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1
Maslowska, Katarzyna H., Karolina Makiela-Dzbenska, Jin-Yao Mo, Iwona J. Fijalkowska, and Roel M. Schaaper. "High-accuracy lagging-strand DNA replication mediated by DNA polymerase dissociation." Proceedings of the National Academy of Sciences 115, no. 16 (April 2, 2018): 4212–17. http://dx.doi.org/10.1073/pnas.1720353115.
Анотація:
The fidelity of DNA replication is a critical factor in the rate at which cells incur mutations. Due to the antiparallel orientation of the two chromosomal DNA strands, one strand (leading strand) is replicated in a mostly processive manner, while the other (lagging strand) is synthesized in short sections called Okazaki fragments. A fundamental question that remains to be answered is whether the two strands are copied with the same intrinsic fidelity. In most experimental systems, this question is difficult to answer, as the replication complex contains a different DNA polymerase for each strand, such as, for example, DNA polymerases δ and ε in eukaryotes. Here we have investigated this question in the bacterium Escherichia coli, in which the replicase (DNA polymerase III holoenzyme) contains two copies of the same polymerase (Pol III, the dnaE gene product), and hence the two strands are copied by the same polymerase. Our in vivo mutagenesis data indicate that the two DNA strands are not copied with the same accuracy, and that, remarkably, the lagging strand has the highest fidelity. We postulate that this effect results from the greater dissociative character of the lagging-strand polymerase, which provides additional options for error removal. Our conclusion is strongly supported by results with dnaE antimutator polymerases characterized by increased dissociation rates.
2
Shi, Jiezhong, Ben Zhang, Tianyi Zheng, Tong Zhou, Min Guo, Ying Wang, and Yuanchen Dong. "DNA Materials Assembled from One DNA Strand." International Journal of Molecular Sciences 24, no. 9 (May 3, 2023): 8177. http://dx.doi.org/10.3390/ijms24098177.
Анотація:
Due to the specific base-pairing recognition, clear nanostructure, programmable sequence and responsiveness of the DNA molecule, DNA materials have attracted extensive attention and been widely used in controlled release, drug delivery and tissue engineering. Generally, the strategies for preparing DNA materials are based on the assembly of multiple DNA strands. The construction of DNA materials using only one DNA strand can not only save time and cost, but also avoid defects in final assemblies generated by the inaccuracy of DNA ratios, which potentially promote the large-scale production and practical application of DNA materials. In order to use one DNA strand to form assemblies, the sequences have to be palindromes with lengths that need to be controlled carefully. In this review, we introduced the development of DNA assembly and mainly summarized current reported materials formed by one DNA strand. We also discussed the principle for the construction of DNA materials using one DNA strand.
3
Jensen, Sarah Ø., Nadia Øgaard, Hans Jørgen Nielsen, Jesper B. Bramsen, and Claus L. Andersen. "Enhanced Performance of DNA Methylation Markers by Simultaneous Measurement of Sense and Antisense DNA Strands after Cytosine Conversion." Clinical Chemistry 66, no. 7 (May 27, 2020): 925–33. http://dx.doi.org/10.1093/clinchem/hvaa100.
Анотація:
Abstract Background Most existing DNA methylation-based methods for detection of circulating tumor DNA (ctDNA) are based on conversion of unmethylated cytosines to uracil. After conversion, the 2 DNA strands are no longer complementary; therefore, targeting only 1 DNA strand merely utilizes half of the available input DNA. We investigated whether the sensitivity of methylation-based ctDNA detection strategies could be increased by targeting both DNA strands after bisulfite conversion. Methods Dual-strand digital PCR assays were designed for the 3 colorectal cancer (CRC)–specific methylation markers KCNQ5, C9orf50, and CLIP4 and compared with previously reported single-strand assays. Performance was tested in tumor and leukocyte DNA, and the ability to detect ctDNA was investigated in plasma from 43 patients with CRC stages I to IV and 42 colonoscopy-confirmed healthy controls. Results Dual-strand assays quantified close to 100% of methylated control DNA input, whereas single-strand assays quantified approximately 50%. Furthermore, dual-strand assays showed a 2-fold increase in the number of methylated DNA copies detected when applied to DNA purified from tumor tissue and plasma from CRC patients. When the results of the 3 DNA methylation markers were combined into a ctDNA detection test and applied to plasma, the dual-strand assay format detected 86% of the cancers compared with 74% for the single-strand assay format. The specificity was 100% for both the dual- and single-strand test formats. Conclusion Dual-strand assays enabled more sensitive detection of methylated ctDNA than single-strand assays.
4
Fan, Xinqing, and Carolyn Mary Price. "Coordinate Regulation of G- and C Strand Length during New Telomere Synthesis." Molecular Biology of the Cell 8, no. 11 (November 1997): 2145–55. http://dx.doi.org/10.1091/mbc.8.11.2145.
Анотація:
We have used the ciliate Euplotes to study the role of DNA polymerase in telomeric C strand synthesis.Euplotes provides a unique opportunity to study C strand synthesis without the complication of simultaneous DNA replication because millions of new telomeres are made at a stage in the life cycle when no general DNA replication takes place. Previously we showed that the C-strands of newly synthesized telomeres have a precisely controlled length while the G-strands are more heterogeneous. This finding suggested that, although synthesis of the G-strand (by telomerase) is the first step in telomere addition, a major regulatory step occurs during subsequent C strand synthesis. We have now examined whether G- and C strand synthesis might be regulated coordinately rather than by two independent mechanisms. We accomplished this by determining what happens to G- and C strand length if C strand synthesis is partially inhibited by aphidicolin. Aphidicolin treatment caused a general lengthening of the G-strands and a large increase in C strand heterogeneity. This concomitant change in both the G- and C strand length indicates that synthesis of the two strands is coordinated. Since aphidicolin is a very specific inhibitor of DNA polα and polδ, our results suggest that this coordinate length regulation is mediated by DNA polymerase.
5
Ma, Jingjing. "Molecular Logic Gate Based on DNA Strand Displacement Reaction." Journal of Nanoelectronics and Optoelectronics 16, no. 6 (June 1, 2021): 974–77. http://dx.doi.org/10.1166/jno.2021.3037.
Анотація:
In this paper, I construct an XOR logic gate based on DNA strand displacement reaction, and verify our design through corresponding biochemical experiment. I designed several different DNA strands. Based on two basic DNA strand displacement reaction mechanisms, by adding different input strands and taking the signal of FAM fluorescent group as the output, the XOR logic gate is realized. The result shows that DNA strand displacement technology has important application value in DNA computing, especially in the construction of DNA molecular logic gates.
6
Sugiman-Marangos, Seiji N., Yoni M. Weiss, and Murray S. Junop. "Mechanism for accurate, protein-assisted DNA annealing by Deinococcus radiodurans DdrB." Proceedings of the National Academy of Sciences 113, no. 16 (April 4, 2016): 4308–13. http://dx.doi.org/10.1073/pnas.1520847113.
