, Susana J.

2012-01-01

Phylogeographic patterns and sex-biased dispersal were studied in riverine populations of West Indian (Trichechus manatus) and Amazonian manatees (T. inunguis) in South America, using 410bp D-loop (Control Region, Mitochondrial DNA) sequences and 15 nuclear microsatellite loci. This multi-locus approach was key to disentangle complex patterns of gene flow among populations. D-loop analyses revealed population structuring among all Colombian rivers for T. manatus, while microsatellite data suggested no structure. Two main populations of T. inunguis separating the Colombian and Peruvian Amazon were supported by analysis of the D-loop and microsatellite data. Overall, we provide molecular evidence for differences in dispersal patterns between sexes, demonstrating male-biased gene flow dispersal in riverine manatees. These results are in contrast with previously reported levels of population structure shown by microsatellite data in marine manatee populations, revealing low habitat restrictions to gene flow in riverine habitats, and more significant dispersal limitations for males in marine environments. PMID:23285054

Phylogeography and sex-biased dispersal across riverine manatee populations (Trichechus inunguis and Trichechus manatus) in South America.

PubMed

Satizábal, Paula; Mignucci-Giannoni, Antonio A; Duchêne, Sebastián; Caicedo-Herrera, Dalila; Perea-Sicchar, Carlos M; García-Dávila, Carmen R; Trujillo, Fernando; Caballero, Susana J

2012-01-01

Phylogeographic patterns and sex-biased dispersal were studied in riverine populations of West Indian (Trichechus manatus) and Amazonian manatees (T. inunguis) in South America, using 410bp D-loop (Control Region, Mitochondrial DNA) sequences and 15 nuclear microsatellite loci. This multi-locus approach was key to disentangle complex patterns of gene flow among populations. D-loop analyses revealed population structuring among all Colombian rivers for T. manatus, while microsatellite data suggested no structure. Two main populations of T. inunguis separating the Colombian and Peruvian Amazon were supported by analysis of the D-loop and microsatellite data. Overall, we provide molecular evidence for differences in dispersal patterns between sexes, demonstrating male-biased gene flow dispersal in riverine manatees. These results are in contrast with previously reported levels of population structure shown by microsatellite data in marine manatee populations, revealing low habitat restrictions to gene flow in riverine habitats, and more significant dispersal limitations for males in marine environments.

Biochemical studies of the Saccharomyces cerevisiae Mph1 helicase on junction-containing DNA structures

PubMed Central

Kang, Young-Hoon; Munashingha, Palinda Ruvan; Lee, Chul-Hwan; Nguyen, Tuan Anh; Seo, Yeon-Soo

2012-01-01

Saccharomyces cerevisiae Mph1 is a 3–5′ DNA helicase, required for the maintenance of genome integrity. In order to understand the ATPase/helicase role of Mph1 in genome stability, we characterized its helicase activity with a variety of DNA substrates, focusing on its action on junction structures containing three or four DNA strands. Consistent with its 3′ to 5′ directionality, Mph1 displaced 3′-flap substrates in double-fixed or equilibrating flap substrates. Surprisingly, Mph1 displaced the 5′-flap strand more efficiently than the 3′ flap strand from double-flap substrates, which is not expected for a 3–5′ DNA helicase. For this to occur, Mph1 required a threshold size (>5 nt) of 5′ single-stranded DNA flap. Based on the unique substrate requirements of Mph1 defined in this study, we propose that the helicase/ATPase activity of Mph1 play roles in converting multiple-stranded DNA structures into structures cleavable by processing enzymes such as Fen1. We also found that the helicase activity of Mph1 was used to cause structural alterations required for restoration of replication forks stalled due to damaged template. The helicase properties of Mph1 reported here could explain how it resolves D-loop structure, and are in keeping with a model proposed for the error-free damage avoidance pathway. PMID:22090425

Single-molecule analysis of DNA cross-links using nanopore technology

NASA Astrophysics Data System (ADS)

Wolna, Anna H.

The alpha-hemolysin (alpha-HL) protein ion channel is a potential next-generation sequencing platform that has been extensively used to study nucleic acids at a single-molecule level. After applying a potential across a lipid bilayer, the imbedded alpha-HL allows monitoring of the duration and current levels of DNA translocation and immobilization. Because this method does not require DNA amplification prior to sequencing, all the DNA damage present in the cell at any given time will be present during the sequencing experiment. The goal of this research is to determine if these damage sites give distinguishable current levels beyond those observed for the canonical nucleobases. Because DNA cross-links are one of the most prevalent types of DNA damage occurring in vivo, the blockage current levels were determined for thymine-dimers, guanine(C8)-thymine(N3) cross-links and platinum adducts. All of these cross-links give a different blockage current level compared to the undamaged strands when immobilized in the ion channel, and they all can easily translocate across the alpha-HL channel. Additionally, the alpha-HL nanopore technique presents a unique opportunity to study the effects of DNA cross-links, such as thymine-dimers, on the secondary structure of DNA G-quadruplexes folded from the human telomere sequence. Using this single-molecule nanopore technique we can detect subtle structural differences that cannot be easily addressed using conventional methods. The human telomere plays crucial roles in maintaining genome stability. In the presence of suitable cations, the repetitive 5'-TTAGGG human telomere sequence can fold into G-quadruplexes that adopt the hybrid fold in vivo. The telomere sequence is hypersensitive to UV-induced thymine-dimer (T=T) formation, and yet the presence of thymine dimers does not cause telomere shortening. The potential structural disruption and thermodynamic stability of the T=T-containing natural telomere sequences were studied to understand how this damage is tolerated in telomeric DNA. The alpha-HL experiments determined that T=Ts disrupt double-chain reversal loop formation but are well tolerated in edgewise and diagonal loops of the hybrid G-quadruplexes. These studies demonstrated the power of the alpha-HL ion channel to analyze DNA modifications and secondary structures at a single-molecule level.

Structural Determination of Functional Domains in Early B-cell Factor (EBF) Family of Transcription Factors Reveals Similarities to Rel DNA-binding Proteins and a Novel Dimerization Motif*

PubMed Central

Siponen, Marina I.; Wisniewska, Magdalena; Lehtiö, Lari; Johansson, Ida; Svensson, Linda; Raszewski, Grzegorz; Nilsson, Lennart; Sigvardsson, Mikael; Berglund, Helena

2010-01-01

The early B-cell factor (EBF) transcription factors are central regulators of development in several organs and tissues. This protein family shows low sequence similarity to other protein families, which is why structural information for the functional domains of these proteins is crucial to understand their biochemical features. We have used a modular approach to determine the crystal structures of the structured domains in the EBF family. The DNA binding domain reveals a striking resemblance to the DNA binding domains of the Rel homology superfamily of transcription factors but contains a unique zinc binding structure, termed zinc knuckle. Further the EBF proteins contain an IPT/TIG domain and an atypical helix-loop-helix domain with a novel type of dimerization motif. The data presented here provide insights into unique structural features of the EBF proteins and open possibilities for detailed molecular investigations of this important transcription factor family. PMID:20592035

Determining the folding and binding free energy of DNA-based nanodevices and nanoswitches using urea titration curves

PubMed Central

Idili, Andrea

2017-01-01

Abstract DNA nanotechnology takes advantage of the predictability of DNA interactions to build complex DNA-based functional nanoscale structures. However, when DNA functional and responsive units that are based on non-canonical DNA interactions are employed it becomes quite challenging to predict, understand and control their thermodynamics. In response to this limitation, here we demonstrate the use of isothermal urea titration experiments to estimate the free energy involved in a set of DNA-based systems ranging from unimolecular DNA-based nanoswitches to more complex DNA folds (e.g. aptamers) and nanodevices. We propose here a set of fitting equations that allow to analyze the urea titration curves of these DNA responsive units based on Watson–Crick and non-canonical interactions (stem-loop, G-quadruplex, triplex structures) and to correctly estimate their relative folding and binding free energy values under different experimental conditions. The results described herein will pave the way toward the use of urea titration experiments in the field of DNA nanotechnology to achieve easier and more reliable thermodynamic characterization of DNA-based functional responsive units. More generally, our results will be of general utility to characterize other complex supramolecular systems based on different biopolymers. PMID:28605461

Molecular dynamics and mutational analysis of the catalytic and translocation cycle of RNA polymerase

PubMed Central

2012-01-01

Background During elongation, multi-subunit RNA polymerases (RNAPs) cycle between phosphodiester bond formation and nucleic acid translocation. In the conformation associated with catalysis, the mobile “trigger loop” of the catalytic subunit closes on the nucleoside triphosphate (NTP) substrate. Closing of the trigger loop is expected to exclude water from the active site, and dehydration may contribute to catalysis and fidelity. In the absence of a NTP substrate in the active site, the trigger loop opens, which may enable translocation. Another notable structural element of the RNAP catalytic center is the “bridge helix” that separates the active site from downstream DNA. The bridge helix may participate in translocation by bending against the RNA/DNA hybrid to induce RNAP forward movement and to vacate the active site for the next NTP loading. The transition between catalytic and translocation conformations of RNAP is not evident from static crystallographic snapshots in which macromolecular motions may be restrained by crystal packing. Results All atom molecular dynamics simulations of Thermus thermophilus (Tt) RNAP reveal flexible hinges, located within the two helices at the base of the trigger loop, and two glycine hinges clustered near the N-terminal end of the bridge helix. As simulation progresses, these hinges adopt distinct conformations in the closed and open trigger loop structures. A number of residues (described as “switch” residues) trade atomic contacts (ion pairs or hydrogen bonds) in response to changes in hinge orientation. In vivo phenotypes and in vitro activities rendered by mutations in the hinge and switch residues in Saccharomyces cerevisiae (Sc) RNAP II support the importance of conformational changes predicted from simulations in catalysis and translocation. During simulation, the elongation complex with an open trigger loop spontaneously translocates forward relative to the elongation complex with a closed trigger loop. Conclusions Switching between catalytic and translocating RNAP forms involves closing and opening of the trigger loop and long-range conformational changes in the atomic contacts of amino acid side chains, some located at a considerable distance from the trigger loop and active site. Trigger loop closing appears to support chemistry and the fidelity of RNA synthesis. Trigger loop opening and limited bridge helix bending appears to promote forward nucleic acid translocation. PMID:22676913

To peep into Pif1 helicase: multifaceted all the way from genome stability to repair-associated DNA synthesis.

PubMed

Chung, Woo-Hyun

2014-02-01

Pif1 DNA helicase is the prototypical member of a 5' to 3' helicase superfamily conserved from bacteria to humans. In Saccharomyces cerevisiae, Pif1 and its homologue Rrm3, localize in both mitochondria and nucleus playing multiple roles in the maintenance of genomic homeostasis. They display relatively weak processivities in vitro, but have largely non-overlapping functions on common genomic loci such as mitochondrial DNA, telomeric ends, and many replication forks especially at hard-to-replicate regions including ribosomal DNA and G-quadruplex structures. Recently, emerging evidence shows that Pif1, but not Rrm3, has a significant new role in repair-associated DNA synthesis with Polδ during homologous recombination stimulating D-loop migration for conservative DNA replication. Comparative genetic and biochemical studies on the structure and function of Pif1 family helicases across different biological systems are further needed to elucidate both diversity and specificity of their mechanisms of action that contribute to genome stability.

Structural and Dynamical Characterization of DNA and RNA Quadruplexes Obtained from the GGGGCC and GGGCCT Hexanucleotide Repeats Associated with C9FTD/ALS and SCA36 Diseases.

PubMed

Zhang, Yuan; Roland, Christopher; Sagui, Celeste

2018-05-16

A (GGGGCC) hexanucleotide repeat (HR) expansion in the C9ORF72 gene has been considered the major cause behind both frontotemporal dementia and amyotrophic lateral sclerosis, while a (GGGCCT) is associated with spinocerebellar ataxia 36. Recent experiments involving NMR, CD, optical melting and 1D 1 H NMR spectroscopy, suggest that the r(GGGGCC) HR can adopt a hairpin structure with G-G mismatches in equilibrium with a G-quadruplex structure. G-Quadruplexes have also been identified for d(GGGGCC). As these experiments lack molecular resolution, we have used molecular dynamics microsecond simulations to obtain a structural characterization of the G-quadruplexes associated with both HRs. All DNA G-quadruplexes, parallel or antiparallel, with or without loops are stable, while only parallel and one antiparallel (stabilized by diagonal loops) RNA G-quadruplexes are stable. It is known that antiparallel G-quadruplexes require alternating guanines to be in a syn conformation that is hindered by the C3'-endo pucker preferred by RNA. Initial RNA antiparallel quadruplexes built with C2'-endo sugars evolve such that the transition (C2'-endo)-to-(C3'-endo) triggers unwinding and buckling of the flat G-tetrads, resulting in the unfolding of the RNA antiparallel quadruplex. Finally, a parallel G-quadruplex stabilizes an adjacent C-tetrad in both DNA and RNA (thus effectively becoming a mixed quadruplex of 5 layers). The C-tetrad is stabilized by the stacking interactions with the preceding G-tetrad, by cyclical hydrogen bonds C(N4)-(O2), and by an ion between the G-tetrad and the C-tetrad. In addition, antiparallel DNA G-quadruplexes also stabilize flat C-layers at the ends of the quadruplexes.

A new photoelectrochemical biosensors based on DNA conformational changes and isothermal circular strand-displacement polymerization reaction.

PubMed

Zhang, Xiaoru; Xu, Yunpeng; Zhao, Yanqing; Song, Weiling

2013-01-15

We report a strategy for the transduction of DNA hybridization into a readily detectable photoelectrochemical signal by means of a conformational change analogous to electrochemical DNA (E-DNA) approach. To demonstrate the effect of distance change for photosensitizer to the surface of electrode on the change of photocurrent, photosensitizer Ru(bpy)(2)(dcbpy)(2+) tagged DNA stem-loop structures were self-assembled onto a nanogold modified ITO electrode. Hybridization induced a large conformational change in DNA structure, which in turn significantly altered the electron-transfer tunneling distance between the electrode and photosensitizer. The resulting change in photocurrent was proportional to the concentration of DNA in the range of 1.0×10(-10)-8.0×10(-9)M. In order to improve the sensitivity of the photoelectrochemical biosensor, an amplified detection method based on isothermal strand displacement polymerization reaction was employed. With multiple rounds of isothermal strand replication, which led to strand displacement and constituted consecutive signal amplification, a detection limit of 9.4×10(-14)M target DNA was achieved. Copyright © 2012 Elsevier B.V. All rights reserved.

Highly sensitive self-complementary DNA nanoswitches triggered by polyelectrolytes.

PubMed

Wu, Jincai; Yu, Feng; Zhang, Zheng; Chen, Yong; Du, Jie; Maruyama, Atsushi

2016-01-07

Dimerization of two homologous strands of genomic DNA/RNA is an essential feature of retroviral replication. Herein we show that a cationic comb-type copolymer (CCC), poly(L-lysine)-graft-dextran, accelerates the dimerization of self-complementary stem-loop DNA, frequently found in functional DNA/RNA molecules, such as aptamers. Furthermore, an anionic polymer poly(sodium vinylsulfonate) (PVS) dissociates CCC from the duplex shortly within a few seconds. Then single stem-loop DNA spontaneously transforms from its dimer. Thus we can easily control the dimer and stem-loop DNA by switching on/off CCC activity. Both polyelectrolytes and DNA concentrations are in the nanomole per liter range. The polyelectrolyte-assisted transconformation and sequences design strategy ensures the reversible state control with rapid response and effective switching under physiologically relevant conditions. A further application of this sensitive assembly is to construct an aptamer-type drug delivery system, bind or release functional molecules responding to its transconformation.

Simulations of Living Cell Origins Using a Cellular Automata Model

NASA Astrophysics Data System (ADS)

Ishida, Takeshi

2014-04-01

Understanding the generalized mechanisms of cell self-assembly is fundamental for applications in various fields, such as mass producing molecular machines in nanotechnology. Thus, the details of real cellular reaction networks and the necessary conditions for self-organized cells must be elucidated. We constructed a 2-dimensional cellular automata model to investigate the emergence of biological cell formation, which incorporated a looped membrane and a membrane-bound information system (akin to a genetic code and gene expression system). In particular, with an artificial reaction system coupled with a thermal system, the simultaneous formation of a looped membrane and an inner reaction process resulted in a more stable structure. These double structures inspired the primitive biological cell formation process from chemical evolution stage. With a model to simulate cellular self-organization in a 2-dimensional cellular automata model, 3 phenomena could be realized: (1) an inner reaction system developed as an information carrier precursor (akin to DNA); (2) a cell border emerged (akin to a cell membrane); and (3) these cell structures could divide into 2. This double-structured cell was considered to be a primary biological cell. The outer loop evolved toward a lipid bilayer membrane, and inner polymeric particles evolved toward precursor information carriers (evolved toward DNA). This model did not completely clarify all the necessary and sufficient conditions for biological cell self-organization. Further, our virtual cells remained unstable and fragile. However, the "garbage bag model" of Dyson proposed that the first living cells were deficient; thus, it would be reasonable that the earliest cells were more unstable and fragile than the simplest current unicellular organisms.

Simulations of living cell origins using a cellular automata model.

PubMed

Ishida, Takeshi

2014-04-01

Understanding the generalized mechanisms of cell self-assembly is fundamental for applications in various fields, such as mass producing molecular machines in nanotechnology. Thus, the details of real cellular reaction networks and the necessary conditions for self-organized cells must be elucidated. We constructed a 2-dimensional cellular automata model to investigate the emergence of biological cell formation, which incorporated a looped membrane and a membrane-bound information system (akin to a genetic code and gene expression system). In particular, with an artificial reaction system coupled with a thermal system, the simultaneous formation of a looped membrane and an inner reaction process resulted in a more stable structure. These double structures inspired the primitive biological cell formation process from chemical evolution stage. With a model to simulate cellular self-organization in a 2-dimensional cellular automata model, 3 phenomena could be realized: (1) an inner reaction system developed as an information carrier precursor (akin to DNA); (2) a cell border emerged (akin to a cell membrane); and (3) these cell structures could divide into 2. This double-structured cell was considered to be a primary biological cell. The outer loop evolved toward a lipid bilayer membrane, and inner polymeric particles evolved toward precursor information carriers (evolved toward DNA). This model did not completely clarify all the necessary and sufficient conditions for biological cell self-organization. Further, our virtual cells remained unstable and fragile. However, the "garbage bag model" of Dyson proposed that the first living cells were deficient; thus, it would be reasonable that the earliest cells were more unstable and fragile than the simplest current unicellular organisms.

Interactions of DNA binding proteins with G-Quadruplex structures at the single molecule level

NASA Astrophysics Data System (ADS)

Ray, Sujay

Guanine-rich nucleic acid (DNA/RNA) sequences can form non-canonical secondary structures, known as G-quadruplex (GQ). Numerous in vivo and in vitro studies have demonstrated formation of these structures in telomeric and non-telomeric regions of the genome. Telomeric GQs protect the chromosome ends whereas non-telomeric GQs either act as road blocks or recognition sites for DNA metabolic machinery. These observations suggest the significance of these structures in regulation of different metabolic processes, such as replication and repair. GQs are typically thermodynamically more stable than the corresponding Watson-Crick base pairing formed by G-rich and C-rich strands, making protein activity a crucial factor for their destabilization. Inside the cell, GQs interact with different proteins and their enzymatic activity is the determining factor for their stability. We studied interactions of several proteins with GQs to understand the underlying principles of protein-GQ interactions using single-molecule FRET and other biophysical techniques. Replication Protein-A (RPA), a single stranded DNA (ssDNA) binding protein, is known to posses GQ unfolding activity. First, we compared the thermal stability of three potentially GQ-forming DNA sequences (PQS) to their stability against RPA-mediated unfolding. One of these sequences is the human telomeric repeat and the other two, located in the promoter region of tyrosine hydroxylase gene, are highly heterogeneous sequences that better represent PQS in the genome. The thermal stability of these structures do not necessarily correlate with their stability against protein-mediated unfolding. We conclude that thermal stability is not necessarily an adequate criterion for predicting the physiological viability of GQ structures. To determine the critical structural factors that influence protein-GQ interactions we studied two groups of GQ structures that have systematically varying loop lengths and number of G-tetrad layers. We observed a linear increase in the steady-state stability of the GQ against RPA-mediated unfolding with increasing number of layers or decreasing loop length. The stability demonstrated by different GQ structures varied by at least three orders of magnitude. Finally, we studied another protein-GQ system where a protein complex works synergistically with a GQ to suppress DNA damage signals by preventing RPA to bind to telomeric DNA. Human telomeres that terminate with a single-stranded 3' G-overhang can be recognized as a DNA damage site by RPA. The protection of telomere-1 (POT1) and POT1-interacting protein (TPP1) heterodimer, binds specifically to telomeric DNA and protects it against RPA binding. Using model telomeric DNA, we studied the competition between POT1/TPP1 and RPA to access telomeric GQs in vitro. Under physiological salt and pH conditions, POT1/TPP1 stably load to a minimal DNA sequence adjacent to a folded GQ and unfolds the anti-parallel GQ as the parallel conformation remains folded. We showed that GQ formation of telomeres enhances the ability of POT1/TPP1 to block RPA's access to telomeres by two orders of magnitude and contributes to suppress DNA damage signals.

Computational prediction of CTCF/cohesin-based intra-TAD loops that insulate chromatin contacts and gene expression in mouse liver

PubMed Central

2018-01-01

CTCF and cohesin are key drivers of 3D-nuclear organization, anchoring the megabase-scale Topologically Associating Domains (TADs) that segment the genome. Here, we present and validate a computational method to predict cohesin-and-CTCF binding sites that form intra-TAD DNA loops. The intra-TAD loop anchors identified are structurally indistinguishable from TAD anchors regarding binding partners, sequence conservation, and resistance to cohesin knockdown; further, the intra-TAD loops retain key functional features of TADs, including chromatin contact insulation, blockage of repressive histone mark spread, and ubiquity across tissues. We propose that intra-TAD loops form by the same loop extrusion mechanism as the larger TAD loops, and that their shorter length enables finer regulatory control in restricting enhancer-promoter interactions, which enables selective, high-level expression of gene targets of super-enhancers and genes located within repressive nuclear compartments. These findings elucidate the role of intra-TAD cohesin-and-CTCF binding in nuclear organization associated with widespread insulation of distal enhancer activity. PMID:29757144

Computational prediction of CTCF/cohesin-based intra-TAD loops that insulate chromatin contacts and gene expression in mouse liver.

PubMed

Matthews, Bryan J; Waxman, David J

2018-05-14

CTCF and cohesin are key drivers of 3D-nuclear organization, anchoring the megabase-scale Topologically Associating Domains (TADs) that segment the genome. Here, we present and validate a computational method to predict cohesin-and-CTCF binding sites that form intra-TAD DNA loops. The intra-TAD loop anchors identified are structurally indistinguishable from TAD anchors regarding binding partners, sequence conservation, and resistance to cohesin knockdown; further, the intra-TAD loops retain key functional features of TADs, including chromatin contact insulation, blockage of repressive histone mark spread, and ubiquity across tissues. We propose that intra-TAD loops form by the same loop extrusion mechanism as the larger TAD loops, and that their shorter length enables finer regulatory control in restricting enhancer-promoter interactions, which enables selective, high-level expression of gene targets of super-enhancers and genes located within repressive nuclear compartments. These findings elucidate the role of intra-TAD cohesin-and-CTCF binding in nuclear organization associated with widespread insulation of distal enhancer activity. © 2018, Matthews et al.

In the loop: how chromatin topology links genome structure to function in mechanisms underlying learning and memory.

PubMed

Watson, L Ashley; Tsai, Li-Huei

2017-04-01

Different aspects of learning, memory, and cognition are regulated by epigenetic mechanisms such as covalent DNA modifications and histone post-translational modifications. More recently, the modulation of chromatin architecture and nuclear organization is emerging as a key factor in dynamic transcriptional regulation of the post-mitotic neuron. For instance, neuronal activity induces relocalization of gene loci to 'transcription factories', and specific enhancer-promoter looping contacts allow for precise transcriptional regulation. Moreover, neuronal activity-dependent DNA double-strand break formation in the promoter of immediate early genes appears to overcome topological constraints on transcription. Together, these findings point to a critical role for genome topology in integrating dynamic environmental signals to define precise spatiotemporal gene expression programs supporting cognitive processes. Copyright © 2016 Elsevier Ltd. All rights reserved.

The biology and polymer physics underlying large‐scale chromosome organization

PubMed Central

2017-01-01

Chromosome large‐scale organization is a beautiful example of the interplay between physics and biology. DNA molecules are polymers and thus belong to the class of molecules for which physicists have developed models and formulated testable hypotheses to understand their arrangement and dynamic properties in solution, based on the principles of polymer physics. Biologists documented and discovered the biochemical basis for the structure, function and dynamic spatial organization of chromosomes in cells. The underlying principles of chromosome organization have recently been revealed in unprecedented detail using high‐resolution chromosome capture technology that can simultaneously detect chromosome contact sites throughout the genome. These independent lines of investigation have now converged on a model in which DNA loops, generated by the loop extrusion mechanism, are the basic organizational and functional units of the chromosome. PMID:29105235

Discrimination of Single Base Pair Differences Among Individual DNA Molecules Using a Nanopore

NASA Technical Reports Server (NTRS)

Vercoutere, Wenonah; DeGuzman, Veronica

2003-01-01

The protein toxin alpha-hemolysin form nanometer scale channels across lipid membranes. Our lab uses a single channel in an artificial lipid bilayer in a patch clamp device to capture and examine individual DNA molecules. This nanopore detector used with a support vector machine (SVM) can analyze DNA hairpin molecules on the millisecond time scale. We distinguish duplex stem length, base pair mismatches, loop length, and single base pair differences. The residual current fluxes also reveal structural molecular dynamics elements. DNA end-fraying (terminal base pair dissociation) can be observed as near full blockades, or spikes, in current. This technique can be used to investigate other biological processes dependent on DNA end-fraying, such as the processing of HIV DNA by HIV integrase.

C9orf72 Nucleotide Repeat Structures Initiate Molecular Cascades of Disease

PubMed Central

Haeusler, Aaron R.; Donnelly, Christopher J.; Periz, Goran; Simko, Eric A.J.; Shaw, Patrick G.; Kim, Min-Sik; Maragakis, Nicholas J.; Troncoso, Juan C.; Pandey, Akhilesh; Sattler, Rita; Rothstein, Jeffrey D.; Wang, Jiou

2014-01-01

Summary A hexanucleotide repeat expansion (HRE), (GGGGCC)n, in C9orf72 is the most common genetic cause of the neurodegenerative diseases amyotrophic lateral sclerosis (ALS) and frontotemporal dementia (FTD). Here we identify a molecular mechanism by which structural polymorphism of the HRE leads to ALS/FTD pathology and defects. The HRE forms DNA and RNA G-quadruplexes with distinct structures and promotes RNA•DNA hybrids (R-loops). The structural polymorphism causes a repeat length-dependent accumulation of transcripts aborted in the HRE region. These transcribed repeats bind to ribonucleoproteins in a conformationdependent manner. Specifically, nucleolin (NCL), an essential nucleolar protein, preferentially binds the HRE G-quadruplex, and patient cells show evidence of nucleolar stress. Our results demonstrate that distinct C9orf72 HRE structural polymorphism at both DNA and RNA levels initiates molecular cascades leading to ALS/FTD pathologies, and provide the basis for a mechanistic model for repeat-associated neurodegenerative diseases. PMID:24598541

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

Regulated Formation of lncRNA-DNA Hybrids Enables Faster Transcriptional Induction and Environmental Adaptation.

PubMed

Cloutier, Sara C; Wang, Siwen; Ma, Wai Kit; Al Husini, Nadra; Dhoondia, Zuzer; Ansari, Athar; Pascuzzi, Pete E; Tran, Elizabeth J

2016-02-04

Long non-coding (lnc)RNAs, once thought to merely represent noise from imprecise transcription initiation, have now emerged as major regulatory entities in all eukaryotes. In contrast to the rapidly expanding identification of individual lncRNAs, mechanistic characterization has lagged behind. Here we provide evidence that the GAL lncRNAs in the budding yeast S. cerevisiae promote transcriptional induction in trans by formation of lncRNA-DNA hybrids or R-loops. The evolutionarily conserved RNA helicase Dbp2 regulates formation of these R-loops as genomic deletion or nuclear depletion results in accumulation of these structures across the GAL cluster gene promoters and coding regions. Enhanced transcriptional induction is manifested by lncRNA-dependent displacement of the Cyc8 co-repressor and subsequent gene looping, suggesting that these lncRNAs promote induction by altering chromatin architecture. Moreover, the GAL lncRNAs confer a competitive fitness advantage to yeast cells because expression of these non-coding molecules correlates with faster adaptation in response to an environmental switch. Copyright © 2016 Elsevier Inc. All rights reserved.

DNA-binding mechanism of the Escherichia coli Ada O6-alkylguanine–DNA alkyltransferase

PubMed Central

Verdemato, Philip E.; Brannigan, James A.; Damblon, Christian; Zuccotto, Fabio; Moody, Peter C. E.; Lian, Lu-Yun

2000-01-01

The C-terminal domain of the Escherichia coli Ada protein (Ada-C) aids in the maintenance of genomic integrity by efficiently repairing pre-mutagenic O6-alkylguanine lesions in DNA. Structural and thermodynamic studies were carried out to obtain a model of the DNA-binding process. Nuclear magnetic resonance (NMR) studies map the DNA-binding site to helix 5, and a loop region (residues 151–160) which form the recognition helix and the ‘wing’ of a helix–turn–wing motif, respectively. The NMR data also suggest the absence of a large conformational change in the protein upon binding to DNA. Hence, an O6-methylguanine (O6meG) lesion would be inaccessible to active site nucleophile Cys146 if the modified base remained stacked within the DNA duplex. The experimentally determined DNA-binding face of Ada-C was used in combination with homology modelling, based on the catabolite activator protein, and the accepted base-flipping mechanism, to construct a model of how Ada-C binds to DNA in a productive manner. To complement the structural studies, thermodynamic data were obtained which demonstrate that binding to unmethylated DNA was entropically driven, whilst the demethylation reaction provoked an exothermic heat change. Methylation of Cys146 leads to a loss of structural integrity of the DNA-binding subdomain. PMID:11000262

NFκB- and AP-1-mediated DNA looping regulates matrix metalloproteinase-9 transcription in TNF-α-treated human leukemia U937 cells.

PubMed

Chen, Ying-Jung; Chang, Long-Sen

2015-10-01

The aim of this study is to explore the spatial association of critical genomic elements in the effect of TNF-α on matrix metalloproteinase-9 (MMP-9) expression in human leukemia U937 cells. TNF-α up-regulated MMP-9 protein expression and mRNA level in U937 cells, and Akt-mediated-NFκB/p65 activation and JNK-mediated c-Jun activation were proven to be involved in TNF-α-induced MMP-9 up-regulation. Promoter luciferase activity assay revealed that NFκB (nt-600) and AP-1 (nt-79) binding sites were crucial for TNF-α-induced transcription of MMP-9 gene. The results of a chromatin immunoprecipitation assay indicated that TNF-α reduced histone deacetylase-1 (HDAC-1) recruitment but increased p300 (a histone acetyltransferase) recruitment to MMP-9 promoter regions surrounding NFκB and AP-1 binding sites. Consistently, TNF-α increased enrichment of the acetylated histone H3 mark on MMP-9 promoter regions. DNA affinity purification assay revealed that p300 and HDAC1 could bind oligonucleotides containing AP-1/c-Jun and NFκB/p65 binding sites. Chromosome conformation capture assay showed that TNF-α stimulated chromosomal loops in the MMP-9 promoter via NFκB/p65 and AP-1/c-Jun. The p300-associated acetyltransferase activity was crucial for p65/c-Jun-mediated DNA looping, and inhibition of HDAC activity increased the level of DNA looping. Reduction in the level of DNA looping eliminated all TNF-α-stimulated MMP-9 up-regulation. Taken together, our data suggest that p65/c-Jun-mediated DNA looping is involved in TNF-α-induced MMP-9 up-regulation and that the recruitment of p300 or HDAC1 to NFκB and AP-1 binding sites modifies the level of DNA looping. Copyright © 2015 Elsevier B.V. All rights reserved.

Phylogenetic Network for European mtDNA

PubMed Central

Finnilä, Saara; Lehtonen, Mervi S.; Majamaa, Kari

2001-01-01

The sequence in the first hypervariable segment (HVS-I) of the control region has been used as a source of evolutionary information in most phylogenetic analyses of mtDNA. Population genetic inference would benefit from a better understanding of the variation in the mtDNA coding region, but, thus far, complete mtDNA sequences have been rare. We determined the nucleotide sequence in the coding region of mtDNA from 121 Finns, by conformation-sensitive gel electrophoresis and subsequent sequencing and by direct sequencing of the D loop. Furthermore, 71 sequences from our previous reports were included, so that the samples represented all the mtDNA haplogroups present in the Finnish population. We found a total of 297 variable sites in the coding region, which allowed the compilation of unambiguous phylogenetic networks. The D loop harbored 104 variable sites, and, in most cases, these could be localized within the coding-region networks, without discrepancies. Interestingly, many homoplasies were detected in the coding region. Nucleotide variation in the rRNA and tRNA genes was 6%, and that in the third nucleotide positions of structural genes amounted to 22% of that in the HVS-I. The complete networks enabled the relationships between the mtDNA haplogroups to be analyzed. Phylogenetic networks based on the entire coding-region sequence in mtDNA provide a rich source for further population genetic studies, and complete sequences make it easier to differentiate between disease-causing mutations and rare polymorphisms. PMID:11349229

Replication Fork Protection Factors Controlling R-Loop Bypass and Suppression.

PubMed

Chang, Emily Yun-Chia; Stirling, Peter C

2017-01-14

Replication-transcription conflicts have been a well-studied source of genome instability for many years and have frequently been linked to defects in RNA processing. However, recent characterization of replication fork-associated proteins has revealed that defects in fork protection can directly or indirectly stabilize R-loop structures in the genome and promote transcription-replication conflicts that lead to genome instability. Defects in essential DNA replication-associated activities like topoisomerase, or the minichromosome maintenance (MCM) helicase complex, as well as fork-associated protection factors like the Fanconi anemia pathway, both appear to mitigate transcription-replication conflicts. Here, we will highlight recent advances that support the concept that normal and robust replisome function itself is a key component of mitigating R-loop coupled genome instability.

Transcript Lifetime Is Balanced between Stabilizing Stem-Loop Structures and Degradation-Promoting Polyadenylation in Plant Mitochondria

PubMed Central

Kuhn, Josef; Tengler, Ulrike; Binder, Stefan

2001-01-01

To determine the influence of posttranscriptional modifications on 3′ end processing and RNA stability in plant mitochondria, pea atp9 and Oenothera atp1 transcripts were investigated for the presence and function of 3′ nonencoded nucleotides. A 3′ rapid amplification of cDNA ends approach initiated at oligo(dT)-adapter primers finds the expected poly(A) tails predominantly attached within the second stem or downstream of the double stem-loop structures at sites of previously mapped 3′ ends. Functional studies in a pea mitochondrial in vitro processing system reveal a rapid removal of the poly(A) tails up to termini at the stem-loop structure but little if any influence on further degradation of the RNA. In contrast 3′ poly(A) tracts at RNAs without such stem-loop structures significantly promote total degradation in vitro. To determine the in vivo identity of 3′ nonencoded nucleotides more accurately, pea atp9 transcripts were analyzed by a direct anchor primer ligation-reverse transcriptase PCR approach. This analysis identified maximally 3-nucleotide-long nonencoded extensions most frequently of adenosines combined with cytidines. Processing assays with substrates containing homopolymer stretches of different lengths showed that 10 or more adenosines accelerate RNA processivity, while 3 adenosines have no impact on RNA life span. Thus polyadenylation can generally stimulate the decay of RNAs, but processivity of degradation is almost annihilated by the stabilizing effect of the stem-loop structures. These antagonistic actions thus result in the efficient formation of 3′ processed and stable transcripts. PMID:11154261

The Chern-Simons current in time series of knots and links in proteins

NASA Astrophysics Data System (ADS)

Capozziello, Salvatore; Pincak, Richard

2018-06-01

A superspace model of knots and links for DNA time series data is proposed to take into account the feedback loop from docking to undocking state of protein-protein interactions. In particular, the direction of interactions between the 8 hidden states of DNA is considered. It is a E8 ×E8 unified spin model where the genotype, from active and inactive side of DNA time data series, can be considered for any living organism. The mathematical model is borrowed from loop-quantum gravity and adapted to biology. It is used to derive equations for gene expression describing transitions from ground to excited states, and for the 8 coupling states between geneon and anti-geneon transposon and retrotransposon in trash DNA. Specifically, we adopt a modified Grothendieck cohomology and a modified Khovanov cohomology for biology. The result is a Chern-Simons current in (8 + 3) extradimensions of a given unoriented supermanifold with ghost fields of protein structures. The 8 dimensions come from the 8 hidden states of spinor field of genetic code. The extradimensions come from the 3 types of principle fiber bundle in the secondary protein.

Characterization of Short Range DNA Looping in Endotoxin-mediated Transcription of the Murine Inducible Nitric-oxide Synthase (iNOS) Gene*

PubMed Central

Guo, Hongtao; Mi, Zhiyong; Kuo, Paul C.

2008-01-01

The local structural properties and spatial conformations of chromosomes are intimately associated with gene expression. The spatial associations of critical genomic elements in inducible nitric-oxide synthase (iNOS) transcription have not been previously examined. In this regard, the murine iNOS promoter contains 2 NF-κB binding sites (nt –86 and nt –972) that are essential for maximal transactivation of iNOS by LPS. Although AP-1 is commonly listed as an essential transcription factor for LPS-mediated iNOS transactivation, the relationship between AP-1 and NF-κB in this setting is not well studied. In this study using a model of LPS-stimulated ANA-1 murine macrophages, we demonstrate that short range DNA looping occurs at the iNOS promoter. This looping requires the presence of AP-1, c-Jun, NF-κB p65, and p300-associated acetyltransferase activity. The distal AP-1 binding site interacts via p300 with the proximal NF-κB binding site to create this DNA loop to participate in iNOS transcription. Other geographically distant AP-1 and NF-κB sites are certainly occupied, but selected sites are critical for iNOS transcription and the formation of the c-Jun, p65, and p300 transcriptional complex. In this “simplified” model of murine iNOS promoter, numerous transcription factors recognize and bind to various response elements, but these locales do not equally contribute to iNOS gene transcription. PMID:18596035

Extended clinical and genetic spectrum associated with biallelic RTEL1 mutations.

PubMed

Touzot, Fabien; Kermasson, Laetitia; Jullien, Laurent; Moshous, Despina; Ménard, Christelle; Ikincioğullari, Aydan; Doğu, Figen; Sari, Sinan; Giacobbi-Milet, Vannina; Etzioni, Amos; Soulier, Jean; Londono-Vallejo, Arturo; Fischer, Alain; Callebaut, Isabelle; de Villartay, Jean-Pierre; Leblanc, Thierry; Kannengiesser, Caroline; Revy, Patrick

2016-11-29

Telomeres are repetitive hexameric sequences located at the end of linear chromosomes. They adopt a lariat-like structure, the T-loop, to prevent them from being recognized as DNA breaks by the DNA repair machinery. RTEL1 is a DNA helicase required for proper telomere replication and stability. In particular, it has been postulated that RTEL1 is involved in the opening of the T-loop during telomere replication to avoid sudden telomere deletion and telomere circle (T-circle) formation. In humans, biallelic RTEL1 mutations cause Hoyeraal-Hreidarsson syndrome (HH), a rare and severe telomere biology disorder characterized by intrauterine growth retardation, bone marrow failure, microcephaly and/or cerebellar hypoplasia, and immunodeficiency. To date, 18 different RTEL1 mutations have been described in 19 cases of HH with short telomeres. The impaired T-loop resolution has been proposed to be a major cause of telomere shortening in RTEL1 deficiency. However, the biological and clinical consequences of this disorder remain incompletely documented. Here, we describe 4 new patients harboring biallelic RTEL1 mutations, including 2 novel missense mutations located in the C-terminal end of RTEL1 (p.Cys1268Arg and p.Val1294Phe). Clinical characteristics from these 4 patients were collected as those from 4 other RTEL1-deficient patients previously reported. In addition, we assessed whether T-circles, the product of improper T-loop resolution, were detected in our RTEL1-deficient patients. Overall, our study broadens and refines the clinical and biological spectrum of human RTEL1 deficiency.

Extended clinical and genetic spectrum associated with biallelic RTEL1 mutations

PubMed Central

Kermasson, Laetitia; Jullien, Laurent; Moshous, Despina; Ménard, Christelle; Ikincioğullari, Aydan; Doğu, Figen; Sari, Sinan; Giacobbi-Milet, Vannina; Etzioni, Amos; Soulier, Jean; Londono-Vallejo, Arturo; Fischer, Alain; Callebaut, Isabelle; de Villartay, Jean-Pierre; Leblanc, Thierry; Kannengiesser, Caroline; Revy, Patrick

2016-01-01

Telomeres are repetitive hexameric sequences located at the end of linear chromosomes. They adopt a lariat-like structure, the T-loop, to prevent them from being recognized as DNA breaks by the DNA repair machinery. RTEL1 is a DNA helicase required for proper telomere replication and stability. In particular, it has been postulated that RTEL1 is involved in the opening of the T-loop during telomere replication to avoid sudden telomere deletion and telomere circle (T-circle) formation. In humans, biallelic RTEL1 mutations cause Hoyeraal-Hreidarsson syndrome (HH), a rare and severe telomere biology disorder characterized by intrauterine growth retardation, bone marrow failure, microcephaly and/or cerebellar hypoplasia, and immunodeficiency. To date, 18 different RTEL1 mutations have been described in 19 cases of HH with short telomeres. The impaired T-loop resolution has been proposed to be a major cause of telomere shortening in RTEL1 deficiency. However, the biological and clinical consequences of this disorder remain incompletely documented. Here, we describe 4 new patients harboring biallelic RTEL1 mutations, including 2 novel missense mutations located in the C-terminal end of RTEL1 (p.Cys1268Arg and p.Val1294Phe). Clinical characteristics from these 4 patients were collected as those from 4 other RTEL1-deficient patients previously reported. In addition, we assessed whether T-circles, the product of improper T-loop resolution, were detected in our RTEL1-deficient patients. Overall, our study broadens and refines the clinical and biological spectrum of human RTEL1 deficiency. PMID:29296694

Structural Variation of Type I-F CRISPR RNA Guided DNA Surveillance.

PubMed

Pausch, Patrick; Müller-Esparza, Hanna; Gleditzsch, Daniel; Altegoer, Florian; Randau, Lennart; Bange, Gert

2017-08-17

CRISPR-Cas systems are prokaryotic immune systems against invading nucleic acids. Type I CRISPR-Cas systems employ highly diverse, multi-subunit surveillance Cascade complexes that facilitate duplex formation between crRNA and complementary target DNA for R-loop formation, retention, and DNA degradation by the subsequently recruited nuclease Cas3. Typically, the large subunit recognizes bona fide targets through the PAM (protospacer adjacent motif), and the small subunit guides the non-target DNA strand. Here, we present the Apo- and target-DNA-bound structures of the I-Fv (type I-F variant) Cascade lacking the small and large subunits. Large and small subunits are functionally replaced by the 5' terminal crRNA cap Cas5fv and the backbone protein Cas7fv, respectively. Cas5fv facilitates PAM recognition from the DNA major groove site, in contrast to all other described type I systems. Comparison of the type I-Fv Cascade with an anti-CRISPR protein-bound I-F Cascade reveals that the type I-Fv structure differs substantially at known anti-CRISPR protein target sites and might therefore be resistant to viral Cascade interception. Copyright © 2017 Elsevier Inc. All rights reserved.

Restriction enzyme cutting site distribution regularity for DNA looping technology.

PubMed

Shang, Ying; Zhang, Nan; Zhu, Pengyu; Luo, Yunbo; Huang, Kunlun; Tian, Wenying; Xu, Wentao

2014-01-25

The restriction enzyme cutting site distribution regularity and looping conditions were studied systematically. We obtained the restriction enzyme cutting site distributions of 13 commonly used restriction enzymes in 5 model organism genomes through two novel self-compiled software programs. All of the average distances between two adjacent restriction sites fell sharply with increasing statistic intervals, and most fragments were 0-499 bp. A shorter DNA fragment resulted in a lower looping rate, which was also directly proportional to the DNA concentration. When the length was more than 500 bp, the concentration did not affect the looping rate. Therefore, the best known fragment length was longer than 500 bp, and did not contain the restriction enzyme cutting sites which would be used for digestion. In order to make the looping efficiencies reach nearly 100%, 4-5 single cohesive end systems were recommended to digest the genome separately. Copyright © 2013 Elsevier B.V. All rights reserved.

Histone H3.3 promotes IgV gene diversification by enhancing formation of AID-accessible single-stranded DNA.

PubMed

Romanello, Marina; Schiavone, Davide; Frey, Alexander; Sale, Julian E

2016-07-01

Immunoglobulin diversification is driven by activation-induced deaminase (AID), which converts cytidine to uracil within the Ig variable (IgV) regions. Central to the recruitment of AID to the IgV genes are factors that regulate the generation of single-stranded DNA (ssDNA), the enzymatic substrate of AID Here, we report that chicken DT40 cells lacking variant histone H3.3 exhibit reduced IgV sequence diversification. We show that this results from impairment of the ability of AID to access the IgV genes due to reduced formation of ssDNA during IgV transcription. Loss of H3.3 also diminishes IgV R-loop formation. However, reducing IgV R-loops by RNase HI overexpression in wild-type cells does not affect IgV diversification, showing that these structures are not necessary intermediates for AID access. Importantly, the reduction in the formation of AID-accessible ssDNA in cells lacking H3.3 is independent of any effect on the level of transcription or the kinetics of RNAPII elongation, suggesting the presence of H3.3 in the nucleosomes of the IgV genes increases the chances of the IgV DNA becoming single-stranded, thereby creating an effective AID substrate. © 2016 MRC Laboratory of Molecular Biology. Published under the terms of the CC BY 4.0 license.

The Agrobacterium tumefaciens Transcription Factor BlcR Is Regulated via Oligomerization

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

Pan, Yi; Fiscus, Valena; Meng, Wuyi

2012-02-08

The Agrobacterium tumefaciens BlcR is a member of the emerging isocitrate lyase transcription regulators that negatively regulates metabolism of {gamma}-butyrolactone, and its repressing function is relieved by succinate semialdehyde (SSA). Our crystal structure showed that BlcR folded into the DNA- and SSA-binding domains and dimerized via the DNA-binding domains. Mutational analysis identified residues, including Phe{sup 147}, that are important for SSA association; BlcR{sup F147A} existed as tetramer. Two BlcR dimers bound to target DNA and in a cooperative manner, and the distance between the two BlcR-binding sequences in DNA was critical for BlcR-DNA association. Tetrameric BlcR{sup F147A} retained DNA bindingmore » activity, and importantly, this activity was not affected by the distance separating the BlcR-binding sequences in DNA. SSA did not dissociate tetrameric BlcR{sup F147A} or BlcR{sup F147A}-DNA. As well as in the SSA-binding site, Phe{sup 147} is located in a structurally flexible loop that may be involved in BlcR oligomerization. We propose that SSA regulates BlcR DNA-binding function via oligomerization.« less

Analysis of mitochondrial DNA in Bolivian llama, alpaca and vicuna populations: a contribution to the phylogeny of the South American camelids.

PubMed

Barreta, J; Gutiérrez-Gil, B; Iñiguez, V; Saavedra, V; Chiri, R; Latorre, E; Arranz, J J

2013-04-01

The objectives of this work were to assess the mtDNA diversity of Bolivian South American camelid (SAC) populations and to shed light on the evolutionary relationships between the Bolivian camelids and other populations of SACs. We have analysed two different mtDNA regions: the complete coding region of the MT-CYB gene and 513 bp of the D-loop region. The populations sampled included Bolivian llamas, alpacas and vicunas, and Chilean guanacos. High levels of genetic diversity were observed in the studied populations. In general, MT-CYB was more variable than D-loop. On a species level, the vicunas showed the lowest genetic variability, followed by the guanacos, alpacas and llamas. Phylogenetic analyses performed by including additional available mtDNA sequences from the studied species confirmed the existence of the two monophyletic clades previously described by other authors for guanacos (G) and vicunas (V). Significant levels of mtDNA hybridization were found in the domestic species. Our sequence analyses revealed significant sequence divergence within clade G, and some of the Bolivian llamas grouped with the majority of the southern guanacos. This finding supports the existence of more than the one llama domestication centre in South America previously suggested on the basis of archaeozoological evidence. Additionally, analysis of D-loop sequences revealed two new matrilineal lineages that are distinct from the previously reported G and V clades. The results presented here represent the first report on the population structure and genetic variability of Bolivian camelids and may help to elucidate the complex and dynamic domestication process of SAC populations. © 2012 The Authors, Animal Genetics © 2012 Stichting International Foundation for Animal Genetics.

Suppression and enhancement of transcriptional noise by DNA looping

NASA Astrophysics Data System (ADS)

Vilar, Jose M. G.; Saiz, Leonor

2014-06-01

DNA looping has been observed to enhance and suppress transcriptional noise but it is uncertain which of these two opposite effects is to be expected for given conditions. Here, we derive analytical expressions for the main quantifiers of transcriptional noise in terms of the molecular parameters and elucidate the role of DNA looping. Our results rationalize paradoxical experimental observations and provide the first quantitative explanation of landmark individual-cell measurements at the single molecule level on the classical lac operon genetic system [Choi, L. Cai, K. Frieda, and X. S. Xie, Science 322, 442 (2008), 10.1126/science.1161427].

Unraveling DNA dynamics using atomic force microscopy.

PubMed

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

2011-01-01

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

Structures and functions in the crowded nucleus: new biophysical insights

NASA Astrophysics Data System (ADS)

Hancock, Ronald

2014-09-01

Concepts and methods from the physical sciences have catalysed remarkable progress in understanding the cell nucleus in recent years. To share this excitement with physicists and encourage their interest in this field, this review offers an overview of how the physics which underlies structures and functions in the nucleus is becoming more clear thanks to methods which have been developed to simulate and study macromolecules, polymers, and colloids. The environment in the nucleus is very crowded with macromolecules, making entropic (depletion) forces major determinants of interactions. Simulation and experiments are consistent with their key role in forming membraneless compartments such as nucleoli, PML and Cajal bodies, and discrete "territories" for chromosomes. The chromosomes, giant linear polyelectrolyte polymers, exist in vivo in a state like a polymer melt. Looped conformations are predicted in crowded conditions, and have been confirmed experimentally and are central to the regulation of gene expression. Polymer theory has revealed how the chromosomes are so highly compacted in the nucleus, forming a "crumpled globule" with fractal properties which avoids knots and entanglements in DNA while allowing facile accessibility for its replication and transcription. Entropic repulsion between looped polymers can explain the confinement of each chromosome to a discrete region of the nucleus. Crowding and looping are predicted to facilitate finding the specific targets of factors which modulate activities of DNA. Simulation shows that entropic effects contribute to finding and repairing potentially lethal double-strand breaks in DNA by increasing the mobility of the broken ends, favouring their juxtaposition for repair. Signaling pathways are strongly influenced by crowding, which favours a processive mode of response (consecutive reactions without releasing substrates). This new information contributes to understanding the sometimes counter-intuitive consequences.