Анотація:
Accurate pairing of DNA strands is essential for repair of DNA double-strand breaks (DSBs). How cells achieve accurate annealing when large regions of single-strand DNA are unpaired has remained unclear despite many efforts focused on understanding proteins, which mediate this process. Here we report the crystal structure of a single-strand annealing protein [DdrB (DNA damage response B)] in complex with a partially annealed DNA intermediate to 2.2 Å. This structure and supporting biochemical data reveal a mechanism for accurate annealing involving DdrB-mediated proofreading of strand complementarity. DdrB promotes high-fidelity annealing by constraining specific bases from unauthorized association and only releases annealed duplex when bound strands are fully complementary. To our knowledge, this mechanism provides the first understanding for how cells achieve accurate, protein-assisted strand annealing under biological conditions that would otherwise favor misannealing.
7
Bolt, Edward L., and Thorsten Allers. "New enzymes, new mechanisms?: DNA repair by recombination in the Archaea." Biochemist 26, no. 3 (June 1, 2004): 19–21. http://dx.doi.org/10.1042/bio02603019.
Анотація:
DNA repair by homologous recombination is highly accurate, since it uses an intact DNA strand to guide repair of its damaged homologue. This article focuses on two key steps in recombination: unwinding of strands by repair helicases, and annealing of homologous strands by strand-exchange enzymes.
8
Domljanovic, Ivana, Alessandro Ianiro, Curzio Rüegg, Michael Mayer, and Maria Taskova. "Natural and Modified Oligonucleotide Sequences Show Distinct Strand Displacement Kinetics and These Are Affected Further by Molecular Crowders." Biomolecules 12, no. 9 (September 6, 2022): 1249. http://dx.doi.org/10.3390/biom12091249.
Анотація:
DNA and RNA strand exchange is a process of fundamental importance in biology. Herein, we used a FRET-based assay to investigate, for the first time, the stand exchange kinetics of natural DNA, natural RNA, and locked nucleic acid (LNA)-modified DNA sequences in vitro in PBS in the absence or presence of molecular additives and macromolecular crowders such as diethylene glycol dimethyl ether (deg), polyethylene glycol (peg), and polyvinylpyrrolidone (pvp). The results show that the kinetics of strand exchange mediated by DNA, RNA, and LNA-DNA oligonucleotide sequences are different. Different molecular crowders further affect the strand displacement kinetics, highlighting the complexity of the process of nucleic acid strand exchange as it occurs in vivo. In a peg-containing buffer, the rate constant of displacement was slightly increased for the DNA displacement strand, while it was slightly decreased for the RNA and the LNA-DNA strands compared with displacement in pure PBS. When we used a deg-containing buffer, the rate constants of displacement for all three sequences were drastically increased compared with displacement in PBS. Overall, we show that interactions of the additives with the duplex strands have a significant effect on the strand displacement kinetics and this effect can exceed the one exerted by the chemical nature of the displacement strand itself.
9
Cronan, Glen E., Elena A. Kouzminova, and Andrei Kuzminov. "Near-continuously synthesized leading strands inEscherichia coliare broken by ribonucleotide excision." Proceedings of the National Academy of Sciences 116, no. 4 (January 7, 2019): 1251–60. http://dx.doi.org/10.1073/pnas.1814512116.
Анотація:
In vitro, purified replisomes drive model replication forks to synthesize continuous leading strands, even without ligase, supporting the semidiscontinuous model of DNA replication. However, nascent replication intermediates isolated from ligase-deficientEscherichia colicomprise only short (on average 1.2-kb) Okazaki fragments. It was long suspected that cells replicate their chromosomal DNA by the semidiscontinuous mode observed in vitro but that, in vivo, the nascent leading strand was artifactually fragmented postsynthesis by excision repair. Here, using high-resolution separation of pulse-labeled replication intermediates coupled with strand-specific hybridization, we show that excision-proficientE. coligenerates leading-strand intermediates >10-fold longer than lagging-strand Okazaki fragments. Inactivation of DNA-repair activities, including ribonucleotide excision, further increased nascent leading-strand size to ∼80 kb, while lagging-strand Okazaki fragments remained unaffected. We conclude that in vivo, repriming occurs ∼70× less frequently on the leading versus lagging strands, and that DNA replication inE. coliis effectively semidiscontinuous.
10
Delagoutte, Emmanuelle, and Giuseppe Baldacci. "5′CAG and 5′CTG Repeats Create Differential Impediment to the Progression of a Minimal Reconstituted T4 Replisome Depending on the Concentration of dNTPs." Molecular Biology International 2011 (August 10, 2011): 1–14. http://dx.doi.org/10.4061/2011/213824.
Анотація:
Instability of repetitive sequences originates from strand misalignment during repair or replicative DNA synthesis. To investigate the activity of reconstituted T4 replisomes across trinucleotide repeats (TNRs) during leading strand DNA synthesis, we developed a method to build replication miniforks containing a TNR unit of defined sequence and length. Each minifork consists of three strands, primer, leading strand template, and lagging strand template with a 5′ single-stranded (ss) tail. Each strand is prepared independently, and the minifork is assembled by hybridization of the three strands. Using these miniforks and a minimal reconstituted T4 replisome, we show that during leading strand DNA synthesis, the dNTP concentration dictates which strand of the structure-forming 5′CAG/5′CTG repeat creates the strongest impediment to the minimal replication complex. We discuss this result in the light of the known fluctuation of dNTP concentration during the cell cycle and cell growth and the known concentration balance among individual dNTPs.
11
Vaitsiankova, Alina, Kamila Burdova, Margarita Sobol, Amit Gautam, Oldrich Benada, Hana Hanzlikova, and Keith W. Caldecott. "PARP inhibition impedes the maturation of nascent DNA strands during DNA replication." Nature Structural & Molecular Biology 29, no. 4 (March 24, 2022): 329–38. http://dx.doi.org/10.1038/s41594-022-00747-1.
Анотація:
AbstractPoly(ADP-ribose) polymerase 1 (PARP1) is implicated in the detection and processing of unligated Okazaki fragments and other DNA replication intermediates, highlighting such structures as potential sources of genome breakage induced by PARP inhibition. Here, we show that PARP1 activity is greatly elevated in chicken and human S phase cells in which FEN1 nuclease is genetically deleted and is highest behind DNA replication forks. PARP inhibitor reduces the integrity of nascent DNA strands in both wild-type chicken and human cells during DNA replication, and does so in FEN1−/− cells to an even greater extent that can be detected as postreplicative single-strand nicks or gaps. Collectively, these data show that PARP inhibitors impede the maturation of nascent DNA strands during DNA replication, and implicate unligated Okazaki fragments and other nascent strand discontinuities in the cytotoxicity of these compounds.
12
Bielawski, Joseph P., and John R. Gold. "Mutation Patterns of Mitochondrial H- and L-Strand DNA in Closely Related Cyprinid Fishes." Genetics 161, no. 4 (August 1, 2002): 1589–97. http://dx.doi.org/10.1093/genetics/161.4.1589.
Анотація:
Abstract Mitochondrial genome replication is asymmetric. Replication starts from the origin of heavy (H)-strand replication, displacing the parental H-strand as it proceeds along the molecule. The H-strand remains single stranded until light (L)-strand replication is initiated from a second origin of replication. It has been suggested that single-stranded H-strand DNA is more sensitive to mutational damage, giving rise to substitutional rate differences between the two strands and among genes in mammalian mitochondrial DNA. In this study, we analyzed sequences of the cytochrome b, ND4, ND4L, and COI genes of cyprinid fishes to investigate rates and patterns of nucleotide substitution in the mitochondrial genome. To test for strand-asymmetric mutation pressure, a likelihood-ratio test was developed and applied to the cyprinid sequences. Patterns of substitution and levels of strand-asymmetric mutation pressure were largely consistent with a mutation gradient between the H- and L-strand origins of replication. Significant strand bias was observed among rates of transitional substitution. However, biological interpretation of the direction and strength of strand asymmetry for specific classes of substitutions is problematic. The problem occurs because the rate of any single class of substitution inferred from one strand is actually a sum of rates on two strands. The validity of the likelihood-ratio test is not affected by this problem.
13
Moore, John D., and Jocelyn E. Krebs. "Histone modifications and DNA double-strand break repair." Biochemistry and Cell Biology 82, no. 4 (August 1, 2004): 446–52. http://dx.doi.org/10.1139/o04-034.
Анотація:
The roles of different histone modifications have been explored extensively in a number of nuclear processes, particularly in transcriptional regulation. Only recently has the role of histone modification in signaling or facilitating DNA repair begun to be elucidated. DNA broken along both strands in the same region, a double-strand break, is damaged in the most severe way possible and can be the most difficult type of damage to repair accurately. To successfully repair the double-strand break, the cell must gain access to the damaged ends of the DNA and recruit repair factors, and in the case of homologous recombination repair, the cell must also find, colocalize, and gain access to a suitable homologous sequence. In the repair of a double-strand break, the cell must also choose between homologous and non-homologous pathways of repair. Here, we will briefly review the mechanisms of double-strand-break repair, and discuss the known roles of histone modifications in signaling and repairing double-strand breaks.Key words: H23A, double strand break repair, histone modification.
14
Hahn, Jaeseung, and William M. Shih. "Thermal cycling of DNA devices via associative strand displacement." Nucleic Acids Research 47, no. 20 (October 4, 2019): 10968–75. http://dx.doi.org/10.1093/nar/gkz844.
Анотація:
Abstract DNA-based devices often operate through a series of toehold-mediated strand-displacement reactions. To achieve cycling, fluidic mixing can be used to introduce ‘recovery’ strands to reset the system. However, such mixing can be cumbersome, non-robust, and wasteful of materials. Here we demonstrate mixing-free thermal cycling of DNA devices that operate through associative strand-displacement cascades. These cascades are favored at low temperatures due to the primacy of a net increase in base pairing, whereas rebinding of ‘recovery’ strands is favored at higher temperatures due to the primacy of a net release of strands. The temperature responses of the devices could be modulated by adjustment of design parameters such as the net increase of base pairs and the concentrations of strands. Degradation of function was not observable even after 500 thermal cycles. We experimentally demonstrated simple digital-logic circuits that evaluate at 35°C and reset after transient heating to 65°C. Thus associative strand displacement enables robust thermal cycling of DNA-based devices in a closed system.
15
Yu, Man, and Warren Masker. "T7 Single Strand DNA Binding Protein but Not T7 Helicase Is Required for DNA Double Strand Break Repair." Journal of Bacteriology 183, no. 6 (March 15, 2001): 1862–69. http://dx.doi.org/10.1128/jb.183.6.1862-1869.2001.
Анотація:
ABSTRACT An in vitro system based on Escherichia coliinfected with bacteriophage T7 was used to test for involvement of host and phage recombination proteins in the repair of double strand breaks in the T7 genome. Double strand breaks were placed in a uniqueXhoI site located approximately 17% from the left end of the T7 genome. In one assay, repair of these breaks was followed by packaging DNA recovered from repair reactions and determining the yield of infective phage. In a second assay, the product of the reactions was visualized after electrophoresis to estimate the extent to which the double strand breaks had been closed. Earlier work demonstrated that in this system double strand break repair takes place via incorporation of a patch of DNA into a gap formed at the break site. In the present study, it was found that extracts prepared from uninfected E. coli were unable to repair broken T7 genomes in this in vitro system, thus implying that phage rather than host enzymes are the primary participants in the predominant repair mechanism. Extracts prepared from an E. coli recA mutant were as capable of double strand break repair as extracts from a wild-type host, arguing that the E. coli recombinase is not essential to the recombinational events required for double strand break repair. In T7 strand exchange during recombination is mediated by the combined action of the helicase encoded by gene 4 and the annealing function of the gene 2.5 single strand binding protein. Although a deficiency in the gene 2.5 protein blocked double strand break repair, a gene 4 deficiency had no effect. This argues that a strand transfer step is not required during recombinational repair of double strand breaks in T7 but that the ability of the gene 2.5 protein to facilitate annealing of complementary single strands of DNA is critical to repair of double strand breaks in T7.
16
Griffith, Jack D., Lorelei D. Harris, and Stephen L. Brenner. "Dna Strand Exchange." Critical Reviews in Biochemistry 23, sup1 (January 1988): S43—S86. http://dx.doi.org/10.3109/10409238809083375.
17
Scalise, Dominic, Nisita Dutta, and Rebecca Schulman. "DNA Strand Buffers." Journal of the American Chemical Society 140, no. 38 (September 11, 2018): 12069–76. http://dx.doi.org/10.1021/jacs.8b05373.
18
Wu, CHUNG-I. "DNA strand asymmetry." Nature 352, no. 6331 (July 1991): 114. http://dx.doi.org/10.1038/352114b0.
19
Weiser, Martin, and Hans-Achim Wagenknecht. "Dynamic DNA architectures: spontaneous DNA strand exchange and self-sorting driven by perylene bisimide interactions." Chemical Communications 51, no. 92 (2015): 16530–33. http://dx.doi.org/10.1039/c5cc06491k.
Анотація:
Three differently bay-substituted perylene bisimides together with the conventional unsubstituted chromophore were synthetically incorporated as homodimers in DNA double strands and undergo spontaneous strand exchange if mixed together.