CasA mediates Cas3-catalyzed target degradation during CRISPR RNA-guided interference.

PubMed

Hochstrasser, Megan L; Taylor, David W; Bhat, Prashant; Guegler, Chantal K; Sternberg, Samuel H; Nogales, Eva; Doudna, Jennifer A

2014-05-06

In bacteria, the clustered regularly interspaced short palindromic repeats (CRISPR)-associated (Cas) DNA-targeting complex Cascade (CRISPR-associated complex for antiviral defense) uses CRISPR RNA (crRNA) guides to bind complementary DNA targets at sites adjacent to a trinucleotide signature sequence called the protospacer adjacent motif (PAM). The Cascade complex then recruits Cas3, a nuclease-helicase that catalyzes unwinding and cleavage of foreign double-stranded DNA (dsDNA) bearing a sequence matching that of the crRNA. Cascade comprises the CasA-E proteins and one crRNA, forming a structure that binds and unwinds dsDNA to form an R loop in which the target strand of the DNA base pairs with the 32-nt RNA guide sequence. Single-particle electron microscopy reconstructions of dsDNA-bound Cascade with and without Cas3 reveal that Cascade positions the PAM-proximal end of the DNA duplex at the CasA subunit and near the site of Cas3 association. The finding that the DNA target and Cas3 colocalize with CasA implicates this subunit in a key target-validation step during DNA interference. We show biochemically that base pairing of the PAM region is unnecessary for target binding but critical for Cas3-mediated degradation. In addition, the L1 loop of CasA, previously implicated in PAM recognition, is essential for Cas3 activation following target binding by Cascade. Together, these data show that the CasA subunit of Cascade functions as an essential partner of Cas3 by recognizing DNA target sites and positioning Cas3 adjacent to the PAM to ensure cleavage.

Crystal Structure of the Minimalist Max-E47 Protein Chimera

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

Ahmadpour, Faraz; Ghirlando, Rodolfo; De Jong, Antonia T.

Max-E47 is a protein chimera generated from the fusion of the DNA-binding basic region of Max and the dimerization region of E47, both members of the basic region/helix-loop-helix (bHLH) superfamily of transcription factors. Like native Max, Max-E47 binds with high affinity and specificity to the E-box site, 5'-CACGTG, both in vivo and in vitro. We have determined the crystal structure of Max-E47 at 1.7 Å resolution, and found that it associates to form a well-structured dimer even in the absence of its cognate DNA. Analytical ultracentrifugation confirms that Max-E47 is dimeric even at low micromolar concentrations, indicating that the Max-E47more » dimer is stable in the absence of DNA. Circular dichroism analysis demonstrates that both non-specific DNA and the E-box site induce similar levels of helical secondary structure in Max-E47. These results suggest that Max-E47 may bind to the E-box following the two-step mechanism proposed for other bHLH proteins. In this mechanism, a rapid step where protein binds to DNA without sequence specificity is followed by a slow step where specific protein:DNA interactions are fine-tuned, leading to sequence-specific recognition. Collectively, these results show that the designed Max-E47 protein chimera behaves both structurally and functionally like its native counterparts.« less

Computational Model for DNA Organization Mediated by Protein Interaction in Prokaryotes

NASA Astrophysics Data System (ADS)

Garimella, Karthik; Kharel, Savan

2016-03-01

In Escherichia Coli, there are several mechanisms that drive chromosomal organization. We know through experiments that the E. Coli chromosome is condensed into highly structured regions known as macrodomains (MDs). One of the regions known as the Terminus undergoes DNA-bridging condensation that form loops between distant DNA sites and it is known to be mediated by a Terminus specific protein, which binds to specific markers within the Terminus region. In the absence of Terminus specific protein, however, the Terminus region is known to not condense nearly as much, which will likely impede several biological processes including DNA replication. In order to understand the molecular basis of protein mediation in vivo several models of Terminus specific segregation have been constructed in silico which model DNA as polymer chains.

DNA Methylation-a Potential Source of Mitochondria DNA Base Mismatch in the Development of Diabetic Retinopathy.

PubMed

Mishra, Manish; Kowluru, Renu A

2018-04-21

In the development of diabetic retinopathy, retinal mitochondria are dysfunctional, and mitochondrial DNA (mtDNA) is damaged with increased base mismatches and hypermethylated cytosines. DNA methylation is also a potential source of mutation, and in diabetes, the noncoding region, the displacement loop (D-loop), experiences more methylation and base mismatches than other regions of the mtDNA. Our aim was to investigate a possible crosstalk between mtDNA methylation and base mismatches in the development of diabetic retinopathy. The effect of inhibition of Dnmts (by 5-aza-2'-deoxycytidine or Dnmt1-siRNA) on glucose-induced mtDNA base mismatches was investigated in human retinal endothelial cells by surveyor endonuclease digestion and validated by Sanger sequencing. The role of deamination factors on increased base mismatches was determined in the cells genetically modulated for mitochondrial superoxide dismutase (Sod2) or cytidine-deaminase (APOBEC3A). The results were confirmed in an in vivo model using retinal microvasculature from diabetic mice overexpressing Sod2. Inhibition of DNA methylation, or regulation of cytosine deamination, significantly inhibited an increase in base mismatches at the D-loop and prevented mitochondrial dysfunction. Overexpression of Sod2 in mice also prevented diabetes-induced D-loop hypermethylation and increase in base mismatches. The crosstalk between DNA methylation and base mismatches continued even after termination of hyperglycemia, suggesting its role in the metabolic memory phenomenon associated with the progression of diabetic retinopathy. Inhibition of DNA methylation limits the availability of methylated cytosine for deamination, suggesting a crosstalk between DNA methylation and base mismatches. Thus, regulation of DNA methylation, or its deamination, should impede the development of diabetic retinopathy by preventing formation of base mismatches and mitochondrial dysfunction.

The biology and polymer physics underlying large-scale chromosome organization.

PubMed

Sazer, Shelley; Schiessel, Helmut

2018-02-01

Chromosome large-scale organization is a beautiful example of the interplay between physics and biology. DNA molecules are polymers and thus belong to the class of molecules for which physicists have developed models and formulated testable hypotheses to understand their arrangement and dynamic properties in solution, based on the principles of polymer physics. Biologists documented and discovered the biochemical basis for the structure, function and dynamic spatial organization of chromosomes in cells. The underlying principles of chromosome organization have recently been revealed in unprecedented detail using high-resolution chromosome capture technology that can simultaneously detect chromosome contact sites throughout the genome. These independent lines of investigation have now converged on a model in which DNA loops, generated by the loop extrusion mechanism, are the basic organizational and functional units of the chromosome. © 2017 The Authors. Traffic published by John Wiley & Sons Ltd.

Towards a physical map of the fertility genes on the heterochromatic Y chromosome of Drosophila hydei: families of repetitive sequences transcribed on the lampbrush loops Nooses and Threads are organized in extended clusters of several hundred kilobases.

PubMed

Trapitz, P; Glätzer, K H; Bünemann, H

1992-11-01

The understanding of structure and function of the so-called fertility genes of Drosophila is very limited due to their unusual size--several megabases--and their location on the heterochromatic Y chromosome. Since mapping of these genes has mainly been done by classical cytogenetic analyses using a small number of cytologically visible lampbrush loops as the sole markers for particular fertility genes, the resolution of the genetic map of the Y chromosome is restricted to 3-5 Mb. Here we demonstrate that a substantially finer subdivision of the megabase-sized fertility genes in the subtelomeric regions of the Y chromosome of Drosophila hydei can be achieved by a combination of digestion with restriction enzymes having 6 bp recognition sequences, and pulsed field gel electrophoresis. The physical subdivision is based upon large conserved fragments of repetitive DNA in the size range from 50 up to 1600 kb and refers to the long-range organization of several families of repetitive DNA involved in Y chromosomal transcription processes in primary spermatocytes. We conclude from our results that at least five different families of repetitive DNA specifically transcribed on the lampbrush loops nooses and threads are organized as extended clusters of several hundred kb, essentially free of interspersed non-repetitive sequences.

Creating Directed Double-strand Breaks with the Ref Protein: A Novel Rec A-Dependent Nuclease from Bacteriophage P1

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

Gruenig, Marielle C.; Lu, Duo; Won, Sang Joon

2012-03-16

The bacteriophage P1-encoded Ref protein enhances RecA-dependent recombination in vivo by an unknown mechanism. We demonstrate that Ref is a new type of enzyme; that is, a RecA-dependent nuclease. Ref binds to ss- and dsDNA but does not cleave any DNA substrate until RecA protein and ATP are added to form RecA nucleoprotein filaments. Ref cleaves only where RecA protein is bound. RecA functions as a co-nuclease in the Ref/RecA system. Ref nuclease activity can be limited to the targeted strands of short RecA-containing D-loops. The result is a uniquely programmable endonuclease activity, producing targeted double-strand breaks at any chosenmore » DNA sequence in an oligonucleotide-directed fashion. We present evidence indicating that cleavage occurs in the RecA filament groove. The structure of the Ref protein has been determined to 1.4 {angstrom} resolution. The core structure, consisting of residues 77-186, consists of a central 2-stranded {beta}-hairpin that is sandwiched between several {alpha}-helical and extended loop elements. The N-terminal 76 amino acid residues are disordered; this flexible region is required for optimal activity. The overall structure of Ref, including several putative active site histidine residues, defines a new subclass of HNH-family nucleases. We propose that enhancement of recombination by Ref reflects the introduction of directed, recombinogenic double-strand breaks.« less

Differential Targeting of Unpaired Bases within Duplex DNA by the Natural Compound Clerocidin: A Valuable Tool to Dissect DNA Secondary Structure

PubMed Central

Nadai, Matteo; Palù, Giorgio; Palumbo, Manlio; Richter, Sara N.

2012-01-01

Non-canonical DNA structures have been postulated to mediate protein-nucleic acid interactions and to function as intermediates in the generation of frame-shift mutations when errors in DNA replication occur, which result in a variety of diseases and cancers. Compounds capable of binding to non-canonical DNA conformations may thus have significant diagnostic and therapeutic potential. Clerocidin is a natural diterpenoid which has been shown to selectively react with single-stranded bases without targeting the double helix. Here we performed a comprehensive analysis on several non-canonical DNA secondary structures, namely mismatches, nicks, bulges, hairpins, with sequence variations in both the single-stranded region and the double-stranded flanking segment. By analysis of clerocidin reactivity, we were able to identify the exposed reactive residues which provided information on both the secondary structure and the accessibility of the non-paired sites. Mismatches longer than 1 base were necessary to be reached by clerocidin reactive groups, while 1-base nicks were promptly targeted by clerocidin; in hairpins, clerocidin reactivity increased with the length of the hairpin loop, while, interestingly, reactivity towards bulges reached a maximum in 3-base-long bulges and declined in longer bulges. Electrophoretic mobility shift analysis demonstrated that bulges longer than 3 bases (i.e. 5- and 7-bases) folded or stacked on the duplex region therefore being less accessible by the compound. Clerocidin thus represents a new valuable diagnostic tool to dissect DNA secondary structures. PMID:23285245

Differential targeting of unpaired bases within duplex DNA by the natural compound clerocidin: a valuable tool to dissect DNA secondary structure.

PubMed

Nadai, Matteo; Palù, Giorgio; Palumbo, Manlio; Richter, Sara N

2012-01-01

Non-canonical DNA structures have been postulated to mediate protein-nucleic acid interactions and to function as intermediates in the generation of frame-shift mutations when errors in DNA replication occur, which result in a variety of diseases and cancers. Compounds capable of binding to non-canonical DNA conformations may thus have significant diagnostic and therapeutic potential. Clerocidin is a natural diterpenoid which has been shown to selectively react with single-stranded bases without targeting the double helix. Here we performed a comprehensive analysis on several non-canonical DNA secondary structures, namely mismatches, nicks, bulges, hairpins, with sequence variations in both the single-stranded region and the double-stranded flanking segment. By analysis of clerocidin reactivity, we were able to identify the exposed reactive residues which provided information on both the secondary structure and the accessibility of the non-paired sites. Mismatches longer than 1 base were necessary to be reached by clerocidin reactive groups, while 1-base nicks were promptly targeted by clerocidin; in hairpins, clerocidin reactivity increased with the length of the hairpin loop, while, interestingly, reactivity towards bulges reached a maximum in 3-base-long bulges and declined in longer bulges. Electrophoretic mobility shift analysis demonstrated that bulges longer than 3 bases (i.e. 5- and 7-bases) folded or stacked on the duplex region therefore being less accessible by the compound. Clerocidin thus represents a new valuable diagnostic tool to dissect DNA secondary structures.

Proteins mediating DNA loops effectively block transcription.

PubMed

Vörös, Zsuzsanna; Yan, Yan; Kovari, Daniel T; Finzi, Laura; Dunlap, David

2017-07-01

Loops are ubiquitous topological elements formed when proteins simultaneously bind to two noncontiguous DNA sites. While a loop-mediating protein may regulate initiation at a promoter, the presence of the protein at the other site may be an obstacle for RNA polymerases (RNAP) transcribing a different gene. To test whether a DNA loop alters the extent to which a protein blocks transcription, the lac repressor (LacI) was used. The outcome of in vitro transcription along templates containing two LacI operators separated by 400 bp in the presence of LacI concentrations that produced both looped and unlooped molecules was visualized with scanning force microscopy (SFM). An analysis of transcription elongation complexes, moving for 60 s at an average of 10 nt/s on unlooped DNA templates, revealed that they more often surpassed LacI bound to the lower affinity O2 operator than to the highest affinity Os operator. However, this difference was abrogated in looped DNA molecules where LacI became a strong roadblock independently of the affinity of the operator. Recordings of transcription elongation complexes, using magnetic tweezers, confirmed that they halted for several minutes upon encountering a LacI bound to a single operator. The average pause lifetime is compatible with RNAP waiting for LacI dissociation, however, the LacI open conformation visualized in the SFM images also suggests that LacI could straddle RNAP to let it pass. Independently of the mechanism by which RNAP bypasses the LacI roadblock, the data indicate that an obstacle with looped topology more effectively interferes with transcription. © 2017 The Authors Protein Science published by Wiley Periodicals, Inc. on behalf of The Protein Society.

Effect of loop structure of bovine lactoferricin on apoptosis in Jurkat cells.

PubMed

Zhang, Tie-nan; Yang, Wei; Liu, Ning

2010-06-01

Bovine lactoferricin (LfcinB) is a cationic peptide that selectively induces apoptosis in Jurkat cells. However less is known about the influence of this kind of apoptosis on the intra-cellular ceramide metabolism and the structure-function relationship between the loop structure of LfcinB and its action of inducing apoptosis in Jurkat cells. In the present study, the artificially synthesized LfcinB and LfcinB-derived peptide (Cys 19 residue in LfcinB was replaced by Ala) was added in Jurkat cells, the nucleolus shape was observed by fluorescent microscopy, the ceramide concentration in Jurkat cells was determined by reversed phase high performance liquid chromatography (RP-HPLC). The results of MTT assay showed that LfcinB inhibited proliferation of Jurkat cells, and the inhibition rate was approximately 18.90%. Moreover, the inhibition rate of LfcinB together with MAPP was upto approximately 59.89%. The RP-HPLC result showed that LfcinB improved the ceramide level in Jurkat cells. By using the DNA fragmentation assay and observing the nucleolus shape, the result displayed deficiency of the loop structure could cause LfcinB losing the biological activity of inducing apoptosis in Jurkat cells.

Structural maintenance of chromosome complexes differentially compact mitotic chromosomes according to genomic context

PubMed Central

Schalbetter, S. A.; Goloborodko, A.; Fudenberg, G.; Belton, J.-M.; Miles, C.; Yu, M.; Dekker, J.; Mirny, L.; Baxter, J.

2017-01-01

Structural Maintenance of Chromosomes (SMC) protein complexes are key determinants of chromosome conformation. Using Hi-C and polymer modeling, we study how cohesin and condensin, two deeply conserved SMC complexes, organize chromosomes in the budding yeast Saccharomyces cerevisiae. The canonical role of cohesin is to co-align sister chromatids whilst condensin generally compacts mitotic chromosomes. We find strikingly different roles for the two complexes in budding yeast mitosis. First, cohesin is responsible for compacting mitotic chromosome arms, independently of sister chromatid cohesion. Polymer simulations demonstrate this role can be fully accounted for through cis-looping of chromatin. Second, condensin is generally dispensable for compaction along chromosome arms. Instead it plays a targeted role compacting the rDNA proximal regions and promoting resolution of peri-centromeric regions. Our results argue that the conserved mechanism of SMC complexes is to form chromatin loops and that distinct SMC-dependent looping activities are selectively deployed to appropriately compact chromosomes. PMID:28825700

DNA binding specificity of the basic-helix-loop-helix protein MASH-1.

PubMed

Meierhan, D; el-Ariss, C; Neuenschwander, M; Sieber, M; Stackhouse, J F; Allemann, R K

1995-09-05

Despite the high degree of sequence similarity in their basic-helix-loop-helix (BHLH) domains, MASH-1 and MyoD are involved in different biological processes. In order to define possible differences between the DNA binding specificities of these two proteins, we investigated the DNA binding properties of MASH-1 by circular dichroism spectroscopy and by electrophoretic mobility shift assays (EMSA). Upon binding to DNA, the BHLH domain of MASH-1 underwent a conformational change from a mainly unfolded to a largely alpha-helical form, and surprisingly, this change was independent of the specific DNA sequence. The same conformational transition could be induced by the addition of 20% 2,2,2-trifluoroethanol. The apparent dissociation constants (KD) of the complexes of full-length MASH-1 with various oligonucleotides were determined from half-saturation points in EMSAs. MASH-1 bound as a dimer to DNA sequences containing an E-box with high affinity KD = 1.4-4.1 x 10(-14) M2). However, the specificity of DNA binding was low. The dissociation constant for the complex between MASH-1 and the highest affinity E-box sequence (KD = 1.4 x 10(-14) M2) was only a factor of 10 smaller than for completely unrelated DNA sequences (KD = approximately 1 x 10(-13) M2). The DNA binding specificity of MASH-1 was not significantly increased by the formation of an heterodimer with the ubiquitous E12 protein. MASH-1 and MyoD displayed similar binding site preferences, suggesting that their different target gene specificities cannot be explained solely by differential DNA binding. An explanation for these findings is provided on the basis of the known crystal structure of the BHLH domain of MyoD.

Exploring the Dynamics of Propeller Loops in Human Telomeric DNA Quadruplexes Using Atomistic Simulations.

PubMed

Islam, Barira; Stadlbauer, Petr; Gil-Ley, Alejandro; Pérez-Hernández, Guillermo; Haider, Shozeb; Neidle, Stephen; Bussi, Giovanni; Banas, Pavel; Otyepka, Michal; Sponer, Jiri

2017-06-13

We have carried out a series of extended unbiased molecular dynamics (MD) simulations (up to 10 μs long, ∼162 μs in total) complemented by replica-exchange with the collective variable tempering (RECT) approach for several human telomeric DNA G-quadruplex (GQ) topologies with TTA propeller loops. We used different AMBER DNA force-field variants and also processed simulations by Markov State Model (MSM) analysis. The slow conformational transitions in the propeller loops took place on a scale of a few μs, emphasizing the need for long simulations in studies of GQ dynamics. The propeller loops sampled similar ensembles for all GQ topologies and for all force-field dihedral-potential variants. The outcomes of standard and RECT simulations were consistent and captured similar spectrum of loop conformations. However, the most common crystallographic loop conformation was very unstable with all force-field versions. Although the loss of canonical γ-trans state of the first propeller loop nucleotide could be related to the indispensable bsc0 α/γ dihedral potential, even supporting this particular dihedral by a bias was insufficient to populate the experimentally dominant loop conformation. In conclusion, while our simulations were capable of providing a reasonable albeit not converged sampling of the TTA propeller loop conformational space, the force-field description still remained far from satisfactory.

Exploring the Dynamics of Propeller Loops in Human Telomeric DNA Quadruplexes Using Atomistic Simulations

PubMed Central

2017-01-01

We have carried out a series of extended unbiased molecular dynamics (MD) simulations (up to 10 μs long, ∼162 μs in total) complemented by replica-exchange with the collective variable tempering (RECT) approach for several human telomeric DNA G-quadruplex (GQ) topologies with TTA propeller loops. We used different AMBER DNA force-field variants and also processed simulations by Markov State Model (MSM) analysis. The slow conformational transitions in the propeller loops took place on a scale of a few μs, emphasizing the need for long simulations in studies of GQ dynamics. The propeller loops sampled similar ensembles for all GQ topologies and for all force-field dihedral-potential variants. The outcomes of standard and RECT simulations were consistent and captured similar spectrum of loop conformations. However, the most common crystallographic loop conformation was very unstable with all force-field versions. Although the loss of canonical γ-trans state of the first propeller loop nucleotide could be related to the indispensable bsc0 α/γ dihedral potential, even supporting this particular dihedral by a bias was insufficient to populate the experimentally dominant loop conformation. In conclusion, while our simulations were capable of providing a reasonable albeit not converged sampling of the TTA propeller loop conformational space, the force-field description still remained far from satisfactory. PMID:28475322

Substrate sequence selectivity of APOBEC3A implicates intra-DNA interactions.

PubMed

Silvas, Tania V; Hou, Shurong; Myint, Wazo; Nalivaika, Ellen; Somasundaran, Mohan; Kelch, Brian A; Matsuo, Hiroshi; Kurt Yilmaz, Nese; Schiffer, Celia A

2018-05-14

The APOBEC3 (A3) family of human cytidine deaminases is renowned for providing a first line of defense against many exogenous and endogenous retroviruses. However, the ability of these proteins to deaminate deoxycytidines in ssDNA makes A3s a double-edged sword. When overexpressed, A3s can mutate endogenous genomic DNA resulting in a variety of cancers. Although the sequence context for mutating DNA varies among A3s, the mechanism for substrate sequence specificity is not well understood. To characterize substrate specificity of A3A, a systematic approach was used to quantify the affinity for substrate as a function of sequence context, length, secondary structure, and solution pH. We identified the A3A ssDNA binding motif as (T/C)TC(A/G), which correlated with enzymatic activity. We also validated that A3A binds RNA in a sequence specific manner. A3A bound tighter to substrate binding motif within a hairpin loop compared to linear oligonucleotide, suggesting A3A affinity is modulated by substrate structure. Based on these findings and previously published A3A-ssDNA co-crystal structures, we propose a new model with intra-DNA interactions for the molecular mechanism underlying A3A sequence preference. Overall, the sequence and structural preferences identified for A3A leads to a new paradigm for identifying A3A's involvement in mutation of endogenous or exogenous DNA.

Interactions of Human Nucleotide Excision Repair Protein XPA with DNA and RPA70 Delta c327: Chemical Shift Mapping and N-15 NMR Relaxation Studies

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

Buchko, Garry W.; Daughdrill, Gary W.; De Lorimier, Robert

1999-12-28

Human XPA is an essential component in the multienzyme nucleotide excision repair (NER) pathway. The solution structure of the minimal DNA binding domain of XPA (XPA-MBD: M98-F219) was recently determined [Buchko et al. (1998) Nucleic Acids Res. 26, 2779-2788, Ikegami et al (1998) Nat. Struct. Biol. 5, 701-706] and shown to consist of a compact zinc-binding core and a loop-rich C-terminal subdomain connected by a linker sequence.

Mutations reducing replication from R-loops suppress the defects of growth, chromosome segregation and DNA supercoiling in cells lacking topoisomerase I and RNase HI activity.

PubMed

Usongo, Valentine; Martel, Makisha; Balleydier, Aurélien; Drolet, Marc

2016-04-01

R-loop formation occurs when the nascent RNA hybridizes with the template DNA strand behind the RNA polymerase. R-loops affect a wide range of cellular processes and their use as origins of replication was the first function attributed to them. In Escherichia coli, R-loop formation is promoted by the ATP-dependent negative supercoiling activity of gyrase (gyrA and gyrB) and is inhibited by topoisomerase (topo) I (topA) relaxing transcription-induced negative supercoiling. RNase HI (rnhA) degrades the RNA moiety of R-loops. The depletion of RNase HI activity in topA null mutants was previously shown to lead to extensive DNA relaxation, due to DNA gyrase inhibition, and to severe growth and chromosome segregation defects that were partially corrected by overproducing topo III (topB). Here, DNA gyrase assays in crude cell extracts showed that the ATP-dependent activity (supercoiling) of gyrase but not its ATP-independent activity (relaxation) was inhibited in topA null cells lacking RNase HI. To characterize the cellular event(s) triggered by the absence of RNase HI, we performed a genetic screen for suppressors of the growth defect of topA rnhA null cells. Suppressors affecting genes in replication (holC2::aph and dnaT18::aph) nucleotide metabolism (dcd49::aph), RNA degradation (rne59::aph) and fimbriae synthesis (fimD22::aph) were found to reduce replication from R-loops and to restore supercoiling, thus pointing to a correlation between R-loop-dependent replication in topA rnhA mutants and the inhibition of gyrase activity and growth. Interestingly, the position of fimD on the E. coli chromosome corresponds to the site of one of the five main putative origins of replication from R-loops in rnhA null cells recently identified by next-generation sequencing, thus suggesting that the fimD22::aph mutation inactivated one of these origins. Furthermore, we show that topo III overproduction is unable to complement the growth defect of topA rnhA null mutants at low temperatures that stabilizes hyper-negatively supercoiled DNA. Copyright © 2016 Elsevier B.V. All rights reserved.

Structure of Peptide Sex Pheromone Receptor PrgX and PrgX/Pheromone Complexes and Regulation of Conjugation in Enterococcus faecalis

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

Shi,K.; Brown, C.; Gu, Z.

2005-01-01

Many bacterial activities, including expression of virulence factors, horizontal genetic transfer, and production of antibiotics, are controlled by intercellular signaling using small molecules. To date, understanding of the molecular mechanisms of peptide-mediated cell-cell signaling has been limited by a dearth of published information about the molecular structures of the signaling components. Here, we present the molecular structure of PrgX, a DNA- and peptide-binding protein that regulates expression of the conjugative transfer genes of the Enterococcus faecalis plasmid pCF10 in response to an intercellular peptide pheromone signal. Comparison of the structures of PrgX and the PrgX/pheromone complex suggests that pheromone bindingmore » destabilizes PrgX tetramers, opening a 70-bp pCF10 DNA loop required for conjugation repression.« less

DNA sequence-dependent mechanics and protein-assisted bending in repressor-mediated loop formation

PubMed Central

Boedicker, James Q.; Garcia, Hernan G.; Johnson, Stephanie; Phillips, Rob

2014-01-01

As the chief informational molecule of life, DNA is subject to extensive physical manipulations. The energy required to deform double-helical DNA depends on sequence, and this mechanical code of DNA influences gene regulation, such as through nucleosome positioning. Here we examine the sequence-dependent flexibility of DNA in bacterial transcription factor-mediated looping, a context for which the role of sequence remains poorly understood. Using a suite of synthetic constructs repressed by the Lac repressor and two well-known sequences that show large flexibility differences in vitro, we make precise statistical mechanical predictions as to how DNA sequence influences loop formation and test these predictions using in vivo transcription and in vitro single-molecule assays. Surprisingly, sequence-dependent flexibility does not affect in vivo gene regulation. By theoretically and experimentally quantifying the relative contributions of sequence and the DNA-bending protein HU to DNA mechanical properties, we reveal that bending by HU dominates DNA mechanics and masks intrinsic sequence-dependent flexibility. Such a quantitative understanding of how mechanical regulatory information is encoded in the genome will be a key step towards a predictive understanding of gene regulation at single-base pair resolution. PMID:24231252

Tip-enhanced fluorescence with radially polarized illumination for monitoring loop-mediated isothermal amplification on Hepatitis C virus cDNA

NASA Astrophysics Data System (ADS)

Wei, Shih-Chung; Chuang, Tsung-Liang; Wang, Da-Shin; Lu, Hui-Hsin; Gu, Frank X.; Sung, Kung-Bin; Lin, Chii-Wann

2015-02-01

A tip nanobiosensor for monitoring DNA replication was presented. The effects of excitation power and polarization on tip-enhanced fluorescence (TEF) were assessed with the tip immersed in fluorescein isothiocyanate solution first. The photon count rose on average fivefold with radially polarized illumination at 50 mW. We then used polymerase-functionalized tips for monitoring loop-mediated isothermal amplification on Hepatitis C virus cDNA. The amplicon-SYBR Green I complex was detected and compared to real-time loop-mediated isothermal amplification. The signals of the reaction using 4 and 0.004 ng/μl templates were detected 10 and 30 min earlier, respectively. The results showed the potential of TEF in developing a nanobiosensor for real-time DNA amplification.

High-resolution three-dimensional NMR structure of the KRAS proto-oncogene promoter reveals key features of a G-quadruplex involved in transcriptional regulation.

PubMed

Kerkour, Abdelaziz; Marquevielle, Julien; Ivashchenko, Stefaniia; Yatsunyk, Liliya A; Mergny, Jean-Louis; Salgado, Gilmar F

2017-05-12

Non-canonical base pairing within guanine-rich DNA and RNA sequences can produce G-quartets, whose stacking leads to the formation of a G-quadruplex (G4). G4s can coexist with canonical duplex DNA in the human genome and have been suggested to suppress gene transcription, and much attention has therefore focused on studying G4s in promotor regions of disease-related genes. For example, the human KRAS proto-oncogene contains a nuclease-hypersensitive element located upstream of the major transcription start site. The KRAS nuclease-hypersensitive element (NHE) region contains a G-rich element (22RT; 5'-AGGGCGGTGTGGGAATAGGGAA-3') and encompasses a Myc-associated zinc finger-binding site that regulates KRAS transcription. The NEH region therefore has been proposed as a target for new drugs that control KRAS transcription, which requires detailed knowledge of the NHE structure. In this study, we report a high-resolution NMR structure of the G-rich element within the KRAS NHE. We found that the G-rich element forms a parallel structure with three G-quartets connected by a four-nucleotide loop and two short one-nucleotide double-chain reversal loops. In addition, a thymine bulge is found between G8 and G9. The loops of different lengths and the presence of a bulge between the G-quartets are structural elements that potentially can be targeted by small chemical ligands that would further stabilize the structure and interfere or block transcriptional regulators such as Myc-associated zinc finger from accessing their binding sites on the KRAS promoter. In conclusion, our work suggests a possible new route for the development of anticancer agents that could suppress KRAS expression. © 2017 by The American Society for Biochemistry and Molecular Biology, Inc.

Mechanism of Microhomology-Mediated End-Joining Promoted by Human DNA Polymerase Theta

PubMed Central

Kent, Tatiana; Chandramouly, Gurushankar; McDevitt, Shane Michael; Ozdemir, Ahmet Y.; Pomerantz, Richard T.

2014-01-01

Microhomology-mediated end-joining (MMEJ) is an error-prone alternative double-strand break repair pathway that utilizes sequence microhomology to recombine broken DNA. Although MMEJ is implicated in cancer development, the mechanism of this pathway is unknown. We demonstrate that purified human DNA polymerase θ (Polθ) performs MMEJ of DNA containing 3’ single-strand DNA overhangs with two or more base-pairs of homology, including DNA modeled after telomeres, and show that MMEJ is dependent on Polθ in human cells. Our data support a mechanism whereby Polθ facilitates end-joining and microhomology annealing then utilizes the opposing overhang as a template in trans which stabilizes the DNA synapse. Polθ exhibits a preference for DNA containing a 5’-terminal phosphate, similar to polymerases involved in non-homologous end-joining. Lastly, we identify a conserved loop domain that is essential for MMEJ and higher-order structures of Polθ which likely promote DNA synapse formation. PMID:25643323

Maternal phylogeny of a newly-found yak population in china.

PubMed

Mipam, Tserang Donko; Wen, Yongli; Fu, Changxiu; Li, Shanrong; Zhao, Hongwen; Ai, Yi; Li, Lu; Zhang, Lei; Zou, Deqiang

2012-01-01

The Jinchuan yak is a new yak population identified in Sichuan, China. This population has a special anatomical characteristic: an additional pair of ribs compared with other yak breeds. The genetic structure of this population is unknown. In the present study, we investigated the maternal phylogeny of this special yak population using the mitochondrial DNA variation. A total of 23 Jinchuan yaks were sequenced for a 823-bp fragment of D-loop control region and three individuals were sequenced for the whole mtDNA genome with a length of 16,371-bp. To compare with the data from other yaks, we extracted sequence data from Genebank, including D-loop of 398 yaks (from 12 breeds) and 55 wild yaks, and whole mitochondrial genomes of 53 yaks (from 12 breeds) and 21 wild yaks. A total of 127 haplotypes were defined, based on the D-loop data. Thirteen haplotypes were defined from 23 mtDNA D-loop sequences of Jinchuan yaks, six of which were shared only by Jinchuan, and one was shared by Jinchuan and wild yaks. The Jinquan yaks were found to carry clades A and B from lineage I and clade C of lineage II, respectively. It was also suggested that the Jinchuan population has no distinct different phylogenetic relationship in maternal inheritance with other breeds of yak. The highly haplotype diversity of the Pali breed, Jinchuan population, Maiwa breed and Jiulong breed suggested that the yak was first domesticated from wild yaks in the middle Himalayan region and the northern Hengduan Mountains. The special anatomic characteristic that we found in the Jinchuan population needs further studies based on nuclear data.

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

PubMed

Belotserkovskii, Boris P; Hanawalt, Philip C

2015-11-01

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

Orthogonal labeling of M13 minor capsid proteins with DNA to self-assemble end-to-end multiphage structures.

PubMed

Hess, Gaelen T; Guimaraes, Carla P; Spooner, Eric; Ploegh, Hidde L; Belcher, Angela M

2013-09-20

M13 bacteriophage has been used as a scaffold to organize materials for various applications. Building more complex multiphage devices requires precise control of interactions between the M13 capsid proteins. Toward this end, we engineered a loop structure onto the pIII capsid protein of M13 bacteriophage to enable sortase-mediated labeling reactions for C-terminal display. Combining this with N-terminal sortase-mediated labeling, we thus created a phage scaffold that can be labeled orthogonally on three capsid proteins: the body and both ends. We show that covalent attachment of different DNA oligonucleotides at the ends of the new phage structure enables formation of multiphage particles oriented in a specific order. These have potential as nanoscale scaffolds for multi-material devices.

Characterization of Non-coding DNA Satellites Associated with Sweepoviruses (Genus Begomovirus, Geminiviridae) – Definition of a Distinct Class of Begomovirus-Associated Satellites

PubMed Central

Lozano, Gloria; Trenado, Helena P.; Fiallo-Olivé, Elvira; Chirinos, Dorys; Geraud-Pouey, Francis; Briddon, Rob W.; Navas-Castillo, Jesús

2016-01-01

Begomoviruses (family Geminiviridae) are whitefly-transmitted, plant-infecting single-stranded DNA viruses that cause crop losses throughout the warmer parts of the World. Sweepoviruses are a phylogenetically distinct group of begomoviruses that infect plants of the family Convolvulaceae, including sweet potato (Ipomoea batatas). Two classes of subviral molecules are often associated with begomoviruses, particularly in the Old World; the betasatellites and the alphasatellites. An analysis of sweet potato and Ipomoea indica samples from Spain and Merremia dissecta samples from Venezuela identified small non-coding subviral molecules in association with several distinct sweepoviruses. The sequences of 18 clones were obtained and found to be structurally similar to tomato leaf curl virus-satellite (ToLCV-sat, the first DNA satellite identified in association with a begomovirus), with a region with significant sequence identity to the conserved region of betasatellites, an A-rich sequence, a predicted stem–loop structure containing the nonanucleotide TAATATTAC, and a second predicted stem–loop. These sweepovirus-associated satellites join an increasing number of ToLCV-sat-like non-coding satellites identified recently. Although sharing some features with betasatellites, evidence is provided to suggest that the ToLCV-sat-like satellites are distinct from betasatellites and should be considered a separate class of satellites, for which the collective name deltasatellites is proposed. PMID:26925037

Mitotic chromosome compaction via active loop extrusion

NASA Astrophysics Data System (ADS)

Goloborodko, Anton; Imakaev, Maxim; Marko, John; Mirny, Leonid; MIT-Northwestern Team

During cell division, two copies of each chromosome are segregated from each other and compacted more than hundred-fold into the canonical X-shaped structures. According to earlier microscopic observations and the recent Hi-C study, chromosomes are compacted into arrays of consecutive loops of ~100 kilobases. Mechanisms that lead to formation of such loop arrays are largely unknown. Here we propose that, during cell division, chromosomes can be compacted by enzymes that extrude loops on chromatin fibers. First, we use computer simulations and analytical modeling to show that a system of loop-extruding enzymes on a chromatin fiber self-organizes into an array of consecutive dynamic loops. Second, we model the process of loop extrusion in 3D and show that, coupled with the topo II strand-passing activity, it leads to robust compaction and segregation of sister chromatids. This mechanism of chromosomal condensation and segregation does not require additional proteins or specific DNA markup and is robust against variations in the number and properties of such loop extruding enzymes. Work at NU was supported by the NSF through Grants DMR-1206868 and MCB-1022117, and by the NIH through Grants GM105847 and CA193419. Work at MIT was supported by the NIH through Grants GM114190 R01HG003143.

The annealing helicase and branch migration activities of Drosophila HARP.

PubMed

Kassavetis, George A; Kadonaga, James T

2014-01-01

HARP (SMARCAL1, MARCAL1) is an annealing helicase that functions in the repair and restart of damaged DNA replication forks through its DNA branch migration and replication fork regression activities. HARP is conserved among metazoans. HARP from invertebrates differs by the absence of one of the two HARP-specific domain repeats found in vertebrates. The annealing helicase and branch migration activity of invertebrate HARP has not been documented. We found that HARP from Drosophila melanogaster retains the annealing helicase activity of human HARP, the ability to disrupt D-loops and to branch migrate Holliday junctions, but fails to regress model DNA replication fork structures. A comparison of human and Drosophila HARP on additional substrates revealed that both HARPs are competent in branch migrating a bidirectional replication bubble composed of either DNA:DNA or RNA:DNA hybrid. Human, but not Drosophila, HARP is also capable of regressing a replication fork structure containing a highly stable poly rG:dC hybrid. Persistent RNA:DNA hybrids in vivo can lead to replication fork arrest and genome instability. The ability of HARP to strand transfer hybrids may signify a hybrid removal function for this enzyme, in vivo.

RPA-mediated unfolding of systematically varying G-quadruplex structures.

PubMed

Ray, Sujay; Qureshi, Mohammad H; Malcolm, Dominic W; Budhathoki, Jagat B; Celik, Uğur; Balci, Hamza

2013-05-21

G-quadruplex (GQ) is a noncanonical nucleic acid structure that is formed by guanine rich sequences. Unless it is destabilized by proteins such as replication protein A (RPA), GQ could interfere with DNA metabolic functions, such as replication or repair. We studied RPA-mediated GQ unfolding using single-molecule FRET on two groups of GQ structures that have different loop lengths and different numbers of G-tetrad layers. We observed a linear increase in the steady-state stability of the GQ against RPA-mediated unfolding with increasing number of layers or decreasing loop length. The stability demonstrated by different GQ structures varied by at least three orders of magnitude. Those with shorter loops (less than three nucleotides long) or a greater number of layers (more than three layers) maintained a significant folded population even at physiological RPA concentration (≈1 μM), raising the possibility of physiological viability of such GQ structures. Finally, we measured the transition time between the start and end of the RPA-mediated GQ unfolding process to be 0.35 ± 0.10 s for all GQ constructs we studied, despite significant differences in their steady-state stabilities. We propose a two-step RPA-mediated GQ unfolding mechanism that is consistent with our observations. Copyright © 2013 Biophysical Society. Published by Elsevier Inc. All rights reserved.

RPA-Mediated Unfolding of Systematically Varying G-Quadruplex Structures

PubMed Central

Ray, Sujay; Qureshi, Mohammad H.; Malcolm, Dominic W.; Budhathoki, Jagat B.; Çelik, Uğur; Balci, Hamza

2013-01-01

G-quadruplex (GQ) is a noncanonical nucleic acid structure that is formed by guanine rich sequences. Unless it is destabilized by proteins such as replication protein A (RPA), GQ could interfere with DNA metabolic functions, such as replication or repair. We studied RPA-mediated GQ unfolding using single-molecule FRET on two groups of GQ structures that have different loop lengths and different numbers of G-tetrad layers. We observed a linear increase in the steady-state stability of the GQ against RPA-mediated unfolding with increasing number of layers or decreasing loop length. The stability demonstrated by different GQ structures varied by at least three orders of magnitude. Those with shorter loops (less than three nucleotides long) or a greater number of layers (more than three layers) maintained a significant folded population even at physiological RPA concentration (≈1 μM), raising the possibility of physiological viability of such GQ structures. Finally, we measured the transition time between the start and end of the RPA-mediated GQ unfolding process to be 0.35 ± 0.10 s for all GQ constructs we studied, despite significant differences in their steady-state stabilities. We propose a two-step RPA-mediated GQ unfolding mechanism that is consistent with our observations. PMID:23708363

Mitochondrial DNA variation in natural populations of endangered Indian feather-back fish, Chitala chitala.

PubMed

Mandal, Anup; Mohindra, Vindhya; Singh, Rajeev Kumar; Punia, Peyush; Singh, Ajay Kumar; Lal, Kuldeep Kumar

2012-02-01

Genetic variation at mitochondrial cytochrome b (cyt b) and D-loop region reveals the evidence of population sub-structuring in Indian populations of highly endangered primitive feather-back fish Chitala chitala. Samples collected through commercial catches from eight riverine populations from different geographical locations of India were analyzed for cyt b region (307 bp) and D-loop region (636-716 bp). The sequences of the both the mitochondrial regions revealed high haplotype diversity and low nucleotide diversity. The patterns of genetic diversity, haplotypes networks clearly indicated two distinct mitochondrial lineages and mismatch distribution strongly suggest a historical influence on the genetic structure of C. chitala populations. The baseline information on genetic variation and the evidence of population sub-structuring generated from this study would be useful for planning effective strategies for conservation and rehabilitation of this highly endangered species.

Identification of Specific DNA Binding Residues in the TCP Family of Transcription Factors in Arabidopsis[W

PubMed Central

Aggarwal, Pooja; Das Gupta, Mainak; Joseph, Agnel Praveen; Chatterjee, Nirmalya; Srinivasan, N.; Nath, Utpal

2010-01-01

The TCP transcription factors control multiple developmental traits in diverse plant species. Members of this family share an ∼60-residue-long TCP domain that binds to DNA. The TCP domain is predicted to form a basic helix-loop-helix (bHLH) structure but shares little sequence similarity with canonical bHLH domain. This classifies the TCP domain as a novel class of DNA binding domain specific to the plant kingdom. Little is known about how the TCP domain interacts with its target DNA. We report biochemical characterization and DNA binding properties of a TCP member in Arabidopsis thaliana, TCP4. We have shown that the 58-residue domain of TCP4 is essential and sufficient for binding to DNA and possesses DNA binding parameters comparable to canonical bHLH proteins. Using a yeast-based random mutagenesis screen and site-directed mutants, we identified the residues important for DNA binding and dimer formation. Mutants defective in binding and dimerization failed to rescue the phenotype of an Arabidopsis line lacking the endogenous TCP4 activity. By combining structure prediction, functional characterization of the mutants, and molecular modeling, we suggest a possible DNA binding mechanism for this class of transcription factors. PMID:20363772

Novel structural features drive DNA binding properties of Cmr, a CRP family protein in TB complex mycobacteria.

PubMed

Ranganathan, Sridevi; Cheung, Jonah; Cassidy, Michael; Ginter, Christopher; Pata, Janice D; McDonough, Kathleen A

2018-01-09

Mycobacterium tuberculosis (Mtb) encodes two CRP/FNR family transcription factors (TF) that contribute to virulence, Cmr (Rv1675c) and CRPMt (Rv3676). Prior studies identified distinct chromosomal binding profiles for each TF despite their recognizing overlapping DNA motifs. The present study shows that Cmr binding specificity is determined by discriminator nucleotides at motif positions 4 and 13. X-ray crystallography and targeted mutational analyses identified an arginine-rich loop that expands Cmr's DNA interactions beyond the classical helix-turn-helix contacts common to all CRP/FNR family members and facilitates binding to imperfect DNA sequences. Cmr binding to DNA results in a pronounced asymmetric bending of the DNA and its high level of cooperativity is consistent with DNA-facilitated dimerization. A unique N-terminal extension inserts between the DNA binding and dimerization domains, partially occluding the site where the canonical cAMP binding pocket is found. However, an unstructured region of this N-terminus may help modulate Cmr activity in response to cellular signals. Cmr's multiple levels of DNA interaction likely enhance its ability to integrate diverse gene regulatory signals, while its novel structural features establish Cmr as an atypical CRP/FNR family member. © The Author(s) 2017. Published by Oxford University Press on behalf of Nucleic Acids Research.

Combing Chromosomal DNA Mediated by the SMC Complex: Structure and Mechanisms.

PubMed

Kamada, Katsuhiko; Barillà, Daniela

2018-02-01

Genome maintenance requires various nucleoid-associated factors in prokaryotes. Among them, the SMC (Structural Maintenance of Chromosomes) protein has been thought to play a static role in the organization and segregation of the chromosome during cell division. However, recent studies have shown that the bacterial SMC is required to align left and right arms of the emerging chromosome and that the protein dynamically travels from origin to Ter region. A rod form of the SMC complex mediates DNA bridging and has been recognized as a machinery responsible for DNA loop extrusion, like eukaryotic condensin or cohesin complexes, which act as chromosome organizers. Attention is now turning to how the prototype of the complex is loaded on the entry site and translocated on chromosomal DNA, explaining its overall conformational changes at atomic levels. Here, we review and highlight recent findings concerning the prokaryotic SMC complex and discuss possible mechanisms from the viewpoint of protein architecture. © 2017 The Authors. BioEssays Published by WILEY Periodicals, Inc.

Characterization of the Caenorhabditis elegans HIM-6/BLM helicase: unwinding recombination intermediates.

PubMed

Jung, Hana; Lee, Jin A; Choi, Seoyoon; Lee, Hyunwoo; Ahn, Byungchan

2014-01-01

Mutations in three human RecQ genes are implicated in heritable human syndromes. Mutations in BLM, a RecQ gene, cause Bloom syndrome (BS), which is characterized by short stature, cancer predisposition, and sensitivity to sunlight. BLM is a RecQ DNA helicase that, with interacting proteins, is able to dissolve various DNA structures including double Holliday junctions. A BLM ortholog, him-6, has been identified in Caenorhabditis elegans, but little is known about its enzymatic activities or its in vivo roles. By purifying recombinant HIM-6 and performing biochemical assays, we determined that the HIM-6 has DNA-dependent ATPase activity HIM-6 and helicase activity that proceeds in the 3'-5' direction and needs at least five 3' overhanging nucleotides. HIM-6 is also able to unwind DNA structures including D-loops and Holliday junctions. Worms with him-6 mutations were defective in recovering the cell cycle arrest after HU treatment. These activities strongly support in vivo roles for HIM-6 in processing recombination intermediates.

Characterization of the Caenorhabditis elegans HIM-6/BLM Helicase: Unwinding Recombination Intermediates

PubMed Central

Choi, Seoyoon; Lee, Hyunwoo; Ahn, Byungchan

2014-01-01

Mutations in three human RecQ genes are implicated in heritable human syndromes. Mutations in BLM, a RecQ gene, cause Bloom syndrome (BS), which is characterized by short stature, cancer predisposition, and sensitivity to sunlight. BLM is a RecQ DNA helicase that, with interacting proteins, is able to dissolve various DNA structures including double Holliday junctions. A BLM ortholog, him-6, has been identified in Caenorhabditis elegans, but little is known about its enzymatic activities or its in vivo roles. By purifying recombinant HIM-6 and performing biochemical assays, we determined that the HIM-6 has DNA-dependent ATPase activity HIM-6 and helicase activity that proceeds in the 3'-5' direction and needs at least five 3' overhanging nucleotides. HIM-6 is also able to unwind DNA structures including D-loops and Holliday junctions. Worms with him-6 mutations were defective in recovering the cell cycle arrest after HU treatment. These activities strongly support in vivo roles for HIM-6 in processing recombination intermediates. PMID:25036527

Evidence and age-related distribution of mtDNA D-loop point mutations in skeletal muscle from healthy subjects and mitochondrial patients.

PubMed

Del Bo, Roberto; Bordoni, Andreina; Martinelli Boneschi, Filippo; Crimi, Marco; Sciacco, Monica; Bresolin, Nereo; Scarlato, Guglielmo; Comi, Giacomo Pietri

2002-10-15

The progressive accumulation of mitochondrial DNA (mtDNA) alterations, ranging from single mutations to large-scale deletions, in both the normal ageing process and pathological conditions is a relevant phenomenon in terms of frequency and heteroplasmic degree. Recently, two point mutations (A189G and T408A) within the Displacement loop (D-loop) region, the control region for mtDNA replication, were shown to occur in skeletal muscles from aged individuals. We evaluated the presence and the heteroplasmy levels of these two mutations in muscle biopsies from 91 unrelated individuals of different ages (21 healthy subjects and 70 patients affected by mitochondrial encephalomyopathies). Overall, both mutations significantly accumulate with age. However, a different relationship was discovered among the different subgroups of patients: a higher number of A189G positive subjects younger than 53 years was detected in the subgroup of multiple-deleted patients; furthermore, a trend towards an increased risk for the mutations was evidenced among patients carrying multiple deletions when compared to healthy controls. These findings support the idea that a common biological mechanism determines the accumulation of somatic point mutations in the D-loop region, both in healthy subjects and in mitochondrial myopathy patients. At the same time, it appears that disorders caused by mutations of nuclear genes controlling mtDNA replication (the "mtDNA multiple deletions" syndromes) present a temporal advantage to mutate in the D-loop region. This observation may be relevant to the definition of the molecular pathogenesis of these latter syndromes. Copyright 2002 Elsevier Science B.V.

Are mutagenic non D-loop direct repeat motifs in mitochondrial DNA under a negative selection pressure?

PubMed Central

Lakshmanan, Lakshmi Narayanan; Gruber, Jan; Halliwell, Barry; Gunawan, Rudiyanto

2015-01-01

Non D-loop direct repeats (DRs) in mitochondrial DNA (mtDNA) have been commonly implicated in the mutagenesis of mtDNA deletions associated with neuromuscular disease and ageing. Further, these DRs have been hypothesized to put a constraint on the lifespan of mammals and are under a negative selection pressure. Using a compendium of 294 mammalian mtDNA, we re-examined the relationship between species lifespan and the mutagenicity of such DRs. Contradicting the prevailing hypotheses, we found no significant evidence that long-lived mammals possess fewer mutagenic DRs than short-lived mammals. By comparing DR counts in human mtDNA with those in selectively randomized sequences, we also showed that the number of DRs in human mtDNA is primarily determined by global mtDNA properties, such as the bias in synonymous codon usage (SCU) and nucleotide composition. We found that SCU bias in mtDNA positively correlates with DR counts, where repeated usage of a subset of codons leads to more frequent DR occurrences. While bias in SCU and nucleotide composition has been attributed to nucleotide mutational bias, mammalian mtDNA still exhibit higher SCU bias and DR counts than expected from such mutational bias, suggesting a lack of negative selection against non D-loop DRs. PMID:25855815

A Mutation Directs the Structural Switch of DNA Binding Proteins under Starvation to a Ferritin-like Protein Cage.

PubMed

Williams, Sunanda Margrett; Chandran, Anu Vijayakumari; Prakash, Sunita; Vijayan, Mamannamana; Chatterji, Dipankar

2017-09-05

Proteins of the ferritin family are ubiquitous in living organisms. With their spherical cage-like structures they are the iron storehouses in cells. Subfamilies of ferritins include 24-meric ferritins and bacterioferritins (maxiferritins), and 12-meric Dps (miniferritins). Dps safeguards DNA by direct binding, affording physical protection and safeguards from free radical-mediated damage by sequestering iron in its core. The maxiferritins can oxidize and store iron but cannot bind DNA. Here we show that a mutation at a critical interface in Dps alters its assembly from the canonical 12-mer to a ferritin-like 24-mer under crystallization. This structural switch was attributed to the conformational alteration of a highly conserved helical loop and rearrangement of the C-terminus. Our results demonstrate a novel concept of mutational switch between related protein subfamilies and corroborate the popular model for evolution by which subtle substitutions in an amino acid sequence lead to diversification among proteins. Copyright © 2017 Elsevier Ltd. All rights reserved.

Testing the Use of Implicit Solvent in the Molecular Dynamics Modelling of DNA Flexibility

NASA Astrophysics Data System (ADS)

Mitchell, J.; Harris, S.

DNA flexibility controls packaging, looping and in some cases sequence specific protein binding. Molecular dynamics simulations carried out with a computationally efficient implicit solvent model are potentially a powerful tool for studying larger DNA molecules than can be currently simulated when water and counterions are represented explicitly. In this work we compare DNA flexibility at the base pair step level modelled using an implicit solvent model to that previously determined from explicit solvent simulations and database analysis. Although much of the sequence dependent behaviour is preserved in implicit solvent, the DNA is considerably more flexible when the approximate model is used. In addition we test the ability of the implicit solvent to model stress induced DNA disruptions by simulating a series of DNA minicircle topoisomers which vary in size and superhelical density. When compared with previously run explicit solvent simulations, we find that while the levels of DNA denaturation are similar using both computational methodologies, the specific structural form of the disruptions is different.

Overcoming a nucleosomal barrier to replication

PubMed Central

Chang, Han-Wen; Pandey, Manjula; Kulaeva, Olga I.; Patel, Smita S.; Studitsky, Vasily M.

2016-01-01

Efficient overcoming and accurate maintenance of chromatin structure and associated histone marks during DNA replication are essential for normal functioning of the daughter cells. However, the molecular mechanisms of replication through chromatin are unknown. We have studied traversal of uniquely positioned mononucleosomes by T7 replisome in vitro. Nucleosomes present a strong, sequence-dependent barrier for replication, with particularly strong pausing of DNA polymerase at the +(31–40) and +(41–65) regions of the nucleosomal DNA. The exonuclease activity of T7 DNA polymerase increases the overall rate of progression of the replisome through a nucleosome, likely by resolving nonproductive complexes. The presence of nucleosome-free DNA upstream of the replication fork facilitates the progression of DNA polymerase through the nucleosome. After replication, at least 50% of the nucleosomes assume an alternative conformation, maintaining their original positions on the DNA. Our data suggest a previously unpublished mechanism for nucleosome maintenance during replication, likely involving transient formation of an intranucleosomal DNA loop. PMID:27847876

Comparison between TRF2 and TRF1 of their telomeric DNA-bound structures and DNA-binding activities

PubMed Central

Hanaoka, Shingo; Nagadoi, Aritaka; Nishimura, Yoshifumi

2005-01-01

Mammalian telomeres consist of long tandem arrays of double-stranded telomeric TTAGGG repeats packaged by the telomeric DNA-binding proteins TRF1 and TRF2. Both contain a similar C-terminal Myb domain that mediates sequence-specific binding to telomeric DNA. In a DNA complex of TRF1, only the single Myb-like domain consisting of three helices can bind specifically to double-stranded telomeric DNA. TRF2 also binds to double-stranded telomeric DNA. Although the DNA binding mode of TRF2 is likely identical to that of TRF1, TRF2 plays an important role in the t-loop formation that protects the ends of telomeres. Here, to clarify the details of the double-stranded telomeric DNA-binding modes of TRF1 and TRF2, we determined the solution structure of the DNA-binding domain of human TRF2 bound to telomeric DNA; it consists of three helices, and like TRF1, the third helix recognizes TAGGG sequence in the major groove of DNA with the N-terminal arm locating in the minor groove. However, small but significant differences are observed; in contrast to the minor groove recognition of TRF1, in which an arginine residue recognizes the TT sequence, a lysine residue of TRF2 interacts with the TT part. We examined the telomeric DNA-binding activities of both DNA-binding domains of TRF1 and TRF2 and found that TRF1 binds more strongly than TRF2. Based on the structural differences of both domains, we created several mutants of the DNA-binding domain of TRF2 with stronger binding activities compared to the wild-type TRF2. PMID:15608118

Genetic distance of Malaysian mousedeer based on mitochondrial DNA cytochrome oxidase I (COI) and D-loop region sequences

NASA Astrophysics Data System (ADS)

Bakar, Mohamad-Azam Akmal Abu; Rovie-Ryan, Jeffrine Japning; Ampeng, Ahmad; Yaakop, Salmah; Nor, Shukor Md; Md-Zain, Badrul Munir

2018-04-01

Mousedeer is one of the primitive mammals that can be found mainly in Southeast-Asia region. There are two species of mousedeer in Malaysia which are Tragulus kanchil and Tragulus napu. Both species can be distinguish by size, coat coloration, and throat pattern but clear diagnosis still cannot be found. The objective of the study is to show the genetic distance relationship between T. kanchil and T. napu and their population based on mitochondrial DNA (mtDNA) cytochrome oxidase I (COI) and D-loop region. There are 42 sample of mousedeer were used in this study collected by PERHILITAN from different locality. Another 29 D-loop sequence were retrieved from Genbank for comparative analysis. All sample were amplified using universal primer and species-specific primer for COI and D-loop genes via PCR process. The amplified sequences were analyzed to determine genetic distance of T. kanchil and T. napu. From the analysis, the average genetic distance between T. kanchil and T. napu based on locus COI and D-loop were 0.145 and 0.128 respectively. The genetic distance between populations of T. kanchil based on locus COI was between 0.003-0.013. For locus D-loop, genetic distance analysis showed distance in relationship between west-coast populations to east-coast population of T. kanchil. COI and D-loop mtDNA region provided a clear picture on the relationship within the mousedeer species. Last but not least, conservation effort toward protecting this species can be done by study the molecular genetics and prevent the extinction of this species.

Principles of Chromosome Architecture Revealed by Hi-C.

PubMed

Eagen, Kyle P

2018-06-01

Chromosomes are folded and compacted in interphase nuclei, but the molecular basis of this folding is poorly understood. Chromosome conformation capture methods, such as Hi-C, combine chemical crosslinking of chromatin with fragmentation, DNA ligation, and high-throughput DNA sequencing to detect neighboring loci genome-wide. Hi-C has revealed the segregation of chromatin into active and inactive compartments and the folding of DNA into self-associating domains and loops. Depletion of CTCF, cohesin, or cohesin-associated proteins was recently shown to affect the majority of domains and loops in a manner that is consistent with a model of DNA folding through extrusion of chromatin loops. Compartmentation was not dependent on CTCF or cohesin. Hi-C contact maps represent the superimposition of CTCF/cohesin-dependent and -independent folding states. Copyright © 2018 Elsevier Ltd. All rights reserved.

The intervening domain from MeCP2 enhances the DNA affinity of the methyl binding domain and provides an independent DNA interaction site.

PubMed

Claveria-Gimeno, Rafael; Lanuza, Pilar M; Morales-Chueca, Ignacio; Jorge-Torres, Olga C; Vega, Sonia; Abian, Olga; Esteller, Manel; Velazquez-Campoy, Adrian

2017-01-31

Methyl-CpG binding protein 2 (MeCP2) preferentially interacts with methylated DNA and it is involved in epigenetic regulation and chromatin remodelling. Mutations in MeCP2 are linked to Rett syndrome, the leading cause of intellectual retardation in girls and causing mental, motor and growth impairment. Unstructured regions in MeCP2 provide the plasticity for establishing interactions with multiple binding partners. We present a biophysical characterization of the methyl binding domain (MBD) from MeCP2 reporting the contribution of flanking domains to its structural stability and dsDNA interaction. The flanking disordered intervening domain (ID) increased the structural stability of MBD, modified its dsDNA binding profile from an entropically-driven moderate-affinity binding to an overwhelmingly enthalpically-driven high-affinity binding. Additionally, ID provided an additional site for simultaneously and autonomously binding an independent dsDNA molecule, which is a key feature linked to the chromatin remodelling and looping activity of MeCP2, as well as its ability to interact with nucleosomes replacing histone H1. The dsDNA interaction is characterized by an unusually large heat capacity linked to a cluster of water molecules trapped within the binding interface. The dynamics of disordered regions together with extrinsic factors are key determinants of MeCP2 global structural properties and functional capabilities.

The intervening domain from MeCP2 enhances the DNA affinity of the methyl binding domain and provides an independent DNA interaction site

PubMed Central

Claveria-Gimeno, Rafael; Lanuza, Pilar M.; Morales-Chueca, Ignacio; Jorge-Torres, Olga C.; Vega, Sonia; Abian, Olga; Esteller, Manel; Velazquez-Campoy, Adrian

2017-01-01

Methyl-CpG binding protein 2 (MeCP2) preferentially interacts with methylated DNA and it is involved in epigenetic regulation and chromatin remodelling. Mutations in MeCP2 are linked to Rett syndrome, the leading cause of intellectual retardation in girls and causing mental, motor and growth impairment. Unstructured regions in MeCP2 provide the plasticity for establishing interactions with multiple binding partners. We present a biophysical characterization of the methyl binding domain (MBD) from MeCP2 reporting the contribution of flanking domains to its structural stability and dsDNA interaction. The flanking disordered intervening domain (ID) increased the structural stability of MBD, modified its dsDNA binding profile from an entropically-driven moderate-affinity binding to an overwhelmingly enthalpically-driven high-affinity binding. Additionally, ID provided an additional site for simultaneously and autonomously binding an independent dsDNA molecule, which is a key feature linked to the chromatin remodelling and looping activity of MeCP2, as well as its ability to interact with nucleosomes replacing histone H1. The dsDNA interaction is characterized by an unusually large heat capacity linked to a cluster of water molecules trapped within the binding interface. The dynamics of disordered regions together with extrinsic factors are key determinants of MeCP2 global structural properties and functional capabilities. PMID:28139759

Molecular Dynamics Simulations and Structural Analysis to Decipher Functional Impact of a Twenty Residue Insert in the Ternary Complex of Mus musculus TdT Isoform

PubMed Central

Mutt, Eshita; Sowdhamini, Ramanathan

2016-01-01

Insertions/deletions are common evolutionary tools employed to alter the structural and functional repertoire of protein domains. An insert situated proximal to the active site or ligand binding site frequently impacts protein function; however, the effect of distal indels on protein activity and/or stability are often not studied. In this paper, we have investigated a distal insert, which influences the function and stability of a unique DNA polymerase, called terminal deoxynucleotidyl transferase (TdT). TdT (EC:2.7.7.31) is a monomeric 58 kDa protein belonging to family X of eukaryotic DNA polymerases and known for its role in V(D)J recombination as well as in non-homologous end-joining (NHEJ) pathways. Two murine isoforms of TdT, with a length difference of twenty residues and having different biochemical properties, have been studied. All-atom molecular dynamics simulations at different temperatures and interaction network analyses were performed on the short and long-length isoforms. We observed conformational changes in the regions distal to the insert position (thumb subdomain) in the longer isoform, which indirectly affects the activity and stability of the enzyme through a mediating loop (Loop1). A structural rationale could be provided to explain the reduced polymerization rate as well as increased thermosensitivity of the longer isoform caused by peripherally located length variations within a DNA polymerase. These observations increase our understanding of the roles of length variants in introducing functional diversity in protein families in general. PMID:27311013

RECQ-like helicases Sgs1 and BLM regulate R-loop–associated genome instability

PubMed Central

Chang, Emily Yun-Chia; Novoa, Carolina A.; Aristizabal, Maria J.; Coulombe, Yan; Segovia, Romulo; Shen, Yaoqing; Keong, Christelle; Tam, Annie S.; Jones, Steven J.M.; Masson, Jean-Yves; Kobor, Michael S.

2017-01-01

Sgs1, the orthologue of human Bloom’s syndrome helicase BLM, is a yeast DNA helicase functioning in DNA replication and repair. We show that SGS1 loss increases R-loop accumulation and sensitizes cells to transcription–replication collisions. Yeast lacking SGS1 accumulate R-loops and γ-H2A at sites of Sgs1 binding, replication pausing regions, and long genes. The mutation signature of sgs1Δ reveals copy number changes flanked by repetitive regions with high R-loop–forming potential. Analysis of BLM in Bloom’s syndrome fibroblasts or by depletion of BLM from human cancer cells confirms a role for Sgs1/BLM in suppressing R-loop–associated genome instability across species. In support of a potential direct effect, BLM is found physically proximal to DNA:RNA hybrids in human cells, and can efficiently unwind R-loops in vitro. Together, our data describe a conserved role for Sgs1/BLM in R-loop suppression and support an increasingly broad view of DNA repair and replication fork stabilizing proteins as modulators of R-loop–mediated genome instability. PMID:29042409

Crystal structure of a c-kit promoter quadruplex reveals the structural role of metal ions and water molecules in maintaining loop conformation.

PubMed

Wei, Dengguo; Parkinson, Gary N; Reszka, Anthony P; Neidle, Stephen

2012-05-01

We report here the 1.62 Å crystal structure of an intramolecular quadruplex DNA formed from a sequence in the promoter region of the c-kit gene. This is the first reported crystal structure of a promoter quadruplex and the first observation of localized magnesium ions in a quadruplex structure. The structure reveals that potassium and magnesium ions have an unexpected yet significant structural role in stabilizing particular quadruplex loops and grooves that is distinct from but in addition to the role of potassium ions in the ion channel at the centre of all quadruplex structures. The analysis also shows how ions cluster together with structured water molecules to stabilize the quadruplex arrangement. This particular quadruplex has been previously studied by NMR methods, and the present X-ray structure is in accord with the earlier topology assignment. However, as well as the observations of potassium and magnesium ions, the crystal structure has revealed a highly significant difference in the dimensions of the large cleft in the structure, which is a plausible target for small molecules. This difference can be understood by the stabilizing role of structured water networks.

The Local Dinucleotide Preference of APOBEC3G Can Be Altered from 5′-CC to 5′-TC by a Single Amino Acid Substitution

PubMed Central

Rathore, Anurag; Carpenter, Michael A; Demir, Özlem; Ikeda, Terumasa; Li, Ming; Shaban, Nadine; Law, Emily K.; Anokhin, Dmitry; Brown, William L.; Amaro, Rommie E.; Harris, Reuben S.

2013-01-01

APOBEC3A and APOBEC3G are DNA cytosine deaminases with biological functions in foreign DNA and retrovirus restriction, respectively. APOBEC3A has an intrinsic preference for cytosine preceded by thymine (5′-TC) in single-stranded DNA substrates, whereas APOBEC3G prefers the target cytosine to be preceded by another cytosine (5′-CC). To determine the amino acids responsible for these strong dinucleotide preferences, we analyzed a series of chimeras in which putative DNA binding loop regions of APOBEC3G were replaced with the corresponding regions from APOBEC3A. Loop 3 replacement enhanced APOBEC3G catalytic activity but did not alter its intrinsic 5′-CC dinucleotide substrate preference. Loop 7 replacement caused APOBEC3G to become APOBEC3A-like and strongly prefer 5′-TC substrates. Simultaneous loop 3/7 replacement resulted in a hyperactive APOBEC3G variant that also preferred 5′-TC dinucleotides. Single amino acid exchanges revealed D317 as a critical determinant of dinucleotide substrate specificity. Multi-copy explicitly solvated all-atom molecular dynamics simulations suggested a model in which D317 acts as a helix-capping residue by constraining the mobility of loop 7, forming a novel binding pocket that favorably accommodates cytosine. All catalytically active APOBEC3G variants, regardless of dinucleotide preference, retained HIV-1 restriction activity. These data support a model in which the loop 7 region governs the selection of local dinucleotide substrates for deamination but is unlikely to be part of the higher level targeting mechanisms that direct these enzymes to biological substrates such as HIV-1 cDNA. PMID:23938202

A vicious loop of fatty acid-binding protein 4 and DNA methyltransferase 1 promotes acute myeloid leukemia and acts as a therapeutic target

PubMed Central

Yan, F; Shen, N; Pang, JX; Zhao, N; Zhang, YW; Bode, AM; Al-Kali, A; Litzow, MR; Li, B; Liu, SJ

2017-01-01

Aberrant DNA methylation mediated by deregulation of DNA methyltransferases (DNMT) is a key hallmark of acute myeloid leukemia (AML), yet efforts to target DNMT deregulation for drug development have lagged. We previously demonstrated that upregulation of fatty acid-binding protein 4 (FABP4) promotes AML aggressiveness through enhanced DNMT1-dependent DNA methylation. Here we demonstrate that FABP4 upregulation in AML cells occurs through vascular endothelial growth factor (VEGF) signaling, thus elucidating a crucial FABP4-DNMT1 regulatory feedback loop in AML biology. We show that FABP4 dysfunction by its selective inhibitor BMS309403 leads to downregulation of DNMT1, decrease of global DNA methylation and re-expression of p15INK4B tumor suppressor gene by promoter DNA hypomethylation in vitro, ex vivo and in vivo. Functionally, BMS309403 suppresses cell colony formation, induces cell differentiation, and, importantly, impairs leukemic disease progression in mouse models of leukemia. Our findings highlight AML-promoting properties of the FABP4-DNMT1 vicious loop, and identify an attractive class of therapeutic agents with a high potential for clinical use in AML patients. The results will also assist in establishing the FABP4-DNMT1 loop as a target for therapeutic discovery to enhance the index of current epigenetic therapies. PMID:28993705

Maternal Betaine Supplementation during Gestation Enhances Expression of mtDNA-Encoded Genes through D-Loop DNA Hypomethylation in the Skeletal Muscle of Newborn Piglets.

PubMed

Jia, Yimin; Song, Haogang; Gao, Guichao; Cai, Demin; Yang, Xiaojing; Zhao, Ruqian

2015-11-25

Betaine has been widely used in animal and human nutrition to promote muscle growth and performance, yet it remains unknown whether maternal betaine supplementation during gestation affects the metabolic characteristics of neonatal skeletal muscles. In the present study, feeding sows with betaine-supplemented diets throughout gestation significantly upregulated the expression of mtDNA-encoded OXPHOS genes (p < 0.05), including COX1, COX2, and ND5, in the muscle of newborn piglets, which was associated with enhanced mitochondrial COX enzyme activity (p < 0.05). Concurrently, maternal betaine supplementation increased the plasma betaine concentration and muscle expression of methyl transfer enzymes (p < 0.05), BHMT and GNMT, in offspring piglets. Nevertheless, Dnmt3a was downregulated at the level of both mRNA and protein, which was associated with a hypomethylated mtDNA D-loop region (p < 0.05). These results suggest that maternal betaine supplementation during gestation enhances expression of mtDNA-encoded genes through D-loop DNA hypomethylation in the skeletal muscle of newborn piglets.

The cell pole: the site of cross talk between the DNA uptake and genetic recombination machinery.

PubMed

Kidane, Dawit; Ayora, Silvia; Sweasy, Joann B; Graumann, Peter L; Alonso, Juan C

2012-01-01

Natural transformation is a programmed mechanism characterized by binding of free double-stranded (ds) DNA from the environment to the cell pole in rod-shaped bacteria. In Bacillus subtilis some competence proteins, which process the dsDNA and translocate single-stranded (ss) DNA into the cytosol, recruit a set of recombination proteins mainly to one of the cell poles. A subset of single-stranded binding proteins, working as "guardians", protects ssDNA from degradation and limit the RecA recombinase loading. Then, the "mediators" overcome the inhibitory role of guardians, and recruit RecA onto ssDNA. A RecA·ssDNA filament searches for homology on the chromosome and, in a process that is controlled by "modulators", catalyzes strand invasion with the generation of a displacement loop (D-loop). A D-loop resolvase or "resolver" cleaves this intermediate, limited DNA replication restores missing information and a DNA ligase seals the DNA ends. However, if any step fails, the "rescuers" will repair the broken end to rescue chromosomal transformation. If the ssDNA does not share homology with resident DNA, but it contains information for autonomous replication, guardian and mediator proteins catalyze plasmid establishment after inhibition of RecA. DNA replication and ligation reconstitute the molecule (plasmid transformation). In this review, the interacting network that leads to a cross talk between proteins of the uptake and genetic recombination machinery will be placed into prospective.

The cell pole: The site of cross talk between the DNA uptake and genetic recombination machinery

PubMed Central

Kidane, Dawit; Ayora, Silvia; Sweasy, Joann; Graumann, Peter L.; Alonso, Juan C.

2012-01-01

Natural transformation is a programmed mechanism characterized by binding of free double-stranded (ds) DNA from the environment to the cell pole in rod-shaped bacteria. In Bacillus subtilis some competence proteins, which process the dsDNA and translocate single-stranded (ss) DNA into the cytosol, recruit a set of recombination proteins mainly to one of the cell poles. A subset of single-stranded binding proteins, working as “guardians”, protect ssDNA from degradation and limit the RecA recombinase loading. Then, the “mediators” overcome the inhibitory role of guardians, and recruit RecA onto ssDNA. A RecA·ssDNA filament searches for homology on the chromosome and, in a process that is controlled by “modulators”, catalyzes strand invasion with the generation of a displacement loop (D-loop). A D-loop resolvase or “resolver” cleaves this intermediate, limited DNA replication restores missing information and a DNA ligase seals the DNA ends. However, if any step fails, the “rescuers” will repair the broken end to rescue chromosomal transformation. If the ssDNA does not share homology with resident DNA, but it contains information for autonomous replication, guardian and mediator proteins catalyze plasmid establishment after inhibition of RecA. DNA replication and ligation reconstitute the molecule (plasmid transformation). In this review, the interacting network that leads to a cross talk between proteins of the uptake and genetic recombination machinery will be placed into prospective. PMID:23046409

Chromatin-Specific Regulation of Mammalian rDNA Transcription by Clustered TTF-I Binding Sites

PubMed Central

Diermeier, Sarah D.; Németh, Attila; Rehli, Michael; Grummt, Ingrid; Längst, Gernot

2013-01-01

Enhancers and promoters often contain multiple binding sites for the same transcription factor, suggesting that homotypic clustering of binding sites may serve a role in transcription regulation. Here we show that clustering of binding sites for the transcription termination factor TTF-I downstream of the pre-rRNA coding region specifies transcription termination, increases the efficiency of transcription initiation and affects the three-dimensional structure of rRNA genes. On chromatin templates, but not on free rDNA, clustered binding sites promote cooperative binding of TTF-I, loading TTF-I to the downstream terminators before it binds to the rDNA promoter. Interaction of TTF-I with target sites upstream and downstream of the rDNA transcription unit connects these distal DNA elements by forming a chromatin loop between the rDNA promoter and the terminators. The results imply that clustered binding sites increase the binding affinity of transcription factors in chromatin, thus influencing the timing and strength of DNA-dependent processes. PMID:24068958

Translocation-coupled DNA cleavage by the Type ISP restriction-modification enzymes

PubMed Central

Chand, Mahesh Kumar; Nirwan, Neha; Diffin, Fiona M.; van Aelst, Kara; Kulkarni, Manasi; Pernstich, Christian; Szczelkun, Mark D.; Saikrishnan, Kayarat

2015-01-01

Endonucleolytic double-strand DNA break production requires separate strand cleavage events. Although catalytic mechanisms for simple dimeric endonucleases are available, there are many complex nuclease machines which are poorly understood in comparison. Here we studied the single polypeptide Type ISP restriction-modification (RM) enzymes, which cleave random DNA between distant target sites when two enzymes collide following convergent ATP-driven translocation. We report the 2.7 Angstroms resolution X-ray crystal structure of a Type ISP enzyme-DNA complex, revealing that both the helicase-like ATPase and nuclease are unexpectedly located upstream of the direction of translocation, inconsistent with simple nuclease domain-dimerization. Using single-molecule and biochemical techniques, we demonstrate that each ATPase remodels its DNA-protein complex and translocates along DNA without looping it, leading to a collision complex where the nuclease domains are distal. Sequencing of single cleavage events suggests a previously undescribed endonuclease model, where multiple, stochastic strand nicking events combine to produce DNA scission. PMID:26389736

Translocation and deletion breakpoints in cancer genomes are associated with potential non-B DNA-forming sequences.

PubMed

Bacolla, Albino; Tainer, John A; Vasquez, Karen M; Cooper, David N

2016-07-08

Gross chromosomal rearrangements (including translocations, deletions, insertions and duplications) are a hallmark of cancer genomes and often create oncogenic fusion genes. An obligate step in the generation of such gross rearrangements is the formation of DNA double-strand breaks (DSBs). Since the genomic distribution of rearrangement breakpoints is non-random, intrinsic cellular factors may predispose certain genomic regions to breakage. Notably, certain DNA sequences with the potential to fold into secondary structures [potential non-B DNA structures (PONDS); e.g. triplexes, quadruplexes, hairpin/cruciforms, Z-DNA and single-stranded looped-out structures with implications in DNA replication and transcription] can stimulate the formation of DNA DSBs. Here, we tested the postulate that these DNA sequences might be found at, or in close proximity to, rearrangement breakpoints. By analyzing the distribution of PONDS-forming sequences within ±500 bases of 19 947 translocation and 46 365 sequence-characterized deletion breakpoints in cancer genomes, we find significant association between PONDS-forming repeats and cancer breakpoints. Specifically, (AT)n, (GAA)n and (GAAA)n constitute the most frequent repeats at translocation breakpoints, whereas A-tracts occur preferentially at deletion breakpoints. Translocation breakpoints near PONDS-forming repeats also recur in different individuals and patient tumor samples. Hence, PONDS-forming sequences represent an intrinsic risk factor for genomic rearrangements in cancer genomes. © The Author(s) 2016. Published by Oxford University Press on behalf of Nucleic Acids Research.

Mediator and Cohesin Connect Gene Expression and Chromatin Architecture

PubMed Central

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

2010-01-01

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

Intersection of calorie restriction and magnesium in the suppression of genome-destabilizing RNA–DNA hybrids

PubMed Central

Abraham, Karan J.; Chan, Janet N.Y.; Salvi, Jayesh S.; Ho, Brandon; Hall, Amanda; Vidya, Elva; Guo, Ru; Killackey, Samuel A.; Liu, Nancy; Lee, Jeffrey E.; Brown, Grant W.; Mekhail, Karim

2016-01-01

Dietary calorie restriction is a broadly acting intervention that extends the lifespan of various organisms from yeast to mammals. On another front, magnesium (Mg2+) is an essential biological metal critical to fundamental cellular processes and is commonly used as both a dietary supplement and treatment for some clinical conditions. If connections exist between calorie restriction and Mg2+ is unknown. Here, we show that Mg2+, acting alone or in response to dietary calorie restriction, allows eukaryotic cells to combat genome-destabilizing and lifespan-shortening accumulations of RNA–DNA hybrids, or R-loops. In an R-loop accumulation model of Pbp1-deficient Saccharomyces cerevisiae, magnesium ions guided by cell membrane Mg2+ transporters Alr1/2 act via Mg2+-sensitive R-loop suppressors Rnh1/201 and Pif1 to restore R-loop suppression, ribosomal DNA stability and cellular lifespan. Similarly, human cells deficient in ATXN2, the human ortholog of Pbp1, exhibit nuclear R-loop accumulations repressible by Mg2+ in a process that is dependent on the TRPM7 Mg2+ transporter and the RNaseH1 R-loop suppressor. Thus, we identify Mg2+ as a biochemical signal of beneficial calorie restriction, reveal an R-loop suppressing function for human ATXN2 and propose that practical magnesium supplementation regimens can be used to combat R-loop accumulation linked to the dysfunction of disease-linked human genes. PMID:27574117

R-loops in bacterial transcription: their causes and consequences.

PubMed

Gowrishankar, J; Leela, J Krishna; Anupama, K

2013-01-01

Nascent untranslated transcripts in bacteria are prone to generating RNA-DNA hybrids (R-loops); Rho-dependent transcription termination acts to reduce their prevalence. Here we discuss the mechanisms of R-loop formation and growth inhibition in bacteria.

Resolved single-molecule detection of individual species within a mixture of anti-biotin antibodies using an engineered monomeric nanopore.

PubMed

Fahie, Monifa; Chisholm, Christina; Chen, Min

2015-02-24

Oligomeric protein nanopores with rigid structures have been engineered for the purpose of sensing a wide range of analytes including small molecules and biological species such as proteins and DNA. We chose a monomeric β-barrel porin, OmpG, as the platform from which to derive the nanopore sensor. OmpG is decorated with seven flexible loops that move dynamically to create a distinct gating pattern when ionic current passes through the pore. Biotin was chemically tethered to the most flexible one of these loops. The gating characteristic of the loop's movement in and out of the porin was substantially altered by analyte protein binding. The gating characteristics of the pore with bound targets were remarkably sensitive to molecular identity, even providing the ability to distinguish between homologues within an antibody mixture. A total of five gating parameters were analyzed for each analyte to create a unique fingerprint for each biotin-binding protein. Our exploitation of gating noise as a molecular identifier may allow more sophisticated sensor design, while OmpG's monomeric structure greatly simplifies nanopore production.

Structure and hydrodynamics of a DNA G-quadruplex with a cytosine bulge.

PubMed

Meier, Markus; Moya-Torres, Aniel; Krahn, Natalie J; McDougall, Matthew D; Orriss, George L; McRae, Ewan K S; Booy, Evan P; McEleney, Kevin; Patel, Trushar R; McKenna, Sean A; Stetefeld, Jörg

2018-06-01

The identification of four-stranded G-quadruplexes (G4s) has highlighted the fact that DNA has additional spatial organisations at its disposal other than double-stranded helices. Recently, it became clear that the formation of G4s is not limited to the traditional G3+NL1G3+NL2G3+NL3G3+ sequence motif. Instead, the G3 triplets can be interrupted by deoxythymidylate (DNA) or uridylate (RNA) where the base forms a bulge that loops out from the G-quadruplex core. Here, we report the first high-resolution X-ray structure of a unique unimolecular DNA G4 with a cytosine bulge. The G4 forms a dimer that is stacked via its 5'-tetrads. Analytical ultracentrifugation, static light scattering and small angle X-ray scattering confirmed that the G4 adapts a predominantly dimeric structure in solution. We provide a comprehensive comparison of previously published G4 structures containing bulges and report a special γ torsion angle range preferentially populated by the G4 core guanylates adjacent to bulges. Since the penalty for introducing bulges appears to be negligible, it should be possible to functionalize G4s by introducing artificial or modified nucleotides at such positions. The presence of the bulge alters the surface of the DNA, providing an opportunity to develop drugs that can specifically target individual G4s.

A Paper-Based Device for Performing Loop-Mediated Isothermal Amplification with Real-Time Simultaneous Detection of Multiple DNA Targets.

PubMed

Seok, Youngung; Joung, Hyou-Arm; Byun, Ju-Young; Jeon, Hyo-Sung; Shin, Su Jeong; Kim, Sanghyo; Shin, Young-Beom; Han, Hyung Soo; Kim, Min-Gon

2017-01-01

Paper-based diagnostic devices have many advantages as a one of the multiple diagnostic test platforms for point-of-care (POC) testing because they have simplicity, portability, and cost-effectiveness. However, despite high sensitivity and specificity of nucleic acid testing (NAT), the development of NAT based on a paper platform has not progressed as much as the others because various specific conditions for nucleic acid amplification reactions such as pH, buffer components, and temperature, inhibitions from technical differences of paper-based device. Here, we propose a paper-based device for performing loop-mediated isothermal amplification (LAMP) with real-time simultaneous detection of multiple DNA targets. We determined the optimal chemical components to enable dry conditions for the LAMP reaction without lyophilization or other techniques. We also devised the simple paper device structure by sequentially stacking functional layers, and employed a newly discovered property of hydroxynaphthol blue fluorescence to analyze real-time LAMP signals in the paper device. This proposed platform allowed analysis of three different meningitis DNA samples in a single device with single-step operation. This LAMP-based multiple diagnostic device has potential for real-time analysis with quantitative detection of 10 2 -10 5 copies of genomic DNA. Furthermore, we propose the transformation of DNA amplification devices to a simple and affordable paper system approach with great potential for realizing a paper-based NAT system for POC testing.

A Paper-Based Device for Performing Loop-Mediated Isothermal Amplification with Real-Time Simultaneous Detection of Multiple DNA Targets

PubMed Central

Seok, Youngung; Joung, Hyou-Arm; Byun, Ju-Young; Jeon, Hyo-Sung; Shin, Su Jeong; Kim, Sanghyo; Shin, Young-Beom; Han, Hyung Soo; Kim, Min-Gon

2017-01-01

Paper-based diagnostic devices have many advantages as a one of the multiple diagnostic test platforms for point-of-care (POC) testing because they have simplicity, portability, and cost-effectiveness. However, despite high sensitivity and specificity of nucleic acid testing (NAT), the development of NAT based on a paper platform has not progressed as much as the others because various specific conditions for nucleic acid amplification reactions such as pH, buffer components, and temperature, inhibitions from technical differences of paper-based device. Here, we propose a paper-based device for performing loop-mediated isothermal amplification (LAMP) with real-time simultaneous detection of multiple DNA targets. We determined the optimal chemical components to enable dry conditions for the LAMP reaction without lyophilization or other techniques. We also devised the simple paper device structure by sequentially stacking functional layers, and employed a newly discovered property of hydroxynaphthol blue fluorescence to analyze real-time LAMP signals in the paper device. This proposed platform allowed analysis of three different meningitis DNA samples in a single device with single-step operation. This LAMP-based multiple diagnostic device has potential for real-time analysis with quantitative detection of 102-105 copies of genomic DNA. Furthermore, we propose the transformation of DNA amplification devices to a simple and affordable paper system approach with great potential for realizing a paper-based NAT system for POC testing. PMID:28740546

The Bacteroides sp. 3_1_23 Pif1 protein is a multifunctional helicase.

PubMed

Liu, Na-Nv; Duan, Xiao-Lei; Ai, Xia; Yang, Yan-Tao; Li, Ming; Dou, Shuo-Xing; Rety, Stephane; Deprez, Eric; Xi, Xu-Guang

2015-10-15

ScPif1 DNA helicase is the prototypical member of a 5'-to-3' helicase superfamily conserved from bacteria to human and plays various roles in the maintenance of genomic homeostasis. While many studies have been performed with eukaryotic Pif1 helicases, including yeast and human Pif1 proteins, the potential functions and biochemical properties of prokaryotic Pif1 helicases remain largely unknown. Here, we report the expression, purification and biochemical analysis of Pif1 helicase from Bacteroides sp. 3_1_23 (BsPif1). BsPif1 binds to a large panel of DNA substrates and, in particular, efficiently unwinds partial duplex DNAs with 5'-overhang, fork-like substrates, D-loop and flap-like substrates, suggesting that BsPif1 may act at stalled DNA replication forks and enhance Okazaki fragment maturation. Like its eukaryotic homologues, BsPif1 resolves R-loop structures and unwinds DNA-RNA hybrids. Furthermore, BsPif1 efficiently unfolds G-quadruplexes and disrupts nucleoprotein complexes. Altogether, these results highlight that prokaryotic Pif1 helicases may resolve common issues that arise during DNA transactions. Interestingly, we found that BsPif1 is different from yeast Pif1, but resembles more human Pif1 with regard to substrate specificity, helicase activity and mode of action. These findings are discussed in the context of the possible functions of prokaryotic Pif1 helicases in vivo. © The Author(s) 2015. Published by Oxford University Press on behalf of Nucleic Acids Research.

Sequence analysis of the mitochondrial DNA control region of ciscoes (genus Coregonus): taxonomic implications for the Great Lakes species flock.

PubMed

Reed, K M; Dorschner, M O; Todd, T N; Phillips, R B

1998-09-01

Sequence variation in the control region (D-loop) of the mitochondrial DNA (mtDNA) was examined to assess the genetic distinctiveness of the shortjaw cisco (Coregonus zenithicus). Individuals from within the Great Lakes Basin as well as inland lakes outside the basin were sampled. DNA fragments containing the entire D-loop were amplified by PCR from specimens of C. zenithicus and the related species C. artedi, C. hoyi, C. kiyi, and C. clupeaformis. DNA sequence analysis revealed high similarity within and among species and shared polymorphism for length variants. Based on this analysis, the shortjaw cisco is not genetically distinct from other cisco species.

Insights into the effects of mutations on Cren7-DNA binding using molecular dynamics simulations and free energy calculations.

PubMed

Chen, Lin; Zheng, Qing-Chuan; Zhang, Hong-Xing

2015-02-28

A novel, highly conserved chromatin protein, Cren7 is involved in regulating essential cellular processes such as transcription, replication and repair. Although mutations in the DNA-binding loop of Cren7 destabilize the structure and reduce DNA-binding activity, the details are not very clear. Focusing on the specific Cren7-dsDNA complex (PDB code ), we applied molecular dynamics (MD) simulations and the molecular mechanics Poisson-Boltzmann surface area (MM-PBSA) free energy calculations to explore the structural and dynamic effects of W26A, L28A, and K53A mutations in comparison to the wild-type protein. The energetic analysis indicated that the intermolecular van der Waals interaction and nonpolar solvation energy play an important role in the binding process of Cren7 and dsDNA. Compared with the wild type Cren7, all the studied mutants W26A, L28A, and K53A have obviously reduced binding free energies with dsDNA in the reduction of the polar and/or nonpolar interactions. These results further elucidated the previous experiments to understand the Cren7-DNA interaction comprehensively. Our work also would provide support for an understanding of the interactions of proteins with nucleic acids.

Linear and Nonlinear Statistical Characterization of DNA

NASA Astrophysics Data System (ADS)

Norio Oiwa, Nestor; Goldman, Carla; Glazier, James

2002-03-01

We find spatial order in the distribution of protein-coding (including RNAs) and control segments of GenBank genomic sequences, irrespective of ATCG content. This is achieved by correlations, histograms, fractal dimensions and singularity spectra. Estimates of these quantities in complete nuclear genome indicate that coding sequences are long-range correlated and their disposition are self-similar (multifractal) for eukaryotes. These characteristics are absent in prokaryotes, where there are few noncoding sequences, suggesting the `junk' DNA play a relevant role to the genome structure and function. Concerning the genetic message of ATCG sequences, we build a random walk (Levy flight), using DNA symmetry arguments, where we associate A, T, C and G as left, right, down and up steps, respectively. Nonlinear analysis of mitochondrial DNA walks reveal multifractal pattern based on palindromic sequences, which fold in hairpins and loops.

Type III restriction-modification enzymes: a historical perspective.

PubMed

Rao, Desirazu N; Dryden, David T F; Bheemanaik, Shivakumara

2014-01-01

Restriction endonucleases interact with DNA at specific sites leading to cleavage of DNA. Bacterial DNA is protected from restriction endonuclease cleavage by modifying the DNA using a DNA methyltransferase. Based on their molecular structure, sequence recognition, cleavage position and cofactor requirements, restriction-modification (R-M) systems are classified into four groups. Type III R-M enzymes need to interact with two separate unmethylated DNA sequences in inversely repeated head-to-head orientations for efficient cleavage to occur at a defined location (25-27 bp downstream of one of the recognition sites). Like the Type I R-M enzymes, Type III R-M enzymes possess a sequence-specific ATPase activity for DNA cleavage. ATP hydrolysis is required for the long-distance communication between the sites before cleavage. Different models, based on 1D diffusion and/or 3D-DNA looping, exist to explain how the long-distance interaction between the two recognition sites takes place. Type III R-M systems are found in most sequenced bacteria. Genome sequencing of many pathogenic bacteria also shows the presence of a number of phase-variable Type III R-M systems, which play a role in virulence. A growing number of these enzymes are being subjected to biochemical and genetic studies, which, when combined with ongoing structural analyses, promise to provide details for mechanisms of DNA recognition and catalysis.

The mitochondrial C16069T polymorphism, not mitochondrial D310 (D-loop) mononucleotide sequence variations, is associated with bladder cancer.

PubMed

Shakhssalim, Nasser; Houshmand, Massoud; Kamalidehghan, Behnam; Faraji, Abolfazl; Sarhangnejad, Reza; Dadgar, Sepideh; Mobaraki, Maryam; Rosli, Rozita; Sanati, Mohammad Hossein

2013-12-05

Bladder cancer is a relatively common and potentially life-threatening neoplasm that ranks ninth in terms of worldwide cancer incidence. The aim of this study was to determine deletions and sequence variations in the mitochondrial displacement loop (D-loop) region from the blood specimens and tumoral tissues of patients with bladder cancer, compared to adjacent non-tumoral tissues. The DNA from blood, tumoral tissues and adjacent non-tumoral tissues of twenty-six patients with bladder cancer and DNA from blood of 504 healthy controls from different ethnicities were investigated to determine sequence variation in the mitochondrial D-loop region using multiplex polymerase chain reaction (PCR), DNA sequencing and southern blotting analysis. From a total of 110 variations, 48 were reported as new mutations. No deletions were detected in tumoral tissues, adjacent non-tumoral tissues and blood samples from patients. Although the polymorphisms at loci 16189, 16261 and 16311 were not significantly correlated with bladder cancer, the C16069T variation was significantly present in patient samples compared to control samples (p < 0.05). Interestingly, there was no significant difference (p > 0.05) of C variations, including C7TC6, C8TC6, C9TC6 and C10TC6, in D310 mitochondrial DNA between patients and control samples. Our study suggests that 16069 mitochondrial DNA D-Loop mutations may play a significant role in the etiology of bladder cancer and facilitate the definition of carcinogenesis-related mutations in human cancer.

DNA Hairpins Containing the Cytidine Analog Pyrrolo-dC: Structural, Thermodynamic, and Spectroscopic Studies

PubMed Central

Zhang, Xu; Wadkins, Randy M.

2009-01-01

Structures formed by single-strand DNA have become increasingly interesting because of their roles in a number of biological processes, particularly transcription and its regulation. Of particular importance is the fact that antitumor drugs such as Actinomycin D can selectively bind DNA hairpins over fully paired, double-strand DNA. A new fluorescent base analog, pyrrolo-deoxycytidine (PdC), can now be routinely incorporated into single-strand DNA. The fluorescence of PdC is particularly useful for studying the formation of single-strand DNA in regions of double-strand DNA. The fluorescence is quenched when PdC is paired with a complementary guanine residue, and thus is greatly enhanced upon formation of single-strand DNA. Hence, any process that results in melting or opening of DNA strands produces an increase in the fluorescence intensity of this base analog. In this study we measured the structural effects of incorporating PdC into DNA hairpins, and the effect of this incorporation on the binding of the hairpins by a fluorescent analog of the drug Actinomycin D. Two hairpin DNAs were used: one with PdC in the stem (basepaired) and one with PdC in the loop (unpaired). The thermal stability, 7-aminoactinomycin D binding, and three-dimensional structures of PdC incorporated into these DNA hairpins were all quite similar as compared to the hairpins containing an unmodified dC residue. Fluorescence lifetime measurements indicate that two lifetimes are present in PdC, and that the increase in fluorescence of the unpaired PdC residue compared to the basepaired PdC is due to an increase in the contribution of the longer lifetime to the average fluorescence lifetime. Our data indicate that PdC can be used effectively to differentiate paired and unpaired bases in DNA hairpin secondary structures, and should be similarly applicable for related structures such as cruciforms and quadruplexes. Further, our data indicate that PdC can act as a fluorescence resonance energy transfer donor for the fluorescent drug 7-aminoactinomycin D. PMID:19254547

DNA hairpins containing the cytidine analog pyrrolo-dC: structural, thermodynamic, and spectroscopic studies.

PubMed

Zhang, Xu; Wadkins, Randy M

2009-03-04

Structures formed by single-strand DNA have become increasingly interesting because of their roles in a number of biological processes, particularly transcription and its regulation. Of particular importance is the fact that antitumor drugs such as Actinomycin D can selectively bind DNA hairpins over fully paired, double-strand DNA. A new fluorescent base analog, pyrrolo-deoxycytidine (PdC), can now be routinely incorporated into single-strand DNA. The fluorescence of PdC is particularly useful for studying the formation of single-strand DNA in regions of double-strand DNA. The fluorescence is quenched when PdC is paired with a complementary guanine residue, and thus is greatly enhanced upon formation of single-strand DNA. Hence, any process that results in melting or opening of DNA strands produces an increase in the fluorescence intensity of this base analog. In this study we measured the structural effects of incorporating PdC into DNA hairpins, and the effect of this incorporation on the binding of the hairpins by a fluorescent analog of the drug Actinomycin D. Two hairpin DNAs were used: one with PdC in the stem (basepaired) and one with PdC in the loop (unpaired). The thermal stability, 7-aminoactinomycin D binding, and three-dimensional structures of PdC incorporated into these DNA hairpins were all quite similar as compared to the hairpins containing an unmodified dC residue. Fluorescence lifetime measurements indicate that two lifetimes are present in PdC, and that the increase in fluorescence of the unpaired PdC residue compared to the basepaired PdC is due to an increase in the contribution of the longer lifetime to the average fluorescence lifetime. Our data indicate that PdC can be used effectively to differentiate paired and unpaired bases in DNA hairpin secondary structures, and should be similarly applicable for related structures such as cruciforms and quadruplexes. Further, our data indicate that PdC can act as a fluorescence resonance energy transfer donor for the fluorescent drug 7-aminoactinomycin D.

Oligomer formation and G-quadruplex binding by purified murine Rif1 protein, a key organizer of higher-order chromatin architecture.

PubMed

Moriyama, Kenji; Yoshizawa-Sugata, Naoko; Masai, Hisao

2018-03-09

Rap1-interacting protein 1 (Rif1) regulates telomere length in budding yeast. We previously reported that, in metazoans and fission yeast, Rif1 also plays pivotal roles in controlling genome-wide DNA replication timing. We proposed that Rif1 may assemble chromatin compartments that contain specific replication-timing domains by promoting chromatin loop formation. Rif1 also is involved in DNA lesion repair, restart after replication fork collapse, anti-apoptosis activities, replicative senescence, and transcriptional regulation. Although multiple physiological functions of Rif1 have been characterized, biochemical and structural information on mammalian Rif1 is limited, mainly because of difficulties in purifying the full-length protein. Here, we expressed and purified the 2418-amino-acid-long, full-length murine Rif1 as well as its partially truncated variants in human 293T cells. Hydrodynamic analyses indicated that Rif1 forms elongated or extended homo-oligomers in solution, consistent with the presence of a HEAT-type helical repeat segment known to adopt an elongated shape. We also observed that the purified murine Rif1 bound G-quadruplex (G4) DNA with high specificity and affinity, as was previously shown for Rif1 from fission yeast. Both the N-terminal (HEAT-repeat) and C-terminal segments were involved in oligomer formation and specifically bound G4 DNA, and the central intrinsically disordered polypeptide segment increased the affinity for G4. Of note, pulldown assays revealed that Rif1 simultaneously binds multiple G4 molecules. Our findings support a model in which Rif1 modulates chromatin loop structures through binding to multiple G4 assemblies and by holding chromatin fibers together. © 2018 by The American Society for Biochemistry and Molecular Biology, Inc.

The Recombinant Inhibitor of DNA Binding Id2 Forms Multimeric Structures via the Helix-Loop-Helix Domain and the Nuclear Export Signal.

PubMed

Roschger, Cornelia; Schubert, Mario; Regl, Christof; Andosch, Ancuela; Marquez, Augusto; Berger, Thomas; Huber, Christian G; Lütz-Meindl, Ursula; Cabrele, Chiara

2018-04-07

The inhibitor of DNA binding and cell differentiation 2 (Id2) is a helix-loop-helix (HLH) protein that acts as negative dominant regulator of basic-HLH transcription factors during development and in cancer. The structural properties of Id2 have been investigated so far by using synthetic or recombinant fragments reproducing single domains (N-terminus, HLH, C-terminus): the HLH domain tends to dimerize into a four-helix bundle, whereas the flanking regions are flexible. In this work, the intact protein was expressed in E. coli , solubilized from inclusion bodies with urea, purified and dissolved in water at pH~4. Under these conditions, Id2 was obtained with both cysteine residues disulfide-bonded to β-mercaptoethanol that was present during the solubilization process. Moreover, it existed in a self-assembled state, in which the N-terminus remained highly flexible, while the HLH domain and, surprisingly, part of the C-terminus, which corresponds to the nuclear export signal (NES), both were involved in slowly tumbling, rigid structures. The protein oligomers also formed twisted fibrils that were several micrometers long and up to 80 nm thick. These results show that self-assembly decreases the backbone flexibility of those two protein regions (HLH and NES) that are important for interaction with basic-HLH transcription factors or for nucleocytoplasmic shuttling.

The p53–Mdm2 feedback loop protects against DNA damage by inhibiting p53 activity but is dispensable for p53 stability, development, and longevity

PubMed Central

Pant, Vinod; Xiong, Shunbin; Jackson, James G.; Post, Sean M.; Abbas, Hussein A.; Quintás-Cardama, Alfonso; Hamir, Amirali N.; Lozano, Guillermina

2013-01-01

The p53–Mdm2 feedback loop is perceived to be critical for regulating stress-induced p53 activity and levels. However, this has never been tested in vivo. Using a genetically engineered mouse with mutated p53 response elements in the Mdm2 P2 promoter, we show that feedback loop-deficient Mdm2P2/P2 mice are viable and aphenotypic and age normally. p53 degradation kinetics after DNA damage in radiosensitive tissues remains similar to wild-type controls. Nonetheless, DNA damage response is elevated in Mdm2P2/P2 mice. Enhanced p53-dependent apoptosis sensitizes hematopoietic stem cells (HSCs), causing drastic myeloablation and lethality. These results suggest that while basal Mdm2 levels are sufficient to regulate p53 in most tissues under homeostatic conditions, the p53–Mdm2 feedback loop is critical for regulating p53 activity and sustaining HSC function after DNA damage. Therefore, transient disruption of p53–Mdm2 interaction could be explored as a potential adjuvant/therapeutic strategy for targeting stem cells in hematological malignancies. PMID:23973961

Genetic Diversity and Phylogenetic Analysis of South-East Asian Duck Populations Based on the mtDNA D-loop Sequences

PubMed Central

Sultana, H.; Seo, D. W.; Bhuiyan, M. S. A.; Choi, N. R.; Hoque, M. R.; Heo, K. N.; Lee, J. H.