20
Hernandez, Alfredo J., Seung-Joo Lee, and Charles C. Richardson. "Primer release is the rate-limiting event in lagging-strand synthesis mediated by the T7 replisome." Proceedings of the National Academy of Sciences 113, no. 21 (May 9, 2016): 5916–21. http://dx.doi.org/10.1073/pnas.1604894113.
Анотація:
DNA replication occurs semidiscontinuously due to the antiparallel DNA strands and polarity of enzymatic DNA synthesis. Although the leading strand is synthesized continuously, the lagging strand is synthesized in small segments designated Okazaki fragments. Lagging-strand synthesis is a complex event requiring repeated cycles of RNA primer synthesis, transfer to the lagging-strand polymerase, and extension effected by cooperation between DNA primase and the lagging-strand polymerase. We examined events controlling Okazaki fragment initiation using the bacteriophage T7 replication system. Primer utilization by T7 DNA polymerase is slower than primer formation. Slow primer release from DNA primase allows the polymerase to engage the complex and is followed by a slow primer handoff step. The T7 single-stranded DNA binding protein increases primer formation and extension efficiency but promotes limited rounds of primer extension. We present a model describing Okazaki fragment initiation, the regulation of fragment length, and their implications for coordinated leading- and lagging-strand DNA synthesis.
21
Prévost, Chantal, and Masayuki Takahashi. "Geometry of the DNA strands within the RecA nucleofilament: role in homologous recombination." Quarterly Reviews of Biophysics 36, no. 4 (November 2003): 429–53. http://dx.doi.org/10.1017/s0033583504003956.
Анотація:
1. Introduction 4302. Transformations of the RecA filament 4312.1 The different forms of the RecA filament 4312.2 Orientation and position of the RecA monomers in the active filament 4332.3 Transmission of structural information along the filament 4333. RecA-induced DNA deformations 4353.1 Characteristics of RecA-bound DNA 4353.2 Stretching properties of double-stranded DNA 4363.3 DNA bound to architectural proteins 4373.4 Implications for RecA-induced DNA deformations 4383.5 Axial distribution of the DNA stretching deformation 4384. Contacts between RecA and the DNA strands 4404.1 The DNA-binding sites 4404.2 Possible arrangement of loops L1 and L2 and the three bound strands of DNA 4425. Strand arrangement during pairing reorganization 4445.1 Hypotheses for DNA strand association 4445.2 Association via major or minor grooves 4465.3 Post-strand exchange geometries 4466. Conclusion 4477. Acknowledgments 4488. References 448Homologous recombination consists of exchanging DNA strands of identical or almost identical sequence. This process is important for both DNA repair and DNA segregation. In prokaryotes, it involves the formation of long helical filaments of the RecA protein on DNA. These filaments incorporate double-stranded DNA from the cell's genetic material, recognize sequence homology and promote strand exchange between the two DNA segments. DNA processing by these nucleofilaments is characterized by large amplitude deformations of the double helix, which is stretched by 50% and unwound by 40% with respect to B-DNA. In this article, information concerning the structure and interactions of the RecA, DNA and ATP molecules involved in DNA strand exchange is gathered and analyzed to present a view of their possible arrangement within the filament, their behavior during strand exchange and during ATP hydrolysis, the mechanism of RecA-promoted DNA deformation and the role of DNA deformation in the process of homologous recombination. In particular, the unusual characteristics of DNA within the RecA filament are compared to the DNA deformations locally induced by architectural proteins which bind in the DNA minor groove. The possible role and location of two flexible loops of RecA are discussed.
22
Loeb, Daniel D., and Ru Tian. "Mutations That Increase In Situ Priming Also Decrease Circularization for Duck Hepatitis B Virus." Journal of Virology 75, no. 14 (July 15, 2001): 6492–97. http://dx.doi.org/10.1128/jvi.75.14.6492-6497.2001.
Анотація:
ABSTRACT The process of hepadnavirus reverse transcription involves two template switches during the synthesis of plus-strand DNA. The first involves translocation of the plus-strand primer from its site of generation, the 3′ end of minus-strand DNA, to the complementary sequence DR2, located near the 5′ end of the minus-strand DNA. Plus strands initiated from DR2 are extended to the 5′ end of the minus-strand DNA. At this point, the 3′ end of the minus strand becomes the template via the second template switch, a process called circularization. Elongation of circularized plus-strand DNA generates relaxed circular DNA. Although most virions contain relaxed circular DNA, some contain duplex linear DNA. Duplex linear genomes are synthesized when the plus-strand primer is used at the site of its generation, the 3′ end of the minus-strand template. This type of synthesis is called in situ priming. Although in situ priming is normally low, in some duck hepatitis B virus mutants this type of priming is elevated. For example, mutations within the 3′ end of the minus-strand DNA can lead to increased levels of in situ priming. We report here that these same mutations result in a second defect, a less efficient template switch that circularizes the genome. Although it is not clear how these mutations affect both steps in DNA replication, our findings suggest a commonality in the mechanism of initiation of plus-strand synthesis and the template switch that circularizes the genome.
23
Thomas, David C., Yegor A. Voronin, Galina N. Nikolenko, Jianbo Chen, Wei-Shau Hu, and Vinay K. Pathak. "Determination of the Ex Vivo Rates of Human Immunodeficiency Virus Type 1 Reverse Transcription by Using Novel Strand-Specific Amplification Analysis." Journal of Virology 81, no. 9 (February 21, 2007): 4798–807. http://dx.doi.org/10.1128/jvi.02471-06.
Анотація:
ABSTRACT Replication of human immunodeficiency virus type 1 (HIV-1), like all organisms, involves synthesis of a minus-strand and a plus-strand of nucleic acid. Currently available PCR methods cannot distinguish between the two strands of nucleic acids. To carry out detailed analysis of HIV-1 reverse transcription from infected cells, we have developed a novel strand-specific amplification (SSA) assay using single-stranded padlock probes that are specifically hybridized to a target strand, ligated, and quantified for sensitive analysis of the kinetics of HIV-1 reverse transcription in cells. Using SSA, we have determined for the first time the ex vivo rates of HIV-1 minus-strand DNA synthesis in 293T and human primary CD4+ T cells (∼68 to 70 nucleotides/min). We also determined the rates of minus-strand DNA transfer (∼4 min), plus-strand DNA transfer (∼26 min), and initiation of plus-strand DNA synthesis (∼9 min) in 293T cells. Additionally, our results indicate that plus-strand DNA synthesis is initiated at multiple sites and that several reverse transcriptase inhibitors influence the kinetics of minus-strand DNA synthesis differently, providing insights into their mechanism of inhibition. The SSA technology provides a novel approach to analyzing DNA replication processes and should facilitate the development of new antiretroviral drugs that target specific steps in HIV-1 reverse transcription.
24
Hentosh, P., and P. Grippo. "2-Chloro-2′-deoxyadenosine monophosphate residues in DNA enhance susceptibility to 3′ → 5′ exonucleases." Biochemical Journal 302, no. 2 (September 1, 1994): 567–71. http://dx.doi.org/10.1042/bj3020567.