2016-01-01

The maternally inherited mitochondrial DNA (mtDNA) D–loop region is widely used for exploring genetic relationships and for investigating the origin of various animal species. Currently, domestic ducks play an important role in animal protein supply. In this study, partial mtDNA D–loop sequences were obtained from 145 samples belonging to six South-East Asian duck populations and commercial duck population. All these populations were closely related to the mallard duck (Anas platyrhynchos), as indicated by their mean overall genetic distance. Sixteen nucleotide substitutions were identified in sequence analyses allowing the distinction of 28 haplotypes. Around 42.76% of the duck sequences were classified as Hap_02, which completely matched with Anas platyrhynchos duck species. The neighbor-joining phylogenetic tree also revealed that South-East Asian duck populations were closely related to Anas platyrhynchos. Network profiles were also traced using the 28 haplotypes. Overall, results showed that those duck populations D-loop haplotypes were shared between several duck breeds from Korea and Bangladesh sub continental regions. Therefore, these results confirmed that South-East Asian domestic duck populations have been domesticated from Anas platyrhynchos duck as the maternal origins. PMID:27004808

Structural insights into the cTAR DNA recognition by the HIV-1 nucleocapsid protein: role of sugar deoxyriboses in the binding polarity of NC

PubMed Central

Bazzi, Ali; Zargarian, Loussiné; Chaminade, Françoise; Boudier, Christian; De Rocquigny, Hughes; René, Brigitte; Mély, Yves; Fossé, Philippe; Mauffret, Olivier

2011-01-01

An essential step of the reverse transcription of the HIV-1 genome is the first strand transfer that requires the annealing of the TAR RNA hairpin to the cTAR DNA hairpin. HIV-1 nucleocapsid protein (NC) plays a crucial role by facilitating annealing of the complementary hairpins. Using nuclear magnetic resonance and gel retardation assays, we investigated the interaction between NC and the top half of the cTAR DNA (mini-cTAR). We show that NC(11-55) binds the TGG sequence in the lower stem that is destabilized by the adjacent internal loop. The 5′ thymine interacts with residues of the N-terminal zinc knuckle and the 3′ guanine is inserted in the hydrophobic plateau of the C-terminal zinc knuckle. The TGG sequence is preferred relative to the apical and internal loops containing unpaired guanines. Investigation of the DNA–protein contacts shows the major role of hydrophobic interactions involving nucleobases and deoxyribose sugars. A similar network of hydrophobic contacts is observed in the published NC:DNA complexes, whereas NC contacts ribose differently in NC:RNA complexes. We propose that the binding polarity of NC is related to these contacts that could be responsible for the preferential binding to single-stranded nucleic acids. PMID:21227929

Long repeating (TTAGGG)n single stranded DNA self-condenses into compact beaded filaments stabilized by G-quadruplex formation.

PubMed

Kar, Anirban; Jones, Nathan; Arat, N Özlem; Fishel, Richard; Griffith, Jack

2018-04-19

Conformations adopted by long stretches of single stranded DNA (ssDNA) are of central interest in understanding the architecture of replication forks, R loops, and other structures generated during DNA metabolism in vivo. This is particularly so if the ssDNA consists of short nucleotide repeats. Such studies have been hampered by the lack of defined substrates greater than ~150 nt, and the absence of high-resolution biophysical approaches. Here we describe the generation of very long ssDNA consisting of the mammalian telomeric repeat (5'-TTAGGG-3')n as well as the interrogation of its structure by electron microscopy (EM) and single molecule magnetic tweezers (smMT). This repeat is of particular interest as it contains a run of 3 contiguous guanine residues capable of forming G quartets as ssDNA. Fluorescent-dye exclusion assays confirmed that this G-strand ssDNA forms ubiquitous G-quadruplex folds. EM revealed thick bead-like filaments that condensed the DNA ~12 fold. The bead-like structures were 5 nm and 8 nm in diameter and linked by thin filaments. The G-strand ssDNA displayed initial stability to smMT force extension that ultimately released in steps that were multiples ~28 nm at forces between 6-12 pN; well below the >20 pN required to unravel G-quadruplexes. Most smMT steps were consistent with the disruption of the beads seen by EM. Binding by RAD51 distinctively altered the force extension properties of the G-strand ssDNA, suggesting a stochastic G-quadruplex-dependent condensation model that is discussed. Published under license by The American Society for Biochemistry and Molecular Biology, Inc.

Computational Study on Full-length Human Ku70 with Double Stranded DNA: Dynamics, Interactions and Functional Implications

NASA Technical Reports Server (NTRS)

Hu, Shaowen; Cucinotta, Francis A.

2009-01-01

The Ku70/80 heterodimer is the first repair protein in the initial binding of double-strand break (DSB) ends following DNA damage, and is a component of nonhomologous end joining repair, the primary pathway for DSB repair in mammalian cells. In this study we constructed a full-length human Ku70 structure based on its crystal structure, and performed 20 ns conventional molecular dynamic (CMD) simulations on this protein and several other complexes with short DNA duplexes of different sequences. The trajectories of these simulations indicated that, without the topological support of Ku80, the residues in the bridge and C-terminal arm of Ku70 are more flexible than other experimentally identified domains. We studied the two missing loops in the crystal structure and predicted that they are also very flexible. Simulations revealed that they make an important contribution to the Ku70 interaction with DNA. Dislocation of the previously studied SAP domain was observed in several systems, implying its role in DNA binding. Targeted molecular dynamic (TMD) simulation was also performed for one system with a far-away 14bp DNA duplex. The TMD trajectory and energetic analysis disclosed detailed interactions of the DNA-binding residues during the DNA dislocation, and revealed a possible conformational transition for a DSB end when encountering Ku70 in solution. Compared to experimentally based analysis, this study identified more detailed interactions between DNA and Ku70. Free energy analysis indicated Ku70 alone is able to bind DNA with relatively high affinity, with consistent contributions from various domains of Ku70 in different systems. The functional implications of these domains in the processes of Ku heterodimerization and DNA damage recognition and repair can be characterized in detail based upon this analysis.

A mitosis-specific and R loop-driven ATR pathway promotes faithful chromosome segregation.

PubMed

Kabeche, Lilian; Nguyen, Hai Dang; Buisson, Rémi; Zou, Lee

2018-01-05

The ataxia telangiectasia mutated and Rad3-related (ATR) kinase is crucial for DNA damage and replication stress responses. Here, we describe an unexpected role of ATR in mitosis. Acute inhibition or degradation of ATR in mitosis induces whole-chromosome missegregation. The effect of ATR ablation is not due to altered cyclin-dependent kinase 1 (CDK1) activity, DNA damage responses, or unscheduled DNA synthesis but to loss of an ATR function at centromeres. In mitosis, ATR localizes to centromeres through Aurora A-regulated association with centromere protein F (CENP-F), allowing ATR to engage replication protein A (RPA)-coated centromeric R loops. As ATR is activated at centromeres, it stimulates Aurora B through Chk1, preventing formation of lagging chromosomes. Thus, a mitosis-specific and R loop-driven ATR pathway acts at centromeres to promote faithful chromosome segregation, revealing functions of R loops and ATR in suppressing chromosome instability. Copyright © 2018, American Association for the Advancement of Science.

The nuclear higher-order structure defined by the set of topological relationships between DNA and the nuclear matrix is species-specific in hepatocytes.

PubMed

Silva-Santiago, Evangelina; Pardo, Juan Pablo; Hernández-Muñoz, Rolando; Aranda-Anzaldo, Armando

2017-01-15

During the interphase the nuclear DNA of metazoan cells is organized in supercoiled loops anchored to constituents of a nuclear substructure or compartment known as the nuclear matrix. The stable interactions between DNA and the nuclear matrix (NM) correspond to a set of topological relationships that define a nuclear higher-order structure (NHOS). Current evidence suggests that the NHOS is cell-type-specific. Biophysical evidence and theoretical models suggest that thermodynamic and structural constraints drive the actualization of DNA-NM interactions. However, if the topological relationships between DNA and the NM were the subject of any biological constraint with functional significance then they must be adaptive and thus be positively selected by natural selection and they should be reasonably conserved, at least within closely related species. We carried out a coarse-grained, comparative evaluation of the DNA-NM topological relationships in primary hepatocytes from two closely related mammals: rat and mouse, by determining the relative position to the NM of a limited set of target sequences corresponding to highly-conserved genomic regions that also represent a sample of distinct chromosome territories within the interphase nucleus. Our results indicate that the pattern of topological relationships between DNA and the NM is not conserved between the hepatocytes of the two closely related species, suggesting that the NHOS, like the karyotype, is species-specific. Copyright © 2016 Elsevier B.V. All rights reserved.

Structural evolution of nrDNA ITS in Pinaceae and its phylogenetic implications.

PubMed

Kan, Xian-Zhao; Wang, Shan-Shan; Ding, Xin; Wang, Xiao-Quan

2007-08-01

Nuclear ribosomal DNA (nrDNA) has been considered as an important tool for inferring phylogenetic relationships at many taxonomic levels. In comparison with its fast concerted evolution in angiosperms, nrDNA is symbolized by slow concerted evolution and substantial ITS region length variation in gymnosperms, particularly in Pinaceae. Here we studied structure characteristics, including subrepeat composition, size, GC content and secondary structure, of nrDNA ITS regions of all Pinaceae genera. The results showed that the ITS regions of all taxa studied contained subrepeat units, ranging from 2 to 9 in number, and these units could be divided into two types, longer subrepeat (LSR) without the motif (5'-GGCCACCCTAGTC) and shorter subrepeat (SSR) with the motif. Phylogenetic analyses indicate that the homology of some SSRs still can be recognized, providing important informations for the evolutionary history of nrDNA ITS and phylogeny of Pinaceae. In particular, the adjacent tandem SSRs are not more closely related to one another than they are to remote SSRs in some genera, which may imply that multiple structure variations such as recombination have occurred in the ITS1 region of these groups. This study also found that GC content in the ITS1 region is relevant to its sequence length and subrepeat number, and could provide some phylogenetic information, especially supporting the close relationships among Picea, Pinus, and Cathaya. Moreover, several characteristics of the secondary structure of Pinaceae ITS1 were found as follows: (1) the structure is dominated by several extended hairpins; (2) the configuration complexity is positively correlated with subrepeat number; (3) paired subrepeats often partially overlap at the conserved motif (5'-GGCCACCCTAGTC), and form a long stem, while other subrepeats fold onto itself, leaving part of the conserved motif exposed in hairpin loops.

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

NASA Technical Reports Server (NTRS)

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

2005-01-01

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

Genome Organization Drives Chromosome Fragility.

PubMed

Canela, Andres; Maman, Yaakov; Jung, Seolkyoung; Wong, Nancy; Callen, Elsa; Day, Amanda; Kieffer-Kwon, Kyong-Rim; Pekowska, Aleksandra; Zhang, Hongliang; Rao, Suhas S P; Huang, Su-Chen; Mckinnon, Peter J; Aplan, Peter D; Pommier, Yves; Aiden, Erez Lieberman; Casellas, Rafael; Nussenzweig, André

2017-07-27

In this study, we show that evolutionarily conserved chromosome loop anchors bound by CCCTC-binding factor (CTCF) and cohesin are vulnerable to DNA double strand breaks (DSBs) mediated by topoisomerase 2B (TOP2B). Polymorphisms in the genome that redistribute CTCF/cohesin occupancy rewire DNA cleavage sites to novel loop anchors. While transcription- and replication-coupled genomic rearrangements have been well documented, we demonstrate that DSBs formed at loop anchors are largely transcription-, replication-, and cell-type-independent. DSBs are continuously formed throughout interphase, are enriched on both sides of strong topological domain borders, and frequently occur at breakpoint clusters commonly translocated in cancer. Thus, loop anchors serve as fragile sites that generate DSBs and chromosomal rearrangements. VIDEO ABSTRACT. Published by Elsevier Inc.

How a short double-stranded DNA bends

NASA Astrophysics Data System (ADS)

Shin, Jaeoh; Lee, O.-Chul; Sung, Wokyung

2015-04-01

A recent experiment using fluorescence microscopy showed that double-stranded DNA fragments shorter than 100 base pairs loop with the probabilities higher by the factor of 102-106 than predicted by the worm-like chain (WLC) model [R. Vafabakhsh and T. Ha, Science 337, 1101(2012)]. Furthermore, the looping probabilities were found to be nearly independent of the loop size. The results signify a breakdown of the WLC model for DNA mechanics which works well on long length scales and calls for fundamental understanding for stressed DNA on shorter length scales. We develop an analytical, statistical mechanical model to investigate what emerges to the short DNA under a tight bending. A bending above a critical level initiates nucleation of a thermally induced bubble, which could be trapped for a long time, in contrast to the bubbles in both free and uniformly bent DNAs, which are either transient or unstable. The trapped bubble is none other than the previously hypothesized kink, which releases the bending energy more easily as the contour length decreases. It leads to tremendous enhancement of the cyclization probabilities, in a reasonable agreement with experiment.

Mitochondrial DNA variation and phylogenetic relationships among five tuna species based on sequencing of D-loop region.

PubMed

Kumar, Girish; Kocour, Martin; Kunal, Swaraj Priyaranjan

2016-05-01

In order to assess the DNA sequence variation and phylogenetic relationship among five tuna species (Auxis thazard, Euthynnus affinis, Katsuwonus pelamis, Thunnus tonggol, and T. albacares) out of all four tuna genera, partial sequences of the mitochondrial DNA (mtDNA) D-loop region were analyzed. The estimate of intra-specific sequence variation in studied species was low, ranging from 0.027 to 0.080 [Kimura's two parameter distance (K2P)], whereas values of inter-specific variation ranged from 0.049 to 0.491. The longtail tuna (T. tonggol) and yellowfin tuna (T. albacares) were found to share a close relationship (K2P = 0.049) while skipjack tuna (K. pelamis) was most divergent studied species. Phylogenetic analysis using Maximum-Likelihood (ML) and Neighbor-Joining (NJ) methods supported the monophyletic origin of Thunnus species. Similarly, phylogeny of Auxis and Euthynnus species substantiate the monophyly. However, results showed a distinct origin of K. pelamis from genus Thunnus as well as Auxis and Euthynnus. Thus, the mtDNA D-loop region sequence data supports the polyphyletic origin of tuna species.

Unfolding and melting of DNA (RNA) hairpins: the concept of structure-specific 2D dynamic landscapes.

PubMed

Lin, Milo M; Meinhold, Lars; Shorokhov, Dmitry; Zewail, Ahmed H

2008-08-07

A 2D free-energy landscape model is presented to describe the (un)folding transition of DNA/RNA hairpins, together with molecular dynamics simulations and experimental findings. The dependence of the (un)folding transition on the stem sequence and the loop length is shown in the enthalpic and entropic contributions to the free energy. Intermediate structures are well defined by the two coordinates of the landscape during (un)zipping. Both the free-energy landscape model and the extensive molecular dynamics simulations totaling over 10 mus predict the existence of temperature-dependent kinetic intermediate states during hairpin (un)zipping and provide the theoretical description of recent ultrafast temperature-jump studies which indicate that hairpin (un)zipping is, in general, not a two-state process. The model allows for lucid prediction of the collapsed state(s) in simple 2D space and we term it the kinetic intermediate structure (KIS) model.

Centromeric DNA replication reconstitution reveals DNA loops and ATR checkpoint suppression

PubMed Central

Aze, Antoine; Sannino, Vincenzo; Soffientini, Paolo; Bachi, Angela; Costanzo, Vincenzo

2016-01-01

Half of human genome is made of repetitive DNA. However, mechanisms underlying replication of chromosome regions containing repetitive DNA are poorly understood. We reconstituted replication of defined human chromosome segments using Bacterial Artificial Chromosomes (BACs) in Xenopus laevis egg extract. Using this approach we characterized chromatin assembly and replication dynamics of centromeric alpha-satellite DNA. Proteomic analysis of centromeric chromatin revealed replication dependent enrichment of a network of DNA repair factors among which the MSH2-6 complex, which was required for efficient centromeric DNA replication. However, contrary to expectations, the ATR dependent checkpoint monitoring DNA replication fork arrest could not be activated on highly repetitive DNA due to inability of single stranded DNA binding protein RPA to accumulate on chromatin. Electron microscopy of centromeric DNA and supercoil mapping revealed the presence of Topoisomerase I dependent DNA loops embedded in a protein matrix enriched for SMC2-4 proteins. This arrangement suppressed ATR signalling by preventing RPA hyper-loading, facilitating replication of centromeric DNA. These findings have important implications on our understanding of repetitive DNA metabolism and centromere organization under normal and stressful conditions. PMID:27111843

Transcription of Gypsy Elements in a Y-Chromosome Male Fertility Gene of Drosophila Hydei

PubMed Central

Hochstenbach, R.; Harhangi, H.; Schouren, K.; Bindels, P.; Suijkerbuijk, R.; Hennig, W.

1996-01-01

We have found that defective gypsy retrotransposons are a major constituent of the lampbrush loop pair Nooses in the short arm of the Y chromosome of Drosophila hydei. The loop pair is formed by male fertility gene Q during the primary spermatocyte stage of spermatogenesis, each loop being a single transcription unit with an estimated length of 260 kb. Using fluorescent in situ hybridization, we show that throughout the loop transcripts gypsy elements are interspersed with blocks of a tandemly repetitive Y-specific DNA sequence, ay1. Nooses transcripts containing both sequence types show a wide size range on Northern blots, do not migrate to the cytoplasm, and are degraded just before the first meiotic division. Only one strand of ay1 and only the coding strand of gypsy can be detected in the loop transcripts. However, as cloned genomic DNA fragments also display opposite orientations of ay1 and gypsy, such DNA sections cannot be part of the Nooses. Hence, they are most likely derived from the flanking heterochromatin. The direction of transcription of ay1 and gypsy thus appears to be of a functional significance. PMID:8852843

Interrogating Key Positions of Size-Reduced TALE Repeats Reveals a Programmable Sensor of 5-Carboxylcytosine.

PubMed

Maurer, Sara; Giess, Mario; Koch, Oliver; Summerer, Daniel

2016-12-16

Transcription-activator-like effector (TALE) proteins consist of concatenated repeats that recognize consecutive canonical nucleobases of DNA via the major groove in a programmable fashion. Since this groove displays unique chemical information for the four human epigenetic cytosine nucleobases, TALE repeats with epigenetic selectivity can be engineered, with potential to establish receptors for the programmable decoding of all human nucleobases. TALE repeats recognize nucleobases via key amino acids in a structurally conserved loop whose backbone is positioned very close to the cytosine 5-carbon. This complicates the engineering of selectivities for large 5-substituents. To interrogate a more promising structural space, we engineered size-reduced repeat loops, performed saturation mutagenesis of key positions, and screened a total of 200 repeat-nucleobase interactions for new selectivities. This provided insight into the structural requirements of TALE repeats for affinity and selectivity, revealed repeats with improved or relaxed selectivity, and resulted in the first selective sensor of 5-carboxylcytosine.

Unfolding and Targeting Thermodynamics of a DNA Intramolecular Complex with Joined Triplex-Duplex Domains.

PubMed

Johnson, Sarah E; Reiling-Steffensmeier, Calliste; Lee, Hui-Ting; Marky, Luis A

2018-01-25

Our laboratory is interested in developing methods that can be used for the control of gene expression. In this work, we are investigating the reaction of an intramolecular complex containing a triplex-duplex junction with partially complementary strands. We used a combination of isothermal titration calorimetry (ITC), differential scanning calorimetry (DSC), and spectroscopy techniques to determine standard thermodynamic profiles for these targeting reactions. Specifically, we have designed single strands to target one loop (CTTTC) or two loops (CTTTC and GCAA) of this complex. Both reactions yielded exothermic enthalpies of -66.3 and -82.8 kcal/mol by ITC, in excellent agreement with the reaction enthalpies of -72.7 and -88.7 kcal/mol, respectively, obtained from DSC Hess cycles. The favorable heat contributions result from the formation of base-pair stacks involving mainly the unpaired bases of the loops. This shows that each complementary strand is able to invade and disrupt the secondary structure. The simultaneous targeting of two loops yielded a more favorable reaction free energy, by approximately -8 kcal/mol, which corresponds to the formation of roughly four base-pair stacks involving the unpaired bases of the 5'-GCAA loop. The main conclusion is that the targeting of loops with a large number of unpaired bases results in a more favorable reaction free energy.

An insight into the active site of a type I DNA topoisomerase from the kinetoplastid protozoan Leishmania donovani

PubMed Central

Das, Aditi; Mandal, Chhabinath; Dasgupta, Arindam; Sengupta, Tanushri; Majumder, Hemanta K.

2002-01-01

DNA topoisomerases are ubiquitous enzymes that govern the topological interconversions of DNA thereby playing a key role in many aspects of nucleic acid metabolism. Recently determined crystal structures of topoisomerase fragments, representing nearly all the known subclasses, have been solved. The type IB enzymes are structurally distinct from other known topoisomerases but are similar to a class of enzymes referred to as tyrosine recombinases. A putative topoisomerase I open reading frame from the kinetoplastid Leishmania donovani was reported which shared a substantial degree of homology with type IB topoisomerases but having a variable C-terminus. Here we present a molecular model of the above parasite gene product, using the human topoisomerase I crystal structure in complex with a 22 bp oligonucleotide as a template. Our studies indicate that the overall structure of the parasite protein is similar to the human enzyme; however, major differences occur in the C-terminal loop, which harbors a serine in place of the usual catalytic tyrosine. Most other structural themes common to type IB topoisomerases, including secondary structural folds, hinged clamps that open and close to bind DNA, nucleophilic attack on the scissile DNA strand and formation of a ternary complex with the topoisomerase I inhibitor camptothecin could be visualized in our homology model. The validity of serine acting as the nucleophile in the case of the parasite protein model was corroborated with our biochemical mapping of the active site with topoisomerase I enzyme purified from L.donovani promastigotes. PMID:11809893

Sequence analysis of the mitochondrial DNA control region of ciscoes (genus Coregonus): Taxonomic implications for the Great Lakes species flock

USGS Publications Warehouse

Reed, Kent M.; Dorschner, Michael O.; Todd, Thomas N.; Phillips, Ruth B.

1998-01-01

Sequence variation in the control region (D-loop) of the mitochondrial DNA (mtDNA) was examined to assess the genetic distinctiveness of the shortjaw cisco (Coregonus zenithicus). Individuals from within the Great Lakes Basin as well as inland lakes outside the basin were sampled. DNA fragments containing the entire D-loop were amplified by PCR from specimens ofC. zenithicus and the related species C. artedi, C. hoyi, C. kiyi, and C. clupeaformis. DNA sequence analysis revealed high similarity within and among species and shared polymorphism for length variants. Based on this analysis, the shortjaw cisco is not genetically distinct from other cisco species.

Crystal structure of four-stranded Oxytricha telomeric DNA

NASA Technical Reports Server (NTRS)

Kang, C.; Zhang, X.; Ratliff, R.; Moyzis, R.; Rich, A.

1992-01-01

The sequence d(GGGGTTTTGGGG) from the 3' overhang of the Oxytricha telomere has been crystallized and its three-dimensional structure solved to 2.5 A resolution. The oligonucleotide forms hairpins, two of which join to make a four-stranded helical structure with the loops containing four thymine residues at either end. The guanine residues are held together by cyclic hydrogen bonding and an ion is located in the centre. The four guanine residues in each segment have a glycosyl conformation that alternates between anti and syn. There are two four-stranded molecules in the asymmetric unit showing that the structure has some intrinsic flexibility.

Physical proximity of chromatin to nuclear pores prevents harmful R loop accumulation contributing to maintain genome stability.

PubMed

García-Benítez, Francisco; Gaillard, Hélène; Aguilera, Andrés

2017-10-10

During transcription, the mRNA may hybridize with DNA, forming an R loop, which can be physiological or pathological, constituting in this case a source of genomic instability. To understand the mechanism by which eukaryotic cells prevent harmful R loops, we used human activation-induced cytidine deaminase (AID) to identify genes preventing R loops. A screening of 400 Saccharomyces cerevisiae selected strains deleted in nuclear genes revealed that cells lacking the Mlp1/2 nuclear basket proteins show AID-dependent genomic instability and replication defects that were suppressed by RNase H1 overexpression. Importantly, DNA-RNA hybrids accumulated at transcribed genes in mlp1/2 mutants, indicating that Mlp1/2 prevents R loops. Consistent with the Mlp1/2 role in gene gating to nuclear pores, artificial tethering to the nuclear periphery of a transcribed locus suppressed R loops in mlp1 ∆ cells. The same occurred in THO-deficient hpr1 ∆ cells. We conclude that proximity of transcribed chromatin to the nuclear pore helps restrain pathological R loops.

Directed nucleation assembly of DNA tile complexes for barcode-patterned lattices

NASA Astrophysics Data System (ADS)

Yan, Hao; Labean, Thomas H.; Feng, Liping; Reif, John H.

2003-07-01

The programmed self-assembly of patterned aperiodic molecular structures is a major challenge in nanotechnology and has numerous potential applications for nanofabrication of complex structures and useful devices. Here we report the construction of an aperiodic patterned DNA lattice (barcode lattice) by a self-assembly process of directed nucleation of DNA tiles around a scaffold DNA strand. The input DNA scaffold strand, constructed by ligation of shorter synthetic oligonucleotides, provides layers of the DNA lattice with barcode patterning information represented by the presence or absence of DNA hairpin loops protruding out of the lattice plane. Self-assembly of multiple DNA tiles around the scaffold strand was shown to result in a patterned lattice containing barcode information of 01101. We have also demonstrated the reprogramming of the system to another patterning. An inverted barcode pattern of 10010 was achieved by modifying the scaffold strands and one of the strands composing each tile. A ribbon lattice, consisting of repetitions of the barcode pattern with expected periodicity, was also constructed by the addition of sticky ends. The patterning of both classes of lattices was clearly observable via atomic force microscopy. These results represent a step toward implementation of a visual readout system capable of converting information encoded on a 1D DNA strand into a 2D form readable by advanced microscopic techniques. A functioning visual output method would not only increase the readout speed of DNA-based computers, but may also find use in other sequence identification techniques such as mutation or allele mapping.

Directed nucleation assembly of DNA tile complexes for barcode-patterned lattices.

PubMed

Yan, Hao; LaBean, Thomas H; Feng, Liping; Reif, John H

2003-07-08

The programmed self-assembly of patterned aperiodic molecular structures is a major challenge in nanotechnology and has numerous potential applications for nanofabrication of complex structures and useful devices. Here we report the construction of an aperiodic patterned DNA lattice (barcode lattice) by a self-assembly process of directed nucleation of DNA tiles around a scaffold DNA strand. The input DNA scaffold strand, constructed by ligation of shorter synthetic oligonucleotides, provides layers of the DNA lattice with barcode patterning information represented by the presence or absence of DNA hairpin loops protruding out of the lattice plane. Self-assembly of multiple DNA tiles around the scaffold strand was shown to result in a patterned lattice containing barcode information of 01101. We have also demonstrated the reprogramming of the system to another patterning. An inverted barcode pattern of 10010 was achieved by modifying the scaffold strands and one of the strands composing each tile. A ribbon lattice, consisting of repetitions of the barcode pattern with expected periodicity, was also constructed by the addition of sticky ends. The patterning of both classes of lattices was clearly observable via atomic force microscopy. These results represent a step toward implementation of a visual readout system capable of converting information encoded on a 1D DNA strand into a 2D form readable by advanced microscopic techniques. A functioning visual output method would not only increase the readout speed of DNA-based computers, but may also find use in other sequence identification techniques such as mutation or allele mapping.

Curated collection of yeast transcription factor DNA binding specificity data reveals novel structural and gene regulatory insights

PubMed Central

2011-01-01

Background Transcription factors (TFs) play a central role in regulating gene expression by interacting with cis-regulatory DNA elements associated with their target genes. Recent surveys have examined the DNA binding specificities of most Saccharomyces cerevisiae TFs, but a comprehensive evaluation of their data has been lacking. Results We analyzed in vitro and in vivo TF-DNA binding data reported in previous large-scale studies to generate a comprehensive, curated resource of DNA binding specificity data for all characterized S. cerevisiae TFs. Our collection comprises DNA binding site motifs and comprehensive in vitro DNA binding specificity data for all possible 8-bp sequences. Investigation of the DNA binding specificities within the basic leucine zipper (bZIP) and VHT1 regulator (VHR) TF families revealed unexpected plasticity in TF-DNA recognition: intriguingly, the VHR TFs, newly characterized by protein binding microarrays in this study, recognize bZIP-like DNA motifs, while the bZIP TF Hac1 recognizes a motif highly similar to the canonical E-box motif of basic helix-loop-helix (bHLH) TFs. We identified several TFs with distinct primary and secondary motifs, which might be associated with different regulatory functions. Finally, integrated analysis of in vivo TF binding data with protein binding microarray data lends further support for indirect DNA binding in vivo by sequence-specific TFs. Conclusions The comprehensive data in this curated collection allow for more accurate analyses of regulatory TF-DNA interactions, in-depth structural studies of TF-DNA specificity determinants, and future experimental investigations of the TFs' predicted target genes and regulatory roles. PMID:22189060

A Looping-Based Model for Quenching Repression

PubMed Central

Pollak, Yaroslav; Goldberg, Sarah; Amit, Roee

2017-01-01

We model the regulatory role of proteins bound to looped DNA using a simulation in which dsDNA is represented as a self-avoiding chain, and proteins as spherical protrusions. We simulate long self-avoiding chains using a sequential importance sampling Monte-Carlo algorithm, and compute the probabilities for chain looping with and without a protrusion. We find that a protrusion near one of the chain’s termini reduces the probability of looping, even for chains much longer than the protrusion–chain-terminus distance. This effect increases with protrusion size, and decreases with protrusion-terminus distance. The reduced probability of looping can be explained via an eclipse-like model, which provides a novel inhibitory mechanism. We test the eclipse model on two possible transcription-factor occupancy states of the D. melanogaster eve 3/7 enhancer, and show that it provides a possible explanation for the experimentally-observed eve stripe 3 and 7 expression patterns. PMID:28085884

Mitochondrial DNA mutation screening of male patients with obstructive sleep apnea-hypopnea syndrome.

PubMed

Huang, Xiao-Ying; Li, Hong; Xu, Xiao-Mei; Wang, Liang-Xing

2014-08-01

The aim of the present study was to analyze the differences between the genes of the mitochondrial DNA (mtDNA) displacement loop (D-loop) region and the Cambridge Reference sequence, in order to screen the mutation sites and investigate the correlation between mutations, clinical parameters and complications associated with obstructive sleep apnea-hypopnea syndrome (OSAHS). mtDNA was obtained from male patients with OSAHS in the Zhejiang Province. In total, 60 male patients with OSAHS and 102 healthy adults were assessed to determine the levels of fasting blood glucose, total cholesterol, triglyceride (TG) and high-density and low-density lipoproteins (LDL). Furthermore, peripheral mtDNA was extracted and bidirectional sequencing was conducted to enable mutation screening. In the mtDNA D-loop region, 178 mutation sites were identified, of which 115 sites were present in the two groups. The number of non-common sites in the OSAHS group was significantly higher compared with the control group (P<0.05). No statistically significant difference was observed in the mutations among the mild, moderate and severe OSAHS groups (P>0.05). A total of 21 cases in the severe OSAHS group exhibited mutation rates of >10%. In the control group, there were 24 cases where the np73A-G and np263A-G mutations were predominant. The np303-np315 region was identified to be the highly variable region and various mutation forms were observed. Statistically significant differences were observed in the neck perimeter, TG and LDL levels among the OSAHS-no-mutation subgroups (P<0.05) and LDL was shown to be associated with an mtDNA mutation in the OSAHS group. Numerous polymorphic mutation sites were identified in the mtDNA D-loop region of the OSAHS group. Therefore, mtDNA mutation sites may be closely associated with the clinical manifestations and complications of OSAHS.

DNA nanostructure-based fluorescence thermometer with silver nanoclusters

NASA Astrophysics Data System (ADS)

Bu, Congcong; Mu, Lixuan; Cao, Xingxing; Chen, Min; She, Guangwei; Shi, Wensheng

2018-07-01

DNA nanostructure-based fluorescence thermometers were fabricated by linking fluorescent silver nanoclusters (AgNCs) and guanine-rich（G-rich）DNA chains via a thermally sensitive DNA stem-loop at terminals 5‧ and 3‧. Variations of temperature alter the distance between the AgNCs and G-rich DNA chain, affecting the interaction between them. As a result, the intensity of fluorescence emission from the AgNCs at 636 nm can be sensitively modulated. It was found that the intensity of such red emission is more temperature sensitive than the equivalent green emission at 543 nm; sensitivity of ‑3.6%/°C was achieved. Through variation of the melting temperature of the DNA stem-loop, the response temperature range of the thermometers could be readily adjusted. Novel DNA nanostructure-based fluorescence thermometers as described in this work are anticipated to be able to measure the temperature of biological systems at small scales—even a single cell.

DNA nanostructure-based fluorescence thermometer with silver nanoclusters.

PubMed

Bu, Congcong; Mu, Lixuan; Cao, XIngxing; Chen, Min; She, Guangwei; Shi, Wensheng

2018-04-27

Linking the fluorescent silver nanoclusters (AgNCs) and guanine-rich（G-rich）DNA chains by the thermal sensitive DNA stem-loop at teminal 5' and 3', DNA nanostructure-based fluorescence thermometers were fabricated. The variations of the temperature alter the distance between AgNCs and G-rich DNA chain, which could affect the interaction between them. As a result, the intensity of fluorescence emission from AgNCs at 636 nm can be sensitively modulated. It was found that such red emission is more sensitive to the temperature comparing with its intrinsic green emission at 543 nm, and sensitivity of -3.6%/℃ was achieved. Varying the melting temperature of the DNA stem-loop could readily adjust the response temperature range of thermometers. Novel DNA nanostructure-based fluorescence thermometers in this work could be anticipated to measure the temperature of biological system, even a single cell. © 2018 IOP Publishing Ltd.

Cyclic Peptidic Mimetics of Apollo Peptides Targeting Telomeric Repeat Binding Factor 2 (TRF2) and Apollo Interaction.

PubMed

Chen, Xia; Liu, Liu; Chen, Yong; Yang, Yuting; Yang, Chao-Yie; Guo, Tianyue; Lei, Ming; Sun, Haiying; Wang, Shaomeng

2018-05-10

Telomeric repeat binding factor 2 (TRF2) is a telomere-associated protein that plays an important role in the formation of the 3' single strand DNA overhang and the "T loop", two structures critical for the stability of the telomeres. Apollo is a 5'-exonuclease recruited by TRF2 to the telomere and contributes to the formation of the 3' single strand DNA overhang. Knocking down of Apollo can induce DNA damage response similar to that caused by the knocking down of TRF2. In this Letter, we report the design and synthesis of a class of cyclic peptidic mimetics of the TRFH binding motif of Apollo (Apollo TBM ). We found conformational control of the C terminal residues of Apollo TBM can effectively improve the binding affinity. We have obtained a crystal structure of a cyclic peptidic Apollo peptide mimetic ( 34 ) complexed with TRF2, which provides valuable guidance to the future design of TRF2 inhibitors.

Rapid and Simple Detection of Hot Spot Point Mutations of Epidermal Growth Factor Receptor, BRAF, and NRAS in Cancers Using the Loop-Hybrid Mobility Shift Assay

PubMed Central

Matsukuma, Shoichi; Yoshihara, Mitsuyo; Kasai, Fumio; Kato, Akinori; Yoshida, Akira; Akaike, Makoto; Kobayashi, Osamu; Nakayama, Haruhiko; Sakuma, Yuji; Yoshida, Tsutomu; Kameda, Yoichi; Tsuchiya, Eiju; Miyagi, Yohei

2006-01-01

A simple and rapid method to detect the epidermal growth factor receptor hot spot mutation L858R in lung adenocarcinoma was developed based on principles similar to the universal heteroduplex generator technology. A single-stranded oligonucleotide with an internal deletion was used to generate heteroduplexes (loop-hybrids) bearing a loop in the complementary strand derived from the polymerase chain reaction product of the normal or mutant allele. By placing deletion in the oligonucleotide adjacent to the mutational site, difference in electrophoretic mobility between loop-hybrids with normal and mutated DNA was distinguishable in a native polyacrylamide gel. The method was also modified to detect in-frame deletion mutations of epidermal growth factor receptor in lung adenocarcinomas. In addition, the method was adapted to detect hot spot mutations in the B-type Raf kinase (BRAF) at V600 and in a Ras-oncogene (NRAS) at Q61, the mutations commonly found in thyroid carcinomas. Our mutation detection system, designated the loop-hybrid mobility shift assay was sensitive enough to detect mutant DNA comprising 7.5% of the total DNA. As a simple and straightforward mutation detection technique, loop-hybrid mobility shift assay may be useful for the molecular diagnosis of certain types of clinical cancers. Other applications are also discussed. PMID:16931592

Recombination of mitochondrial DNA in skeletal muscle of individuals with multiple mitochondrial DNA heteroplasmy.

PubMed

Zsurka, Gábor; Kraytsberg, Yevgenia; Kudina, Tatiana; Kornblum, Cornelia; Elger, Christian E; Khrapko, Konstantin; Kunz, Wolfram S

2005-08-01

Experimental evidence for human mitochondrial DNA (mtDNA) recombination was recently obtained in an individual with paternal inheritance of mtDNA and in an in vitro cell culture system. Whether mtDNA recombination is a common event in humans remained to be determined. To detect mtDNA recombination in human skeletal muscle, we analyzed the distribution of alleles in individuals with multiple mtDNA heteroplasmy using single-cell PCR and allele-specific PCR. In all ten individuals who carried a heteroplasmic D-loop mutation and a distantly located tRNA point mutation or a large deletion, we observed a mixture of four allelic combinations (tetraplasmy), a hallmark of recombination. Twelve of 14 individuals with closely located heteroplasmic D-loop mutation pairs contained a mixture of only three types of mitochondrial genomes (triplasmy), consistent with the absence of recombination between adjacent markers. These findings indicate that mtDNA recombination is common in human skeletal muscle.

Protein Self-Assembly and Protein-Induced DNA Morphologies

NASA Astrophysics Data System (ADS)

Mawhinney, Matthew T.

The ability of biomolecules to associate into various structural configurations has a substantial impact on human physiology. The synthesis of protein polypeptide chains using the information encoded by DNA is mediated through the use of regulatory proteins, known as transcription factors. Some transcription factors perform function by inducing local curvature in deoxyribonucleic acid (DNA) strands, the mechanisms of which are not entirely known. An important architectural protein, eleven zinc finger CTCF (11 ZF CTCF) is involved in genome organization and hypothesized to mediate DNA loop formation. Direct evidence for these CTCF-induced DNA loops has yet to be observed. In this thesis, the effect of 11 ZF CTCF on DNA morphology is examined using atomic force microscopy, a powerful technique for visualizing biomolecules with nanometer resolution. The presence of CTCF is revealed to induce a variety of morphologies deviating from the relaxed state of control DNA samples, including compact circular complexes, meshes, and networks. Images reveal quasi-circular DNA/CTCF complexes consistent with a single DNA molecule twice wrapped around the protein. The structures of DNA and proteins are highly important for operations in the cell. Structural irregularities may lead to a variety of issues, including more than twenty human pathologies resulting from aberrant protein misfolding into amyloid aggregates of elongated fibrils. Insulin deficiency and resistance characterizing type 2 diabetes often requires administration of insulin. Injectable and inhalable delivery methods have been documented to result in the deposition of amyloid fibrils. Oligomers, soluble multiprotein assemblies, are believed to play an important role in this process. Insulin aggregation under physiological conditions is not well understood and oligomers have not yet been fully characterized. In this thesis, in vitro insulin aggregation at acidic and neutral pH is explored using a variety of techniques, including kinetic thioflavin T fluorescence, circular dichroism spectroscopy, atomic force and electron microscopy imaging. The size distribution of insulin oligomers at different assembly stages is characterized through covalent cross-linking and gel electrophoresis. Results show that at the earliest assembly stage, oligomers comprise up to 40% and 70% of soluble insulin at acidic and neutral pH, respectively. While the highest oligomer order increases with insulin concentration at acidic pH, the opposite tendency is observed at neutral pH, with heptamers formed in 10 muM insulin. These findings suggest that oligomers may be on- and off- pathway assemblies for insulin at acidic and neutral pH, respectively. Agitation, required to induce insulin aggregation at neutral pH, increases fibril formation rate and fibrillar mass by an order of magnitude each. Insulin incubated under agitated conditions at neutral pH rapidly aggregates into large micrometer-sized aggregates, which provides insight into injection-site amyloidosis and toxic pulmonary aggregates induced by administration of extraneous insulin.

Stress and transcriptional regulation of tick ferritin HC.

PubMed

Mulenga, A; Simser, J A; Macaluso, K R; Azad, A F

2004-08-01

We previously identified a partial Dermacentor variabilis cDNA encoding ferritin HC (HC) subunit homolog (DVFER) that was differentially upregulated in Rickettsia montanensis infected ticks (Mulenga et al., 2003a). We have used rapid amplification of cDNA ends to clone full-length DVFER cDNA and its apparent ortholog from the wood tick, D. andersoni (DAFER), both of which show high sequence similarity to vertebrate than insect ferritin. Both DVFER and DAFER contain the stem-loop structure of a putative iron responsive element in the 5' untranslated region (nucleotide positions, 16-42) and the feroxidase centre loop typical for vertebrate ferritin HC subunits. Quantitative Western and Northern blotting analyses of protein and RNA from unfed and partially fed whole tick as well as dissected tick tissues demonstrated that DVFER is constitutively and ubiquitously expressed. Based on densitometric analysis of detected protein and mRNA bands, DVFER is predominantly expressed in the midgut, and to a lesser extent in the salivary glands, ovary and fatbody. Sham treatment (mechanical injury) and Escherichia coli challenge of D. variabilis ticks stimulated statistically significant (approximately 1.5- and approximately 3.0-fold, respectively) increases in DVFER mRNA abundance over time point matched naive control ticks. These data suggest that DVFER mRNA is nonspecifically up regulated in response to mechanical injury or bacterial infection induced stress.

The effects of DNA supercoiling on G-quadruplex formation.

PubMed

Sekibo, Doreen A T; Fox, Keith R

2017-12-01

Guanine-rich DNAs can fold into four-stranded structures that contain stacks of G-quartets. Bioinformatics studies have revealed that G-rich sequences with the potential to adopt these structures are unevenly distributed throughout genomes, and are especially found in gene promoter regions. With the exception of the single-stranded telomeric DNA, all genomic G-rich sequences will always be present along with their C-rich complements, and quadruplex formation will be in competition with the corresponding Watson-Crick duplex. Quadruplex formation must therefore first require local dissociation (melting) of the duplex strands. Since negative supercoiling is known to facilitate the formation of alternative DNA structures, we have investigated G-quadruplex formation within negatively supercoiled DNA plasmids. Plasmids containing multiple copies of (G3T)n and (G3T4)n repeats, were probed with dimethylsulphate, potassium permanganate and S1 nuclease. While dimethylsulphate footprinting revealed some evidence for G-quadruplex formation in (G3T)n sequences, this was not affected by supercoiling, and permanganate failed to detect exposed thymines in the loop regions. (G3T4)n sequences were not protected from DMS and showed no reaction with permanganate. Similarly, both S1 nuclease and 2D gel electrophoresis of DNA topoisomers did not detect any supercoil-dependent structural transitions. These results suggest that negative supercoiling alone is not sufficient to drive G-quadruplex formation. © The Author(s) 2017. Published by Oxford University Press on behalf of Nucleic Acids Research.

A Structural Model for the Single-Stranded DNA Genome of Filamentous Bacteriophage Pf1†

PubMed Central

Tsuboi, Masamichi; Tsunoda, Masaru; Overman, Stacy A.; Benevides, James M.; Thomas, George J.

2010-01-01

The filamentous bacteriophage Pf1, which infects strain PAK of Pseudomonas aeruginosa, is a flexible filament (~2000 × 6.5 nm) consisting of a covalently closed DNA loop of 7349 nucleotides sheathed by 7350 copies of a 46-residue α-helical subunit. The subunit α-helices, which are inclined at a small average angle (~16°) from the virion axis, are arranged compactly around the DNA core. Orientations of the Pf1 DNA nucleotides with respect to the filament axis are not known. In this work we report and interpret the polarized Raman spectra of oriented Pf1 filaments. We demonstrate that the polarizations of DNA Raman band intensities establish that the nucleotide bases of packaged Pf1 DNA are well ordered within the virion and that the base planes are positioned close to parallel to the filament axis. The present results are combined with a previously proposed projection of the intraviral path of Pf1 DNA (1) to develop a novel molecular model for the Pf1 assembly. PMID:20078135

Loop-mediated isothermal amplification (LAMP) shield for Arduino DNA detection.

PubMed

Velders, Aldrik H; Schoen, Cor; Saggiomo, Vittorio

2018-02-01

Loop-mediated isothermal amplification (LAMP) of DNA is gaining relevance as a method to detect nucleic acids, as it is easier, faster, and more powerful than conventional Polymerase Chain Reaction. However, LAMP is still mostly used in laboratory settings, because of the lack of a cheap and easy, one-button device that can perform LAMP experiments. Here we show how to build and program an Arduino shield for a LAMP and detection of DNA. The here described Arduino Shield is cheap, easy to assemble, to program and use, it is battery operated and the detection of DNA is done by naked-eye so that it can be used in field.

Tetrahelical structural family adopted by AGCGA-rich regulatory DNA regions

NASA Astrophysics Data System (ADS)

Kocman, Vojč; Plavec, Janez

2017-05-01

Here we describe AGCGA-quadruplexes, an unexpected addition to the well-known tetrahelical families, G-quadruplexes and i-motifs, that have been a focus of intense research due to their potential biological impact in G- and C-rich DNA regions, respectively. High-resolution structures determined by solution-state nuclear magnetic resonance (NMR) spectroscopy demonstrate that AGCGA-quadruplexes comprise four 5'-AGCGA-3' tracts and are stabilized by G-A and G-C base pairs forming GAGA- and GCGC-quartets, respectively. Residues in the core of the structure are connected with edge-type loops. Sequences of alternating 5'-AGCGA-3' and 5'-GGG-3' repeats could be expected to form G-quadruplexes, but are shown herein to form AGCGA-quadruplexes instead. Unique structural features of AGCGA-quadruplexes together with lower sensitivity to cation and pH variation imply their potential biological relevance in regulatory regions of genes responsible for basic cellular processes that are related to neurological disorders, cancer and abnormalities in bone and cartilage development.

Analysis of the crystal structure of an active MCM hexamer.

PubMed

Miller, Justin M; Arachea, Buenafe T; Epling, Leslie B; Enemark, Eric J

2014-09-29

In a previous Research article (Froelich et al., 2014), we suggested an MCM helicase activation mechanism, but were limited in discussing the ATPase domain because it was absent from the crystal structure. Here we present the crystal structure of a nearly full-length MCM hexamer that is helicase-active and thus has all features essential for unwinding DNA. The structure is a chimera of Sulfolobus solfataricus N-terminal domain and Pyrococcus furiosus ATPase domain. We discuss three major findings: 1) a novel conformation for the A-subdomain that could play a role in MCM regulation; 2) interaction of a universally conserved glutamine in the N-terminal Allosteric Communication Loop with the AAA+ domain helix-2-insert (h2i); and 3) a recessed binding pocket for the MCM ssDNA-binding motif influenced by the h2i. We suggest that during helicase activation, the h2i clamps down on the leading strand to facilitate strand retention and regulate ATP hydrolysis.

Evidence for the role of Mycobacterium tuberculosis RecG helicase in DNA repair and recombination.

PubMed

Thakur, Roshan S; Basavaraju, Shivakumar; Somyajit, Kumar; Jain, Akshatha; Subramanya, Shreelakshmi; Muniyappa, Kalappa; Nagaraju, Ganesh

2013-04-01

In order to survive and replicate in a variety of stressful conditions during its life cycle, Mycobacterium tuberculosis must possess mechanisms to safeguard the integrity of the genome. Although DNA repair and recombination related genes are thought to play key roles in the repair of damaged DNA in all organisms, so far only a few of them have been functionally characterized in the tubercle bacillus. In this study, we show that M. tuberculosis RecG (MtRecG) expression was induced in response to different genotoxic agents. Strikingly, expression of MtRecG in Escherichia coli ∆recG mutant strain provided protection against mitomycin C, methyl methane sulfonate and UV induced cell death. Purified MtRecG exhibited higher binding affinity for the Holliday junction (HJ) compared with a number of canonical recombinational DNA repair intermediates. Notably, although MtRecG binds at the core of the mobile and immobile HJs, and with higher binding affinity for the immobile HJ, branch migration was evident only in the case of the mobile HJ. Furthermore, immobile HJs stimulate MtRecG ATPase activity less efficiently than mobile HJs. In addition to HJ substrates, MtRecG exhibited binding affinity for a variety of branched DNA structures including three-way junctions, replication forks, flap structures, forked duplex and a D-loop structure, but demonstrated strong unwinding activity on replication fork and flap DNA structures. Together, these results support that MtRecG plays an important role in processes related to DNA metabolism under normal as well as stress conditions. © 2013 The Authors Journal compilation © 2013 FEBS.

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

Patel, Asmita; Shuman, Stewart; Mondragon, Alfonso

Type IB DNA topoisomerases are found in all eukarya, two families of eukaryotic viruses (poxviruses and mimivirus), and many genera of bacteria. They alter DNA topology by cleaving and resealing one strand of duplex DNA via a covalent DNA-(3-phosphotyrosyl)-enzyme intermediate. Bacterial type IB enzymes were discovered recently and are described as poxvirus-like with respect to their small size, primary structures, and bipartite domain organization. Here we report the 1.75-{angstrom} crystal structure of Deinococcus radiodurans topoisomerase IB (DraTopIB), a prototype of the bacterial clade. DraTopIB consists of an amino-terminal (N) {beta}-sheet domain (amino acids 1-90) and a predominantly {alpha}-helical carboxyl-terminal (C)more » domain (amino acids 91-346) that closely resemble the corresponding domains of vaccinia virus topoisomerase IB. The five amino acids of DraTopIB that comprise the catalytic pentad (Arg-137, Lys-174, Arg-239, Asn-280, and Tyr-289) are preassembled into the active site in the absence of DNA in a manner nearly identical to the pentad configuration in human topoisomerase I bound to DNA. This contrasts with the apoenzyme of vaccinia topoisomerase, in which three of the active site constituents are either displaced or disordered. The N and C domains of DraTopIB are splayed apart in an 'open' conformation, in which the surface of the catalytic domain containing the active site is exposed for DNA binding. A comparison with the human topoisomerase I-DNA cocrystal structure suggests how viral and bacterial topoisomerase IB enzymes might bind DNA circumferentially via movement of the N domain into the major groove and clamping of a disordered loop of the C domain around the helix.« less

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

PubMed Central

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

2011-01-01

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

Plasmid DNA Delivery: Nanotopography Matters.