Анотація:
2-Chloro-2′-deoxyadenosine triphosphate, a purine nucleotide analogue and potent antileukaemic agent, was incorporated into double-stranded 36-mers in place of dATP to investigate the effects of 2-chloroadenine (ClAde) on DNA polymerase-associated 3′-->5′ exonuclease activity. ClAde residues within one strand of duplex DNA did not inhibit exonuclease activity; on the contrary, ClAde-containing minus strands were digested to a greater extent than was control DNA in the absence of deoxyribonucleoside triphosphates by Escherichia coli Klenow fragment, yeast DNA polymerase II and T4 DNA polymerase. After a 30 min incubation with 5 units of Klenow fragment, approximately 65% of control DNA remained in DNA fragments of 26 bases or larger compared with only approximately 25% of ClAde-substituted substrates. Unsubstituted plus strands opposite a ClAde-containing strand were likewise digested more quickly by 3′-->5′ exonuclease, but only in the vicinity of the ClAde sites. Approx. 63% of the plus strands from ClAde-containing oligomers were less than 24 bases in length after a 25 min digestion period with Klenow fragment compared with only approximately 32% of control DNA. Such results indicate that, unlike other base modifications such as pyrimidine dimers, methoxy psoralen adducts and certain nucleoside analogues, all of which inhibit or decrease the rate of strand degradation by 3′-->5′ exonucleases, incorporated ClAde enhances strand degradation of duplex DNA.
25
Lestienne, Patrick P. "Priming DNA Replication from Triple Helix Oligonucleotides: Possible Threestranded DNA in DNA Polymerases." Molecular Biology International 2011 (September 14, 2011): 1–9. http://dx.doi.org/10.4061/2011/562849.
Анотація:
Triplex associate with a duplex DNA presenting the same polypurine or polypyrimidine-rich sequence in an antiparallel orientation. So far, triplex forming oligonucleotides (TFOs) are known to inhibit transcription, replication, and to induce mutations. A new property of TFO is reviewed here upon analysis of DNA breakpoint yielding DNA rearrangements; the synthesized sequence of the first direct repeat displays a skewed polypurine- rich sequence. This synthesized sequence can bind the second homologous duplex sequence through the formation of a triple helix, which is able to prime further DNA replication. In these case, the d(G)-rich Triple Helix Primers (THP) bind the homologous strand in a parallel manner, possibly via a RecA-like mechanism. This novel property is shared by all tested DNA polymerases: phage, retrovirus, bacteria, and human. These features may account for illegitimate initiation of replication upon single-strand breakage and annealing to a homologous sequence where priming may occur. Our experiments suggest that DNA polymerases can bind three instead of two polynucleotide strands in their catalytic centre.
26
Lin, D. C., B. Yurke, and N. A. Langrana. "Inducing Reversible Stiffness Changes in DNA-crosslinked Gels." Journal of Materials Research 20, no. 6 (June 1, 2005): 1456–64. http://dx.doi.org/10.1557/jmr.2005.0186.
Анотація:
Researchers have constructed a number of DNA-based nanodevices that undergo stepped configuration changes through the application of single-stranded DNA oligomers. Such devices can be incorporated into gel networks to create new classes of active materials with controllable bulk mechanical properties. This concept was demonstrated in a DNA-crosslinked gel, the stiffness of which was modulated through the application of DNA strands. Each crosslink incorporated a single-stranded region to which a DNA strand with a complementary base sequence (called the fuel strand) bound, thereby changing the nanostructure of the gel network. The gel was restored to its initial stiffness through the application of the complement of the fuel strand, which cleared the fuel strand from the crosslink via competitive binding. Stiffness changes in excess of a factor of three were observed. The ability to switch the mechanical properties of these gels without changing temperature, buffer composition, or other environmental conditions, apart from the application of DNA, makes these materials attractive candidates for biotechnology applications.
27
Lo, Chen-Yu, and Yang Gao. "DNA Helicase–Polymerase Coupling in Bacteriophage DNA Replication." Viruses 13, no. 9 (August 31, 2021): 1739. http://dx.doi.org/10.3390/v13091739.
Анотація:
Bacteriophages have long been model systems to study the molecular mechanisms of DNA replication. During DNA replication, a DNA helicase and a DNA polymerase cooperatively unwind the parental DNA. By surveying recent data from three bacteriophage replication systems, we summarized the mechanistic basis of DNA replication by helicases and polymerases. Kinetic data have suggested that a polymerase or a helicase alone is a passive motor that is sensitive to the base-pairing energy of the DNA. When coupled together, the helicase–polymerase complex is able to unwind DNA actively. In bacteriophage T7, helicase and polymerase reside right at the replication fork where the parental DNA is separated into two daughter strands. The two motors pull the two daughter strands to opposite directions, while the polymerase provides a separation pin to split the fork. Although independently evolved and containing different replisome components, bacteriophage T4 replisome shares mechanistic features of Hel–Pol coupling that are similar to T7. Interestingly, in bacteriophages with a limited size of genome like Φ29, DNA polymerase itself can form a tunnel-like structure, which encircles the DNA template strand and facilitates strand displacement synthesis in the absence of a helicase. Studies on bacteriophage replication provide implications for the more complicated replication systems in bacteria, archaeal, and eukaryotic systems, as well as the RNA genome replication in RNA viruses.
28
Boyer, Benjamin, Claudia Danilowicz, Mara Prentiss, and Chantal Prévost. "Weaving DNA strands: structural insight on ATP hydrolysis in RecA-induced homologous recombination." Nucleic Acids Research 47, no. 15 (August 2, 2019): 7798–808. http://dx.doi.org/10.1093/nar/gkz667.
Анотація:
Abstract Homologous recombination is a fundamental process in all living organisms that allows the faithful repair of DNA double strand breaks, through the exchange of DNA strands between homologous regions of the genome. Results of three decades of investigation and recent fruitful observations have unveiled key elements of the reaction mechanism, which proceeds along nucleofilaments of recombinase proteins of the RecA family. Yet, one essential aspect of homologous recombination has largely been overlooked when deciphering the mechanism: while ATP is hydrolyzed in large quantity during the process, how exactly hydrolysis influences the DNA strand exchange reaction at the structural level remains to be elucidated. In this study, we build on a previous geometrical approach that studied the RecA filament variability without bound DNA to examine the putative implication of ATP hydrolysis on the structure, position, and interactions of up to three DNA strands within the RecA nucleofilament. Simulation results on modeled intermediates in the ATP cycle bring important clues about how local distortions in the DNA strand geometries resulting from ATP hydrolysis can aid sequence recognition by promoting local melting of already formed DNA heteroduplex and transient reverse strand exchange in a weaving type of mechanism.
29
Gao, Yang, Yanxiang Cui, Tara Fox, Shiqiang Lin, Huaibin Wang, Natalia de Val, Z. Hong Zhou, and Wei Yang. "Structures and operating principles of the replisome." Science 363, no. 6429 (January 24, 2019): eaav7003. http://dx.doi.org/10.1126/science.aav7003.
Анотація:
Visualization in atomic detail of the replisome that performs concerted leading– and lagging–DNA strand synthesis at a replication fork has not been reported. Using bacteriophage T7 as a model system, we determined cryo–electron microscopy structures up to 3.2-angstroms resolution of helicase translocating along DNA and of helicase-polymerase-primase complexes engaging in synthesis of both DNA strands. Each domain of the spiral-shaped hexameric helicase translocates sequentially hand-over-hand along a single-stranded DNA coil, akin to the way AAA+ ATPases (adenosine triphosphatases) unfold peptides. Two lagging-strand polymerases are attached to the primase, ready for Okazaki fragment synthesis in tandem. A β hairpin from the leading-strand polymerase separates two parental DNA strands into a T-shaped fork, thus enabling the closely coupled helicase to advance perpendicular to the downstream DNA duplex. These structures reveal the molecular organization and operating principles of a replisome.
30
Lukac, David, Zuzana Machacova, and Pavel Moudry. "Emetine blocks DNA replication via proteosynthesis inhibition not by targeting Okazaki fragments." Life Science Alliance 5, no. 12 (September 9, 2022): e202201560. http://dx.doi.org/10.26508/lsa.202201560.
Анотація:
DNA synthesis of the leading and lagging strands works independently and cells tolerate single-stranded DNA generated during strand uncoupling if it is protected by RPA molecules. Natural alkaloid emetine is used as a specific inhibitor of lagging strand synthesis, uncoupling leading and lagging strand replication. Here, by analysis of lagging strand synthesis inhibitors, we show that despite emetine completely inhibiting DNA replication: it does not induce the generation of single-stranded DNA and chromatin-bound RPA32 (CB-RPA32). In line with this, emetine does not activate the replication checkpoint nor DNA damage response. Emetine is also an inhibitor of proteosynthesis and ongoing proteosynthesis is essential for the accurate replication of DNA. Mechanistically, we demonstrate that the acute block of proteosynthesis by emetine temporally precedes its effects on DNA replication. Thus, our results are consistent with the hypothesis that emetine affects DNA replication by proteosynthesis inhibition. Emetine and mild POLA1 inhibition prevent S-phase poly(ADP-ribosyl)ation. Collectively, our study reveals that emetine is not a specific lagging strand synthesis inhibitor with implications for its use in molecular biology.
31
Masai, Hisao, Naoko Kakusho, Rino Fukatsu, Yue Ma, Keisuke Iida, Yutaka Kanoh, and Kazuo Nagasawa. "Molecular architecture of G-quadruplex structures generated on duplex Rif1-binding sequences." Journal of Biological Chemistry 293, no. 44 (September 14, 2018): 17033–49. http://dx.doi.org/10.1074/jbc.ra118.005240.
Анотація:
G-quadruplexes (G4s) are four-stranded DNA structures comprising stacks of four guanines, are prevalent in genomes, and have diverse biological functions in various chromosomal structures. A conserved protein, Rap1-interacting factor 1 (Rif1) from fission yeast (Schizosaccharomyces pombe), binds to Rif1-binding sequence (Rif1BS) and regulates DNA replication timing. Rif1BS is characterized by the presence of multiple G-tracts, often on both strands, and their unusual spacing. Although previous studies have suggested generation of G4-like structures on duplex Rif1BS, its precise molecular architecture remains unknown. Using gel-shift DNA binding assays and DNA footprinting with various nuclease probes, we show here that both of the Rif1BS strands adopt specific higher-order structures upon heat denaturation. We observed that the structure generated on the G-strand is consistent with a G4 having unusually long loop segments and that the structure on the complementary C-strand does not have an intercalated motif (i-motif). Instead, we found that the formation of the C-strand structure depends on the G4 formation on the G-strand. Thus, the higher-order structure generated at Rif1BS involved both DNA strands, and in some cases, G4s may form on both of these strands. The presence of multiple G-tracts permitted the formation of alternative structures when some G-tracts were mutated or disrupted by deazaguanine replacement, indicating the robust nature of DNA higher-order structures generated at Rif1BS. Our results provide general insights into DNA structures generated at G4-forming sequences on duplex DNA.
32
Kapadia, Jay Bhakti, Nawwaf Kharma, Alen Nellikulam Davis, Nicolas Kamel, and Jonathan Perreault. "Toehold-mediated strand displacement to measure released product from self-cleaving ribozymes." RNA 28, no. 2 (December 3, 2021): 263–73. http://dx.doi.org/10.1261/rna.078823.121.
Анотація:
This paper presents a probe comprising a fluorophore and a quencher, enabling measurement of released product from self-cleaving hammerhead ribozyme, without labeled RNA molecules, regular sampling or use of polyacrylamide gels. The probe is made of two DNA strands; one strand is labeled with a fluorophore at its 5′-end, while the other strand is labeled with a quencher at its 3′-end. These two DNA strands are perfectly complementary, but with a 3′-overhang of the fluorophore strand. These unpaired nucleotides act as a toehold, which is utilized by a detached cleaved fragment (coming from a self-cleaving hammerhead ribozyme) as the starting point for a strand displacement reaction. This reaction causes the separation of the fluorophore strand from the quencher strand, culminating in fluorescence, detectable in a plate reader. Notably, the emitted fluorescence is proportional to the amount of detached cleaved-off RNAs, displacing the DNA quencher strand. This method can replace or complement radio-hazardous unstable 32P as a method of measurement of the product release from ribozyme cleavage reactions; it also eliminates the need for polyacrylamide gels, for the same purpose. Critically, this method allows to distinguish between the total amount of cleaved ribozymes and the amount of detached fragments, resulting from that cleavage reaction.
33
Meagher, Martin, Alexander Myasnikov, and Eric J. Enemark. "Two Distinct Modes of DNA Binding by an MCM Helicase Enable DNA Translocation." International Journal of Molecular Sciences 23, no. 23 (November 24, 2022): 14678. http://dx.doi.org/10.3390/ijms232314678.
Анотація:
A six-subunit ATPase ring forms the central hub of the replication forks in all domains of life. This ring performs a helicase function to separate the two complementary DNA strands to be replicated and drives the replication machinery along the DNA. Disruption of this helicase/ATPase ring is associated with genetic instability and diseases such as cancer. The helicase/ATPase rings of eukaryotes and archaea consist of six minichromosome maintenance (MCM) proteins. Prior structural studies have shown that MCM rings bind one encircled strand of DNA in a spiral staircase, suggesting that the ring pulls this strand of DNA through its central pore in a hand-over-hand mechanism where the subunit at the bottom of the staircase dissociates from DNA and re-binds DNA one step above the staircase. With high-resolution cryo-EM, we show that the MCM ring of the archaeal organism Saccharolobus solfataricus binds an encircled DNA strand in two different modes with different numbers of subunits engaged to DNA, illustrating a plausible mechanism for the alternating steps of DNA dissociation and re-association that occur during DNA translocation.