PubMed

Song, Hao; Yu, Meihua; Lu, Yao; Gu, Zhengying; Yang, Yannan; Zhang, Min; Fu, Jianye; Yu, Chengzhong

2017-12-20

Plasmid DNA molecules with unique loop structures have widespread bioapplications, in many cases relying heavily on delivery vehicles to introduce them into cells and achieve their functions. Herein, we demonstrate that control over delicate nanotopography of silica nanoparticles as plasmid DNA vectors has significant impact on the transfection efficacy. For silica nanoparticles with rambutan-, raspberry-, and flower-like morphologies composed of spike-, hemisphere-, and bowl-type subunit nanotopographies, respectively, the rambutan-like nanoparticles with spiky surfaces demonstrate the highest plasmid DNA binding capability and transfection efficacy of 88%, higher than those reported for silica-based nanovectors. Moreover, it is shown that the surface spikes of rambutan nanoparticles provide a continuous open space to bind DNA chains via multivalent interactions and protect the gene molecules sheltered in the spiky layer against nuclease degradation, exhibiting no significant transfection decay. This unique protection feature is in great contrast to a commercial transfection agent with similar transfection performance but poor protection capability against enzymatic cleavage. Our study provides new understandings in the rational design of nonviral vectors for efficient gene delivery.

Highly conserved D-loop-like nuclear mitochondrial sequences (Numts) in tiger (Panthera tigris).

PubMed

Zhang, Wenping; Zhang, Zhihe; Shen, Fujun; Hou, Rong; Lv, Xiaoping; Yue, Bisong

2006-08-01

Using oligonucleotide primers designed to match hypervariable segments I (HVS-1) of Panthera tigris mitochondrial DNA (mtDNA), we amplified two different PCR products (500 bp and 287 bp) in the tiger (Panthera tigris), but got only one PCR product (287 bp) in the leopard (Panthera pardus). Sequence analyses indicated that the sequence of 287 bp was a D-loop-like nuclear mitochondrial sequence (Numts), indicating a nuclear transfer that occurred approximately 4.8-17 million years ago in the tiger and 4.6-16 million years ago in the leopard. Although the mtDNA D-loop sequence has a rapid rate of evolution, the 287-bp Numts are highly conserved; they are nearly identical in tiger subspecies and only 1.742% different between tiger and leopard. Thus, such sequences represent molecular 'fossils' that can shed light on evolution of the mitochondrial genome and may be the most appropriate outgroup for phylogenetic analysis. This is also proved by comparing the phylogenetic trees reconstructed using the D-loop sequence of snow leopard and the 287-bp Numts as outgroup.

Mutational Hotspots in the Mitochondrial D-Loop Region of Cancerous and Precancerous Colorectal Lesions in Egyptian Patients

PubMed Central

El-Guendy, Nadia; Tantawy, Marwa; Abdelhady, Hala; El-Ghor, Akmal; Abdel Wahab, Abdel Hady

2011-01-01

Mutations in the mitochondrial genome (mtDNA) are associated with different types of cancer, specifically colorectal cancer (CRC). However, few studies have been performed on precancerous lesions, such as ulcerative colitis (UC) lesions and adenomatous polyps (AP). The aim of this study was to identify mtDNA mutations in the cancerous and precancerous lesions of Egyptian patients. An analysis of the mutations found in six regions of the mtDNA genome (ND1, ND5, COI, tRNAser, D-loop 1, and 2) in 80 Egyptian patients (40 CRC, 20 UC, and 20 AP) was performed using polymerase chain reaction–single-strand conformational polymorphism techniques and followed up by direct sequencing. The overall incidence of mutations was 25%, 25%, and 35% in CRC, UC, and AP cases, respectively. Although there was no common mutation pattern within each group, a large number of mutations were detected in the D-loop region in all of the groups. Some mutations (e.g., T414G) were detected repeatedly in precancerous (UC and AP) and cancerous lesions. Mutations detected in patients with CRC were predominantly found in the ND1 gene (40%). Our preliminary study suggests that Egyptian patients with CRC have a large number of mtDNA mutations, especially in the D-loop region, which have not been previously reported. Mutations in the mtDNA of precancerous lesions (i.e., AP and UC) may contribute to transformation events that lead to CRC. PMID:21612400

Construction of Bacteriophage Phi29 DNA Packaging Motor and its Applications in Nanotechnology and Therapy

PubMed Central

Lee, Tae Jin; Schwartz, Chad; Guo, Peixuan

2010-01-01

Nanobiotechnology involves the creation, characterization, and modification of organized nanomaterials to serve as building blocks for constructing nanoscale devices in technology and medicine. Living systems contain a wide variety of nanomachines and highly ordered structures of macromolecules. The novelty and ingenious design of the bacterial virus phi29 DNA packaging motor and its parts inspired the synthesis of this motor and its components as biomimetics. This 30-nm nanomotor uses six copies of an ATP-binding pRNA to gear the motor. The structural versatility of pRNA has been utilized to construct dimers, trimers, hexamers, and patterned superstructures via the interaction of two interlocking loops. The approach, based on bottom-up assembly, has also been applied to nanomachine fabrication, pathogen detection and the delivery of drugs, siRNA, ribozymes, and genes to specific cells in vitro and in vivo. Another essential component of the motor is the connector, which contains 12 copies of a protein gp10 to form a 3.6-nm central channel as a path for DNA. This article will review current studies of the structure and function of the phi29 DNA packaging motor, as well as the mechanism of motion, the principle of in vitro construction, and its potential nanotechnological and medical applications. PMID:19495981

Centromeric DNA replication reconstitution reveals DNA loops and ATR checkpoint suppression.

PubMed

Aze, Antoine; Sannino, Vincenzo; Soffientini, Paolo; Bachi, Angela; Costanzo, Vincenzo

2016-06-01

Half of the human genome is made up of repetitive DNA. However, mechanisms underlying replication of chromosome regions containing repetitive DNA are poorly understood. We reconstituted replication of defined human chromosome segments using bacterial artificial chromosomes in Xenopus laevis egg extract. Using this approach we characterized the chromatin assembly and replication dynamics of centromeric alpha-satellite DNA. Proteomic analysis of centromeric chromatin revealed replication-dependent enrichment of a network of DNA repair factors including the MSH2-6 complex, which was required for efficient centromeric DNA replication. However, contrary to expectations, the ATR-dependent checkpoint monitoring DNA replication fork arrest could not be activated on highly repetitive DNA due to the inability of the single-stranded DNA binding protein RPA to accumulate on chromatin. Electron microscopy of centromeric DNA and supercoil mapping revealed the presence of topoisomerase I-dependent DNA loops embedded in a protein matrix enriched for SMC2-4 proteins. This arrangement suppressed ATR signalling by preventing RPA hyper-loading, facilitating replication of centromeric DNA. These findings have important implications for our understanding of repetitive DNA metabolism and centromere organization under normal and stressful conditions.

Changes in 5S rDNA Chromatin Organization and Transcription during Heterochromatin Establishment in Arabidopsis

PubMed Central

Mathieu, Olivier; Jasencakova, Zuzana; Vaillant, Isabelle; Gendrel, Anne-Valérie; Colot, Vincent; Schubert, Ingo; Tourmente, Sylvette

2003-01-01

In the Arabidopsis accession Columbia, 5S rDNA is located in the pericentromeric heterochromatin of chromosomes 3, 4, and 5. Both a major and some minor 5S rRNA species are expressed from chromosomes 4 and 5, whereas the genes on chromosome 3 are not transcribed. Here, we show that 5S rDNA methylation is reduced in 2-day-old seedlings versus 4-day-old or older aerial plant tissues, and the minor 5S rRNA species are expressed most abundantly at this stage. Similarly, when 5S rDNA is demethylated by 5-azacytidine treatment or via the decrease in DNA methylation1 (ddm1) mutation, the expression of minor 5S rRNA species is increased. We also show that in leaf nuclei of mature wild-type plants, the transcribed fraction of 5S rDNA forms loops that emanate from chromocenters. These loops, which are enlarged in nuclei of mature ddm1 plants, are enriched for histone H3 acetylated at Lys-9 and methylated at Lys-4 compared with the heterochromatic chromocenters. Up to 4 days after germination, heterochromatin is not fully developed: the 5S rDNA resides in prechromocenters, does not form conspicuous loops, and shows the lowest transcription level. Our results indicate that the expression and chromatin organization of 5S rRNA genes change during heterochromatin establishment. PMID:14630972

Unexpected sequences and structures of mtDNA required for efficient transcription from the first heavy-strand promoter

PubMed Central

Uchida, Akira; Murugesapillai, Divakaran; Kastner, Markus; Wang, Yao; Lodeiro, Maria F; Prabhakar, Shaan; Oliver, Guinevere V; Arnold, Jamie J; Maher, L James; Williams, Mark C; Cameron, Craig E

2017-01-01

Human mtDNA contains three promoters, suggesting a need for differential expression of the mitochondrial genome. Studies of mitochondrial transcription have used a reductionist approach, perhaps masking differential regulation. Here we evaluate transcription from light-strand (LSP) and heavy-strand (HSP1) promoters using templates that mimic their natural context. These studies reveal sequences upstream, hypervariable in the human population (HVR3), and downstream of the HSP1 transcription start site required for maximal yield. The carboxy-terminal tail of TFAM is essential for activation of HSP1 but not LSP. Images of the template obtained by atomic force microscopy show that TFAM creates loops in a discrete region, the formation of which correlates with activation of HSP1; looping is lost in tail-deleted TFAM. Identification of HVR3 as a transcriptional regulatory element may contribute to between-individual variability in mitochondrial gene expression. The unique requirement of HSP1 for the TFAM tail may enable its regulation by post-translational modifications. DOI: http://dx.doi.org/10.7554/eLife.27283.001 PMID:28745586

The haloarchaeal MCM proteins: bioinformatic analysis and targeted mutagenesis of the β7-β8 and β9-β10 hairpin loops and conserved zinc binding domain cysteines.

PubMed

Kristensen, Tatjana P; Maria Cherian, Reeja; Gray, Fiona C; MacNeill, Stuart A

2014-01-01

The hexameric MCM complex is the catalytic core of the replicative helicase in eukaryotic and archaeal cells. Here we describe the first in vivo analysis of archaeal MCM protein structure and function relationships using the genetically tractable haloarchaeon Haloferax volcanii as a model system. Hfx. volcanii encodes a single MCM protein that is part of the previously identified core group of haloarchaeal MCM proteins. Three structural features of the N-terminal domain of the Hfx. volcanii MCM protein were targeted for mutagenesis: the β7-β8 and β9-β10 β-hairpin loops and putative zinc binding domain. Five strains carrying single point mutations in the β7-β8 β-hairpin loop were constructed, none of which displayed impaired cell growth under normal conditions or when treated with the DNA damaging agent mitomycin C. However, short sequence deletions within the β7-β8 β-hairpin were not tolerated and neither was replacement of the highly conserved residue glutamate 187 with alanine. Six strains carrying paired alanine substitutions within the β9-β10 β-hairpin loop were constructed, leading to the conclusion that no individual amino acid within that hairpin loop is absolutely required for MCM function, although one of the mutant strains displays greatly enhanced sensitivity to mitomycin C. Deletions of two or four amino acids from the β9-β10 β-hairpin were tolerated but mutants carrying larger deletions were inviable. Similarly, it was not possible to construct mutants in which any of the conserved zinc binding cysteines was replaced with alanine, underlining the likely importance of zinc binding for MCM function. The results of these studies demonstrate the feasibility of using Hfx. volcanii as a model system for reverse genetic analysis of archaeal MCM protein function and provide important confirmation of the in vivo importance of conserved structural features identified by previous bioinformatic, biochemical and structural studies.

Mechanism of retinoic acid-induced transcription: histone code, DNA oxidation and formation of chromatin loops.

PubMed

Zuchegna, Candida; Aceto, Fabiana; Bertoni, Alessandra; Romano, Antonella; Perillo, Bruno; Laccetti, Paolo; Gottesman, Max E; Avvedimento, Enrico V; Porcellini, Antonio

2014-01-01

Histone methylation changes and formation of chromatin loops involving enhancers, promoters and 3' end regions of genes have been variously associated with active transcription in eukaryotes. We have studied the effect of activation of the retinoic A receptor, at the RARE-promoter chromatin of CASP9 and CYP26A1 genes, 15 and 45 min following RA exposure, and we found that histone H3 lysines 4 and 9 are demethylated by the lysine-specific demethylase, LSD1 and by the JMJ-domain containing demethylase, D2A. The action of the oxidase (LSD1) and a dioxygenase (JMJD2A) in the presence of Fe++ elicits an oxidation wave that locally modifies the DNA and recruits the enzymes involved in base and nucleotide excision repair (BER and NER). These events are essential for the formation of chromatin loop(s) that juxtapose the RARE element with the 5' transcription start site and the 3' end of the genes. The RARE bound-receptor governs the 5' and 3' end selection and directs the productive transcription cycle of RNA polymerase. These data mechanistically link chromatin loops, histone methylation changes and localized DNA repair with transcription. © The Author(s) 2014. Published by Oxford University Press on behalf of Nucleic Acids Research.

All-atomic Molecular Dynamic Studies of Human CDK8: Insight into the A-loop, Point Mutations and Binding with Its Partner CycC

PubMed Central

Xu, Wu; Amire-Brahimi, Benjamin; Xie, Xiao-Jun; Huang, Liying; Ji, Jun-Yuan

2014-01-01

The Mediator, a conserved multisubunit protein complex in eukaryotic organisms, regulates gene expression by bridging sequence-specific DNA-binding transcription factors to the general RNA polymerase II machinery. In yeast, Mediator complex is organized in three core modules (head, middle and tail) and a separable ‘CDK8 submodule’ consisting of four subunits including Cyclin-dependent kinase CDK8 (CDK8), Cyclin C (CycC), MED12, and MED13. The 3-D structure of human CDK8-CycC complex has been recently experimentally determined. To take advantage of this structure and the improved theoretical calculation methods, we have performed molecular dynamic simulations to study dynamics of CDK8 and two CDK8 point mutations (D173A and D189N), which have been identified in human cancers, with and without full length of the A-loop as well as the binding between CDK8 and CycC. We found that CDK8 structure gradually loses two helical structures during the 50-ns molecular dynamic simulation, likely due to the presence of the full-length A-loop. In addition, our studies showed the hydrogen bond occupation of the CDK8 A-loop increases during the first 20-ns MD simulation and stays stable during the later 30-ns MD simulation. Four residues in the A-loop of CDK8 have high hydrogen bond occupation, while the rest residues have low or no hydrogen bond occupation. The hydrogen bond dynamic study of the A-loop residues exhibits three types of changes: increasing, decreasing, and stable. Furthermore, the 3-D structures of CDK8 point mutations D173A, D189N, T196A and T196D have been built by molecular modeling and further investigated by 50-ns molecular dynamic simulations. D173A has the highest average potential energy, while T196D has the lowest average potential energy, indicating that T196D is the most stable structure. Finally, we calculated theoretical binding energy of CDK8 and CycC by MM/PBSA and MM/GBSA methods, and the negative values obtained from both methods demonstrate stability of CDK8-CycC complex. Taken together, these analyses will improve our understanding of the exact functions of CDK8 and the interaction with its partner CycC. PMID:24754906

The detailed 3D multi-loop aggregate/rosette chromatin architecture and functional dynamic organization of the human and mouse genomes.

PubMed

Knoch, Tobias A; Wachsmuth, Malte; Kepper, Nick; Lesnussa, Michael; Abuseiris, Anis; Ali Imam, A M; Kolovos, Petros; Zuin, Jessica; Kockx, Christel E M; Brouwer, Rutger W W; van de Werken, Harmen J G; van IJcken, Wilfred F J; Wendt, Kerstin S; Grosveld, Frank G

2016-01-01

The dynamic three-dimensional chromatin architecture of genomes and its co-evolutionary connection to its function-the storage, expression, and replication of genetic information-is still one of the central issues in biology. Here, we describe the much debated 3D architecture of the human and mouse genomes from the nucleosomal to the megabase pair level by a novel approach combining selective high-throughput high-resolution chromosomal interaction capture ( T2C ), polymer simulations, and scaling analysis of the 3D architecture and the DNA sequence. The genome is compacted into a chromatin quasi-fibre with ~5 ± 1 nucleosomes/11 nm, folded into stable ~30-100 kbp loops forming stable loop aggregates/rosettes connected by similar sized linkers. Minor but significant variations in the architecture are seen between cell types and functional states. The architecture and the DNA sequence show very similar fine-structured multi-scaling behaviour confirming their co-evolution and the above. This architecture, its dynamics, and accessibility, balance stability and flexibility ensuring genome integrity and variation enabling gene expression/regulation by self-organization of (in)active units already in proximity. Our results agree with the heuristics of the field and allow "architectural sequencing" at a genome mechanics level to understand the inseparable systems genomic properties.

The Affinity of the S9.6 Antibody for Double-Stranded RNAs Impacts the Accurate Mapping of R-Loops in Fission Yeast.

PubMed

Hartono, Stella R; Malapert, Amélie; Legros, Pénélope; Bernard, Pascal; Chédin, Frédéric; Vanoosthuyse, Vincent

2018-02-02

R-loops, which result from the formation of stable DNA:RNA hybrids, can both threaten genome integrity and act as physiological regulators of gene expression and chromatin patterning. To characterize R-loops in fission yeast, we used the S9.6 antibody-based DRIPc-seq method to sequence the RNA strand of R-loops and obtain strand-specific R-loop maps at near nucleotide resolution. Surprisingly, preliminary DRIPc-seq experiments identified mostly RNase H-resistant but exosome-sensitive RNAs that mapped to both DNA strands and resembled RNA:RNA hybrids (dsRNAs), suggesting that dsRNAs form widely in fission yeast. We confirmed in vitro that S9.6 can immuno-precipitate dsRNAs and provide evidence that dsRNAs can interfere with its binding to R-loops. dsRNA elimination by RNase III treatment prior to DRIPc-seq allowed the genome-wide and strand-specific identification of genuine R-loops that responded in vivo to RNase H levels and displayed classical features associated with R-loop formation. We also found that most transcripts whose levels were altered by in vivo manipulation of RNase H levels did not form detectable R-loops, suggesting that prolonged manipulation of R-loop levels could indirectly alter the transcriptome. We discuss the implications of our work in the design of experimental strategies to probe R-loop functions. Copyright © 2017 Elsevier Ltd. All rights reserved.

Analysis of the Functions of Recombination-Related Genes in the Generation of Large Chromosomal Deletions by Loop-Out Recombination in Aspergillus oryzae

PubMed Central

Ogawa, Masahiro; Koyama, Yasuji

2012-01-01

Loop-out-type recombination is a type of intrachromosomal recombination followed by the excision of a chromosomal region. The detailed mechanism underlying this recombination and the genes involved in loop-out recombination remain unknown. In the present study, we investigated the functions of ku70, ligD, rad52, rad54, and rdh54 in the construction of large chromosomal deletions via loop-out recombination and the effect of the position of the targeted chromosomal region on the efficiency of loop-out recombination in Aspergillus oryzae. The efficiency of generation of large chromosomal deletions in the near-telomeric region of chromosome 3, including the aflatoxin gene cluster, was compared with that in the near-centromeric region of chromosome 8, including the tannase gene. In the Δku70 and Δku70-rdh54 strains, only precise loop-out recombination occurred in the near-telomeric region. In contrast, in the ΔligD, Δku70-rad52, and Δku70-rad54 strains, unintended chromosomal deletions by illegitimate loop-out recombination occurred in the near-telomeric region. In addition, large chromosomal deletions via loop-out recombination were efficiently achieved in the near-telomeric region, but barely achieved in the near-centromeric region, in the Δku70 strain. Induction of DNA double-strand breaks by I-SceI endonuclease facilitated large chromosomal deletions in the near-centromeric region. These results indicate that ligD, rad52, and rad54 play a role in the generation of large chromosomal deletions via precise loop-out-type recombination in the near-telomeric region and that loop-out recombination between distant sites is restricted in the near-centromeric region by chromosomal structure. PMID:22286092

Unraveling secrets of telomeres: one molecule at a time

PubMed Central

Lin, Jiangguo; Kaur, Parminder; Countryman, Preston; Opresko, Patricia L.; Wang, Hong

2016-01-01

Telomeres play important roles in maintaining the stability of linear chromosomes. Telomere maintenance involves dynamic actions of multiple proteins interacting with long repetitive sequences and complex dynamic DNA structures, such as G-quadruplexes, T-loops and t-circles. Given the heterogeneity and complexity of telomeres, single-molecule approaches are essential to fully understand the structure-function relationships that govern telomere maintenance. In this review, we present a brief overview of the principles of single-molecule imaging and manipulation techniques. We then highlight results obtained from applying these single-molecule techniques for studying structure, dynamics and functions of G-quadruplexes, telomerase, and shelterin proteins. PMID:24569170

Computational and Experimental Characterization of Ribosomal DNA and RNA G-Quadruplexes

NASA Astrophysics Data System (ADS)

Cho, Samuel

DNA G-quadruplexes in human telomeres and gene promoters are being extensively studied for their role in controlling the growth of cancer cells. Recent studies strongly suggest that guanine (G)-rich genes encoding pre-ribosomal RNA (pre-rRNA) are a potential anticancer target through the inhibition of RNA polymerase I (Pol I) in ribosome biogenesis. However, the structures of ribosomal G-quadruplexes at atomic resolution are unknown, and very little biophysical characterization has been performed on them to date. Here, we have modeled two putative rDNA G-quadruplex structures, NUC 19P and NUC 23P, which we observe via circular dichroism (CD) spectroscopy to adopt a predominantly parallel topology, and their counterpart rRNA. To validate and refine the putative ribosomal G-quadruplex structures, we performed all-atom molecular dynamics (MD) simulations using the CHARMM36 force field in the presence and absence of stabilizing K + or Na + ions. We optimized the CHARMM36 force field K + parameters to be more consistent with quantum mechanical calculations (and the polarizable Drude model force field) so that the K + ion is predominantly in the G-quadruplex channel. Our MD simulations show that the rDNA G-quadruplex have more well-defined, predominantly parallel-topology structures than rRNA and NUC 19P is more structured than NUC 23P, which features extended loops. Our study demonstrates that they are both potential targets for the design of novel chemotherapeutics.

Mitochondria DNA replication and DNA methylation in the metabolic memory associated with continued progression of diabetic retinopathy.

PubMed

Tewari, Shikha; Zhong, Qing; Santos, Julia M; Kowluru, Renu A

2012-07-24

Diabetic retinopathy fails to halt after cessation of hyperglycemic insult, and a vicious cycle of mitochondria damage continues. The aim of our study was to investigate the effect of termination of hyperglycemia on retinal mtDNA replication, and elucidate the mechanism responsible for the continued mtDNA damage. Polymerase gamma 1 (POLG1), the catalytic subunit of the mitochondrial DNA replication enzyme, and the damage to the displacement loop region of mtDNA (D-loop) were analyzed in the retina from streptozotocin-diabetic rats maintained in poor glycemic control (PC, glycated hemoglobin ∼11%) or in good glycemic control (GC, glycated hemoglobin ∼6%) for 6 months, or in PC for three months followed by GC for three months (Rev). To understand the mechanism DNA methylation status of POLG1 promoter was investigated by methylation-specific PCR. The key parameters were confirmed in the isolated retinal endothelial cells exposed to high glucose, followed by normal glucose. POLG1 continued to be down-regulated, the D-loop region damaged, and the CpG islands at the regulatory region of POLG hyper-methylated even after three months of GC that had followed three months of PC (Rev group). Similar results were observed in the retinal endothelial cells exposed to normal glucose after being exposed to high glucose. Continued hypermethylation of the CpG sites at the regulatory region of POLG affects its binding to the mtDNA, compromising the transcriptional activity. Modulation of DNA methylation using pharmaceutic or molecular means could help maintain mitochondria homeostasis, and prevent further progression of diabetic retinopathy.

Directed nucleation assembly of DNA tile complexes for barcode-patterned lattices

PubMed Central

Yan, Hao; LaBean, Thomas H.; Feng, Liping; Reif, John H.

2003-01-01

The programmed self-assembly of patterned aperiodic molecular structures is a major challenge in nanotechnology and has numerous potential applications for nanofabrication of complex structures and useful devices. Here we report the construction of an aperiodic patterned DNA lattice (barcode lattice) by a self-assembly process of directed nucleation of DNA tiles around a scaffold DNA strand. The input DNA scaffold strand, constructed by ligation of shorter synthetic oligonucleotides, provides layers of the DNA lattice with barcode patterning information represented by the presence or absence of DNA hairpin loops protruding out of the lattice plane. Self-assembly of multiple DNA tiles around the scaffold strand was shown to result in a patterned lattice containing barcode information of 01101. We have also demonstrated the reprogramming of the system to another patterning. An inverted barcode pattern of 10010 was achieved by modifying the scaffold strands and one of the strands composing each tile. A ribbon lattice, consisting of repetitions of the barcode pattern with expected periodicity, was also constructed by the addition of sticky ends. The patterning of both classes of lattices was clearly observable via atomic force microscopy. These results represent a step toward implementation of a visual readout system capable of converting information encoded on a 1D DNA strand into a 2D form readable by advanced microscopic techniques. A functioning visual output method would not only increase the readout speed of DNA-based computers, but may also find use in other sequence identification techniques such as mutation or allele mapping. PMID:12821776

G-Quadruplex conformational change driven by pH variation with potential application as a nanoswitch.

PubMed

Yan, Yi-Yong; Tan, Jia-Heng; Lu, Yu-Jing; Yan, Siu-Cheong; Wong, Kwok-Yin; Li, Ding; Gu, Lian-Quan; Huang, Zhi-Shu

2013-10-01

G-Quadruplex is a highly polymorphic structure, and its behavior in acidic condition has not been well studied. Circular dichroism (CD) spectra were used to study the conformational change of G-quadruplex. The thermal stabilities of the G-quadruplex were measured with CD melting. Interconversion kinetics profiles were investigated by using CD kinetics. The fluorescence of the inserted 2-Aminopurine (Ap) was monitored during pH change and acrylamide quenching, indicating the status of the loop. Proton NMR was adopted to help illustrate the change of the conformation. G-Quadruplex of specific loop was found to be able to transform upon pH variation. The transformation was resulted from the loop rearrangement. After screening of a library of diverse G-quadruplex, a sequence exhibiting the best transformation property was found. A pH-driven nanoswitch with three gears was obtained based on this transition cycle. Certain G-quadruplex was found to go through conformational change at low pH. Loop was the decisive factor controlling the interconversion upon pH variation. G-Quadruplex with TT central loop could be converted in a much milder condition than the one with TTA loop. It can be used to design pH-driven nanodevices such as a nanoswitch. These results provide more insights into G-quadruplex polymorphism, and also contribute to the design of DNA-based nanomachines and logic gates. © 2013.

A short autocomplementary sequence plays an essential role in avian sarcoma-leukosis virus RNA dimerization.

PubMed

Fossé, P; Motté, N; Roumier, A; Gabus, C; Muriaux, D; Darlix, J L; Paoletti, J

1996-12-24

Retroviral genomes consist of two identical RNA molecules joined noncovalently near their 5'-ends. Recently, two models have been proposed for RNA dimer formation on the basis of results obtained in vitro with human immunodeficiency virus type 1 RNA and Moloney murine leukemia virus RNA. It was first proposed that viral RNA dimerizes by forming an interstrand quadruple helix with purine tetrads. The second model postulates that RNA dimerization is initiated by a loop-loop interaction between the two RNA molecules. In order to better characterize the dimerization process of retroviral genomic RNA, we analyzed the in vitro dimerization of avian sarcoma-leukosis virus (ASLV) RNA using different transcripts. We determined the requirements for heterodimer formation, the thermal dissociation of RNA dimers, and the influence of antisense DNA oligonucleotides on dimer formation. Our results strongly suggest that purine tetrads are not involved in dimer formation. Data show that an autocomplementary sequence located upstream from the splice donor site and within a major packaging signal plays a crucial role in ASLV RNA dimer formation in vitro. This sequence is able to form a stem-loop structure, and phylogenetic analysis reveals that it is conserved in 28 different avian sarcoma and leukosis viruses. These results suggest that dimerization of ASLV RNA is initiated by a loop-loop interaction between two RNA molecules and provide an additional argument for the ubiquity of the dimerization process via loop-loop interaction.

Telomere Length Determines TERRA and R-Loop Regulation through the Cell Cycle.

PubMed

Graf, Marco; Bonetti, Diego; Lockhart, Arianna; Serhal, Kamar; Kellner, Vanessa; Maicher, André; Jolivet, Pascale; Teixeira, Maria Teresa; Luke, Brian

2017-06-29

Maintenance of a minimal telomere length is essential to prevent cellular senescence. When critically short telomeres arise in the absence of telomerase, they can be repaired by homology-directed repair (HDR) to prevent premature senescence onset. It is unclear why specifically the shortest telomeres are targeted for HDR. We demonstrate that the non-coding RNA TERRA accumulates as HDR-promoting RNA-DNA hybrids (R-loops) preferentially at very short telomeres. The increased level of TERRA and R-loops, exclusively at short telomeres, is due to a local defect in RNA degradation by the Rat1 and RNase H2 nucleases, respectively. Consequently, the coordination of TERRA degradation with telomere replication is altered at shortened telomeres. R-loop persistence at short telomeres contributes to activation of the DNA damage response (DDR) and promotes recruitment of the Rad51 recombinase. Thus, the telomere length-dependent regulation of TERRA and TERRA R-loops is a critical determinant of the rate of replicative senescence. Copyright © 2017 Elsevier Inc. All rights reserved.

Detection of early and single infections of Schistosoma japonicum in the intermediate host snail, Oncomelania hupensis, by PCR and loop-mediated isothermal amplification (LAMP) assay.

PubMed

Kumagai, Takashi; Furushima-Shimogawara, Rieko; Ohmae, Hiroshi; Wang, Tian-Ping; Lu, Shaohong; Chen, Rui; Wen, Liyong; Ohta, Nobuo

2010-09-01

Polymerase chain reaction (PCR) with the specific primer set amplifying 28S ribosomal DNA (rDNA) of Schistosoma japonicum was able to detect genomic DNA of S. japonicum, but not S. mansoni, at 100 fg. This procedure enabled us to detect the DNA from a single miracidium and a snail infected with one miracidium at just 1 day after infection. We compared these results with those from loop-mediated isothermal amplification (LAMP) targeting 28S rDNA and found similar results. The LAMP could amplify the specific DNA from a group of 100 normal snails mixed with one infected snail A PCR screening of infected snails from endemic regions in Anhui Province revealed schistosomal DNA even in snails found negative by microscopy. PCR and LAMP show promise for monitoring the early infection rate in snails, and they may be useful for predicting the risk of infection in the endemic places.

The structure and DNA-binding properties of Mgm101 from a yeast with a linear mitochondrial genome

PubMed Central

Pevala, Vladimír; Truban, Dominika; Bauer, Jacob A.; Košťan, Július; Kunová, Nina; Bellová, Jana; Brandstetter, Marlene; Marini, Victoria; Krejčí, Lumír; Tomáška, Ľubomír; Nosek, Jozef; Kutejová, Eva

2016-01-01

To study the mechanisms involved in the maintenance of a linear mitochondrial genome we investigated the biochemical properties of the recombination protein Mgm101 from Candida parapsilosis. We show that CpMgm101 complements defects associated with the Saccharomyces cerevisiae mgm101–1ts mutation and that it is present in both the nucleus and mitochondrial nucleoids of C. parapsilosis. Unlike its S. cerevisiae counterpart, CpMgm101 is associated with the entire nucleoid population and is able to bind to a broad range of DNA substrates in a non-sequence specific manner. CpMgm101 is also able to catalyze strand annealing and D-loop formation. CpMgm101 forms a roughly C-shaped trimer in solution according to SAXS. Electron microscopy of a complex of CpMgm101 with a model mitochondrial telomere revealed homogeneous, ring-shaped structures at the telomeric single-stranded overhangs. The DNA-binding properties of CpMgm101, together with its DNA recombination properties, suggest that it can play a number of possible roles in the replication of the mitochondrial genome and the maintenance of its telomeres. PMID:26743001

Chromatin Structure and Replication Origins: Determinants Of Chromosome Replication And Nuclear Organization

PubMed Central

Smith, Owen K.; Aladjem, Mirit I.

2014-01-01

The DNA replication program is, in part, determined by the epigenetic landscape that governs local chromosome architecture and directs chromosome duplication. Replication must coordinate with other biochemical processes occurring concomitantly on chromatin, such as transcription and remodeling, to insure accurate duplication of both genetic and epigenetic features and to preserve genomic stability. The importance of genome architecture and chromatin looping in coordinating cellular processes on chromatin is illustrated by two recent sets of discoveries. First, chromatin-associated proteins that are not part of the core replication machinery were shown to affect the timing of DNA replication. These chromatin-associated proteins could be working in concert, or perhaps in competition, with the transcriptional machinery and with chromatin modifiers to determine the spatial and temporal organization of replication initiation events. Second, epigenetic interactions are mediated by DNA sequences that determine chromosomal replication. In this review we summarize recent findings and current models linking spatial and temporal regulation of the replication program with epigenetic signaling. We discuss these issues in the context of the genome’s three-dimensional structure with an emphasis on events occurring during the initiation of DNA replication. PMID:24905010

Topological Interaction by Entangled DNA Loops

NASA Astrophysics Data System (ADS)

Feng, Lang; Sha, Ruojie; Seeman, Nadrian. C.; Chaikin, Paul. M.

2012-11-01

We have discovered a new type of interaction between micro- or nanoscale particles that results from the entanglement of strands attached to their surfaces. Self-complementary DNA single strands on a particle can hybridize to form loops. A similar proximal particle can have its loops catenate with those of the first. Unlike conventional thermodynamic interparticle interactions, the catenation interaction is strongly history and protocol dependent, allowing for nonequilibrium particle assembly. The interactions can be controlled by an interesting combination of forces, temperature, light sensitive cross-linking and enzymatic unwinding of the topological links. This novel topological interaction may lead to new materials and phenomena such as particles strung on necklaces, confined motions on designed contours and surfaces, and colloidal Olympic gels.

Integrated Circuit Wear out Prediction and Recycling Detection using Radio Frequency Distinct Native Attribute Features

DTIC Science & Technology

2016-12-22

105 A.1 Main Loop ... loop monitoring for preventative maintenance rather than early replacement based on statistical projections or replacement-after- failure schemes. IC...estimates, RF-DNA may provide a means to track an IC’s physical degradation during actual use. Monitoring an IC’s degradation in a closed loop fashion

Single nucleotide polymorphisms in the mitochondrial displacement loop and outcome of esophageal squamous cell carcinoma.

PubMed

Zhang, Ruixing; Wang, Rui; Zhang, Fengbin; Wu, Chensi; Fan, Haiyan; Li, Yan; Wang, Cuiju; Guo, Zhanjun

2010-11-26

Accumulation of single nucleotide polymorphisms (SNPs) in the displacement loop (D-loop) of mitochondrial DNA (mtDNA) has been described for different types of cancers and might be associated with cancer risk and disease outcome. We used a population-based series of esophageal squamous cell carcinoma (ESCC) patients for investigating the prediction power of SNPs in mitochondrial D-loop. The D-loop region of mtDNA was sequenced for 60 ESCC patients recorded in the Fourth Hospital of Hebei Medical University between 2003 and 2004. The 5 year survival curve were calculated with the Kaplan-Meier method and compared by the log-rank test at each SNP site, a multivariate survival analysis was also performed with the Cox proportional hazards method. The SNP sites of nucleotides 16274G/A, 16278C/T and 16399A/G were identified for prediction of post-operational survival by the log-rank test. In an overall multivariate analysis, the 16278 and 16399 alleles were identified as independent predictors of ESCC outcome. The length of survival of patients with the minor allele 16278T genotype was significantly shorter than that of patients with 16278C at the 16278 site (relative risk, 3.001; 95% CI, 1.029 - 8.756; p = 0.044). The length of survival of patients with the minor allele 16399G genotype was significantly shorter than that of patients with the more frequent allele 16399A at the 16399 site in ESCC patients (relative risk, 3.483; 95% CI, 1.068 - 11.359; p = 0.039). Genetic polymorphisms in the D-loop are independent prognostic markers for patients with ESCC. Accordingly, the analysis of genetic polymorphisms in the mitochondrial D-loop can help identify patient subgroups at high risk of a poor disease outcome.

Dissecting the hybridization of oligonucleotides to structured complementary sequences.

PubMed

Peracchi, Alessio

2016-06-01

When oligonucleotides hybridize to long target molecules, the process is slowed by the secondary structure in the targets. The phenomenon has been analyzed in several previous studies, but many details remain poorly understood. I used a spectrofluorometric strategy, focusing on the formation/breaking of individual base pairs, to study the kinetics of association between a DNA hairpin and >20 complementary oligonucleotides ('antisenses'). Hybridization rates differed by over three orders of magnitude. Association was toehold-mediated, both for antisenses binding to the target's ends and for those designed to interact with the loop. Binding of these latter, besides being consistently slower, was affected to variable, non-uniform extents by the asymmetric loop structure. Divalent metal ions accelerated hybridization, more pronouncedly when nucleation occurred at the loop. Incorporation of locked nucleic acid (LNA) residues in the antisenses substantially improved the kinetics only when LNAs participated to the earliest hybridization steps. The effects of individual LNAs placed along the antisense indicated that the reaction transition state occurred after invading at least the first base pair of the stem. The experimental approach helps dissect hybridization reactions involving structured nucleic acids. Toehold-dependent, nucleation-invasion models appear fully appropriate for describing such reactions. Estimating the stability of nucleation complexes formed at internal toeholds is the major hurdle for the quantitative prediction of hybridization rates. While analyzing the mechanisms of a fundamental biochemical process (hybridization), this work also provides suggestions for the improvement of technologies that rely on such process. Copyright © 2016 Elsevier B.V. All rights reserved.

Stochastic properties of radiation-induced DSB: DSB distributions in large scale chromatin loops, the HPRT gene and within the visible volumes of DNA repair foci.

PubMed

Ponomarev, Artem L; Costes, Sylvain V; Cucinotta, Francis A

2008-11-01

We computed probabilities to have multiple double-strand breaks (DSB), which are produced in DNA on a regional scale, and not in close vicinity, in volumes matching the size of DNA damage foci, of a large chromatin loop, and in the physical volume of DNA containing the HPRT (human hypoxanthine phosphoribosyltransferase) locus. The model is based on a Monte Carlo description of DSB formation by heavy ions in the spatial context of the entire human genome contained within the cell nucleus, as well as at the gene sequence level. We showed that a finite physical volume corresponding to a visible DNA repair focus, believed to be associated with one DSB, can contain multiple DSB due to heavy ion track structure and the DNA supercoiled topography. A corrective distribution was introduced, which was a conditional probability to have excess DSB in a focus volume, given that there was already one present. The corrective distribution was calculated for 19.5 MeV/amu N ions, 3.77 MeV/amu alpha-particles, 1000 MeV/amu Fe ions, and X-rays. The corrected initial DSB yield from the experimental data on DNA repair foci was calculated. The DSB yield based on the corrective function converts the focus yield into the DSB yield, which is comparable with the DSB yield based on the earlier PFGE experiments. The distribution of DSB within the physical limits of the HPRT gene was analyzed by a similar method as well. This corrective procedure shows the applicability of the model and empowers the researcher with a tool to better analyze focus statistics. The model enables researchers to analyze the DSB yield based on focus statistics in real experimental situations that lack one-to-one focus-to-DSB correspondance.

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

PubMed Central

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

2015-01-01

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

Mitochondrial D-loop analysis for uncovering the population structure and genetic diversity among the indigenous duck (Anas platyrhynchos) populations of India.

PubMed

Gaur, Uma; Tantia, Madhu Sudan; Mishra, Bina; Bharani Kumar, Settypalli Tirumala; Vijh, Ramesh Kumar; Chaudhury, Ashok

2018-03-01

The indigenous domestic duck (Anas platyrhynchos domestica) which is domesticated from Mallard (Anas platyrhynchos) contributes significantly to poor farming community in coastal and North Eastern regions of India. For conservation and maintenance of indigenous duck populations it is very important to know the existing genetic diversity and population structure. To unravel the population structure and genetic diversity among the five indigenous duck populations of India, the mitochondrial D-loop sequences of 120 ducks were analyzed. The sequence analysis by comparison of mtDNA D-loop region (470 bp) of five Indian duck populations revealed 25 mitochondrial haplotypes. Pairwise F ST value among populations was 0.4243 (p < .01) and the range of nucleotide substitution per site (Dxy) between the five Indian duck populations was 0.00034-0.00555, and the net divergence (Da) was 0-0.00355. The phylogenetic analysis in the present study unveiled three clades. The analysis revealed genetic continuity among ducks of coastal region of the country which formed a separate group from the ducks of the inland area. Both coastal as well as the land birds revealed introgression of the out group breed Khaki Campbell, which is used for breed improvement programs in India. The observations revealed very less selection and a single matrilineal lineage of indigenous domestic ducks.

RNA-DNA hybrid (R-loop) immunoprecipitation mapping: an analytical workflow to evaluate inherent biases

PubMed Central

Halász, László; Karányi, Zsolt; Boros-Oláh, Beáta; Kuik-Rózsa, Tímea; Sipos, Éva; Nagy, Éva; Mosolygó-L, Ágnes; Mázló, Anett; Rajnavölgyi, Éva; Halmos, Gábor; Székvölgyi, Lóránt

2017-01-01

The impact of R-loops on the physiology and pathology of chromosomes has been demonstrated extensively by chromatin biology research. The progress in this field has been driven by technological advancement of R-loop mapping methods that largely relied on a single approach, DNA-RNA immunoprecipitation (DRIP). Most of the DRIP protocols use the experimental design that was developed by a few laboratories, without paying attention to the potential caveats that might affect the outcome of RNA-DNA hybrid mapping. To assess the accuracy and utility of this technology, we pursued an analytical approach to estimate inherent biases and errors in the DRIP protocol. By performing DRIP-sequencing, qPCR, and receiver operator characteristic (ROC) analysis, we tested the effect of formaldehyde fixation, cell lysis temperature, mode of genome fragmentation, and removal of free RNA on the efficacy of RNA-DNA hybrid detection and implemented workflows that were able to distinguish complex and weak DRIP signals in a noisy background with high confidence. We also show that some of the workflows perform poorly and generate random answers. Furthermore, we found that the most commonly used genome fragmentation method (restriction enzyme digestion) led to the overrepresentation of lengthy DRIP fragments over coding ORFs, and this bias was enhanced at the first exons. Biased genome sampling severely compromised mapping resolution and prevented the assignment of precise biological function to a significant fraction of R-loops. The revised workflow presented herein is established and optimized using objective ROC analyses and provides reproducible and highly specific RNA-DNA hybrid detection. PMID:28341774

Asian affinities and continental radiation of the four founding Native American mtDNAs.

PubMed Central

Torroni, A; Schurr, T G; Cabell, M F; Brown, M D; Neel, J V; Larsen, M; Smith, D G; Vullo, C M; Wallace, D C

1993-01-01

The mtDNA variation of 321 individuals from 17 Native American populations was examined by high-resolution restriction endonuclease analysis. All mtDNAs were amplified from a variety of sources by using PCR. The mtDNA of a subset of 38 of these individuals was also analyzed by D-loop sequencing. The resulting data were combined with previous mtDNA data from five other Native American tribes, as well as with data from a variety of Asian populations, and were used to deduce the phylogenetic relationships between mtDNAs and to estimate sequence divergences. This analysis revealed the presence of four haplotype groups (haplogroups A, B, C, and D) in the Amerind, but only one haplogroup (A) in the Na-Dene, and confirmed the independent origins of the Amerinds and the Na-Dene. Further, each haplogroup appeared to have been founded by a single mtDNA haplotype, a result which is consistent with a hypothesized founder effect. Most of the variation within haplogroups was tribal specific, that is, it occurred as tribal private polymorphisms. These observations suggest that the process of tribalization began early in the history of the Amerinds, with relatively little intertribal genetic exchange occurring subsequently. The sequencing of 341 nucleotides in the mtDNA D-loop revealed that the D-loop sequence variation correlated strongly with the four haplogroups defined by restriction analysis, and it indicated that the D-loop variation, like the haplotype variation, arose predominantly after the migration of the ancestral Amerinds across the Bering land bridge. Images Figure 4 PMID:7688932

Molecular and functional characterization of single-box high-mobility group B (HMGB) chromosomal protein from Aedes aegypti.

PubMed

de Abreu da Silva, Isabel Caetano; Vicentino, Amanda Roberta Revoredo; Dos Santos, Renata Coutinho; da Fonseca, Rodrigo Nunes; de Mendonça Amarante, Anderson; Carneiro, Vitor Coutinho; de Amorim Pinto, Marcia; Aguilera, Estefania Anahi; Mohana-Borges, Ronaldo; Bisch, Paulo Mascarello; da Silva-Neto, Mario Alberto Cardoso; Fantappié, Marcelo Rosado

2018-05-30

High-mobility group B (HMGB) proteins have highly conserved, unique DNA-binding domains, HMG boxes, that can bind non-B-type DNA structures, such as bent, kinked and unwound structures, with high affinity. HMGB proteins also promote DNA bending, looping and unwinding. In this study, we determined the role of the Aedes aegypti single HMG-box domain protein AaHMGB; characterized its structure, spatiotemporal expression levels, subcellular localization, and nucleic acid binding activities; and compared these properties with those of its double-HMG-box counterpart protein, AaHMGB1. Via qRT-PCR, we showed that AaHMGB is expressed at much higher levels than AaHMGB1 throughout mosquito development. In situ hybridization results suggested a role for AaHMGB and AaHMGB1 during embryogenesis. Immunolocalization in the midgut revealed that AaHMGB is exclusively nuclear. Circular dichroism and fluorescence spectroscopy analyses showed that AaHMGB exhibits common features of α-helical structures and is more stably folded than AaHMGB1, likely due to the presence of one or two HMG boxes. Using several DNA substrates or single-stranded RNAs as probes, we observed significant differences between AaHMGB and AaHMGB1 in terms of their binding patterns, activity and/or specificity. Importantly, we showed that the phosphorylation of AaHMGB plays a critical role in its DNA-binding activity. Our study provides additional insight into the roles of single- versus double-HMG-box-containing proteins in nucleic acid interactions for better understanding of mosquito development, physiology and homeostasis. Copyright © 2017. Published by Elsevier B.V.

The Role of elF4E Activity in Breast Cancer

DTIC Science & Technology

2011-08-01

protein; ORF, open reading frame; qPCR, quantitative PCR; RACE, rapid amplification of cDNA ends; RT, reverse transcriptase ; uORF, upstream ORF; UTR...Reactions were also performed using template lacking RT ( reverse transcriptase ): products were either undetectable or greatly reduced (>30000-fold less...that a 5’UTR expressed from the human AXIN2 gene contains a sixty nucleotide sequence that is predicted to form a stable stem-loop structure6. This

Manipulation of a DNA aptamer-protein binding site through arylation of internal guanine residues.

PubMed

Van Riesen, Abigail J; Fadock, Kaila L; Deore, Prashant S; Desoky, Ahmed; Manderville, Richard A; Sowlati-Hashjin, Shahin; Wetmore, Stacey D

2018-05-23

Chemically modified aptamers have the opportunity to increase aptamer target binding affinity and provide structure-activity relationships to enhance our understanding of molecular target recognition by the aptamer fold. In the current study, 8-aryl-2'-deoxyguanosine nucleobases have been inserted into the G-tetrad and central TGT loop of the thrombin binding aptamer (TBA) to determine their impact on antiparallel G-quadruplex (GQ) folding and thrombin binding affinity. The aryl groups attached to the dG nucleobase vary greatly in aryl ring size and impact on GQ stability (∼20 °C change in GQ thermal melting (Tm) values) and thrombin binding affinity (17-fold variation in dissociation constant (Kd)). At G8 of the central TGT loop that is distal from the aptamer recognition site, the probes producing the most stable GQ structure exhibited the strongest thrombin binding affinity. However, within the G-tetrad, changes to the electron density of the dG component within the modified nucleobase can diminish thrombin binding affinity. Detailed molecular dynamics (MD) simulations on the modified TBA (mTBA) and mTBA-protein complexes demonstrate how the internal 8-aryl-dG modification can manipulate the interactions between the DNA nucleobases and the amino acid residues of thrombin. These results highlight the potential of internal fluorescent nuclobase analogs (FBAs) to broaden design options for aptasensor development.

Population Structure of mtDNA Variation due to Pleistocene Fluctuations in the South American Maned Wolf (Chrysocyon brachyurus, Illiger, 1815): Management Units for Conservation.

PubMed

González, Susana; Cosse, Mariana; Franco, María del Rosario; Emmons, Louise; Vynne, Carly; Duarte, José Maurício Barbanti; Beccacesi, Marcelo D; Maldonado, Jesús E

2015-01-01

The maned wolf (Chrysocyon brachyurus) is one of the largest South American canids, and conservation across this charismatic carnivore's large range is presently hampered by a lack of knowledge about possible natural subdivisions which could influence the population's viability. To elucidate the phylogeographic patterns and demographic history of the species, we used 2 mtDNA markers (D-loop and cytochrome b) from 87 individuals collected throughout their range, in Argentina, Bolivia, Brazil, and Uruguay. We found moderate levels of haplotype and nucleotide diversity, and the 14 D-loop haplotypes were closely related. Genetic structure results revealed 4 groups, and when coupled with model inferences from a coalescent analysis, suggested that maned wolves have undergone demographic fluctuations due to changes in climate and habitat during the Pleistocene glaciation period approximately 24000 years before present (YBP). This genetic signature points to an event that occurred within the timing estimated for the start of the contraction of the Cerrado around 50000 YBP. Our results reveal a genetic signature of population size expansion followed by contraction during Pleistocene interglaciations, which had similar impacts on other South American mammals. The 4 groups should for now be considered management units, within which future monitoring efforts should be conducted independently. © The American Genetic Association 2015. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.

DNA aptamer beacon assay for C-telopeptide and handheld fluorometer to monitor bone resorption.

PubMed

Bruno, John Gordon; Carrillo, Maria P; Phillips, Taylor; Hanson, Douglas; Bohmann, Jonathan A

2011-09-01

A novel DNA aptamer beacon is described for quantification of a 26-amino acid C-telopeptide (CTx) of human type I bone collagen. One aptamer sequence and its reverse complement dominated the aptamer pool (31.6% of sequenced clones). Secondary structures of these aptamers were examined for potential binding pockets. Three-dimensional computer models which analyzed docking topologies and binding energies were in agreement with empirical fluorescence experiments used to select one candidate loop for beacon assay development. All loop structures from the aptamer finalists were end-labeled with TYE 665 and Iowa Black quencher for comparison of beacon fluorescence levels as a function of CTx concentration. The optimal beacon, designated CTx 2R-2h yielded a low ng/ml limit of detection using a commercially available handheld fluorometer. The CTx aptamer beacon bound full-length 26-amino acid CTx peptide, but not a shorter 8-amino acid segment of CTx peptide which is a common target for commercial CTx ELISA kits. The prototype assay was shown to detect CTx peptide from human urine after creatinine and urea were removed by size-exclusion chromatography to prevent nonspecific denaturing of the aptamer beacon. This work demonstrates the potential of aptamer beacons to be utilized for rapid and sensitive bone health monitoring in a handheld or point-of-care format.

Near-Atomic Resolution Structure of a Plant Geminivirus Determined by Electron Cryomicroscopy.

PubMed

Hipp, Katharina; Grimm, Clemens; Jeske, Holger; Böttcher, Bettina

2017-08-01

African cassava mosaic virus is a whitefly-transmitted geminivirus which forms unique twin particles of incomplete icosahedra that are joined at five-fold vertices, building an unusual waist. How its 22 capsomers interact within a half-capsid or across the waist is unknown thus far. Using electron cryo-microscopy and image processing, we determined the virion structure with a resolution of 4.2 Å and built an atomic model for its capsid protein. The inter-capsomer contacts mediated by the flexible N termini and loop regions differed within the half-capsids and at the waist, explaining partly the unusual twin structure. The tip of the pentameric capsomer is sealed by a plug formed by a turn region harboring the evolutionary conserved residue Y193. Basic amino acid residues inside the capsid form a positively charged pocket next to the five-fold axis of the capsomer suitable for binding DNA. Within this pocket, density most likely corresponding to DNA was resolved. Copyright © 2017 Elsevier Ltd. All rights reserved.

Solution structure of telomere binding domain of AtTRB2 derived from Arabidopsis thaliana

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

Yun, Ji-Hye; Lee, Won Kyung; Kim, Heeyoun

Highlights: • We have determined solution structure of Myb domain of AtTRB2. • The Myb domain of AtTRB2 is located in the N-terminal region. • The Myb domain of AtTRB2 binds to plant telomeric DNA without fourth helix. • Helix 2 and 3 of the Myb domain of AtTRB2 are involved in DNA recognition. • AtTRB2 is a novel protein distinguished from other known plant TBP. - Abstract: Telomere homeostasis is regulated by telomere-associated proteins, and the Myb domain is well conserved for telomere binding. AtTRB2 is a member of the SMH (Single-Myb-Histone)-like family in Arabidopsis thaliana, having an N-terminalmore » Myb domain, which is responsible for DNA binding. The Myb domain of AtTRB2 contains three α-helices and loops for DNA binding, which is unusual given that other plant telomere-binding proteins have an additional fourth helix that is essential for DNA binding. To understand the structural role for telomeric DNA binding of AtTRB2, we determined the solution structure of the Myb domain of AtTRB2 (AtTRB2{sub 1–64}) using nuclear magnetic resonance (NMR) spectroscopy. In addition, the inter-molecular interaction between AtTRB2{sub 1–64} and telomeric DNA has been characterized by the electrophoretic mobility shift assay (EMSA) and NMR titration analyses for both plant (TTTAGGG)n and human (TTAGGG)n telomere sequences. Data revealed that Trp28, Arg29, and Val47 residues located in Helix 2 and Helix 3 are crucial for DNA binding, which are well conserved among other plant telomere binding proteins. We concluded that although AtTRB2 is devoid of the additional fourth helix in the Myb-extension domain, it is able to bind to plant telomeric repeat sequences as well as human telomeric repeat sequences.« less

R-loops cause genomic instability in T helper lymphocytes from patients with Wiskott-Aldrich syndrome.

PubMed

Sarkar, Koustav; Han, Seong-Su; Wen, Kuo-Kuang; Ochs, Hans D; Dupré, Loïc; Seidman, Michael M; Vyas, Yatin M

2017-12-15

Wiskott-Aldrich syndrome (WAS), X-linked thrombocytopenia (XLT), and X-linked neutropenia, which are caused by WAS mutations affecting Wiskott-Aldrich syndrome protein (WASp) expression or activity, manifest in immunodeficiency, autoimmunity, genomic instability, and lymphoid and other cancers. WASp supports filamentous actin formation in the cytoplasm and gene transcription in the nucleus. Although the genetic basis for XLT/WAS has been clarified, the relationships between mutant forms of WASp and the diverse features of these disorders remain ill-defined. We sought to define how dysfunctional gene transcription is causally linked to the degree of T H cell deficiency and genomic instability in the XLT/WAS clinical spectrum. In human T H 1- or T H 2-skewing cell culture systems, cotranscriptional R-loops (RNA/DNA duplex and displaced single-stranded DNA) and DNA double-strand breaks (DSBs) were monitored in multiple samples from patients with XLT and WAS and in normal T cells depleted of WASp. WASp deficiency provokes increased R-loops and R-loop-mediated DSBs in T H 1 cells relative to T H 2 cells. Mechanistically, chromatin occupancy of serine 2-unphosphorylated RNA polymerase II is increased, and that of topoisomerase 1, an R-loop preventing factor, is decreased at R-loop-enriched regions of IFNG and TBX21 (T H 1 genes) in T H 1 cells. These aberrations accompany increased unspliced (intron-retained) and decreased spliced mRNA of IFNG and TBX21 but not IL13 (T H 2 gene). Significantly, increased cellular load of R-loops and DSBs, which are normalized on RNaseH1-mediated suppression of ectopic R-loops, inversely correlates with disease severity scores. Transcriptional R-loop imbalance is a novel molecular defect causative in T H 1 immunodeficiency and genomic instability in patients with WAS. The study proposes that cellular R-loop load could be used as a potential biomarker for monitoring symptom severity and prognostic outcome in the XLT-WAS clinical spectrum and could be targeted therapeutically. Copyright © 2017 American Academy of Allergy, Asthma & Immunology. All rights reserved.

Genetic diversity of mtDNA D-loop sequences in four native Chinese chicken breeds.

PubMed

Guo, H W; Li, C; Wang, X N; Li, Z J; Sun, G R; Li, G X; Liu, X J; Kang, X T; Han, R L

2017-10-01

1. To explore the genetic diversity of Chinese indigenous chicken breeds, a 585 bp fragment of the mitochondrial DNA (mtDNA) region was sequenced in 102 birds from the Xichuan black-bone chicken, Yunyang black-bone chicken and Lushi chicken. In addition, 30 mtDNA D-loop sequences of Silkie fowls were downloaded from NCBI. The mtDNA D-loop sequence polymorphism and maternal origin of 4 chicken breeds were analysed in this study. 2. The results showed that a total of 33 mutation sites and 28 haplotypes were detected in the 4 chicken breeds. The haplotype diversity and nucleotide diversity of these 4 native breeds were 0.916 ± 0.014 and 0.012 ± 0.002, respectively. Three clusters were formed in 4 Chinese native chickens and 12 reference breeds. Both the Xichuan black-bone chicken and Yunyang black-bone chicken were grouped into one cluster. Four haplogroups (A, B, C and E) emerged in the median-joining network in these breeds. 3. It was concluded that these 4 Chinese chicken breeds had high genetic diversity. The phylogenetic tree and median network profiles showed that Chinese native chickens and its neighbouring countries had at least two maternal origins, one from Yunnan, China and another from Southeast Asia or its surrounding area.

Arsenic Induces Polyadenylation of Canonical Histone mRNA by Down-regulating Stem-Loop-binding Protein Gene Expression*

PubMed Central

Brocato, Jason; Fang, Lei; Chervona, Yana; Chen, Danqi; Kiok, Kathrin; Sun, Hong; Tseng, Hsiang-Chi; Xu, Dazhong; Shamy, Magdy; Jin, Chunyuan; Costa, Max

2014-01-01

The replication-dependent histone genes are the only metazoan genes whose messenger RNA (mRNA) does not terminate with a poly(A) tail at the 3′-end. Instead, the histone mRNAs display a stem-loop structure at their 3′-end. Stem-loop-binding protein (SLBP) binds the stem-loop and regulates canonical histone mRNA metabolism. Here we report that exposure to arsenic, a carcinogenic metal, decreased cellular levels of SLBP by inducing its proteasomal degradation and inhibiting SLBP transcription via epigenetic mechanisms. Notably, arsenic exposure dramatically increased polyadenylation of canonical histone H3.1 mRNA possibly through down-regulation of SLBP expression. The polyadenylated H3.1 mRNA induced by arsenic was not susceptible to normal degradation that occurs at the end of S phase, resulting in continued presence into mitosis, increased total H3.1 mRNA, and increased H3 protein levels. Excess expression of canonical histones have been shown to increase sensitivity to DNA damage as well as increase the frequency of missing chromosomes and induce genomic instability. Thus, polyadenylation of canonical histone mRNA following arsenic exposure may contribute to arsenic-induced carcinogenesis. PMID:25266719

DNA-binding regulates site-specific ubiquitination of IRF-1.

PubMed

Landré, Vivien; Pion, Emmanuelle; Narayan, Vikram; Xirodimas, Dimitris P; Ball, Kathryn L

2013-02-01

Understanding the determinants for site-specific ubiquitination by E3 ligase components of the ubiquitin machinery is proving to be a challenge. In the present study we investigate the role of an E3 ligase docking site (Mf2 domain) in an intrinsically disordered domain of IRF-1 [IFN (interferon) regulatory factor-1], a short-lived IFNγ-regulated transcription factor, in ubiquitination of the protein. Ubiquitin modification of full-length IRF-1 by E3 ligases such as CHIP [C-terminus of the Hsc (heat-shock cognate) 70-interacting protein] and MDM2 (murine double minute 2), which dock to the Mf2 domain, was specific for lysine residues found predominantly in loop structures that extend from the DNA-binding domain, whereas no modification was detected in the more conformationally flexible C-terminal half of the protein. The E3 docking site was not available when IRF-1 was in its DNA-bound conformation and cognate DNA-binding sequences strongly suppressed ubiquitination, highlighting a strict relationship between ligase binding and site-specific modification at residues in the DNA-binding domain. Hyperubiquitination of a non-DNA-binding mutant supports a mechanism where an active DNA-bound pool of IRF-1 is protected from polyubiquitination and degradation.

Loop-mediated isothermal amplification (LAMP) assay-A rapid detection tool for identifying red fox (Vulpes vulpes) DNA in the carcasses of harbour porpoises (Phocoena phocoena).

PubMed

Heers, Teresa; van Neer, Abbo; Becker, André; Grilo, Miguel Luca; Siebert, Ursula; Abdulmawjood, Amir

2017-01-01

Carcasses of wild animals are often visited by different scavengers. However, determining which scavenger caused certain types of bite marks is particularly difficult and knowledge thereof is lacking. Therefore, a loop-mediated isothermal amplification (LAMP) assay (target sequence cytochrome b) was developed to detect red fox DNA in carcasses of harbour porpoises. The MSwab™ method for direct testing without prior DNA isolation was validated. As a detection device, the portable real-time fluorometer Genie® II was used, which yields rapid results and can be used in field studies without huge laboratory equipment. In addition to in vitro evaluation and validation, a stranded and scavenged harbour porpoise carcass was successfully examined for red fox DNA residues. The developed LAMP method is a valuable diagnostic tool for confirming presumable red fox bite wounds in harbour porpoises without further DNA isolation steps.

[Preinvasive vulvar and cervical cancer in a 32-year-old woman, DNA HPV 16 positive with mtDNA mutation--case study].

PubMed

Kedzia, Witold; Malkowska-Walczak, Blanka; Józefiak, Agata; Wadowicka, Alicja; Guglas, Bogna; Pruski, Dominik; Kedzia, Helena; Spaczyński, Marek

2009-07-01

Coincidence of preinvasive vulvar and cervical cancer in young women is very rare. Lesions like VIN 3/preinvasive vulvar cancer and CIN 3/preinvasive cervical cancer are strictly connected with viral infection and are multilocular. In the presented case the following tests have been performed: HPV DNA test for the presence of 13 oncogenic HPV types, mRNA HPV test for the presence of transcripts for HPV 16, 18, 31, 33, 45 and the analysis of mtDNA D-Loop region. In the examined patient HPV 16 infection, as well as the presence of transcripts for HPV 16 E6/7 were diagnosed. The analysis of mtDNA D-Loop region showed nucleotide lesions like: T>C 16.192, T>C 16.223, T>C 16.292, C>T 16.325, C>T 16.579.

Urine sample used for detection of toxoplasma gondii infection by loop-mediated isothermal amplification (LAMP).

PubMed

Hu, Xin; Pan, Chang-Wang; Li, Ya-Fei; Wang, Han; Tan, Feng

2012-02-01

In this study, a loop-mediated isothermal amplification (LAMP) assay was established to detect Toxoplasma gondii DNA in mice infected with T. gondii PRU strain. This LAMP assay was based on the sequence of highly repetitive B1 gene. The detection limit of T. gondii LAMP assay was 1 pg of T. gondii DNA, which was evaluated using 10-fold serially diluted DNA of cultured parasites. The LAMP assay was also highly specific for T. gondii and able to detect T. gondii DNA in urine of mice treated with dexamethasone at 90 day post infection (p.i.), although this assay could not detect the DNA in mice urine 2-6 days p.i. These results demonstrated that LAMP is effective for evaluation of therapy effectiveness for T. gondii infection. The established LAMP assay may represent a useful and practical tool for the routine diagnosis and therapeutic evaluation of human toxoplasmosis.

Sequence-structure relationships in RNA loops: establishing the basis for loop homology modeling.

PubMed

Schudoma, Christian; May, Patrick; Nikiforova, Viktoria; Walther, Dirk

2010-01-01

The specific function of RNA molecules frequently resides in their seemingly unstructured loop regions. We performed a systematic analysis of RNA loops extracted from experimentally determined three-dimensional structures of RNA molecules. A comprehensive loop-structure data set was created and organized into distinct clusters based on structural and sequence similarity. We detected clear evidence of the hallmark of homology present in the sequence-structure relationships in loops. Loops differing by <25% in sequence identity fold into very similar structures. Thus, our results support the application of homology modeling for RNA loop model building. We established a threshold that may guide the sequence divergence-based selection of template structures for RNA loop homology modeling. Of all possible sequences that are, under the assumption of isosteric relationships, theoretically compatible with actual sequences observed in RNA structures, only a small fraction is contained in the Rfam database of RNA sequences and classes implying that the actual RNA loop space may consist of a limited number of unique loop structures and conserved sequences. The loop-structure data sets are made available via an online database, RLooM. RLooM also offers functionalities for the modeling of RNA loop structures in support of RNA engineering and design efforts.

Programmable disorder in random DNA tilings

NASA Astrophysics Data System (ADS)

Tikhomirov, Grigory; Petersen, Philip; Qian, Lulu

2017-03-01

Scaling up the complexity and diversity of synthetic molecular structures will require strategies that exploit the inherent stochasticity of molecular systems in a controlled fashion. Here we demonstrate a framework for programming random DNA tilings and show how to control the properties of global patterns through simple, local rules. We constructed three general forms of planar network—random loops, mazes and trees—on the surface of self-assembled DNA origami arrays on the micrometre scale with nanometre resolution. Using simple molecular building blocks and robust experimental conditions, we demonstrate control of a wide range of properties of the random networks, including the branching rules, the growth directions, the proximity between adjacent networks and the size distribution. Much as combinatorial approaches for generating random one-dimensional chains of polymers have been used to revolutionize chemical synthesis and the selection of functional nucleic acids, our strategy extends these principles to random two-dimensional networks of molecules and creates new opportunities for fabricating more complex molecular devices that are organized by DNA nanostructures.

Probing Human Telomeric DNA and RNA Topology and Ligand Binding in a Cellular Model by Using Responsive Fluorescent Nucleoside Probes.

PubMed

Manna, Sudeshna; Panse, Cornelia H; Sontakke, Vyankat A; Sangamesh, Sarangamath; Srivatsan, Seergazhi G

2017-08-17

The development of biophysical systems that enable an understanding of the structure and ligand-binding properties of G-quadruplex (GQ)-forming nucleic acid sequences in cells or models that mimic the cellular environment would be highly beneficial in advancing GQ-directed therapeutic strategies. Herein, the establishment of a biophysical platform to investigate the structure and recognition properties of human telomeric (H-Telo) DNA and RNA repeats in a cell-like confined environment by using conformation-sensitive fluorescent nucleoside probes and a widely used cellular model, bis(2-ethylhexyl) sodium sulfosuccinate reverse micelles (RMs), is described. The 2'-deoxy and ribonucleoside probes, composed of a 5-benzofuran uracil base analogue, faithfully report the aqueous micellar core through changes in their fluorescence properties. The nucleoside probes incorporated into different loops of H-Telo DNA and RNA oligonucleotide repeats are minimally perturbing and photophysically signal the formation of respective GQ structures in both aqueous buffer and RMs. Furthermore, these sensors enable a direct comparison of the binding affinity of a ligand to H-Telo DNA and RNA GQ structures in the bulk and confined environment of RMs. These results demonstrate that this combination of a GQ nucleoside probe and easy-to-handle RMs could provide new opportunities to study and devise screening-compatible assays in a cell-like environment to discover GQ binders of clinical potential. © 2017 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim.

Structural Basis for Intrinsic Thermosensing by the Master Virulence Regulator RovA of Yersinia*

PubMed Central

Quade, Nick; Mendonca, Chriselle; Herbst, Katharina; Heroven, Ann Kathrin; Ritter, Christiane; Heinz, Dirk W.; Dersch, Petra

2012-01-01

Pathogens often rely on thermosensing to adjust virulence gene expression. In yersiniae, important virulence-associated traits are under the control of the master regulator RovA, which uses a built-in thermosensor to control its activity. Thermal upshifts encountered upon host entry induce conformational changes in the RovA dimer that attenuate DNA binding and render the protein more susceptible to proteolysis. Here, we report the crystal structure of RovA in the free and DNA-bound forms and provide evidence that thermo-induced loss of RovA activity is promoted mainly by a thermosensing loop in the dimerization domain and residues in the adjacent C-terminal helix. These determinants allow partial unfolding of the regulator upon an upshift to 37 °C. This structural distortion is transmitted to the flexible DNA-binding domain of RovA. RovA contacts mainly the DNA backbone in a low-affinity binding mode, which allows the immediate release of RovA from its operator sites. We also show that SlyA, a close homolog of RovA from Salmonella with a very similar structure, is not a thermosensor and remains active and stable at 37 °C. Strikingly, changes in only three amino acids, reflecting evolutionary replacements in SlyA, result in a complete loss of the thermosensing properties of RovA and prevent degradation. In conclusion, only minor alterations can transform a thermotolerant regulator into a thermosensor that allows adjustment of virulence and fitness determinants to their thermal environment. PMID:22936808

Target Site Recognition by a Diversity-Generating Retroelement

PubMed Central

Guo, Huatao; Tse, Longping V.; Nieh, Angela W.; Czornyj, Elizabeth; Williams, Steven; Oukil, Sabrina; Liu, Vincent B.; Miller, Jeff F.

2011-01-01

Diversity-generating retroelements (DGRs) are in vivo sequence diversification machines that are widely distributed in bacterial, phage, and plasmid genomes. They function to introduce vast amounts of targeted diversity into protein-encoding DNA sequences via mutagenic homing. Adenine residues are converted to random nucleotides in a retrotransposition process from a donor template repeat (TR) to a recipient variable repeat (VR). Using the Bordetella bacteriophage BPP-1 element as a prototype, we have characterized requirements for DGR target site function. Although sequences upstream of VR are dispensable, a 24 bp sequence immediately downstream of VR, which contains short inverted repeats, is required for efficient retrohoming. The inverted repeats form a hairpin or cruciform structure and mutational analysis demonstrated that, while the structure of the stem is important, its sequence can vary. In contrast, the loop has a sequence-dependent function. Structure-specific nuclease digestion confirmed the existence of a DNA hairpin/cruciform, and marker coconversion assays demonstrated that it influences the efficiency, but not the site of cDNA integration. Comparisons with other phage DGRs suggested that similar structures are a conserved feature of target sequences. Using a kanamycin resistance determinant as a reporter, we found that transplantation of the IMH and hairpin/cruciform-forming region was sufficient to target the DGR diversification machinery to a heterologous gene. In addition to furthering our understanding of DGR retrohoming, our results suggest that DGRs may provide unique tools for directed protein evolution via in vivo DNA diversification. PMID:22194701

Structural Insights into the Assembly of the Adeno-associated Virus Type 2 Rep68 Protein on the Integration Site AAVS1*

PubMed Central

Musayev, Faik N.; Zarate-Perez, Francisco; Bishop, Clayton; Burgner, John W.; Escalante, Carlos R.

2015-01-01

Adeno-associated virus (AAV) is the only eukaryotic virus with the property of establishing latency by integrating site-specifically into the human genome. The integration site known as AAVS1 is located in chromosome 19 and contains multiple GCTC repeats that are recognized by the AAV non-structural Rep proteins. These proteins are multifunctional, with an N-terminal origin-binding domain (OBD) and a helicase domain joined together by a short linker. As a first step to understand the process of site-specific integration, we proceeded to characterize the recognition and assembly of Rep68 onto the AAVS1 site. We first determined the x-ray structure of AAV-2 Rep68 OBD in complex with the AAVS1 DNA site. Specificity is achieved through the interaction of a glycine-rich loop that binds the major groove and an α-helix that interacts with a downstream minor groove on the same face of the DNA. Although the structure shows a complex with three OBD molecules bound to the AAVS1 site, we show by using analytical centrifugation and electron microscopy that the full-length Rep68 forms a heptameric complex. Moreover, we determined that a minimum of two direct repeats is required to form a stable complex and to melt DNA. Finally, we show that although the individual domains bind DNA poorly, complex assembly requires oligomerization and cooperation between its OBD, helicase, and the linker domains. PMID:26370092

Probing long-range interactions by extracting free energies from genome-wide chromosome conformation capture data.

PubMed

Saberi, Saeed; Farré, Pau; Cuvier, Olivier; Emberly, Eldon

2015-05-23

A variety of DNA binding proteins are involved in regulating and shaping the packing of chromatin. They aid the formation of loops in the DNA that function to isolate different structural domains. A recent experimental technique, Hi-C, provides a method for determining the frequency of such looping between all distant parts of the genome. Given that the binding locations of many chromatin associated proteins have also been measured, it has been possible to make estimates for their influence on the long-range interactions as measured by Hi-C. However, a challenge in this analysis is the predominance of non-specific contacts that mask out the specific interactions of interest. We show that transforming the Hi-C contact frequencies into free energies gives a natural method for separating out the distance dependent non-specific interactions. In particular we apply Principal Component Analysis (PCA) to the transformed free energy matrix to identify the dominant modes of interaction. PCA identifies systematic effects as well as high frequency spatial noise in the Hi-C data which can be filtered out. Thus it can be used as a data driven approach for normalizing Hi-C data. We assess this PCA based normalization approach, along with several other normalization schemes, by fitting the transformed Hi-C data using a pairwise interaction model that takes as input the known locations of bound chromatin factors. The result of fitting is a set of predictions for the coupling energies between the various chromatin factors and their effect on the energetics of looping. We show that the quality of the fit can be used as a means to determine how much PCA filtering should be applied to the Hi-C data. We find that the different normalizations of the Hi-C data vary in the quality of fit to the pairwise interaction model. PCA filtering can improve the fit, and the predicted coupling energies lead to biologically meaningful insights for how various chromatin bound factors influence the stability of DNA loops in chromatin.

Recognition of AT-Rich DNA Binding Sites by the MogR Repressor

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

Shen, Aimee; Higgins, Darren E.; Panne, Daniel

2009-07-22

The MogR transcriptional repressor of the intracellular pathogen Listeria monocytogenes recognizes AT-rich binding sites in promoters of flagellar genes to downregulate flagellar gene expression during infection. We describe here the 1.8 A resolution crystal structure of MogR bound to the recognition sequence 5' ATTTTTTAAAAAAAT 3' present within the flaA promoter region. Our structure shows that MogR binds as a dimer. Each half-site is recognized in the major groove by a helix-turn-helix motif and in the minor groove by a loop from the symmetry-related molecule, resulting in a 'crossover' binding mode. This oversampling through minor groove interactions is important for specificity.more » The MogR binding site has structural features of A-tract DNA and is bent by approximately 52 degrees away from the dimer. The structure explains how MogR achieves binding specificity in the AT-rich genome of L. monocytogenes and explains the evolutionary conservation of A-tract sequence elements within promoter regions of MogR-regulated flagellar genes.« less

Metadynamics Simulation Study on the Conformational Transformation of HhaI Methyltransferase: An Induced-Fit Base-Flipping Hypothesis

PubMed Central

Ye, Fei; Zhao, Dan; Chen, Shijie; Jiang, Ren-Wang; Jiang, Hualiang; Luo, Cheng

2014-01-01

DNA methyltransferases play crucial roles in establishing and maintenance of DNA methylation, which is an important epigenetic mark. Flipping the target cytosine out of the DNA helical stack and into the active site of protein provides DNA methyltransferases with an opportunity to access and modify the genetic information hidden in DNA. To investigate the conversion process of base flipping in the HhaI methyltransferase (M.HhaI), we performed different molecular simulation approaches on M.HhaI-DNA-S-adenosylhomocysteine ternary complex. The results demonstrate that the nonspecific binding of DNA to M.HhaI is initially induced by electrostatic interactions. Differences in chemical environment between the major and minor grooves determine the orientation of DNA. Gln237 at the target recognition loop recognizes the GCGC base pair from the major groove side by hydrogen bonds. In addition, catalytic loop motion is a key factor during this process. Our study indicates that base flipping is likely to be an “induced-fit” process. This study provides a solid foundation for future studies on the discovery and development of mechanism-based DNA methyltransferases regulators. PMID:25045662

Srs2 promotes synthesis-dependent strand annealing by disrupting DNA polymerase δ-extending D-loops

PubMed Central

Liu, Jie; Ede, Christopher; Wright, William D; Gore, Steven K; Jenkins, Shirin S; Freudenthal, Bret D; Todd Washington, M; Veaute, Xavier; Heyer, Wolf-Dietrich

2017-01-01

Synthesis-dependent strand annealing (SDSA) is the preferred mode of homologous recombination in somatic cells leading to an obligatory non-crossover outcome, thus avoiding the potential for chromosomal rearrangements and loss of heterozygosity. Genetic analysis identified the Srs2 helicase as a prime candidate to promote SDSA. Here, we demonstrate that Srs2 disrupts D-loops in an ATP-dependent fashion and with a distinct polarity. Specifically, we partly reconstitute the SDSA pathway using Rad51, Rad54, RPA, RFC, DNA Polymerase δ with different forms of PCNA. Consistent with genetic data showing the requirement for SUMO and PCNA binding for the SDSA role of Srs2, Srs2 displays a slight but significant preference to disrupt extending D-loops over unextended D-loops when SUMOylated PCNA is present, compared to unmodified PCNA or monoubiquitinated PCNA. Our data establish a biochemical mechanism for the role of Srs2 in crossover suppression by promoting SDSA through disruption of extended D-loops. DOI: http://dx.doi.org/10.7554/eLife.22195.001 PMID:28535142

A Rolling Circle Replication Mechanism Produces Multimeric Lariats of Mitochondrial DNA in Caenorhabditis elegans

PubMed Central

Lewis, Samantha C.; Joers, Priit; Willcox, Smaranda; Griffith, Jack D.; Jacobs, Howard T.; Hyman, Bradley C.

2015-01-01

Mitochondrial DNA (mtDNA) encodes respiratory complex subunits essential to almost all eukaryotes; hence respiratory competence requires faithful duplication of this molecule. However, the mechanism(s) of its synthesis remain hotly debated. Here we have developed Caenorhabditis elegans as a convenient animal model for the study of metazoan mtDNA synthesis. We demonstrate that C. elegans mtDNA replicates exclusively by a phage-like mechanism, in which multimeric molecules are synthesized from a circular template. In contrast to previous mammalian studies, we found that mtDNA synthesis in the C. elegans gonad produces branched-circular lariat structures with multimeric DNA tails; we were able to detect multimers up to four mtDNA genome unit lengths. Further, we did not detect elongation from a displacement-loop or analogue of 7S DNA, suggesting a clear difference from human mtDNA in regard to the site(s) of replication initiation. We also identified cruciform mtDNA species that are sensitive to cleavage by the resolvase RusA; we suggest these four-way junctions may have a role in concatemer-to-monomer resolution. Overall these results indicate that mtDNA synthesis in C. elegans does not conform to any previously documented metazoan mtDNA replication mechanism, but instead are strongly suggestive of rolling circle replication, as employed by bacteriophages. As several components of the metazoan mitochondrial DNA replisome are likely phage-derived, these findings raise the possibility that the rolling circle mtDNA replication mechanism may be ancestral among metazoans. PMID:25693201

Mimicking an Enzyme-Based Colorimetric Aptasensor for Antibiotic Residue Detection in Milk Combining Magnetic Loop-DNA Probes and CHA-Assisted Target Recycling Amplification.

PubMed

Luan, Qian; Gan, Ning; Cao, Yuting; Li, Tianhua

2017-07-19

A mimicking-enzyme-based colorimetric aptasensor was developed for the detection of kanamycin (KANA) in milk using magnetic loop-DNA-NMOF-Pt (m-L-DNA) probes and catalytic hairpin assembly (CHA)-assisted target recycling for signal amplification. The m-L-DNA probes were constructed via hybridization of hairpin DNA H1 (containing aptamer sequence) immobilized magnetic beads (m-H1) and signal DNA (sDNA, partial hybridization with H1) labeled nano Fe-MIL-88NH 2 -Pt (NMOF-Pt-sDNA). In the presence of KANA and complementary hairpin DNA H2, the m-L-DNA probes decomposed and formed an m-H1/KANA intermediate, which triggered the CHA reaction to form a stable duplex strand (m-H1-H2) while releasing KANA again for recycling. Consequently, numerous NMOF-Pt-sDNA as mimicking enzymes can synergistically catalyze 3,3',5,5'-tetramethylbenzidine (TMB) for color development. The aptasensor exhibited high selectivity and sensitivity for KANA in milk with a detection limit of 0.2 pg mL -1 within 30 min. The assay can be conveniently extended for on-site screening of other antibiotics in foods by simply changing the base sequence of the probes.

Structural and functional characterization of two unusual endonuclease III enzymes from Deinococcus radiodurans.

PubMed

Sarre, Aili; Ökvist, Mats; Klar, Tobias; Hall, David R; Smalås, Arne O; McSweeney, Sean; Timmins, Joanna; Moe, Elin

2015-08-01

While most bacteria possess a single gene encoding the bifunctional DNA glycosylase Endonuclease III (EndoIII) in their genomes, Deinococcus radiodurans possesses three: DR2438 (DrEndoIII1), DR0289 (DrEndoIII2) and DR0982 (DrEndoIII3). Here we have determined the crystal structures of DrEndoIII1 and an N-terminally truncated form of DrEndoIII3 (DrEndoIII3Δ76). We have also generated a homology model of DrEndoIII2 and measured activity of the three enzymes. All three structures consist of two all α-helical domains, one of which exhibits a [4Fe-4S] cluster and the other a HhH-motif, separated by a DNA binding cleft, similar to previously determined structures of endonuclease III from Escherichia coli and Geobacillus stearothermophilus. However, both DrEndoIII1 and DrEndoIII3 possess an extended HhH motif with extra helical features and an altered electrostatic surface potential. In addition, the DNA binding cleft of DrEndoIII3 seems to be less accessible for DNA interactions, while in DrEndoIII1 it seems to be more open. Analysis of the enzyme activities shows that DrEndoIII2 is most similar to the previously studied enzymes, while DrEndoIII1 seems to be more distant with a weaker activity towards substrate DNA containing either thymine glycol or an abasic site. DrEndoIII3 is the most distantly related enzyme and displays no detectable activity towards these substrates even though the suggested catalytic residues are conserved. Based on a comparative structural analysis, we suggest that the altered surface potential, shape of the substrate-binding pockets and specific amino acid substitutions close to the active site and in the DNA interacting loops may underlie the unexpected differences in activity. Copyright © 2015 Elsevier Inc. All rights reserved.

Protein loop modeling using a new hybrid energy function and its application to modeling in inaccurate structural environments.

PubMed

Park, Hahnbeom; Lee, Gyu Rie; Heo, Lim; Seok, Chaok

2014-01-01

Protein loop modeling is a tool for predicting protein local structures of particular interest, providing opportunities for applications involving protein structure prediction and de novo protein design. Until recently, the majority of loop modeling methods have been developed and tested by reconstructing loops in frameworks of experimentally resolved structures. In many practical applications, however, the protein loops to be modeled are located in inaccurate structural environments. These include loops in model structures, low-resolution experimental structures, or experimental structures of different functional forms. Accordingly, discrepancies in the accuracy of the structural environment assumed in development of the method and that in practical applications present additional challenges to modern loop modeling methods. This study demonstrates a new strategy for employing a hybrid energy function combining physics-based and knowledge-based components to help tackle this challenge. The hybrid energy function is designed to combine the strengths of each energy component, simultaneously maintaining accurate loop structure prediction in a high-resolution framework structure and tolerating minor environmental errors in low-resolution structures. A loop modeling method based on global optimization of this new energy function is tested on loop targets situated in different levels of environmental errors, ranging from experimental structures to structures perturbed in backbone as well as side chains and template-based model structures. The new method performs comparably to force field-based approaches in loop reconstruction in crystal structures and better in loop prediction in inaccurate framework structures. This result suggests that higher-accuracy predictions would be possible for a broader range of applications. The web server for this method is available at http://galaxy.seoklab.org/loop with the PS2 option for the scoring function.

A Novel DDB2-ATM Feedback Loop Regulates Human Cytomegalovirus Replication

PubMed Central

E, Xiaofei; Savidis, George; Chin, Christopher R.; Wang, Shixia; Lu, Shan; Brass, Abraham L.

2014-01-01

Human cytomegalovirus (HCMV) genome replication requires host DNA damage responses (DDRs) and raises the possibility that DNA repair pathways may influence viral replication. We report here that a nucleotide excision repair (NER)-associated-factor is required for efficient HCMV DNA replication. Mutations in genes encoding NER factors are associated with xeroderma pigmentosum (XP). One of the XP complementation groups, XPE, involves mutation in ddb2, which encodes DNA damage binding protein 2 (DDB2). Infectious progeny virus production was reduced by >2 logs in XPE fibroblasts compared to levels in normal fibroblasts. The levels of immediate early (IE) (IE2), early (E) (pp65), and early/late (E/L) (gB55) proteins were decreased in XPE cells. These replication defects were rescued by infection with a retrovirus expressing DDB2 cDNA. Similar patterns of reduced viral gene expression and progeny virus production were also observed in normal fibroblasts that were depleted for DDB2 by RNA interference (RNAi). Mature replication compartments (RCs) were nearly absent in XPE cells, and there were 1.5- to 2.0-log reductions in viral DNA loads in infected XPE cells relative to those in normal fibroblasts. The expression of viral genes (UL122, UL44, UL54, UL55, and UL84) affected by DDB2 status was also sensitive to a viral DNA replication inhibitor, phosphonoacetic acid (PAA), suggesting that DDB2 affects gene expression upstream of or events associated with the initiation of DNA replication. Finally, a novel, infection-associated feedback loop between DDB2 and ataxia telangiectasia mutated (ATM) was observed in infected cells. Together, these results demonstrate that DDB2 and a DDB2-ATM feedback loop influence HCMV replication. PMID:24335308

Single-molecule multiparameter fluorescence spectroscopy reveals directional MutS binding to mismatched bases in DNA

PubMed Central

Cristóvão, Michele; Sisamakis, Evangelos; Hingorani, Manju M.; Marx, Andreas D.; Jung, Caroline P.; Rothwell, Paul J.; Seidel, Claus A. M.; Friedhoff, Peter

2012-01-01

Mismatch repair (MMR) corrects replication errors such as mismatched bases and loops in DNA. The evolutionarily conserved dimeric MMR protein MutS recognizes mismatches by stacking a phenylalanine of one subunit against one base of the mismatched pair. In all crystal structures of G:T mismatch-bound MutS, phenylalanine is stacked against thymine. To explore whether these structures reflect directional mismatch recognition by MutS, we monitored the orientation of Escherichia coli MutS binding to mismatches by FRET and anisotropy with steady state, pre-steady state and single-molecule multiparameter fluorescence measurements in a solution. The results confirm that specifically bound MutS bends DNA at the mismatch. We found additional MutS–mismatch complexes with distinct conformations that may have functional relevance in MMR. The analysis of individual binding events reveal significant bias in MutS orientation on asymmetric mismatches (G:T versus T:G, A:C versus C:A), but not on symmetric mismatches (G:G). When MutS is blocked from binding a mismatch in the preferred orientation by positioning asymmetric mismatches near the ends of linear DNA substrates, its ability to authorize subsequent steps of MMR, such as MutH endonuclease activation, is almost abolished. These findings shed light on prerequisites for MutS interactions with other MMR proteins for repairing the appropriate DNA strand. PMID:22367846

In vitro selection and characterization of single stranded DNA aptamers for luteolin: A possible recognition tool.

PubMed

Tuma Sabah, Jinan; Zulkifli, Razauden Mohamed; Shahir, Shafinaz; Ahmed, Farediah; Abdul Kadir, Mohammed Rafiq; Zakaria, Zarita

2018-05-15

Distinctive bioactivities possessed by luteolin (3', 4', 5, 7-tetrahydroxy-flavone) are advantageous for sundry practical applications. This paper reports the in vitro selection and characterization of single stranded-DNA (ssDNA) aptamers, specific for luteolin (LUT). 76-mer library containing 1015 randomized ssDNA were screened via systematic evolution of ligands by exponential enrichment (SELEX). The recovered ssDNA pool from the 8th round was amplified with unlabeled primers and cloned into PSTBlue-1 vector prior to sequencing. 22 of LUT-binding aptamer variants were further classified into one of the seven groups based on their N40 random sequence regions, wherein one representative from each group was characterized. The dissociation constant of aptamers designated as LUT#28, LUT#20 and LUT#3 was discerned to be 107, 214 and 109 nM, respectively with high binding affinity towards LUT. Prediction analysis of the secondary structure suggested discrete features with typical loop and stem motifs. Furthermore, LUT#3 displayed higher specificity with insignificant binding toward kaempferol and quercetin despite its structural and functional similarity compared to LUT#28 and LUT#20. Further LUT#3 can detect free luteolin within 0.2-1 mM in solution. It was suggested that LUT#3 aptamer were the most suitable for LUT recognition tool at laboratory scale based on the condition tested. Copyright © 2018 Elsevier Inc. All rights reserved.

SMC1-Mediated Intra-S-Phase Arrest Facilitates Bocavirus DNA Replication

PubMed Central

Luo, Yong; Deng, Xuefeng; Cheng, Fang; Li, Yi

2013-01-01

Activation of a host DNA damage response (DDR) is essential for DNA replication of minute virus of canines (MVC), a member of the genus Bocavirus of the Parvoviridae family; however, the mechanism by which DDR contributes to viral DNA replication is unknown. In the current study, we demonstrate that MVC infection triggers the intra-S-phase arrest to slow down host cellular DNA replication and to recruit cellular DNA replication factors for viral DNA replication. The intra-S-phase arrest is regulated by ATM (ataxia telangiectasia-mutated kinase) signaling in a p53-independent manner. Moreover, we demonstrate that SMC1 (structural maintenance of chromosomes 1) is the key regulator of the intra-S-phase arrest induced during infection. Either knockdown of SMC1 or complementation with a dominant negative SMC1 mutant blocks both the intra-S-phase arrest and viral DNA replication. Finally, we show that the intra-S-phase arrest induced during MVC infection was caused neither by damaged host cellular DNA nor by viral proteins but by replicating viral genomes physically associated with the DNA damage sensor, the Mre11-Rad50-Nbs1 (MRN) complex. In conclusion, the feedback loop between MVC DNA replication and the intra-S-phase arrest is mediated by ATM-SMC1 signaling and plays a critical role in MVC DNA replication. Thus, our findings unravel the mechanism underlying DDR signaling-facilitated MVC DNA replication and demonstrate a novel strategy of DNA virus-host interaction. PMID:23365434

Interaction of the replication terminator protein of Bacillus subtilis with DNA probed by NMR spectroscopy

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

Hastings, Adam F.; Otting, Gottfried; Folmer, Rutger H.A.

2005-09-23

Termination of DNA replication in Bacillus subtilis involves the polar arrest of replication forks by a specific complex formed between the dimeric 29 kDa replication terminator protein (RTP) and DNA terminator sites. We have used NMR spectroscopy to probe the changes in {sup 1}H-{sup 15}N correlation spectra of a {sup 15}N-labelled RTP.C110S mutant upon the addition of a 21 base pair symmetrical DNA binding site. Assignment of the {sup 1}H-{sup 15}N correlations was achieved using a suite of triple resonance NMR experiments with {sup 15}N,{sup 13}C,70% {sup 2}H enriched protein recorded at 800 MHz and using TROSY pulse sequences. Perturbationsmore » to {sup 1}H-{sup 15}N spectra revealed that the N-termini, {alpha}3-helices and several loops are affected by the binding interaction. An analysis of this data in light of the crystallographically determined apo- and DNA-bound forms of RTP.C110S revealed that the NMR spectral perturbations correlate more closely to protein structural changes upon complex formation rather than to interactions at the protein-DNA interface.« less

Structure of Rot, a global regulator of virulence genes in Staphylococcus aureus.

PubMed

Zhu, Yuwei; Fan, Xiaojiao; Zhang, Xu; Jiang, Xuguang; Niu, Liwen; Teng, Maikun; Li, Xu

2014-09-01

Staphylococcus aureus is a highly versatile pathogen that can infect human tissue by producing a large arsenal of virulence factors that are tightly regulated by a complex regulatory network. Rot, which shares sequence similarity with SarA homologues, is a global regulator that regulates numerous virulence genes. However, the recognition model of Rot for the promoter region of target genes and the putative regulation mechanism remain elusive. In this study, the 1.77 Å resolution X-ray crystal structure of Rot is reported. The structure reveals that two Rot molecules form a compact homodimer, each of which contains a typical helix-turn-helix module and a β-hairpin motif connected by a flexible loop. Fluorescence polarization results indicate that Rot preferentially recognizes AT-rich dsDNA with ~30-base-pair nucleotides and that the conserved positively charged residues on the winged-helix motif are vital for binding to the AT-rich dsDNA. It is proposed that the DNA-recognition model of Rot may be similar to that of SarA, SarR and SarS, in which the helix-turn-helix motifs of each monomer interact with the major grooves of target dsDNA and the winged motifs contact the minor grooves. Interestingly, the structure shows that Rot adopts a novel dimerization model that differs from that of other SarA homologues. As expected, perturbation of the dimer interface abolishes the dsDNA-binding ability of Rot, suggesting that Rot functions as a dimer. In addition, the results have been further confirmed in vivo by measuring the transcriptional regulation of α-toxin, a major virulence factor produced by most S. aureus strains.

Complete sequence and analysis of the mitochondrial genome of Hemiselmis andersenii CCMP644 (Cryptophyceae).

PubMed

Kim, Eunsoo; Lane, Christopher E; Curtis, Bruce A; Kozera, Catherine; Bowman, Sharen; Archibald, John M

2008-05-12

Cryptophytes are an enigmatic group of unicellular eukaryotes with plastids derived by secondary (i.e., eukaryote-eukaryote) endosymbiosis. Cryptophytes are unusual in that they possess four genomes-a host cell-derived nuclear and mitochondrial genome and an endosymbiont-derived plastid and 'nucleomorph' genome. The evolutionary origins of the host and endosymbiont components of cryptophyte algae are at present poorly understood. Thus far, a single complete mitochondrial genome sequence has been determined for the cryptophyte Rhodomonas salina. Here, the second complete mitochondrial genome of the cryptophyte alga Hemiselmis andersenii CCMP644 is presented. The H. andersenii mtDNA is 60,553 bp in size and encodes 30 structural RNAs and 36 protein-coding genes, all located on the same strand. A prominent feature of the genome is the presence of a approximately 20 Kbp long intergenic region comprised of numerous tandem and dispersed repeat units of between 22-336 bp. Adjacent to these repeats are 27 copies of palindromic sequences predicted to form stable DNA stem-loop structures. One such stem-loop is located near a GC-rich and GC-poor region and may have a regulatory function in replication or transcription. The H. andersenii mtDNA shares a number of features in common with the genome of the cryptophyte Rhodomonas salina, including general architecture, gene content, and the presence of a large repeat region. However, the H. andersenii mtDNA is devoid of inverted repeats and introns, which are present in R. salina. Comparative analyses of the suite of tRNAs encoded in the two genomes reveal that the H. andersenii mtDNA has lost or converted its original trnK(uuu) gene and possesses a trnS-derived 'trnK(uuu)', which appears unable to produce a functional tRNA. Mitochondrial protein coding gene phylogenies strongly support a variety of previously established eukaryotic groups, but fail to resolve the relationships among higher-order eukaryotic lineages. Comparison of the H. andersenii and R. salina mitochondrial genomes reveals a number of cryptophyte-specific genomic features, most notably the presence of a large repeat-rich intergenic region. However, unlike R. salina, the H. andersenii mtDNA does not possess introns and lacks a Lys-tRNA, which is presumably imported from the cytosol.

Complete Sequence and Analysis of the Mitochondrial Genome of Hemiselmis andersenii CCMP644 (Cryptophyceae)

PubMed Central

Kim, Eunsoo; Lane, Christopher E; Curtis, Bruce A; Kozera, Catherine; Bowman, Sharen; Archibald, John M

2008-01-01

Background Cryptophytes are an enigmatic group of unicellular eukaryotes with plastids derived by secondary (i.e., eukaryote-eukaryote) endosymbiosis. Cryptophytes are unusual in that they possess four genomes–a host cell-derived nuclear and mitochondrial genome and an endosymbiont-derived plastid and 'nucleomorph' genome. The evolutionary origins of the host and endosymbiont components of cryptophyte algae are at present poorly understood. Thus far, a single complete mitochondrial genome sequence has been determined for the cryptophyte Rhodomonas salina. Here, the second complete mitochondrial genome of the cryptophyte alga Hemiselmis andersenii CCMP644 is presented. Results The H. andersenii mtDNA is 60,553 bp in size and encodes 30 structural RNAs and 36 protein-coding genes, all located on the same strand. A prominent feature of the genome is the presence of a ~20 Kbp long intergenic region comprised of numerous tandem and dispersed repeat units of between 22–336 bp. Adjacent to these repeats are 27 copies of palindromic sequences predicted to form stable DNA stem-loop structures. One such stem-loop is located near a GC-rich and GC-poor region and may have a regulatory function in replication or transcription. The H. andersenii mtDNA shares a number of features in common with the genome of the cryptophyte Rhodomonas salina, including general architecture, gene content, and the presence of a large repeat region. However, the H. andersenii mtDNA is devoid of inverted repeats and introns, which are present in R. salina. Comparative analyses of the suite of tRNAs encoded in the two genomes reveal that the H. andersenii mtDNA has lost or converted its original trnK(uuu) gene and possesses a trnS-derived 'trnK(uuu)', which appears unable to produce a functional tRNA. Mitochondrial protein coding gene phylogenies strongly support a variety of previously established eukaryotic groups, but fail to resolve the relationships among higher-order eukaryotic lineages. Conclusion Comparison of the H. andersenii and R. salina mitochondrial genomes reveals a number of cryptophyte-specific genomic features, most notably the presence of a large repeat-rich intergenic region. However, unlike R. salina, the H. andersenii mtDNA does not possess introns and lacks a Lys-tRNA, which is presumably imported from the cytosol. PMID:18474103

The haloarchaeal MCM proteins: bioinformatic analysis and targeted mutagenesis of the β7-β8 and β9-β10 hairpin loops and conserved zinc binding domain cysteines

PubMed Central

Kristensen, Tatjana P.; Maria Cherian, Reeja; Gray, Fiona C.; MacNeill, Stuart A.

2014-01-01

The hexameric MCM complex is the catalytic core of the replicative helicase in eukaryotic and archaeal cells. Here we describe the first in vivo analysis of archaeal MCM protein structure and function relationships using the genetically tractable haloarchaeon Haloferax volcanii as a model system. Hfx. volcanii encodes a single MCM protein that is part of the previously identified core group of haloarchaeal MCM proteins. Three structural features of the N-terminal domain of the Hfx. volcanii MCM protein were targeted for mutagenesis: the β7-β8 and β9-β10 β-hairpin loops and putative zinc binding domain. Five strains carrying single point mutations in the β7-β8 β-hairpin loop were constructed, none of which displayed impaired cell growth under normal conditions or when treated with the DNA damaging agent mitomycin C. However, short sequence deletions within the β7-β8 β-hairpin were not tolerated and neither was replacement of the highly conserved residue glutamate 187 with alanine. Six strains carrying paired alanine substitutions within the β9-β10 β-hairpin loop were constructed, leading to the conclusion that no individual amino acid within that hairpin loop is absolutely required for MCM function, although one of the mutant strains displays greatly enhanced sensitivity to mitomycin C. Deletions of two or four amino acids from the β9-β10 β-hairpin were tolerated but mutants carrying larger deletions were inviable. Similarly, it was not possible to construct mutants in which any of the conserved zinc binding cysteines was replaced with alanine, underlining the likely importance of zinc binding for MCM function. The results of these studies demonstrate the feasibility of using Hfx. volcanii as a model system for reverse genetic analysis of archaeal MCM protein function and provide important confirmation of the in vivo importance of conserved structural features identified by previous bioinformatic, biochemical and structural studies. PMID:24723920

Probe DNA-Cisplatin Interaction with Solid-State Nanopores

NASA Astrophysics Data System (ADS)

Zhou, Zhi; Hu, Ying; Li, Wei; Xu, Zhi; Wang, Pengye; Bai, Xuedong; Shan, Xinyan; Lu, Xinghua; Nanopore Collaboration

2014-03-01

Understanding the mechanism of DNA-cisplatin interaction is essential for clinical application and novel drug design. As an emerging single-molecule technology, solid-state nanopore has been employed in biomolecule detection and probing DNA-molecule interactions. Herein, we reported a real-time monitoring of DNA-cisplatin interaction by employing solid-state SiN nanopores. The DNA-cisplatin interacting process is clearly classified into three stages by measuring the capture rate of DNA-cisplatin adducts. In the first stage, the negative charged DNA molecules were partially discharged due to the bonding of positive charged cisplatin and forming of mono-adducts. In the second stage, forming of DNA-cisplatin di-adducts with the adjacent bases results in DNA bending and softening. The capture rate increases since the softened bi-adducts experience a lower barrier to thread into the nanopores. In the third stage, complex structures, such as micro-loop, are formed and the DNA-cisplatin adducts are aggregated. The capture rate decreases to zero as the aggregated adduct grows to the size of the pore. The characteristic time of this stage was found to be linear with the diameter of the nanopore and this dynamic process can be described with a second-order reaction model. We are grateful to Laboratory of Microfabrication, Dr. Y. Yao, and Prof. R.C. Yu (Institute of Physics, Chinese Academy of Sciences) for technical assistance.