34
Nelson, W. G., and M. B. Kastan. "DNA strand breaks: the DNA template alterations that trigger p53-dependent DNA damage response pathways." Molecular and Cellular Biology 14, no. 3 (March 1994): 1815–23. http://dx.doi.org/10.1128/mcb.14.3.1815-1823.1994.
Анотація:
The tumor suppressor protein p53 serves as a critical regulator of a G1 cell cycle checkpoint and of apoptosis following exposure of cells to DNA-damaging agents. The mechanism by which DNA-damaging agents elevate p53 protein levels to trigger G1/S arrest or cell death remains to be elucidated. In fact, whether damage to the DNA template itself participates in transducing the signal leading to p53 induction has not yet been demonstrated. We exposed human cell lines containing wild-type p53 alleles to several different DNA-damaging agents and found that agents which rapidly induce DNA strand breaks, such as ionizing radiation, bleomycin, and DNA topoisomerase-targeted drugs, rapidly triggered p53 protein elevations. In addition, we determined that camptothecin-stimulated trapping of topoisomerase I-DNA complexes was not sufficient to elevate p53 protein levels; rather, replication-associated DNA strand breaks were required. Furthermore, treatment of cells with the antimetabolite N(phosphonoacetyl)-L-aspartate (PALA) did not cause rapid p53 protein increases but resulted in delayed increases in p53 protein levels temporally correlated with the appearance of DNA strand breaks. Finally, we concluded that DNA strand breaks were sufficient for initiating p53-dependent signal transduction after finding that introduction of nucleases into cells by electroporation stimulated rapid p53 protein elevations. While DNA strand breaks appeared to be capable of triggering p53 induction, DNA lesions other than strand breaks did not. Exposure of normal cells and excision repair-deficient xeroderma pigmentosum cells to low doses of UV light, under conditions in which thymine dimers appear but DNA replication-associated strand breaks were prevented, resulted in p53 induction attributable to DNA strand breaks associated with excision repair. Our data indicate that DNA strand breaks are sufficient and probably necessary for p53 induction in cells with wild-type p53 alleles exposed to DNA-damaging agents.
35
Nelson, W. G., and M. B. Kastan. "DNA strand breaks: the DNA template alterations that trigger p53-dependent DNA damage response pathways." Molecular and Cellular Biology 14, no. 3 (March 1994): 1815–23. http://dx.doi.org/10.1128/mcb.14.3.1815.
Анотація:
The tumor suppressor protein p53 serves as a critical regulator of a G1 cell cycle checkpoint and of apoptosis following exposure of cells to DNA-damaging agents. The mechanism by which DNA-damaging agents elevate p53 protein levels to trigger G1/S arrest or cell death remains to be elucidated. In fact, whether damage to the DNA template itself participates in transducing the signal leading to p53 induction has not yet been demonstrated. We exposed human cell lines containing wild-type p53 alleles to several different DNA-damaging agents and found that agents which rapidly induce DNA strand breaks, such as ionizing radiation, bleomycin, and DNA topoisomerase-targeted drugs, rapidly triggered p53 protein elevations. In addition, we determined that camptothecin-stimulated trapping of topoisomerase I-DNA complexes was not sufficient to elevate p53 protein levels; rather, replication-associated DNA strand breaks were required. Furthermore, treatment of cells with the antimetabolite N(phosphonoacetyl)-L-aspartate (PALA) did not cause rapid p53 protein increases but resulted in delayed increases in p53 protein levels temporally correlated with the appearance of DNA strand breaks. Finally, we concluded that DNA strand breaks were sufficient for initiating p53-dependent signal transduction after finding that introduction of nucleases into cells by electroporation stimulated rapid p53 protein elevations. While DNA strand breaks appeared to be capable of triggering p53 induction, DNA lesions other than strand breaks did not. Exposure of normal cells and excision repair-deficient xeroderma pigmentosum cells to low doses of UV light, under conditions in which thymine dimers appear but DNA replication-associated strand breaks were prevented, resulted in p53 induction attributable to DNA strand breaks associated with excision repair. Our data indicate that DNA strand breaks are sufficient and probably necessary for p53 induction in cells with wild-type p53 alleles exposed to DNA-damaging agents.
36
Szambowska, Anna, Ingrid Tessmer, Petri Kursula, Christian Usskilat, Piotr Prus, Helmut Pospiech, and Frank Grosse. "DNA binding properties of human Cdc45 suggest a function as molecular wedge for DNA unwinding." Nucleic Acids Research 42, no. 4 (November 28, 2013): 2308–19. http://dx.doi.org/10.1093/nar/gkt1217.
Анотація:
Abstract The cell division cycle protein 45 (Cdc45) represents an essential replication factor that, together with the Mcm2-7 complex and the four subunits of GINS, forms the replicative DNA helicase in eukaryotes. Recombinant human Cdc45 (hCdc45) was structurally characterized and its DNA-binding properties were determined. Synchrotron radiation circular dichroism spectroscopy, dynamic light scattering, small-angle X-ray scattering and atomic force microscopy revealed that hCdc45 exists as an alpha-helical monomer and possesses a structure similar to its bacterial homolog RecJ. hCdc45 bound long (113-mer or 80-mer) single-stranded DNA fragments with a higher affinity than shorter ones (34-mer). hCdc45 displayed a preference for 3′ protruding strands and bound tightly to single-strand/double-strand DNA junctions, such as those presented by Y-shaped DNA, bubbles and displacement loops, all of which appear transiently during the initiation of DNA replication. Collectively, our findings suggest that hCdc45 not only binds to but also slides on DNA with a 3′–5′ polarity and, thereby acts as a molecular ‘wedge’ to initiate DNA strand displacement.
37
Zhang, Zi-Mou, Peng-Cheng Gao, Zhi-Fei Wang, Bai-Wang Sun, and Yong Jiang. "DNA-caged gold nanoparticles for controlled release of doxorubicin triggered by a DNA enzyme and pH." Chemical Communications 51, no. 65 (2015): 12996–99. http://dx.doi.org/10.1039/c5cc05164a.
Анотація:
DNA polyhedron-caged AuNPs were self-assembled using four-point-star DNAs, with three strands hybridizing to each other and the fourth strand attaching to the AuNPs. The caged AuNPs can act as doxorubicin nanocarriers; a DNA enzyme and pH can trigger controlled release.