DNA unzipping with asymmetric periodic forces: Robustness of the scaling behavior of hysteresis loop

NASA Astrophysics Data System (ADS)

Pal, Tanmoy; Kumar, Sanjay

2018-01-01

We study the effect of periodic unzipping forces (symmetric and asymmetric) on the steady-state hysteresis loop area of force-extension curves of DNA. For the triangular force, we get back the previously reported scaling exponents but for the ratchet force, we find that the scaling exponents deviate from the reported ones. We also study the temperature dependence of the scaling exponents for the triangular force. At the low-frequency regime, the choice of the scaling form determines whether the scaling exponents depend on the temperature or not.

Mitochondrial sequence analysis for forensic identification using pyrosequencing technology.

PubMed

Andréasson, H; Asp, A; Alderborn, A; Gyllensten, U; Allen, M

2002-01-01

Over recent years, requests for mtDNA analysis in the field of forensic medicine have notably increased, and the results of such analyses have proved to be very useful in forensic cases where nuclear DNA analysis cannot be performed. Traditionally, mtDNA has been analyzed by DNA sequencing of the two hypervariable regions, HVI and HVII, in the D-loop. DNA sequence analysis using the conventional Sanger sequencing is very robust but time consuming and labor intensive. By contrast, mtDNA analysis based on the pyrosequencing technology provides fast and accurate results from the human mtDNA present in many types of evidence materials in forensic casework. The assay has been developed to determine polymorphic sites in the mitochondrial D-loop as well as the coding region to further increase the discrimination power of mtDNA analysis. The pyrosequencing technology for analysis of mtDNA polymorphisms has been tested with regard to sensitivity, reproducibility, and success rate when applied to control samples and actual casework materials. The results show that the method is very accurate and sensitive; the results are easily interpreted and provide a high success rate on casework samples. The panel of pyrosequencing reactions for the mtDNA polymorphisms were chosen to result in an optimal discrimination power in relation to the number of bases determined.

Palindromic Sequence Artifacts Generated during Next Generation Sequencing Library Preparation from Historic and Ancient DNA

PubMed Central

Star, Bastiaan; Nederbragt, Alexander J.; Hansen, Marianne H. S.; Skage, Morten; Gilfillan, Gregor D.; Bradbury, Ian R.; Pampoulie, Christophe; Stenseth, Nils Chr; Jakobsen, Kjetill S.; Jentoft, Sissel

2014-01-01

Degradation-specific processes and variation in laboratory protocols can bias the DNA sequence composition from samples of ancient or historic origin. Here, we identify a novel artifact in sequences from historic samples of Atlantic cod (Gadus morhua), which forms interrupted palindromes consisting of reverse complementary sequence at the 5′ and 3′-ends of sequencing reads. The palindromic sequences themselves have specific properties – the bases at the 5′-end align well to the reference genome, whereas extensive misalignments exists among the bases at the terminal 3′-end. The terminal 3′ bases are artificial extensions likely caused by the occurrence of hairpin loops in single stranded DNA (ssDNA), which can be ligated and amplified in particular library creation protocols. We propose that such hairpin loops allow the inclusion of erroneous nucleotides, specifically at the 3′-end of DNA strands, with the 5′-end of the same strand providing the template. We also find these palindromes in previously published ancient DNA (aDNA) datasets, albeit at varying and substantially lower frequencies. This artifact can negatively affect the yield of endogenous DNA in these types of samples and introduces sequence bias. PMID:24608104

Effect of Molecular Crowding on the Response of an Electrochemical DNA Sensor

PubMed Central

Ricci, Francesco; Lai, Rebecca Y.; Heeger, Alan J.; Plaxco, Kevin W.; Sumner, James J.

2009-01-01

E-DNA sensors, the electrochemical equivalent of molecular beacons, appear to be a promising means of detecting oligonucleotides. E-DNA sensors are comprised of a redox-modified (here, methylene blue or ferrocene) DNA stem-loop covalently attached to an interrogating electrode. Because E-DNA signaling arises due to binding-induced changes in the conformation of the stem-loop probe, it is likely sensitive to the nature of the molecular packing on the electrode surface. Here we detail the effects of probe density, target length, and other aspects of molecular crowding on the signaling properties, specificity, and response time of a model E-DNA sensor. We find that the highest signal suppression is obtained at the highest probe densities investigated, and that greater suppression is observed with longer and bulkier targets. In contrast, sensor equilibration time slows monotonically with increasing probe density, and the specificity of hybridization is not significantly affected. In addition to providing insight into the optimization of electrochemical DNA sensors, these results suggest that E-DNA signaling arises due to hybridization-linked changes in the rate, and thus efficiency, with which the redox moiety collides with the electrode and transfers electrons. PMID:17488132

A compositional segmentation of the human mitochondrial genome is related to heterogeneities in the guanine mutation rate

PubMed Central

Samuels, David C.; Boys, Richard J.; Henderson, Daniel A.; Chinnery, Patrick F.

2003-01-01

We applied a hidden Markov model segmentation method to the human mitochondrial genome to identify patterns in the sequence, to compare these patterns to the gene structure of mtDNA and to see whether these patterns reveal additional characteristics important for our understanding of genome evolution, structure and function. Our analysis identified three segmentation categories based upon the sequence transition probabilities. Category 2 segments corresponded to the tRNA and rRNA genes, with a greater strand-symmetry in these segments. Category 1 and 3 segments covered the protein- coding genes and almost all of the non-coding D-loop. Compared to category 1, the mtDNA segments assigned to category 3 had much lower guanine abundance. A comparison to two independent databases of mitochondrial mutations and polymorphisms showed that the high substitution rate of guanine in human mtDNA is largest in the category 3 segments. Analysis of synonymous mutations showed the same pattern. This suggests that this heterogeneity in the mutation rate is partly independent of respiratory chain function and is a direct property of the genome sequence itself. This has important implications for our understanding of mtDNA evolution and its use as a ‘molecular clock’ to determine the rate of population and species divergence. PMID:14530452

The structure and DNA-binding properties of Mgm101 from a yeast with a linear mitochondrial genome.

PubMed

Pevala, Vladimír; Truban, Dominika; Bauer, Jacob A; Košťan, Július; Kunová, Nina; Bellová, Jana; Brandstetter, Marlene; Marini, Victoria; Krejčí, Lumír; Tomáška, Ľubomír; Nosek, Jozef; Kutejová, Eva

2016-03-18

To study the mechanisms involved in the maintenance of a linear mitochondrial genome we investigated the biochemical properties of the recombination protein Mgm101 from Candida parapsilosis. We show that CpMgm101 complements defects associated with the Saccharomyces cerevisiae mgm101-1(ts) mutation and that it is present in both the nucleus and mitochondrial nucleoids of C. parapsilosis. Unlike its S. cerevisiae counterpart, CpMgm101 is associated with the entire nucleoid population and is able to bind to a broad range of DNA substrates in a non-sequence specific manner. CpMgm101 is also able to catalyze strand annealing and D-loop formation. CpMgm101 forms a roughly C-shaped trimer in solution according to SAXS. Electron microscopy of a complex of CpMgm101 with a model mitochondrial telomere revealed homogeneous, ring-shaped structures at the telomeric single-stranded overhangs. The DNA-binding properties of CpMgm101, together with its DNA recombination properties, suggest that it can play a number of possible roles in the replication of the mitochondrial genome and the maintenance of its telomeres. © The Author(s) 2016. Published by Oxford University Press on behalf of Nucleic Acids Research.

Threading dynamics of a polymer through parallel pores: Potential applications to DNA size separation

NASA Astrophysics Data System (ADS)

Åkerman, Björn

1997-04-01

DNA orientation measurements by linear dichroism (LD) spectroscopy and single molecule imaging by fluorescence microscopy are used to investigate the effect of DNA size (71-740 kilo base pairs) and field strength E (1-5.9 V/cm) on the conformation dynamics during the field-driven threading of DNA molecules through a set of parallel pores in agarose gels, with average pore radii between 380 Å and 1400 Å. Locally relaxed but globally oriented DNA molecules are subjected to a perpendicular field, and the observed LD time profile is compared with a recent theory for the threading [D. Long and J.-L. Viovy, Phys. Rev. E 53, 803 (1996)] which assumes the same initial state. As predicted the DNA is driven by the ends into a U-form, leading to an overshoot in the LD. The overshoot-time scales as E-(1.2-1.4) as predicted, but grows more slowly with DNA size than the predicted linear dependence. For long molecules loops form initially in the threading process but are finally consumed by the ends, and the process of transfer of DNA segments, from the loops to the arms of the U, leads to a shoulder in the LD as predicted. The critical size below which loops do not form (as indicated by the LD shoulder being absent) is between 71 and 105 kbp (0.5% agarose, 5.9 V/cm), and considerably larger than predicted because in the initial state the DNA molecules are housed in gel cavities with effective pore sizes about four times larger than the average pore size. From the data, the separation of DNA by exploiting the threading dynamics in pulsed fields [D. Long et al., CR Acad. Sci. Paris, Ser. IIb 321, 239 (1995)] is shown to be feasible in principle in an agarose-based system.

Conformational Sampling in Template-Free Protein Loop Structure Modeling: An Overview

PubMed Central

Li, Yaohang

2013-01-01

Accurately modeling protein loops is an important step to predict three-dimensional structures as well as to understand functions of many proteins. Because of their high flexibility, modeling the three-dimensional structures of loops is difficult and is usually treated as a “mini protein folding problem” under geometric constraints. In the past decade, there has been remarkable progress in template-free loop structure modeling due to advances of computational methods as well as stably increasing number of known structures available in PDB. This mini review provides an overview on the recent computational approaches for loop structure modeling. In particular, we focus on the approaches of sampling loop conformation space, which is a critical step to obtain high resolution models in template-free methods. We review the potential energy functions for loop modeling, loop buildup mechanisms to satisfy geometric constraints, and loop conformation sampling algorithms. The recent loop modeling results are also summarized. PMID:24688696

Conformational sampling in template-free protein loop structure modeling: an overview.

PubMed

Li, Yaohang

2013-01-01

Accurately modeling protein loops is an important step to predict three-dimensional structures as well as to understand functions of many proteins. Because of their high flexibility, modeling the three-dimensional structures of loops is difficult and is usually treated as a "mini protein folding problem" under geometric constraints. In the past decade, there has been remarkable progress in template-free loop structure modeling due to advances of computational methods as well as stably increasing number of known structures available in PDB. This mini review provides an overview on the recent computational approaches for loop structure modeling. In particular, we focus on the approaches of sampling loop conformation space, which is a critical step to obtain high resolution models in template-free methods. We review the potential energy functions for loop modeling, loop buildup mechanisms to satisfy geometric constraints, and loop conformation sampling algorithms. The recent loop modeling results are also summarized.

Nuclear scaffold attachment stimulates, but is not essential for ARS activity in Saccharomyces cerevisiae: analysis of the Drosophila ftz SAR.

PubMed Central

Amati, B; Pick, L; Laroche, T; Gasser, S M

1990-01-01

Nuclei isolated from eukaryotic cells can be depleted of histones and most soluble nuclear proteins to isolate a structural framework called the nuclear scaffold. This structure maintains specific interactions with genomic DNA at sites known as scaffold attached regions (SARs), which are thought to be the bases of DNA loops. In both Saccharomyces cerevisiae and Schizosaccharomyces pombe, genomic ARS elements are recovered as SARs. In addition, SARs from Drosophila melanogaster bind to yeast nuclear scaffolds in vitro and a subclass of these promotes autonomous replication of plasmids in yeast. In the present report, we present fine mapping studies of the Drosophila ftz SAR, which has both SAR and ARS activities in yeast. The data establish a close relationship between the sequences involved in ARS activity and scaffold binding: ARS elements that can bind the nuclear scaffold in vitro promote more efficient plasmid replication in vivo, but scaffold association is not a strict prerequisite for ARS function. Efficient interaction with nuclear scaffolds from both yeast and Drosophila requires a minimal length of SAR DNA that contains reiteration of a narrow minor groove structure of the double helix. Images Fig. 1. Fig. 2. Fig. 3. Fig. 4. PMID:2123454

The Control Region of Mitochondrial DNA Shows an Unusual CpG and Non-CpG Methylation Pattern

PubMed Central

Bellizzi, Dina; D'Aquila, Patrizia; Scafone, Teresa; Giordano, Marco; Riso, Vincenzo; Riccio, Andrea; Passarino, Giuseppe

2013-01-01

DNA methylation is a common epigenetic modification of the mammalian genome. Conflicting data regarding the possible presence of methylated cytosines within mitochondrial DNA (mtDNA) have been reported. To clarify this point, we analysed the methylation status of mtDNA control region (D-loop) on human and murine DNA samples from blood and cultured cells by bisulphite sequencing and methylated/hydroxymethylated DNA immunoprecipitation assays. We found methylated and hydroxymethylated cytosines in the L-strand of all samples analysed. MtDNA methylation particularly occurs within non-C-phosphate-G (non-CpG) nucleotides, mainly in the promoter region of the heavy strand and in conserved sequence blocks, suggesting its involvement in regulating mtDNA replication and/or transcription. We observed DNA methyltransferases within the mitochondria, but the inactivation of Dnmt1, Dnmt3a, and Dnmt3b in mouse embryonic stem (ES) cells results in a reduction of the CpG methylation, while the non-CpG methylation shows to be not affected. This suggests that D-loop epigenetic modification is only partially established by these enzymes. Our data show that DNA methylation occurs in the mtDNA control region of mammals, not only at symmetrical CpG dinucleotides, typical of nuclear genome, but in a peculiar non-CpG pattern previously reported for plants and fungi. The molecular mechanisms responsible for this pattern remain an open question. PMID:23804556

A Bowman-Birk protease inhibitor purified, cloned, sequenced and characterized from the seeds of Maclura pomifera (Raf.) Schneid.

PubMed

Indarte, Martín; Lazza, Cristian M; Assis, Diego; Caffini, Néstor O; Juliano, María A; Avilés, Francesc X; Daura, Xavier; López, Laura M I; Trejo, Sebastián A

2017-02-01

A new BBI-type protease inhibitor with remarkable structural characteristics was purified, cloned, and sequenced from seeds of Maclura pomifera , a dicotyledonous plant belonging to the Moraceae family. In this work, we report a Bowman-Birk inhibitor (BBI) isolated, purified, cloned, and characterized from Maclura pomifera seeds (MpBBI), the first of this type from a species belonging to Moraceae family. MpBBI was purified to homogeneity by RP-HPLC, total RNA was extracted from seeds of M. pomifera, and the 3'RACE-PCR method was applied to obtain the cDNA, which was cloned and sequenced. Peptide mass fingerprinting (PMF) analysis showed correspondence between the in silico-translated protein and MpBBI, confirming that it corresponds to a new plant protease inhibitor. The obtained cDNA encoded a polypeptide of 65 residues and possesses 10 cysteine residues, with molecular mass of 7379.27, pI 6.10, and extinction molar coefficient of 9105 M -1  cm -1 . MpBBI inhibits strongly trypsin with K i in the 10 -10 M range and was stable in a wide array of pH and extreme temperatures. MpBBI comparative modeling was applied to gain insight into its 3D structure and highlighted some distinguishing features: (1) two non-identical loops, (2) loop 1 (CEEESRC) is completely different from any known BBI, and (3) the amount of disulphide bonds is also different from any reported BBI from dicot plants.

Translocation by T7 RNA polymerase: a sensitively poised Brownian ratchet.

PubMed

Guo, Qing; Sousa, Rui

2006-04-21

Studies of halted T7 RNA polymerase (T7RNAP) elongation complexes (ECs) or of T7RNAP transcription against roadblocks due to DNA-bound proteins indicate that T7RNAP translocates via a passive Brownian ratchet mechanism. Crystal structures of T7RNAP ECs suggest that translocation involves an active power-stroke. However, neither solution studies of halted or slowed T7RNAP ECs, nor crystal structures of static complexes, are necessarily relevant to how T7RNAP translocates during rapid elongation. A recent single molecule study of actively elongating T7RNAPs provides support for the Brownian ratchet mechanism. Here, we obtain additional evidence for the existence of a Brownian ratchet during active T7RNAP elongation by showing that both rapidly elongating and halted complexes are equally sensitive to pyrophosphate. Using chemical nucleases tethered to the polymerase we achieve sub-ångström resolution in measuring the average position of halted T7RNAP ECs and find that the positional equilibrium of the EC is sensitively poised between pre-translocated and post-translocated states. This may be important in maximizing the sensitivity of the polymerase to sequences that cause pausing or termination. We also confirm that a crystallographically observed disorder to order transition in a loop formed by residues 589-612 also occurs in solution and is coupled to pyrophosphate or NTP release. This transition allows the loop to make interactions with the DNA that help stabilize the laterally mobile, ligand-free EC against dissociation.

Intracellular modifications induced by poliovirus reduce the requirement for structural motifs in the 5' noncoding region of the genome involved in internal initiation of protein synthesis.

PubMed Central

Percy, N; Belsham, G J; Brangwyn, J K; Sullivan, M; Stone, D M; Almond, J W

1992-01-01

A series of genetic deletions based partly on two RNA secondary structure models (M. A. Skinner, V. R. Racaniello, G. Dunn, J. Cooper, P. D. Minor, and J. W. Almond, J. Mol. Biol. 207:379-392, 1989; E. V. Pilipenko, V. M. Blinov, L. I. Romanova, A. N. Sinyakov, S. V. Maslova, and V. I. Agol, Virology 168:201-209, 1989) was made in the cDNA encoding the 5' noncoding region (5' NCR) of the poliovirus genome in order to study the sequences that direct the internal entry of ribosomes. The modified cDNAs were placed between two open reading frames in a single transcriptional unit and used to transfect cells in culture. Internal entry of ribosomes was detected by measuring translation from the second open reading frame in the bicistronic mRNA. When assayed alone, a large proportion of the poliovirus 5' NCR superstructure including several well-defined stem-loops was required for ribosome entry and efficient translation. However, in cells cotransfected with a complete infectious poliovirus cDNA, the requirement for the stem-loops in this large superstructure was reduced. The results suggest that virus infection modifies the cellular translational machinery, so that shortened forms of the 5' NCR are sufficient for cap-independent translation, and that the internal entry of ribosomes occurs by two distinct modes during the virus replication cycle. Images PMID:1310772

Ddx19 links mRNA nuclear export with progression of transcription and replication and suppresses genomic instability upon DNA damage in proliferating cells.

PubMed

Hodroj, Dana; Serhal, Kamar; Maiorano, Domenico

2017-09-03

The DEAD-box Helicase 19 (Ddx19) gene codes for an RNA helicase involved in both mRNA (mRNA) export from the nucleus into the cytoplasm and in mRNA translation. In unperturbed cells, Ddx19 localizes in the cytoplasm and at the cytoplasmic face of the nuclear pore. Here we review recent findings related to an additional Ddx19 function in the nucleus in resolving RNA:DNA hybrids (R-loops) generated during collision between transcription and replication, and upon DNA damage. Activation of a DNA damage response pathway dependent upon the ATR kinase, a major regulator of replication fork progression, stimulates translocation of the Ddx19 protein from the cytoplasm into the nucleus. Only nuclear Ddx19 is competent to resolve R-loops, and down regulation of Ddx19 expression induces DNA double strand breaks only in proliferating cells. Overall these observations put forward Ddx19 as an important novel mediator of the crosstalk between transcription and replication.

In vitro molecular machine learning algorithm via symmetric internal loops of DNA.

PubMed

Lee, Ji-Hoon; Lee, Seung Hwan; Baek, Christina; Chun, Hyosun; Ryu, Je-Hwan; Kim, Jin-Woo; Deaton, Russell; Zhang, Byoung-Tak

2017-08-01

Programmable biomolecules, such as DNA strands, deoxyribozymes, and restriction enzymes, have been used to solve computational problems, construct large-scale logic circuits, and program simple molecular games. Although studies have shown the potential of molecular computing, the capability of computational learning with DNA molecules, i.e., molecular machine learning, has yet to be experimentally verified. Here, we present a novel molecular learning in vitro model in which symmetric internal loops of double-stranded DNA are exploited to measure the differences between training instances, thus enabling the molecules to learn from small errors. The model was evaluated on a data set of twenty dialogue sentences obtained from the television shows Friends and Prison Break. The wet DNA-computing experiments confirmed that the molecular learning machine was able to generalize the dialogue patterns of each show and successfully identify the show from which the sentences originated. The molecular machine learning model described here opens the way for solving machine learning problems in computer science and biology using in vitro molecular computing with the data encoded in DNA molecules. Copyright © 2017. Published by Elsevier B.V.

Structure-guided Design and Immunological Characterization of Immunogens Presenting the HIV-1 gp120 V3 Loop on a CTB Scaffold

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

M Totrov; X Jiang; X Kong

2011-12-31

V3 loop is a major neutralizing determinant of the HIV-1 gp120. Using 3D structures of cholera toxin B subunit (CTB), complete V3 in the gp120 context, and V3 bound to a monoclonal antibody (mAb), we designed two V3-scaffold immunogen constructs (V3-CTB). The full-length V3-CTB presenting the complete V3 in a structural context mimicking gp120 was recognized by the large majority of our panel of 24 mAbs. The short V3-CTB presenting a V3 fragment in the conformation observed in the complex with the 447-52D Fab, exhibited high-affinity binding to this mAb. The immunogens were evaluated in rabbits using DNA-prime/protein-boost protocol. Boostingmore » with the full-length V3-CTB induced high anti-V3 titers in sera that potently neutralize multiple HIV virus strains. The short V3-CTB was ineffective. The results suggest that very narrow antigenic profile of an immunogen is associated with poor Ab response. An immunogen with broader antigenic activity elicits robust Ab response.« less

TALEs from a spring--superelasticity of Tal effector protein structures.

PubMed

Flechsig, Holger

2014-01-01

Transcription activator-like effectors (TALEs) are DNA-related proteins that recognise and bind specific target sequences to manipulate gene expression. Recently determined crystal structures show that their common architecture reveals a superhelical overall structure that may undergo drastic conformational changes. To establish a link between structure and dynamics in TALE proteins we have employed coarse-grained elastic-network modelling of currently available structural data and implemented a force-probe setup that allowed us to investigate their mechanical behaviour in computer experiments. Based on the measured force-extension curves we conclude that TALEs exhibit superelastic dynamical properties allowing for large-scale global conformational changes along their helical axis, which represents the soft direction in such proteins. For moderate external forcing the TALE models behave like linear springs, obeying Hooke's law, and the investigated structures can be characterised and compared by a corresponding spring constant. We show that conformational flexibility underlying the large-scale motions is not homogeneously distributed over the TALE structure, but instead soft spot residues around which strain is accumulated and which turn out to represent key agents in the transmission of conformational motions are identified. They correspond to the RVD loop residues that have been experimentally determined to play an eminent role in the binding process of target DNA.

TALEs from a Spring – Superelasticity of Tal Effector Protein Structures

PubMed Central

Flechsig, Holger

2014-01-01

Transcription activator-like effectors (TALEs) are DNA-related proteins that recognise and bind specific target sequences to manipulate gene expression. Recently determined crystal structures show that their common architecture reveals a superhelical overall structure that may undergo drastic conformational changes. To establish a link between structure and dynamics in TALE proteins we have employed coarse-grained elastic-network modelling of currently available structural data and implemented a force-probe setup that allowed us to investigate their mechanical behaviour in computer experiments. Based on the measured force-extension curves we conclude that TALEs exhibit superelastic dynamical properties allowing for large-scale global conformational changes along their helical axis, which represents the soft direction in such proteins. For moderate external forcing the TALE models behave like linear springs, obeying Hooke's law, and the investigated structures can be characterised and compared by a corresponding spring constant. We show that conformational flexibility underlying the large-scale motions is not homogeneously distributed over the TALE structure, but instead soft spot residues around which strain is accumulated and which turn out to represent key agents in the transmission of conformational motions are identified. They correspond to the RVD loop residues that have been experimentally determined to play an eminent role in the binding process of target DNA. PMID:25313859

A quantitative measure of chirality inside nucleic acid databank.

PubMed

Pietropaolo, Adriana; Parrinello, Michele

2011-08-01

We show the capability of a chirality index (Pietropaolo et al., Proteins 2008;70:667-677) to investigate nucleic acid structures because of its high sensitivity to helical conformations. By analyzing selected structures of DNA and RNA, we have found that sequences rich in cytosine and guanine have a tendency to left-handed chirality, in contrast to regions rich in adenine or thymine which show strong negative, right-handed, chirality values. We also analyze RNA structures, where specific loops and hairpin motifs are characterized by a well-defined chirality value. We find that in nucleosome the chirality is exalted, whereas in ribosome it is reduced. Our results illustrate the sensitivity of this descriptor for nucleic acid conformations. Copyright © 2011 Wiley-Liss, Inc.

Mitochondrial DNA maintenance is regulated in human hepatoma cells by glycogen synthase kinase 3β and p53 in response to tumor necrosis factor α.

PubMed

Vadrot, Nathalie; Ghanem, Sarita; Braut, Françoise; Gavrilescu, Laura; Pilard, Nathalie; Mansouri, Abdellah; Moreau, Richard; Reyl-Desmars, Florence

2012-01-01

During chronic liver inflammation, up-regulated Tumor Necrosis Factor alpha (TNF-α) targets hepatocytes and induces abnormal reactive oxygen species (ROS) production responsible for mitochondrial DNA (mtDNA) alterations. The serine/threonine Glycogen Synthase Kinase 3 beta (GSK3β) plays a pivotal role during inflammation but its involvement in the maintenance of mtDNA remains unknown. The aim of this study was to investigate its involvement in TNF-α induced mtDNA depletion and its interrelationship with p53 a protein known to maintain mtDNA copy numbers. Using quantitative polymerase chain reaction (qPCR) we found that at 30 min in human hepatoma HepG2 cells TNF-α induced 0.55±0.10 mtDNA lesions per 10 Kb and a 52.4±2.8% decrease in mtDNA content dependent on TNF-R1 receptor and ROS production. Both lesions and depletion returned to baseline from 1 to 6 h after TNF-α exposure. Luminol-amplified chemiluminescence (LAC) was used to measure the rapid (10 min) and transient TNF-α induced increase in ROS production (168±15%). A transient 8-oxo-dG level of 1.4±0.3 ng/mg DNA and repair of abasic sites were also measured by ELISA assays. Translocation of p53 to mitochondria was observed by Western Blot and co-immunoprecipitations showed that TNF-α induced p53 binding to GSK3β and mitochondrial transcription factor A (TFAM). In addition, mitochondrial D-loop immunoprecipitation (mtDIP) revealed that TNF-α induced p53 binding to the regulatory D-loop region of mtDNA. The knockdown of p53 by siRNAs, inhibition by the phosphoSer(15)p53 antibody or transfection of human mutant active GSK3βS9A pcDNA3 plasmid inhibited recovery of mtDNA content while blockade of GSK3β activity by SB216763 inhibitor or knockdown by siRNAs suppressed mtDNA depletion. This study is the first to report the involvement of GSK3β in TNF-α induced mtDNA depletion. We suggest that p53 binding to GSK3β, TFAM and D-loop could induce recovery of mtDNA content through mtDNA repair.

Transcription profiling suggests that mitochondrial topoisomerase IB acts as a topological barrier and regulator of mitochondrial DNA transcription.

PubMed

Dalla Rosa, Ilaria; Zhang, Hongliang; Khiati, Salim; Wu, Xiaolin; Pommier, Yves

2017-12-08

Mitochondrial DNA (mtDNA) is essential for cell viability because it encodes subunits of the respiratory chain complexes. Mitochondrial topoisomerase IB (TOP1MT) facilitates mtDNA replication by removing DNA topological tensions produced during mtDNA transcription, but it appears to be dispensable. To test whether cells lacking TOP1MT have aberrant mtDNA transcription, we performed mitochondrial transcriptome profiling. To that end, we designed and implemented a customized tiling array, which enabled genome-wide, strand-specific, and simultaneous detection of all mitochondrial transcripts. Our technique revealed that Top1mt KO mouse cells process the mitochondrial transcripts normally but that protein-coding mitochondrial transcripts are elevated. Moreover, we found discrete long noncoding RNAs produced by H-strand transcription and encompassing the noncoding regulatory region of mtDNA in human and murine cells and tissues. Of note, these noncoding RNAs were strongly up-regulated in the absence of TOP1MT. In contrast, 7S DNA, produced by mtDNA replication, was reduced in the Top1mt KO cells. We propose that the long noncoding RNA species in the D-loop region are generated by the extension of H-strand transcripts beyond their canonical stop site and that TOP1MT acts as a topological barrier and regulator for mtDNA transcription and D-loop formation.

Multifunctional Dumbbell-Shaped DNA-Templated Selective Formation of Fluorescent Silver Nanoclusters or Copper Nanoparticles for Sensitive Detection of Biomolecules.

PubMed

Chen, Jinyang; Ji, Xinghu; Tinnefeld, Philip; He, Zhike

2016-01-27

In this work, a multifunctional template for selective formation of fluorescent silver nanoclusters (AgNCs) or copper nanoparticles (CuNPs) is put forward. This dumbbell-shaped (DS) DNA template is made up of two cytosine hairpin loops and an adenine-thymine-rich double-helical stem which is closed by the loops. The cytosine loops act as specific regions for the growth of AgNCs, and the double-helical stem serves as template for the CuNPs formation. By carefully investigating the sequence and length of DS DNA, we present the optimal design of the template. Benefiting from the smart design and facile synthesis, a simple, label-free, and ultrasensitive fluorescence strategy for adenosine triphosphate (ATP) detection is proposed. Through the systematic comparison, it is found that the strategy based on CuNPs formation is more sensitive for ATP assay than that based on AgNCs synthesis, and the detection limitation was found to be 81 pM. What's more, the CuNPs formation-based method is successfully applied in the detection of ATP in human serum as well as the determination of cellular ATP. In addition to small target molecule, the sensing strategy was also extended to the detection of biomacromolecule (DNA), which illustrates the generality of this biosensor.

A Single Molecule Study of Two Bacteriophage Epigenetic Switches

NASA Astrophysics Data System (ADS)

Wang, Haowei

Epigenetic switches allow organisms to evolve into different states by activating/repressing different sets of genes without mutations of the underlying DNA sequence. The study of epigenetic switches is very important to understand the mechanism of human development, the origin of cancer, mental illness and fundamental processes such as gene regulation. The coliphage lambda epigenetic switch, which allows switching from lysogeny to lysis, has been studied for more than 50 years as a paradigm, and has recently received renewed attention. Atomic force microscopy (AFM) was used here to show that the lambda repressor oligomerizes on DNA, primarily as a dodecamer, to secure a DNA loop, which is the basis of the lambda switch. This study also provides support for the idea that specifically bound repressor stabilizes adjacent, non-specifically bound repressor molecules, which confers robustness to the switch. 186 is a member of a different coliphage family. One of the major differences between the two coliphage families is that lambda phages can be induced to switch from the lysogenic to the lytic state by UV radiation, but most coliphages of P2 family, to which 186 belongs, cannot. Interaction between coliphage 186 repressor and DNA is characterized by AFM and tethered particle motion (TPM). To expedite analysis of the AFM data, MatLab codes were written to automate the laborious, manual tracing procedures. The programs automatically recognize DNA segments and protein particles in an image, in order to measure the DNA length and position of bound particles as well as their height, diameter and volume. Application of these algorithms greatly improved the efficiency of AFM analysis. It was showed that 186 CI dimers form heptameric wheels, which induce DNA wrapping and different kinds of DNA looping producing various conformations of nucleoprotein complexes. Information about the dynamics of DNA wrapping and looping on 186 CI particles was also obtained by TPM.

Molecular organization of the 5S rDNA gene type II in elasmobranchs.

PubMed

Castro, Sergio I; Hleap, Jose S; Cárdenas, Heiber; Blouin, Christian

2016-01-01

The 5S rDNA gene is a non-coding RNA that can be found in 2 copies (type I and type II) in bony and cartilaginous fish. Previous studies have pointed out that type II gene is a paralog derived from type I. We analyzed the molecular organization of 5S rDNA type II in elasmobranchs. Although the structure of the 5S rDNA is supposed to be highly conserved, our results show that the secondary structure in this group possesses some variability and is different than the consensus secondary structure. One of these differences in Selachii is an internal loop at nucleotides 7 and 112. These mutations observed in the transcribed region suggest an independent origin of the gene among Batoids and Selachii. All promoters were highly conserved with the exception of BoxA, possibly due to its affinity to polymerase III. This latter enzyme recognizes a dT4 sequence as stop signal, however in Rajiformes this signal was doubled in length to dT8. This could be an adaptation toward a higher efficiency in the termination process. Our results suggest that there is no TATA box in elasmobranchs in the NTS region. We also provide some evidence suggesting that the complexity of the microsatellites present in the NTS region play an important role in the 5S rRNA gene since it is significantly correlated with the length of the NTS.

Molecular organization of the 5S rDNA gene type II in elasmobranchs

PubMed Central

Castro, Sergio I.; Hleap, Jose S.; Cárdenas, Heiber; Blouin, Christian

2016-01-01

ABSTRACT The 5S rDNA gene is a non-coding RNA that can be found in 2 copies (type I and type II) in bony and cartilaginous fish. Previous studies have pointed out that type II gene is a paralog derived from type I. We analyzed the molecular organization of 5S rDNA type II in elasmobranchs. Although the structure of the 5S rDNA is supposed to be highly conserved, our results show that the secondary structure in this group possesses some variability and is different than the consensus secondary structure. One of these differences in Selachii is an internal loop at nucleotides 7 and 112. These mutations observed in the transcribed region suggest an independent origin of the gene among Batoids and Selachii. All promoters were highly conserved with the exception of BoxA, possibly due to its affinity to polymerase III. This latter enzyme recognizes a dT4 sequence as stop signal, however in Rajiformes this signal was doubled in length to dT8. This could be an adaptation toward a higher efficiency in the termination process. Our results suggest that there is no TATA box in elasmobranchs in the NTS region. We also provide some evidence suggesting that the complexity of the microsatellites present in the NTS region play an important role in the 5S rRNA gene since it is significantly correlated with the length of the NTS. PMID:26488198

Loss of Topoisomerase I leads to R-loop-mediated transcriptional blocks during ribosomal RNA synthesis

PubMed Central

El Hage, Aziz; French, Sarah L.; Beyer, Ann L.; Tollervey, David

2010-01-01

Pre-rRNA transcription by RNA Polymerase I (Pol I) is very robust on active rDNA repeats. Loss of yeast Topoisomerase I (Top1) generated truncated pre-rRNA fragments, which were stabilized in strains lacking TRAMP (Trf4/Trf5–Air1/Air2–Mtr4 polyadenylation complexes) or exosome degradation activities. Loss of both Top1 and Top2 blocked pre-rRNA synthesis, with pre-rRNAs truncated predominately in the 18S 5′ region. Positive supercoils in front of Pol I are predicted to slow elongation, while rDNA opening in its wake might cause R-loop formation. Chromatin immunoprecipitation analysis showed substantial levels of RNA/DNA hybrids in the wild type, particularly over the 18S 5′ region. The absence of RNase H1 and H2 in cells depleted of Top1 increased the accumulation of RNA/DNA hybrids and reduced pre-rRNA truncation and pre-rRNA synthesis. Hybrid accumulation over the rDNA was greatly exacerbated when Top1, Top2, and RNase H were all absent. Electron microscopy (EM) analysis revealed Pol I pileups in the wild type, particularly over the 18S. Pileups were longer and more frequent in the absence of Top1, and their frequency was exacerbated when RNase H activity was also lacking. We conclude that the loss of Top1 enhances inherent R-loop formation, particularly over the 5′ region of the rDNA, imposing persistent transcription blocks when RNase H is limiting. PMID:20634320

Elucidating the role of transcription in shaping the 3D structure of the bacterial genome

NASA Astrophysics Data System (ADS)

Brandao, Hugo B.; Wang, Xindan; Rudner, David Z.; Mirny, Leonid

Active transcription has been linked to several genome conformation changes in bacteria, including the recruitment of chromosomal DNA to the cell membrane and formation of nucleoid clusters. Using genomic and imaging data as input into mathematical models and polymer simulations, we sought to explore the extent to which bacterial 3D genome structure could be explained by 1D transcription tracks. Using B. subtilis as a model organism, we investigated via polymer simulations the role of loop extrusion and DNA super-coiling on the formation of interaction domains and other fine-scale features that are visible in chromosome conformation capture (Hi-C) data. We then explored the role of the condensin structural maintenance of chromosome complex on the alignment of chromosomal arms. A parameter-free transcription traffic model demonstrated that mean chromosomal arm alignment can be quantitatively explained, and the effects on arm alignment in genomically rearranged strains of B. subtilis were accurately predicted. H.B. acknowledges support from the Natural Sciences and Engineering Research Council of Canada for a PGS-D fellowship.

An asymmetric mesoscopic model for single bulges in RNA

NASA Astrophysics Data System (ADS)

de Oliveira Martins, Erik; Weber, Gerald

2017-10-01

Simple one-dimensional DNA or RNA mesoscopic models are of interest for their computational efficiency while retaining the key elements of the molecular interactions. However, they only deal with perfectly formed DNA or RNA double helices and consider the intra-strand interactions to be the same on both strands. This makes it difficult to describe highly asymmetric structures such as bulges and loops and, for instance, prevents the application of mesoscopic models to determine RNA secondary structures. Here we derived the conditions for the Peyrard-Bishop mesoscopic model to overcome these limitations and applied it to the calculation of single bulges, the smallest and simplest of these asymmetric structures. We found that these theoretical conditions can indeed be applied to any situation where stacking asymmetry needs to be considered. The full set of parameters for group I RNA bulges was determined from experimental melting temperatures using an optimization procedure, and we also calculated average opening profiles for several RNA sequences. We found that guanosine bulges show the strongest perturbation on their neighboring base pairs, considerably reducing the on-site interactions of their neighboring base pairs.

Hairpin DNA Switch for Ultrasensitive Spectrophotometric Detection of DNA Hybridization Based on Gold Nanoparticles and Enzyme Signal Amplification

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

Zhang, Youyu; Tang, Zhiwen; Wang, Jun

2010-08-01

A novel DNA detection platform based on a hairpin-DNA switch, nanoparticles, and enzyme signal amplification for ultrasensitive detection of DNA hybridization has been developed in this work. In this DNA assay, a “stem-loop” DNA probe dually labeled with a thiol at its 5’ end and a biotin at its 3’ end, respectively, was used. This probe was immobilized on the gold nanoparticles (AuNPs) anchored by a protein, globulin, on a 96-well microplate. In the absence of target DNA, the immobilized probe with the stem-loop structure shields the biotin from being approached by a bulky horseradish peroxidase linked-avidin (avidin-HRP) conjugate duemore » to the steric hindrance. However, in the presence of target DNA, the hybridization between the hairpin DNA probe and the target DNA causes significant conformational change of the probe, which forces biotin away from the surface of AuNPs. As a result, the biotin becomes accessible by the avidin-HRP, and the target hybridization event can be sensitively detected via the HRP catalyzed substrate 3, 3', 5, 5'-tetramethylbenzidine using spectrophometric method. Some experimental parameters governing the performance of the assay have been optimized. At optimal conditions, this DNA assay can detect DNA at the concentration of femtomolar level by means of a signal amplification strategy based on the combination of enzymes and nanoparticles. This approach also has shown excellent specificity to distinguish single-base mismatches of DNA targets because of the intrinsic high selectivity of the hairpin DNA probe.« less

NASA Radiation Track Image GUI for Assessing Space Radiation Biological Effects

NASA Technical Reports Server (NTRS)

Ponomarev, Artem L.; Cucinotta, Francis A.

2006-01-01

The high-charge high-energy (HZE) ion components of the galactic cosmic rays when compared to terrestrial forms of radiations present unique challenges to biological systems. In this paper we present a deoxyribonucleic acid (DNA) breakage model to visualize and analyze the impact of chromatin domains and DNA loops on clustering of DNA damage from X rays, protons, and HZE ions. Our model of DNA breakage is based on a stochastic process of DNA double-strand break (DSB) formulation that includes the amorphous model of the radiation track and a polymer model of DNA packed in the cell nucleus. Our model is a Monte-Carlo simulation based on a randomly located DSB cluster formulation that accomodates both high- and low-linear energy transfer radiations. We demonstrate that HZE ions have a strong impact on DSB clustering, both along the chromosome length and in the nucleus volume. The effects of chromosomal domains and DNA loops on the DSB fragment-size distribution and the spatial distribution of DSB in the nucleus were studied. We compare our model predictions with the spatial distribution of DSB obtained from experiments. The implications of our model predictions for radiation protection are discussed.

Molecular cloning of a novel receptor tyrosine kinase, tif, highly expressed in human ovary and testis.

PubMed

Dai, W; Pan, H; Hassanain, H; Gupta, S L; Murphy, M J

1994-03-01

Using a combination of polymerase chain reaction and conventional cDNA library screening approaches, we have cloned and characterized a putative receptor tyrosine kinase termed tif. The extracellular domain of tif has an immunoglobulin-like loop and a fibronectin type III structure. The intracellular domain contains a tyrosine kinase domain. Compared with ryk, a ubiquitously expressed receptor tyrosine kinase, tif expression is tissue-specific with human ovary and testis containing the highest amount of tif mRNA. Many other tested human tissues such as heart, liver, pancreas and thymus do not contain detectable levels of tif mRNA. The molecular cloning and characterization of tif cDNA will facilitate the identification of a potential ligand(s) for the putative receptor and the study of its biological role.

Molecular insights into the colonization and chromosomal diversification of Madeiran house mice.

PubMed

Förster, D W; Gündüz, I; Nunes, A C; Gabriel, S; Ramalhinho, M G; Mathias, M L; Britton-Davidian, J; Searle, J B

2009-11-01

The colonization history of Madeiran house mice was investigated by analysing the complete mitochondrial (mt) D-loop sequences of 156 mice from the island of Madeira and mainland Portugal, extending on previous studies. The numbers of mtDNA haplotypes from Madeira and mainland Portugal were substantially increased (17 and 14 new haplotypes respectively), and phylogenetic analysis confirmed the previously reported link between the Madeiran archipelago and northern Europe. Sequence analysis revealed the presence of four mtDNA lineages in mainland Portugal, of which one was particularly common and widespread (termed the 'Portugal Main Clade'). There was no support for population bottlenecks during the formation of the six Robertsonian chromosome races on the island of Madeira, and D-loop sequence variation was not found to be structured according to karyotype. The colonization time of the Madeiran archipelago by Mus musculus domesticus was approached using two molecular dating methods (mismatch distribution and Bayesian skyline plot). Time estimates based on D-loop sequence variation at mainland sites (including previously published data from France and Turkey) were evaluated in the context of the zooarchaeological record of M. m. domesticus. A range of values for mutation rate (mu) and number of mouse generations per year was considered in these analyses because of the uncertainty surrounding these two parameters. The colonization of Portugal and Madeira by house mice is discussed in the context of the best-supported parameter values. In keeping with recent studies, our results suggest that mutation rate estimates based on interspecific divergence lead to gross overestimates concerning the timing of recent within-species events.

A Method for WD40 Repeat Detection and Secondary Structure Prediction

PubMed Central

Wang, Yang; Jiang, Fan; Zhuo, Zhu; Wu, Xian-Hui; Wu, Yun-Dong

2013-01-01

WD40-repeat proteins (WD40s), as one of the largest protein families in eukaryotes, play vital roles in assembling protein-protein/DNA/RNA complexes. WD40s fold into similar β-propeller structures despite diversified sequences. A program WDSP (WD40 repeat protein Structure Predictor) has been developed to accurately identify WD40 repeats and predict their secondary structures. The method is designed specifically for WD40 proteins by incorporating both local residue information and non-local family-specific structural features. It overcomes the problem of highly diversified protein sequences and variable loops. In addition, WDSP achieves a better prediction in identifying multiple WD40-domain proteins by taking the global combination of repeats into consideration. In secondary structure prediction, the average Q3 accuracy of WDSP in jack-knife test reaches 93.7%. A disease related protein LRRK2 was used as a representive example to demonstrate the structure prediction. PMID:23776530

Folding dynamics of linear emulsion polymers into 3D architectures

NASA Astrophysics Data System (ADS)

McMullen, Angus; Bargteil, Dylan; Brujic, Jasna

Colloidal polymers have been limited to inflexible, solid colloids. Here we show that the fluidity of emulsion droplets allows for the self-assembly of flexible droplet chains, which can subsequently be folded into 3D structures via secondary interactions. We achieve this using DNA-guided interactions, to initially form the chain, and then program its folding pathways. When two emulsion droplets labeled with complementary DNA meet, the balance of hybridization energy and droplet deformation yields an equilibrium patch size. Therefore, the concentration of DNA on the surface determines the number of droplet-droplet bonds in the assembly. We find that 96 % of bound droplets successfully self-assemble into chains. Droplet binding is a stochastic process, following a Poisson distribution of lengths. Since the fluid droplets can rearrange, we compare the dynamics of emulsion chains to that of polymers. We also trigger secondary interactions along the chain, causing the formation of specific loops or compact clusters. This approach will allow us to fold our emulsion polymers into a wide array of soft structures, giving us a powerful biomimetic colloidal system to investigate protein folding on the mesoscopic scale. This work was supported by the NSF MRSEC Program (DMR-0820341).

Kinetics of interior loop formation in semiflexible chains.

PubMed

Hyeon, Changbong; Thirumalai, D

2006-03-14

Loop formation between monomers in the interior of semiflexible chains describes elementary events in biomolecular folding and DNA bending. We calculate analytically the interior distance distribution function for semiflexible chains using a mean field approach. Using the potential of mean force derived from the distance distribution function we present a simple expression for the kinetics of interior looping by adopting Kramers theory. For the parameters, that are appropriate for DNA, the theoretical predictions in comparison with the case are in excellent agreement with explicit Brownian dynamics simulations of wormlike chain (WLC) model. The interior looping times (tauIC) can be greatly altered in the cases when the stiffness of the loop differs from that of the dangling ends. If the dangling end is stiffer than the loop then tauIC increases for the case of the WLC with uniform persistence length. In contrast, attachment of flexible dangling ends enhances rate of interior loop formation. The theory also shows that if the monomers are charged and interact via screened Coulomb potential then both the cyclization (tauc) and interior looping (tauIC) times greatly increase at low ionic concentration. Because both tauc and tauIC are determined essentially by the effective persistence length [lp(R)] we computed lp(R) by varying the range of the repulsive interaction between the monomers. For short range interactions lp(R) nearly coincides with the bare persistence length which is determined largely by the backbone chain connectivity. This finding rationalizes the efficacy of describing a number of experimental observations (response of biopolymers to force and cyclization kinetics) in biomolecules using WLC model with an effective persistence length.

Structure and activity of the Cas3 HD nuclease MJ0384, an effector enzyme of the CRISPR interference

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

Beloglazova, Natalia; Petit, Pierre; Flick, Robert

Clustered regularly interspaced short palindromic repeats (CRISPRs) and Cas proteins represent an adaptive microbial immunity system against viruses and plasmids. Cas3 proteins have been proposed to play a key role in the CRISPR mechanism through the direct cleavage of invasive DNA. Here, we show that the Cas3 HD domain protein MJ0384 from Methanocaldococcus jannaschii cleaves endonucleolytically and exonucleolytically (3'-5') single-stranded DNAs and RNAs, as well as 3'-flaps, splayed arms, and R-loops. The degradation of branched DNA substrates by MJ0384 is stimulated by the Cas3 helicase MJ0383 and ATP. The crystal structure of MJ0384 revealed the active site with two boundmore » metal cations and together with site-directed mutagenesis suggested a catalytic mechanism. Our studies suggest that the Cas3 HD nucleases working together with the Cas3 helicases can completely degrade invasive DNAs through the combination of endo- and exonuclease activities.« less

NPAS1-ARNT and NPAS3-ARNT crystal structures implicate the bHLH-PAS family as multi-ligand binding transcription factors

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

Wu, Dalei; Su, Xiaoyu; Potluri, Nalini

Here, the neuronal PAS domain proteins NPAS1 and NPAS3 are members of the basic helix-loop-helix-PER-ARNT-SIM (bHLH-PAS) family, and their genetic deficiencies are linked to a variety of human psychiatric disorders including schizophrenia, autism spectrum disorders and bipolar disease. NPAS1 and NPAS3 must each heterodimerize with the aryl hydrocarbon receptor nuclear translocator (ARNT), to form functional transcription complexes capable of DNA binding and gene regulation. Here we examined the crystal structures of multi-domain NPAS1-ARNT and NPAS3-ARNT-DNA complexes, discovering each to contain four putative ligand-binding pockets. Through expanded architectural comparisons between these complexes and HIF-1α-ARNT, HIF-2α-ARNT and CLOCK-BMAL1, we show the widermore » mammalian bHLH-PAS family is capable of multi-ligand-binding and presents as an ideal class of transcription factors for direct targeting by small-molecule drugs.« less

NPAS1-ARNT and NPAS3-ARNT crystal structures implicate the bHLH-PAS family as multi-ligand binding transcription factors

DOE PAGES

Wu, Dalei; Su, Xiaoyu; Potluri, Nalini; ...

2016-10-26

Here, the neuronal PAS domain proteins NPAS1 and NPAS3 are members of the basic helix-loop-helix-PER-ARNT-SIM (bHLH-PAS) family, and their genetic deficiencies are linked to a variety of human psychiatric disorders including schizophrenia, autism spectrum disorders and bipolar disease. NPAS1 and NPAS3 must each heterodimerize with the aryl hydrocarbon receptor nuclear translocator (ARNT), to form functional transcription complexes capable of DNA binding and gene regulation. Here we examined the crystal structures of multi-domain NPAS1-ARNT and NPAS3-ARNT-DNA complexes, discovering each to contain four putative ligand-binding pockets. Through expanded architectural comparisons between these complexes and HIF-1α-ARNT, HIF-2α-ARNT and CLOCK-BMAL1, we show the widermore » mammalian bHLH-PAS family is capable of multi-ligand-binding and presents as an ideal class of transcription factors for direct targeting by small-molecule drugs.« less

RNA Nanotechnology: Engineering, Assembly and Applications in Detection, Gene Delivery and Therapy

PubMed Central

Guo, Peixuan

2010-01-01

Biological macromolecules including DNA, RNA, and proteins, have intrinsic features that make them potential building blocks for the bottom-up fabrication of nanodevices. RNA is unique in nanoscale fabrication due to its amazing diversity of function and structure. RNA molecules can be designed and manipulated with a level of simplicity characteristic of DNA while possessing versatility in structure and function similar to that of proteins. RNA molecules typically contain a large variety of single stranded loops suitable for inter- and intra-molecular interaction. These loops can serve as mounting dovetails obviating the need for external linking dowels in fabrication and assembly. The self-assembly of nanoparticles from RNA involves cooperative interaction of individual RNA molecules that spontaneously assemble in a predefined manner to form a larger two- or three-dimensional structure. Within the realm of self-assembly there are two main categories, namely template and non-template. Template assembly involves interaction of RNA molecules under the influence of specific external sequence, forces, or spatial constraints such as RNA transcription, hybridization, replication, annealing, molding, or replicas. In contrast, non-template assembly involves formation of a larger structure by individual components without the influence of external forces. Examples of non-template assembly are ligation, chemical conjugation, covalent linkage, and loop/loop interaction of RNA, especially the formation of RNA multimeric complexes. The best characterized RNA multiplier and the first to be described in RNA nanotechnological application is the motor pRNA of bacteriophage phi29 which form dimers, trimers, and hexamers, via hand-in-hand interaction. phi29 pRNA can be redesigned to form a variety of structures and shapes including twins, tetramers, rods, triangles, and 3D arrays several microns in size via interaction of programmed helical regions and loops. 3D RNA array formation requires a defined nucleotide number for twisting and a palindromic sequence. Such arrays are unusually stable and resistant to a wide range of temperatures, salt concentrations, and pH. Both the therapeutic siRNA or ribozyme and a receptor-binding RNA aptamer or other ligands have been engineered into individual pRNAs. Individual chimeric RNA building blocks harboring siRNA or other therapeutic molecules have been fabricated subsequently into a trimer through hand-in-hand interaction of the engineered right and left interlocking RNA loops. The incubation of these particles containing the receptor-binding aptamer or other ligands results in the binding and co-entry of trivalent therapeutic particles into cells. Such particles were subsequently shown to modulate the apoptosis of cancer cells in both cell cultures and animal trials. The use of such antigen-free 20–40 nm particles holds promise for the repeated long-term treatment of chronic diseases. Other potentially useful RNA molecules that form multimers include HIV RNA that contain kissing loop to form dimers, tecto-RNA that forms a “jigsaw puzzle,” and the Drosophila bicoid mRNA that forms multimers via “hand-by-arm” interactions. Applications of RNA molecules involving replication, molding, embossing, and other related techniques, have recently been described that allow the utilization of a variety of materials to enhance diversity and resolution of nanomaterials. It should eventually be possible to adapt RNA to facilitate construction of ordered, patterned, or pre-programmed arrays or superstructures. Given the potential for 3D fabrication, the chance to produce reversible self-assembly, and the ability of self-repair, editing and replication, RNA self-assembly will play an increasingly significant role in integrated biological nanofabrication. A random 100-nucleotide RNA library may exist in 1.6 × 1060 varieties with multifarious structure to serve as a vital system for efficient fabrication, with a complexity and diversity far exceeding that of any current nanoscale system. This review covers the basic concepts of RNA structure and function, certain methods for the study of RNA structure, the approaches for engineering or fabricating RNA into nanoparticles or arrays, and special features of RNA molecules that form multimers. The most recent development in exploration of RNA nanoparticles for pathogen detection, drug/gene delivery, and therapeutic application is also introduced in this review. PMID:16430131

Action at a Distance in the Cell's Nucleus

NASA Astrophysics Data System (ADS)

Kondev, Jane

Various functions performed by chromosomes involve long-range communication between DNA sequences that are tens of thousands of bases apart along the genome, and microns apart in the nucleus. In this talk I will discuss experiments and theory relating to two distinct modes of long-range communication in the nucleus, chromosome looping and protein hopping along the chromosome, both in the context of DNA-break repair in yeast. Yeast is an excellent model system for studies that link chromosome conformations to their function as there is ample experimental evidence that yeast chromosome conformations are well described by a simple, random-walk polymer model. Using a combination of polymer physics theory and experiments on yeast cells, I will demonstrate that loss of polymer entropy due to chromosome looping is the driving force for homology search during repair of broken DNA by homologous recombination. I will also discuss the spread of histone modifications along the chromosome and away from the DNA break point in the context of simple physics models based on chromosome looping and kinase hopping, and show how combining physics theory and cell-biology experiment can be used to dissect the molecular mechanism of the spreading process. These examples demonstrate how combined theoretical and experimental studies can reveal physical principles of long-range communication in the nucleus, which play important roles in regulation of gene expression, DNA recombination, and chromatin modification. This work was supported by the NSF DMR-1206146.

Replication origins oriGNAI3 and oriB of the mammalian AMPD2 locus nested in a region of straight DNA flanked by intrinsically bent DNA sites.

PubMed

Balani, Valério Américo; de Lima Neto, Quirino Alves; Takeda, Karen Izumi; Gimenes, Fabrícia; Fiorini, Adriana; Debatisse, Michelle; Fernandez, Maria Aparecida

2010-11-01

The aim of this work was to determine whether intrinsically bent DNA sites are present at, or close to, the mammalian replication origins oriGNAI3 and oriB in the Chinese hamster AMPD2 locus. Using an electrophoretic mobility shift assay and in silico analysis, we located four intrinsically bent DNA sites (b1 to b4) in a fragment that contains the oriGNAI3 and one site (b5) proximal to oriB. The helical parameters show that each bent DNA site is curved in a left-handed superhelical writhe. A 2D projection of 3D fragment trajectories revealed that oriGNAI3 is located in a relatively straight segment flanked by bent sites b1 and b2, which map in previously identified Scaffold/Matrix Attachment Region. Sites b3 and b4 are located approximately 2 kb downstream and force the fragment into a strong closed loop structure. The b5 site is also located in an S/MAR that is found just downstream of oriB.

Full expression of Bacillus anthracis toxin gene in the presence of bicarbonate requires a 2.7-kb-long atxA mRNA that contains a terminator structure.

PubMed

Bertin, Marine; Château, Alice; Fouet, Agnès

2010-05-01

Bacillus anthracis toxin gene expression requires AtxA, a virulence regulator that also activates capsule gene transcription and controls expression of more than a hundred genes. Here we report that atxA mRNA is 2.7-kb-long and ends, after a 500 nt-long 3' untranslated region, with a stem loop structure followed by a run of U's. The presence of this structure stabilizes atxA mRNA and is necessary for AtxA maximal accumulation, full expression of the PA toxin gene, pagA and optimal PA accumulation. This structure displays terminator activity independently of its orientation when cloned between an inducible promoter and a reporter gene. The 3.6-kb-long DNA fragment carrying both AtxA promoters and the terminator is sufficient for full expression of pagA in the presence of bicarbonate. No pXO1-encoded element other than the DNA fragment encompassing the 2.7 kb atxA transcript and the pagA promoter is required for bicarbonate induction of pagA transcription. (c) 2010 Elsevier Masson SAS. All rights reserved.

Digital LAMP in a sample self-digitization (SD) chip

PubMed Central

Herrick, Alison M.; Dimov, Ivan K.; Lee, Luke P.; Chiu, Daniel T.

2012-01-01

This paper describes the realization of digital loop-mediated DNA amplification (dLAMP) in a sample self-digitization (SD) chip. Digital DNA amplification has become an attractive technique to quantify absolute concentrations of DNA in a sample. While digital polymerase chain reaction is still the most widespread implementation, its use in resource—limited settings is impeded by the need for thermal cycling and robust temperature control. In such situations, isothermal protocols that can amplify DNA or RNA without thermal cycling are of great interest. Here, we showed the successful amplification of single DNA molecules in a stationary droplet array using isothermal digital loop-mediated DNA amplification. Unlike most (if not all) existing methods for sample discretization, our design allows for automated, loss-less digitization of sample volumes on-chip. We demonstrated accurate quantification of relative and absolute DNA concentrations with sample volumes of less than 2 μl. We assessed the homogeneity of droplet size during sample self-digitization in our device, and verified that the size variation was small enough such that straightforward counting of LAMP-active droplets sufficed for data analysis. We anticipate that the simplicity and robustness of our SD chip make it attractive as an inexpensive and easy-to-operate device for DNA amplification, for example in point-of-care settings. PMID:22399016

Comparison of DNA Microarray, Loop-Mediated Isothermal Amplification (LAMP) and Real-Time PCR with DNA Sequencing for Identification of Fusarium spp. Obtained from Patients with Hematologic Malignancies.

PubMed

de Souza, Marcela; Matsuzawa, Tetsuhiro; Sakai, Kanae; Muraosa, Yasunori; Lyra, Luzia; Busso-Lopes, Ariane Fidelis; Levin, Anna Sara Shafferman; Schreiber, Angélica Zaninelli; Mikami, Yuzuru; Gonoi, Tohoru; Kamei, Katsuhiko; Moretti, Maria Luiza; Trabasso, Plínio

2017-08-01

The performance of three molecular biology techniques, i.e., DNA microarray, loop-mediated isothermal amplification (LAMP), and real-time PCR were compared with DNA sequencing for properly identification of 20 isolates of Fusarium spp. obtained from blood stream as etiologic agent of invasive infections in patients with hematologic malignancies. DNA microarray, LAMP and real-time PCR identified 16 (80%) out of 20 samples as Fusarium solani species complex (FSSC) and four (20%) as Fusarium spp. The agreement among the techniques was 100%. LAMP exhibited 100% specificity, while DNA microarray, LAMP and real-time PCR showed 100% sensitivity. The three techniques had 100% agreement with DNA sequencing. Sixteen isolates were identified as FSSC by sequencing, being five Fusarium keratoplasticum, nine Fusarium petroliphilum and two Fusarium solani. On the other hand, sequencing identified four isolates as Fusarium non-solani species complex (FNSSC), being three isolates as Fusarium napiforme and one isolate as Fusarium oxysporum. Finally, LAMP proved to be faster and more accessible than DNA microarray and real-time PCR, since it does not require a thermocycler. Therefore, LAMP signalizes as emerging and promising methodology to be used in routine identification of Fusarium spp. among cases of invasive fungal infections.

A Role for the Fifth G-Track in G-Quadruplex Forming Oncogene Promoter Sequences during Oxidative Stress: Do These "Spare Tires" Have an Evolved Function?

PubMed

Fleming, Aaron M; Zhou, Jia; Wallace, Susan S; Burrows, Cynthia J

2015-08-26

Uncontrolled inflammation or oxidative stress generates electron-deficient species that oxidize the genome increasing its instability in cancer. The G-quadruplex (G4) sequences regulating the c-MYC , KRAS , VEGF , BCL-2 , HIF-1α , and RET oncogenes, as examples, are targets for oxidation at loop and 5'-core guanines (G) as showcased in this study by CO 3 •- oxidation of the VEGF G4. Products observed include 8-oxo-7,8-dihydroguanine (OG), spiroiminodihydantoin (Sp), and 5-guanidinohydantoin (Gh). Our previous studies found that OG and Gh, when present in the four G-tracks of the solved structure for VEGF and c-MY C, were not substrates for the base excision repair (BER) DNA glycosylases in biologically relevant KCl solutions. We now hypothesize that a fifth G-track found a few nucleotides distant from the G4 tracks involved in folding can act as a "spare tire," facilitating extrusion of a damaged G-run into a large loop that then becomes a substrate for BER. Thermodynamic, spectroscopic, and DMS footprinting studies verified the fifth domain replacing a damaged G-track with OG or Gh at a loop or core position in the VEGF G4. These new "spare tire"-containing strands with Gh in loops are now found to be substrates for initiation of BER with the NEIL1, NEIL2, and NEIL3 DNA glycosylases. The results support a hypothesis in which regulatory G4s carry a "spare-tire" fifth G-track for aiding in the repair process when these sequences are damaged by radical oxygen species, a feature observed in a large number of these sequences. Furthermore, formation and repair of oxidized bases in promoter regions may constitute an additional example of epigenetic modification, in this case of guanine bases, to regulate gene expression in which the G4 sequences act as sensors of oxidative stress.

Human lactoferricin derived di-peptides deploying loop structures induce apoptosis specifically in cancer cells through targeting membranous phosphatidylserine.

PubMed

Riedl, Sabrina; Leber, Regina; Rinner, Beate; Schaider, Helmut; Lohner, Karl; Zweytick, Dagmar

2015-11-01

Host defense-derived peptides have emerged as a novel strategy for the development of alternative anticancer therapies. In this study we report on characteristic features of human lactoferricin (hLFcin) derivatives which facilitate specific killing of cancer cells of melanoma, glioblastoma and rhabdomyosarcoma compared with non-specific derivatives and the synthetic peptide RW-AH. Changes in amino acid sequence of hLFcin providing 9-11 amino acids stretched derivatives LF11-316, -318 and -322 only yielded low antitumor activity. However, the addition of the repeat (di-peptide) and the retro-repeat (di-retro-peptide) sequences highly improved cancer cell toxicity up to 100% at 20 μM peptide concentration. Compared to the complete parent sequence hLFcin the derivatives showed toxicity on the melanoma cell line A375 increased by 10-fold and on the glioblastoma cell line U-87mg by 2-3-fold. Reduced killing velocity, apoptotic blebbing, activation of caspase 3/7 and formation of apoptotic DNA fragments proved that the active and cancer selective peptides, e.g. R-DIM-P-LF11-322, trigger apoptosis, whereas highly active, though non-selective peptides, such as DIM-LF11-318 and RW-AH seem to kill rapidly via necrosis inducing membrane lyses. Structural studies revealed specific toxicity on cancer cells by peptide derivatives with loop structures, whereas non-specific peptides comprised α-helical structures without loop. Model studies with the cancer membrane mimic phosphatidylserine (PS) gave strong evidence that PS only exposed by cancer cells is an important target for specific hLFcin derivatives. Other negatively charged membrane exposed molecules as sialic acid, heparan and chondroitin sulfate were shown to have minor impact on peptide activity. Copyright © 2015. Published by Elsevier B.V.

LoopX: A Graphical User Interface-Based Database for Comprehensive Analysis and Comparative Evaluation of Loops from Protein Structures.

PubMed

Kadumuri, Rajashekar Varma; Vadrevu, Ramakrishna

2017-10-01

Due to their crucial role in function, folding, and stability, protein loops are being targeted for grafting/designing to create novel or alter existing functionality and improve stability and foldability. With a view to facilitate a thorough analysis and effectual search options for extracting and comparing loops for sequence and structural compatibility, we developed, LoopX a comprehensively compiled library of sequence and conformational features of ∼700,000 loops from protein structures. The database equipped with a graphical user interface is empowered with diverse query tools and search algorithms, with various rendering options to visualize the sequence- and structural-level information along with hydrogen bonding patterns, backbone φ, ψ dihedral angles of both the target and candidate loops. Two new features (i) conservation of the polar/nonpolar environment and (ii) conservation of sequence and conformation of specific residues within the loops have also been incorporated in the search and retrieval of compatible loops for a chosen target loop. Thus, the LoopX server not only serves as a database and visualization tool for sequence and structural analysis of protein loops but also aids in extracting and comparing candidate loops for a given target loop based on user-defined search options.

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.

The structure of the L3 loop from the hepatitis delta virus ribozyme: a syn cytidine.

PubMed Central

Lynch, S R; Tinoco, I

1998-01-01

The structure of the L3 central hairpin loop isolated from the antigenomic sequence of the hepatitis delta virus ribozyme with the P2 and P3 stems from the ribozyme stacked on top of the loop has been determined by NMR spectroscopy. The 26 nt stem-loop structure contains nine base pairs and a 7 nt loop (5'-UCCUCGC-3'). This hairpin loop is critical for efficient catalysis in the intact ribozyme. The structure was determined using homonuclear and heteronuclear NMR techniques on non-labeled and15N-labeled RNA oligonucleotides. The overall root mean square deviation for the structure was 1.15 A (+/- 0.28 A) for the loop and the closing C.G base pair and 0.90 A (+/- 0.18 A) for the loop and the closing C.G base pair but without the lone purine in the loop, which is not well defined in the structure. The structure indicates a U.C base pair between the nucleotides on the 5'- and 3'-ends of the loop. This base pair is formed with a single hydrogen bond involving the cytosine exocyclic amino proton and the carbonyl O4 of the uracil. The most unexpected finding in the loop is a syn cytidine. While not unprecedented, syn pyrimidines are highly unusual. This one can be confidently established by intranucleotide distances between the ribose and the base determined by NMR spectroscopy. A similar study of the structure of this loop showed a somewhat different three-dimensional structure. A discussion of differences in the two structures, as well as possible sites of interaction with the cleavage site, will be presented. PMID:9461457

Exact calculation of loop formation probability identifies folding motifs in RNA secondary structures

PubMed Central

Sloma, Michael F.; Mathews, David H.

2016-01-01

RNA secondary structure prediction is widely used to analyze RNA sequences. In an RNA partition function calculation, free energy nearest neighbor parameters are used in a dynamic programming algorithm to estimate statistical properties of the secondary structure ensemble. Previously, partition functions have largely been used to estimate the probability that a given pair of nucleotides form a base pair, the conditional stacking probability, the accessibility to binding of a continuous stretch of nucleotides, or a representative sample of RNA structures. Here it is demonstrated that an RNA partition function can also be used to calculate the exact probability of formation of hairpin loops, internal loops, bulge loops, or multibranch loops at a given position. This calculation can also be used to estimate the probability of formation of specific helices. Benchmarking on a set of RNA sequences with known secondary structures indicated that loops that were calculated to be more probable were more likely to be present in the known structure than less probable loops. Furthermore, highly probable loops are more likely to be in the known structure than the set of loops predicted in the lowest free energy structures. PMID:27852924

Mathematical model of alternative mechanism of telomere length maintenance

NASA Astrophysics Data System (ADS)

Kollár, Richard; Bod'ová, Katarína; Nosek, Jozef; Tomáška, L'ubomír

2014-03-01

Biopolymer length regulation is a complex process that involves a large number of biological, chemical, and physical subprocesses acting simultaneously across multiple spatial and temporal scales. An illustrative example important for genomic stability is the length regulation of telomeres—nucleoprotein structures at the ends of linear chromosomes consisting of tandemly repeated DNA sequences and a specialized set of proteins. Maintenance of telomeres is often facilitated by the enzyme telomerase but, particularly in telomerase-free systems, the maintenance of chromosomal termini depends on alternative lengthening of telomeres (ALT) mechanisms mediated by recombination. Various linear and circular DNA structures were identified to participate in ALT, however, dynamics of the whole process is still poorly understood. We propose a chemical kinetics model of ALT with kinetic rates systematically derived from the biophysics of DNA diffusion and looping. The reaction system is reduced to a coagulation-fragmentation system by quasi-steady-state approximation. The detailed treatment of kinetic rates yields explicit formulas for expected size distributions of telomeres that demonstrate the key role played by the J factor, a quantitative measure of bending of polymers. The results are in agreement with experimental data and point out interesting phenomena: an appearance of very long telomeric circles if the total telomere density exceeds a critical value (excess mass) and a nonlinear response of the telomere size distributions to the amount of telomeric DNA in the system. The results can be of general importance for understanding dynamics of telomeres in telomerase-independent systems as this mode of telomere maintenance is similar to the situation in tumor cells lacking telomerase activity. Furthermore, due to its universality, the model may also serve as a prototype of an interaction between linear and circular DNA structures in various settings.

Loop-mediated isothermal amplification for detection of the tomato and potato late blight pathogen, Phytophthora infestans

USDA-ARS?s Scientific Manuscript database

Aims: To design and validate a colorimetric loop-mediated isothermal amplification assay for rapid detection of P. infestans DNA. Methods and Results: Two sets of LAMP primers were designed and evaluated for their sensitivity and specificity for P. infestans. ITSII primers targeted a portion of the ...

Development of a novel loop-mediated isothermal amplification (LAMP) assay for the detection of Salmonella ser. Enteritidis from egg products

USDA-ARS?s Scientific Manuscript database

Salmonella ser. Enteritidis is a major public health concern worldwide. Loop-mediated isothermal amplification (LAMP) is a novel simple, easy-to-operate detection technology that amplifies DNA with high speed, efficiency, and specificity under isothermal conditions. The objective of this study was t...

DNA damage mediated transcription arrest: Step back to go forward.

PubMed

Mullenders, Leon

2015-12-01

The disturbance of DNA helix conformation by bulky DNA damage poses hindrance to transcription elongating due to stalling of RNA polymerase at transcription blocking lesions. Stalling of RNA polymerase provokes the formation of R-loops, i.e. the formation of a DNA-RNA hybrid and a displaced single stranded DNA strand as well as displacement of spliceosomes. R-loops are processed into DNA single and double strand breaks by NER factors depending on TC-NER factors leading to genome instability. Moreover, stalling of RNA polymerase induces a strong signal for cell cycle arrest and apoptosis. These toxic and mutagenic effects are counteracted by a rapid recruitment of DNA repair proteins to perform transcription coupled nucleotide excision repair (TC-NER) to remove the blocking DNA lesions and to restore transcription. Recent studies have highlighted the role of backtracking of RNA polymerase to facilitate TC-NER and identified novel factors that play key roles in TC-NER and in restoration of transcription. On the molecular level these factors facilitate stability of the repair complex by promotion and regulation of various post-translational modifications of NER factors and chromatin substrate. In addition, the continuous flow of new factors that emerge from screening assays hints to several regulatory levels to safeguard the integrity of transcription elongation after disturbance by DNA damage that have yet to be explored. Copyright © 2015 Elsevier B.V. All rights reserved.

Mitochondrial DNA haplogroup phylogeny of the dog: Proposal for a cladistic nomenclature.

PubMed

Fregel, Rosa; Suárez, Nicolás M; Betancor, Eva; González, Ana M; Cabrera, Vicente M; Pestano, José

2015-05-01

Canis lupus familiaris mitochondrial DNA analysis has increased in recent years, not only for the purpose of deciphering dog domestication but also for forensic genetic studies or breed characterization. The resultant accumulation of data has increased the need for a normalized and phylogenetic-based nomenclature like those provided for human maternal lineages. Although a standardized classification has been proposed, haplotype names within clades have been assigned gradually without considering the evolutionary history of dog mtDNA. Moreover, this classification is based only on the D-loop region, proven to be insufficient for phylogenetic purposes due to its high number of recurrent mutations and the lack of relevant information present in the coding region. In this study, we design 1) a refined mtDNA cladistic nomenclature from a phylogenetic tree based on complete sequences, classifying dog maternal lineages into haplogroups defined by specific diagnostic mutations, and 2) a coding region SNP analysis that allows a more accurate classification into haplogroups when combined with D-loop sequencing, thus improving the phylogenetic information obtained in dog mitochondrial DNA studies. Copyright © 2015 Elsevier B.V. All rights reserved.

Schizosaccharomyces pombe MutSα and MutLα Maintain Stability of Tetra-Nucleotide Repeats and Msh3 of Hepta-Nucleotide Repeats

PubMed Central

Villahermosa, Desirée; Christensen, Olaf; Knapp, Karen; Fleck, Oliver

2017-01-01

Defective mismatch repair (MMR) in humans is associated with colon cancer and instability of microsatellites, that is, DNA sequences with one or several nucleotides repeated. Key factors of eukaryotic MMR are the heterodimers MutSα (Msh2-Msh6), which recognizes base-base mismatches and unpaired nucleotides in DNA, and MutLα (Mlh1-Pms1), which facilitates downstream steps. In addition, MutSβ (Msh2-Msh3) recognizes DNA loops of various sizes, although our previous data and the data presented here suggest that Msh3 of Schizosaccharomyces pombe does not play a role in MMR. To test microsatellite stability in S. pombe and hence DNA loop repair, we have inserted tetra-, penta-, and hepta-nucleotide repeats in the ade6 gene and determined their Ade+ reversion rates and spectra in wild type and various mutants. Our data indicate that loops with four unpaired nucleotides in the nascent and the template strand are the upper limit of MutSα- and MutLα-mediated MMR in S. pombe. Stability of hepta-nucleotide repeats requires Msh3 and Exo1 in MMR-independent processes as well as the DNA repair proteins Rad50, Rad51, and Rad2FEN1. Most strikingly, mutation rates in the double mutants msh3 exo1 and msh3 rad51 were decreased when compared to respective single mutants, indicating that Msh3 prevents error prone processes carried out by Exo1 and Rad51. We conclude that Msh3 has no obvious function in MMR in S. pombe, but contributes to DNA repeat stability in MMR-independent processes. PMID:28341698

Schizosaccharomyces pombe MutSα and MutLα Maintain Stability of Tetra-Nucleotide Repeats and Msh3 of Hepta-Nucleotide Repeats.

PubMed

Villahermosa, Desirée; Christensen, Olaf; Knapp, Karen; Fleck, Oliver

2017-05-05

Defective mismatch repair (MMR) in humans is associated with colon cancer and instability of microsatellites, that is, DNA sequences with one or several nucleotides repeated. Key factors of eukaryotic MMR are the heterodimers MutSα (Msh2-Msh6), which recognizes base-base mismatches and unpaired nucleotides in DNA, and MutLα (Mlh1-Pms1), which facilitates downstream steps. In addition, MutSβ (Msh2-Msh3) recognizes DNA loops of various sizes, although our previous data and the data presented here suggest that Msh3 of Schizosaccharomyces pombe does not play a role in MMR. To test microsatellite stability in S. pombe and hence DNA loop repair, we have inserted tetra-, penta-, and hepta-nucleotide repeats in the ade6 gene and determined their Ade + reversion rates and spectra in wild type and various mutants. Our data indicate that loops with four unpaired nucleotides in the nascent and the template strand are the upper limit of MutSα- and MutLα-mediated MMR in S. pombe Stability of hepta-nucleotide repeats requires Msh3 and Exo1 in MMR-independent processes as well as the DNA repair proteins Rad50, Rad51, and Rad2 FEN1 Most strikingly, mutation rates in the double mutants msh3 exo1 and msh3 rad51 were decreased when compared to respective single mutants, indicating that Msh3 prevents error prone processes carried out by Exo1 and Rad51. We conclude that Msh3 has no obvious function in MMR in S. pombe , but contributes to DNA repeat stability in MMR-independent processes. Copyright © 2017 Villahermosa et al.

Detection and differentiation of Fusarium oxysporum f. sp. lycopersici race 1 using loop-mediated isothermal amplification with three primer sets.

PubMed

Ayukawa, Y; Komatsu, K; Kashiwa, T; Akai, K; Yamada, M; Teraoka, T; Arie, T

2016-09-01

Fusarium oxysporum f. sp. lycopersici (Fol) causes tomato wilt. Based on the difference in pathogenicity towards tomato cultivars, Fol is classified into three races. In this study, a rapid method is developed for the detection and discrimination of Fol race 1 using a loop-mediated isothermal amplification (LAMP) assay with two primer sets targeting a region of the nucleotide sequence of the SIX4 gene specific for race 1 and a primer set targeting the SIX5 gene, conserved in all known Fol isolates. Upon LAMP reaction, amplification using all three primer sets was observed only when DNA of Fol race 1 was used as a template, and not when DNA of other Fol races or other fungal species was used. This method could detect 300 fg of Fol race 1 DNA, a 100-fold higher sensitivity than that obtained by conventional PCR. The method can also detect DNA extracted from soil artificially infested with Fol race 1. It is now possible to detect Fol race 1 in colonies and infected tomato stems without DNA isolation. This method is a rapid and simple tool for discrimination of Fol race 1. This study developed a loop-mediated isothermal amplification (LAMP) assay for detection and differentiation of Fusarium oxysporum f. sp. lycopersici (Fol) race 1 by using three primer sets targeting for the SIX4 and SIX5 genes. These genes are present together only in Fol race 1. This method can detect Fol race 1 in infected tomato stems without DNA extraction, affording an efficient diagnosis of Fusarium wilt on tomatoes in the field. © 2016 The Society for Applied Microbiology.

Initiator-catalyzed self-assembly of duplex-looped DNA hairpin motif based on strand displacement reaction for logic operations and amplified biosensing.

PubMed

Bi, Sai; Yue, Shuzhen; Wu, Qiang; Ye, Jiayan

2016-09-15

Here we program an initiator-catalyzed self-assembly of duplex-looped DNA hairpin motif based on strand displacement reaction. Due to the recycling of initiator and performance in a cascade manner, this system is versatilely extended to logic operations, including the construction of concatenated logic circuits with a feedback function and a biocomputing keypad-lock security system. Compared with previously reported molecular security systems, the prominent feature of our keypad lock is that it can be spontaneously reset and recycled with no need of any external stimulus and human intervention. Moreover, through integrating with an isothermal amplification technique of rolling circle amplification (RCA), this programming catalytic DNA self-assembly strategy readily achieves sensitive and selective biosensing of initiator. Importantly, a magnetic graphene oxide (MGO) is introduced to remarkably reduced background, which plays an important role in enhancing the signal-to-noise ratio and improving the detection sensitivity. Therefore, the proposed sophisticated DNA strand displacement-based methodology with engineering dynamic functions may find broad applications in the construction of programming DNA nanostructures, amplification biosensing platform, and large-scale DNA circuits. Copyright © 2016 Elsevier B.V. All rights reserved.

Net (ERP/SAP2) one of the Ras-inducible TCFs, has a novel inhibitory domain with resemblance to the helix-loop-helix motif.

PubMed Central

Maira, S M; Wurtz, J M; Wasylyk, B

1996-01-01

The three ternary complex factors (TCFs), Net (ERP/ SAP-2), ELK-1 and SAP-1, are highly related ets oncogene family members that participate in the response of the cell to Ras and growth signals. Understanding the different roles of these factors will provide insights into how the signals result in coordinate regulation of the cell. We show that Net inhibits transcription under basal conditions, in which SAP-1a is inactive and ELK-1 stimulates. Repression is mediated by the NID, the Net Inhibitory Domain of about 50 amino acids, which autoregulates the Net protein and also inhibits when it is isolated in a heterologous fusion protein. Net is particularly sensitive to Ras activation. Ras activates Net through the C-domain, which is conserved between the three TCFs, and the NID is an efficient inhibitor of Ras activation. The NID, as well as more C-terminal sequences, inhibit DNA binding. Net is more refractory to DNA binding than the other TCFs, possibly due to the presence of multiple inhibitory elements. The NID may adopt a helix-loop-helix (HLH) structure, as evidenced by homology to other HLH motifs, structure predictions, model building and mutagenesis of critical residues. The sequence resemblance with myogenic factors suggested that Net may form complexes with the same partners. Indeed, we found that Net can interact in vivo with the basic HLH factor, E47. We propose that Net is regulated at the level of its latent DNA-binding activity by protein interactions and/or phosphorylation. Net may form complexes with HLH proteins as well as SRF on specific promotor sequences. The identification of the novel inhibitory domain provides a new inroad into exploring the different roles of the ternary complex factors in growth control and transformation. Images PMID:8918463

Net (ERP/SAP2) one of the Ras-inducible TCFs, has a novel inhibitory domain with resemblance to the helix-loop-helix motif.

PubMed

Maira, S M; Wurtz, J M; Wasylyk, B

1996-11-01

The three ternary complex factors (TCFs), Net (ERP/ SAP-2), ELK-1 and SAP-1, are highly related ets oncogene family members that participate in the response of the cell to Ras and growth signals. Understanding the different roles of these factors will provide insights into how the signals result in coordinate regulation of the cell. We show that Net inhibits transcription under basal conditions, in which SAP-1a is inactive and ELK-1 stimulates. Repression is mediated by the NID, the Net Inhibitory Domain of about 50 amino acids, which autoregulates the Net protein and also inhibits when it is isolated in a heterologous fusion protein. Net is particularly sensitive to Ras activation. Ras activates Net through the C-domain, which is conserved between the three TCFs, and the NID is an efficient inhibitor of Ras activation. The NID, as well as more C-terminal sequences, inhibit DNA binding. Net is more refractory to DNA binding than the other TCFs, possibly due to the presence of multiple inhibitory elements. The NID may adopt a helix-loop-helix (HLH) structure, as evidenced by homology to other HLH motifs, structure predictions, model building and mutagenesis of critical residues. The sequence resemblance with myogenic factors suggested that Net may form complexes with the same partners. Indeed, we found that Net can interact in vivo with the basic HLH factor, E47. We propose that Net is regulated at the level of its latent DNA-binding activity by protein interactions and/or phosphorylation. Net may form complexes with HLH proteins as well as SRF on specific promotor sequences. The identification of the novel inhibitory domain provides a new inroad into exploring the different roles of the ternary complex factors in growth control and transformation.

Thermodynamic stability of RNA structures formed by CNG trinucleotide repeats. Implication for prediction of RNA structure.

PubMed

Broda, Magdalena; Kierzek, Elzbieta; Gdaniec, Zofia; Kulinski, Tadeusz; Kierzek, Ryszard

2005-08-16

Trinucleotide repeat expansion diseases (TREDs) are correlated with elongation of CNG DNA and RNA repeats to pathological level. This paper shows, for the first time, complete data concerning thermodynamic stabilities of RNA with CNG trinucleotide repeats. Our studies include the stability of oligoribonucleotides composed of two to seven of CAG, CCG, CGG, and CUG repeats. The thermodynamic parameters of helix propagation correlated with the presence of multiple N-N mismatches within CNG RNA duplexes were also determined. Moreover, the total stability of CNG RNA hairpins, as well as the contribution of trinucleotide repeats placed only in the stem or loop regions, was evaluated. The improved thermodynamic parameters allow to predict much more accurately the thermodynamic stabilities and structures of CNG RNAs.

A novel mode for transcription inhibition mediated by PNA-induced R-loops with a model in vitro system.

PubMed

D'Souza, Alicia D; Belotserkovskii, Boris P; Hanawalt, Philip C

2018-02-01

The selective inhibition of transcription of a chosen gene by an artificial agent has numerous applications. Usually, these agents are designed to bind a specific nucleotide sequence in the promoter or within the transcribed region of the chosen gene. However, since optimal binding sites might not exist within the gene, it is of interest to explore the possibility of transcription inhibition when the agent is designed to bind at other locations. One of these possibilities arises when an additional transcription initiation site (e.g. secondary promoter) is present upstream from the primary promoter of the target gene. In this case, transcription inhibition might be achieved by inducing the formation of an RNA-DNA hybrid (R-loop) upon transcription from the secondary promoter. The R-loop could extend into the region of the primary promoter, to interfere with promoter recognition by RNA polymerase and thereby inhibit transcription. As a sequence-specific R-loop-inducing agent, a peptide nucleic acid (PNA) could be designed to facilitate R-loop formation by sequestering the non-template DNA strand. To investigate this mode for transcription inhibition, we have employed a model system in which a PNA binding site is localized between the T3 and T7 phage RNA polymerase promoters, which respectively assume the roles of primary and secondary promoters. In accord with our model, we have demonstrated that with PNA-bound DNA substrates, transcription from the T7 promoter reduces transcription from the T3 promoter by 30-fold, while in the absence of PNA binding there is no significant effect of T7 transcription upon T3 transcription. Copyright © 2018 Elsevier B.V. All rights reserved.

DNA strand-exchange patterns associated with double-strand break-induced and spontaneous mitotic crossovers in Saccharomyces cerevisiae

PubMed Central

2018-01-01

Mitotic recombination can result in loss of heterozygosity and chromosomal rearrangements that shape genome structure and initiate human disease. Engineered double-strand breaks (DSBs) are a potent initiator of recombination, but whether spontaneous events initiate with the breakage of one or both DNA strands remains unclear. In the current study, a crossover (CO)-specific assay was used to compare heteroduplex DNA (hetDNA) profiles, which reflect strand exchange intermediates, associated with DSB-induced versus spontaneous events in yeast. Most DSB-induced CO products had the two-sided hetDNA predicted by the canonical DSB repair model, with a switch in hetDNA position from one product to the other at the position of the break. Approximately 40% of COs, however, had hetDNA on only one side of the initiating break. This anomaly can be explained by a modified model in which there is frequent processing of an early invasion (D-loop) intermediate prior to extension of the invading end. Finally, hetDNA tracts exhibited complexities consistent with frequent expansion of the DSB into a gap, migration of strand-exchange junctions, and template switching during gap-filling reactions. hetDNA patterns in spontaneous COs isolated in either a wild-type background or in a background with elevated levels of reactive oxygen species (tsa1Δ mutant) were similar to those associated with the DSB-induced events, suggesting that DSBs are the major instigator of spontaneous mitotic recombination in yeast. PMID:29579095

Genome-wide analysis of basic helix-loop-helix (bHLH) transcription factors in Brachypodium distachyon.

PubMed

Niu, Xin; Guan, Yuxiang; Chen, Shoukun; Li, Haifeng

2017-08-15

As a superfamily of transcription factors (TFs), the basic helix-loop-helix (bHLH) proteins have been characterized functionally in many plants with a vital role in the regulation of diverse biological processes including growth, development, response to various stresses, and so on. However, no systemic analysis of the bHLH TFs has been reported in Brachypodium distachyon, an emerging model plant in Poaceae. A total of 146 bHLH TFs were identified in the Brachypodium distachyon genome and classified into 24 subfamilies. BdbHLHs in the same subfamily share similar protein motifs and gene structures. Gene duplication events showed a close relationship to rice, maize and sorghum, and segment duplications might play a key role in the expansion of this gene family. The amino acid sequence of the bHLH domains were quite conservative, especially Leu-27 and Leu-54. Based on the predicted binding activities, the BdbHLHs were divided into DNA binding and non-DNA binding types. According to the gene ontology (GO) analysis, BdbHLHs were speculated to function in homodimer or heterodimer manner. By integrating the available high throughput data in public database and results of quantitative RT-PCR, we found the expression profiles of BdbHLHs were different, implying their differentiated functions. One hundred fourty-six BdbHLHs were identified and their conserved domains, sequence features, phylogenetic relationship, chromosomal distribution, GO annotations, gene structures, gene duplication and expression profiles were investigated. Our findings lay a foundation for further evolutionary and functional elucidation of BdbHLH genes.

Rapid and sensitive identification of the herbal tea ingredient Taraxacum formosanum using loop-mediated isothermal amplification.

PubMed

Lai, Guan-Hua; Chao, Jung; Lin, Ming-Kuem; Chang, Wen-Te; Peng, Wen-Huang; Sun, Fang-Chun; Lee, Meng-Shiunn; Lee, Meng-Shiou

2015-01-09

Taraxacum formosanum (TF) is a medicinal plant used as an important component of health drinks in Taiwan. In this study, a rapid, sensitive and specific loop-mediated isothermal amplification (LAMP) assay for authenticating TF was established. A set of four specific LAMP primers was designed based on the nucleotide sequence of the internal transcribed spacer 2 (ITS2) nuclear ribosomal DNA (nrDNA) of TF. LAMP amplicons were successfully amplified and detected when purified genomic DNA of TF was added in the LAMP reaction under isothermal condition (65 °C) within 45 min. These specific LAMP primers have high specificity and can accurately discriminate Taraxacum formosanum from other adulterant plants; 1 pg of genomic DNA was determined to be the detection limit of the LAMP assay. In conclusion, using this novel approach, TF and its misused plant samples obtained from herbal tea markets were easily identified and discriminated by LAMP assay for quality control.

Rapid and Sensitive Identification of the Herbal Tea Ingredient Taraxacum formosanum Using Loop-Mediated Isothermal Amplification

PubMed Central

Lai, Guan-Hua; Chao, Jung; Lin, Ming-Kuem; Chang, Wen-Te; Peng, Wen-Huang; Sun, Fang-Chun; Lee, Meng-Shiunn; Lee, Meng-Shiou

2015-01-01

Taraxacum formosanum (TF) is a medicinal plant used as an important component of health drinks in Taiwan. In this study, a rapid, sensitive and specific loop-mediated isothermal amplification (LAMP) assay for authenticating TF was established. A set of four specific LAMP primers was designed based on the nucleotide sequence of the internal transcribed spacer 2 (ITS2) nuclear ribosomal DNA (nrDNA) of TF. LAMP amplicons were successfully amplified and detected when purified genomic DNA of TF was added in the LAMP reaction under isothermal condition (65 °C) within 45 min. These specific LAMP primers have high specificity and can accurately discriminate Taraxacum formosanum from other adulterant plants; 1 pg of genomic DNA was determined to be the detection limit of the LAMP assay. In conclusion, using this novel approach, TF and its misused plant samples obtained from herbal tea markets were easily identified and discriminated by LAMP assay for quality control. PMID:25584616

Maintenance of a functional higher order chromatin structure: The role of the nuclear matrix in normal and disease states

PubMed Central

Linnemann, Amelia K.; Krawetz, Stephen A.

2010-01-01

Summary The ordered packaging of DNA within the nucleus of somatic cells reflects a dynamic supportive structure that facilitates stable transcription interrupted by intermittent cycles of extreme condensation. This dynamic mode of packing and unpacking chromatin is intimately linked to the ability of the genome to specifically complex with both histones and non-histone proteins. Understanding the underlying mechanism that governs the formation of higher order chromatin structures is a key to understanding how local architecture modulates transcription. In part, the formation of these structures appears to be regulated through genomic looping that is dynamically mediated by attachment to the nuclear scaffold/matrix at S/MARs, i.e., Scaffold/Matrix Attachment Regions. Although the mechanism guiding the formation and use of these higher-ordered structures remains unknown, S/MARs continue to reveal a multitude of roles in development and the pathogenesis of disease. PMID:20948980

Maintenance of a functional higher order chromatin structure: The role of the nuclear matrix in normal and disease states.

PubMed

Linnemann, Amelia K; Krawetz, Stephen A

2009-01-01

The ordered packaging of DNA within the nucleus of somatic cells reflects a dynamic supportive structure that facilitates stable transcription interrupted by intermittent cycles of extreme condensation. This dynamic mode of packing and unpacking chromatin is intimately linked to the ability of the genome to specifically complex with both histones and non-histone proteins. Understanding the underlying mechanism that governs the formation of higher order chromatin structures is a key to understanding how local architecture modulates transcription. In part, the formation of these structures appears to be regulated through genomic looping that is dynamically mediated by attachment to the nuclear scaffold/matrix at S/MARs, i.e., Scaffold/Matrix Attachment Regions. Although the mechanism guiding the formation and use of these higher-ordered structures remains unknown, S/MARs continue to reveal a multitude of roles in development and the pathogenesis of disease.

Back to the Origin

PubMed Central

Evertts, Adam G.

2012-01-01

In bacteria, replication is a carefully orchestrated event that unfolds the same way for each bacterium and each cell division. The process of DNA replication in bacteria optimizes cell growth and coordinates high levels of simultaneous replication and transcription. In metazoans, the organization of replication is more enigmatic. The lack of a specific sequence that defines origins of replication has, until recently, severely limited our ability to define the organizing principles of DNA replication. This question is of particular importance as emerging data suggest that replication stress is an important contributor to inherited genetic damage and the genomic instability in tumors. We consider here the replication program in several different organisms including recent genome-wide analyses of replication origins in humans. We review recent studies on the role of cytosine methylation in replication origins, the role of transcriptional looping and gene gating in DNA replication, and the role of chromatin’s 3-dimensional structure in DNA replication. We use these new findings to consider several questions surrounding DNA replication in metazoans: How are origins selected? What is the relationship between replication and transcription? How do checkpoints inhibit origin firing? Why are there early and late firing origins? We then discuss whether oncogenes promote cancer through a role in DNA replication and whether errors in DNA replication are important contributors to the genomic alterations and gene fusion events observed in cancer. We conclude with some important areas for future experimentation. PMID:23634256

Whole-loop mitochondrial DNA D-loop sequence variability in Egyptian Arabian equine matrilines

PubMed Central

Hudson, William

2017-01-01

Background Egyptian Arabian horses have been maintained in a state of genetic isolation for over a hundred years. There is only limited genetic proof that the studbook records of female lines of Egyptian Arabian pedigrees are reliable. This study characterized the mitochondrial DNA (mtDNA) signatures of 126 horses representing 14 matrilines in the Egyptian Agricultural Organization (EAO) horse-breeding program. Findings Analysis of the whole D-loop sequence yielded additional information compared to hypervariable region-1 (HVR1) analysis alone, with 42 polymorphic sites representing ten haplotypes compared to 16 polymorphic sites representing nine haplotypes, respectively. Most EAO haplotypes belonged to ancient haplogroups, suggesting origin from a wide geographical area over many thousands of years, although one haplotype was novel. Conclusions Historical families share haplotypes and some individuals from different strains belonged to the same haplogroup: the classical EAO strain designation is not equivalent to modern monophyletic matrilineal groups. Phylogenetic inference showed that the foundation mares of the historical haplotypes were highly likely to have the same haplotypes as the animals studied (p > 0.998 in all cases), confirming the reliability of EAO studbook records and providing the opportunity for breeders to confirm the ancestry of their horses. PMID:28859174

Exact calculation of loop formation probability identifies folding motifs in RNA secondary structures.

PubMed

Sloma, Michael F; Mathews, David H

2016-12-01

RNA secondary structure prediction is widely used to analyze RNA sequences. In an RNA partition function calculation, free energy nearest neighbor parameters are used in a dynamic programming algorithm to estimate statistical properties of the secondary structure ensemble. Previously, partition functions have largely been used to estimate the probability that a given pair of nucleotides form a base pair, the conditional stacking probability, the accessibility to binding of a continuous stretch of nucleotides, or a representative sample of RNA structures. Here it is demonstrated that an RNA partition function can also be used to calculate the exact probability of formation of hairpin loops, internal loops, bulge loops, or multibranch loops at a given position. This calculation can also be used to estimate the probability of formation of specific helices. Benchmarking on a set of RNA sequences with known secondary structures indicated that loops that were calculated to be more probable were more likely to be present in the known structure than less probable loops. Furthermore, highly probable loops are more likely to be in the known structure than the set of loops predicted in the lowest free energy structures. © 2016 Sloma and Mathews; Published by Cold Spring Harbor Laboratory Press for the RNA Society.

Interaction of the host protein NbDnaJ with Potato virus X minus-strand stem-loop 1 RNA and capsid protein affects viral replication and movement.

PubMed

Cho, Sang-Yun; Cho, Won Kyong; Sohn, Seong-Han; Kim, Kook-Hyung

2012-01-06

Plant viruses must interact with host cellular components to replicate and move from cell to cell. In the case of Potato virus X (PVX), it carries stem-loop 1 (SL1) RNA essential for viral replication and movement. Using two-dimensional electrophoresis northwestern blot analysis, we previously identified several host proteins that bind to SL1 RNA. Of those, we further characterized a DnaJ-like protein from Nicotiana benthamiana named NbDnaJ. An electrophoretic mobility shift assay confirmed that NbDnaJ binds only to SL1 minus-strand RNA, and bimolecular fluorescence complementation (BiFC) indicated that NbDnaJ interacts with PVX capsid protein (CP). Using a series of deletion mutants, the C-terminal region of NbDnaJ was found to be essential for the interaction with PVX CP. The expression of NbDnaJ significantly changed upon infection with different plant viruses such as PVX, Tobacco mosaic virus, and Cucumber mosaic virus, but varied depending on the viral species. In transient experiments, both PVX replication and movement were inhibited in plants that over-expressed NbDnaJ but accelerated in plants in which NbDnaJ was silenced. In summary, we suggest that the newly identified NbDnaJ plays a role in PVX replication and movement by interacting with SL1(-) RNA and PVX CP. Copyright © 2011 Elsevier Inc. All rights reserved.

Disruption of a stem-loop structure located upstream of pseudoknot domain in Tobacco mosaic virus enhanced its infectivity and viral RNA accumulation.

PubMed

Guo, Song; Wong, Sek-Man

2018-06-01

A predicted stem-loop structure of 25 nucleotides, located in the coat protein (CP) gene and 3'-UTR sequences of Tobacco mosaic virus (TMV), was validated previously (Guo et al., 2015). In this study, both disrupted stem-loop and nucleotide deletion mutants of TMV replicated more rapidly in Nicotiana benthamiana protoplasts. The TMV mutant with a complete mirrored stem-loop structure showed similar level of viral RNA accumulation as TMV. Recovering the stem-loop structure also resulted in a similar replication level as TMV. All these mutants induced necrosis in N. benthamiana and assembled into typical rigid rod-shaped virions. TMV mutant without the stem-loop structure induced more local lesions in Chenopodium quinoa. When the putative stem-loop structure in Tomato mosaic virus (ToMV) was disrupted, the mutant also showed an enhanced virus replication. This suggests that the stem-loop structure of TMV is a new cis-acting element with a role in virus replication. Copyright © 2018 Elsevier Inc. All rights reserved.

Channel Size Conversion of Phi29 DNA-Packaging Nanomotor for Discrimination of Single- and Double-Stranded Nucleic Acids

PubMed Central

Geng, Jia; Wang, Shaoying; Fang, Huaming; Guo, Peixuan

2013-01-01

Nanopores have been utilized to detect the conformation and dynamics of polymers, including DNA and RNA. Biological pores are extremely reproducible at the atomic level with uniform channel sizes. The channel of the bacterial virus phi29 DNA packaging motor is a natural conduit for the transportation of double-stranded DNA (dsDNA), and has the largest diameter among the well-studied biological channels. The larger channel facilitates translocation of dsDNA, and offers more space for further channel modification and conjugation. Interestingly, the relatively large wild type channel, which translocates dsDNA, cannot detect single-stranded nucleic acids (ssDNA or ssRNA) under the current experimental conditions. Herein, we reengineered this motor channel by removing the internal loop segment of the channel. The modification resulted in two classes of channels. One class was the same size as the wild type channel, while the other class had a cross-sectional area about 60% of the wild type. This smaller channel was able to detect the real-time translocation of single stranded nucleic acids at single-molecule level. While the wild type connector exhibited a one-way traffic property with respect to dsDNA translocation, the loop deleted connector was able to translocate ssDNA and ssRNA with equal competencies from both termini. This finding of size alterations in reengineered motor channels expands the potential application of the phi29 DNA packaging motor in nanomedicine, nanobiotechnology, and high-throughput single pore DNA sequencing. PMID:23488809

[Correlation of codon biases and potential secondary structures with mRNA translation efficiency in unicellular organisms].

PubMed

Vladimirov, N V; Likhoshvaĭ, V A; Matushkin, Iu G

2007-01-01

Gene expression is known to correlate with degree of codon bias in many unicellular organisms. However, such correlation is absent in some organisms. Recently we demonstrated that inverted complementary repeats within coding DNA sequence must be considered for proper estimation of translation efficiency, since they may form secondary structures that obstruct ribosome movement. We have developed a program for estimation of potential coding DNA sequence expression in defined unicellular organism using its genome sequence. The program computes elongation efficiency index. Computation is based on estimation of coding DNA sequence elongation efficiency, taking into account three key factors: codon bias, average number of inverted complementary repeats, and free energy of potential stem-loop structures formed by the repeats. The influence of these factors on translation is numerically estimated. An optimal proportion of these factors is computed for each organism individually. Quantitative translational characteristics of 384 unicellular organisms (351 bacteria, 28 archaea, 5 eukaryota) have been computed using their annotated genomes from NCBI GenBank. Five potential evolutionary strategies of translational optimization have been determined among studied organisms. A considerable difference of preferred translational strategies between Bacteria and Archaea has been revealed. Significant correlations between elongation efficiency index and gene expression levels have been shown for two organisms (S. cerevisiae and H. pylori) using available microarray data. The proposed method allows to estimate numerically the coding DNA sequence translation efficiency and to optimize nucleotide composition of heterologous genes in unicellular organisms. http://www.mgs.bionet.nsc.ru/mgs/programs/eei-calculator/.