38
Aldag, Pierre, Fabian Welzel, Leonhard Jakob, Andreas Schmidbauer, Marius Rutkauskas, Fergus Fettes, Dina Grohmann, and Ralf Seidel. "Probing the stability of the SpCas9–DNA complex after cleavage." Nucleic Acids Research 49, no. 21 (November 18, 2021): 12411–21. http://dx.doi.org/10.1093/nar/gkab1072.
Анотація:
Abstract CRISPR–Cas9 is a ribonucleoprotein complex that sequence-specifically binds and cleaves double-stranded DNA. Wildtype Cas9 and its nickase and cleavage-incompetent mutants have been used in various biological techniques due to their versatility and programmable specificity. Cas9 has been shown to bind very stably to DNA even after cleavage of the individual DNA strands, inhibiting further turnovers and considerably slowing down in-vivo repair processes. This poses an obstacle in genome editing applications. Here, we employed single-molecule magnetic tweezers to investigate the binding stability of different Streptococcus pyogenes Cas9 variants after cleavage by challenging them with supercoiling. We find that different release mechanisms occur depending on which DNA strand is cleaved. After initial target strand cleavage, supercoils are only removed after the collapse of the R-loop. We identified several states with different stabilities of the R-loop. Most importantly, we find that the post-cleavage state of Cas9 exhibits a higher stability than the pre-cleavage state. After non-target strand cleavage, supercoils are immediately but slowly released by swiveling of the non-target strand around Cas9 bound to the target strand. Consequently, Cas9 and its non-target strand nicking mutant stay stably bound to the DNA for many hours even at elevated torsional stress.
39
Yu, Chuanhe, Haiyun Gan, Albert Serra-Cardona, Lin Zhang, Songlin Gan, Sushma Sharma, Erik Johansson, Andrei Chabes, Rui-Ming Xu, and Zhiguo Zhang. "A mechanism for preventing asymmetric histone segregation onto replicating DNA strands." Science 361, no. 6409 (August 16, 2018): 1386–89. http://dx.doi.org/10.1126/science.aat8849.
Анотація:
How parental histone (H3-H4)2 tetramers, the primary carriers of epigenetic modifications, are transferred onto leading and lagging strands of DNA replication forks for epigenetic inheritance remains elusive. Here we show that parental (H3-H4)2 tetramers are assembled into nucleosomes onto both leading and lagging strands, with a slight preference for lagging strands. The lagging-strand preference increases markedly in budding yeast cells lacking Dpb3 and Dpb4, two subunits of the leading strand DNA polymerase, Pol ε, owing to the impairment of parental (H3-H4)2 transfer to leading strands. Dpb3-Dpb4 binds H3-H4 in vitro and participates in the inheritance of heterochromatin. These results indicate that different proteins facilitate the transfer of parental (H3-H4)2 onto leading versus lagging strands and that Dbp3-Dpb4 plays an important role in this poorly understood process.
40
Thompson, Shannon J., Aoife Rooney, Kevin M. Prise, and Stephen J. McMahon. "Evaluating Iodine-125 DNA Damage Benchmarks of Monte Carlo DNA Damage Models." Cancers 14, no. 3 (January 18, 2022): 463. http://dx.doi.org/10.3390/cancers14030463.
Анотація:
A wide range of Monte Carlo models have been applied to predict yields of DNA damage based on nanoscale track structure calculations. While often similar on the macroscopic scale, these models frequently employ different assumptions which lead to significant differences in nanoscale dose deposition. However, the impact of these differences on key biological readouts remains unclear. A major challenge in this area is the lack of robust datasets which can be used to benchmark models, due to a lack of resolution at the base pair level required to deeply test nanoscale dose deposition. Studies investigating the distribution of strand breakage in short DNA strands following the decay of incorporated 125I offer one of the few benchmarks for model predictions on this scale. In this work, we have used TOPAS-nBio to evaluate the performance of three Geant4-DNA physics models at predicting the distribution and yield of strand breaks in this irradiation scenario. For each model, energy and OH radical distributions were simulated and used to generate predictions of strand breakage, varying energy thresholds for strand breakage and OH interaction rates to fit to the experimental data. All three models could fit well to the observed data, although the best-fitting strand break energy thresholds ranged from 29.5 to 32.5 eV, significantly higher than previous studies. However, despite well describing the resulting DNA fragment distribution, these fit models differed significantly with other endpoints, such as the total yield of breaks, which varied by 70%. Limitations in the underlying data due to inherent normalisation mean it is not possible to distinguish clearly between the models in terms of total yield. This suggests that, while these physics models can effectively fit some biological data, they may not always generalise in the same way to other endpoints, requiring caution in their extrapolation to new systems and the use of multiple different data sources for robust model benchmarking.
41
Lin, Maoxuan, and Jun-tao Guo. "New insights into protein–DNA binding specificity from hydrogen bond based comparative study." Nucleic Acids Research 47, no. 21 (October 30, 2019): 11103–13. http://dx.doi.org/10.1093/nar/gkz963.
Анотація:
Abstract Knowledge of protein–DNA binding specificity has important implications in understanding DNA metabolism, transcriptional regulation and developing therapeutic drugs. Previous studies demonstrated hydrogen bonds between amino acid side chains and DNA bases play major roles in specific protein–DNA interactions. In this paper, we investigated the roles of individual DNA strands and protein secondary structure types in specific protein–DNA recognition based on side chain-base hydrogen bonds. By comparing the contribution of each DNA strand to the overall binding specificity between DNA-binding proteins with different degrees of binding specificity, we found that highly specific DNA-binding proteins show balanced hydrogen bonding with each of the two DNA strands while multi-specific DNA binding proteins are generally biased towards one strand. Protein-base pair hydrogen bonds, in which both bases of a base pair are involved in forming hydrogen bonds with amino acid side chains, are more prevalent in the highly specific protein–DNA complexes than those in the multi-specific group. Amino acids involved in side chain-base hydrogen bonds favor strand and coil secondary structure types in highly specific DNA-binding proteins while multi-specific DNA-binding proteins prefer helices.
42
Gao, Rui, Zhuang Cai, Jianbang Wang, and Huajie Liu. "Condensed DNA Nanosphere for DNA Origami Cryptography." Chemistry 5, no. 4 (November 8, 2023): 2406–17. http://dx.doi.org/10.3390/chemistry5040159.
Анотація:
Maintaining the confidentiality and integrity of the messages during a transmission is one of the most important aims of encrypted communication systems. Many achievements were made using biomolecules to improve the quality of the messages in communication. At the same time, it is still a challenge to construct cooperative communications based on the interactions between biomolecules to achieve the confidentiality and integrity of the transmitted messages. DNA-based encrypted communications have been developed, and in particular, DNA-origami-based message encryption can combine steganography and pattern encryption and exhibits extremely high confidentiality. Nevertheless, limited by biological characteristics, encrypted messages based on DNA require a strict storage environment in the process of transmission. The integrity of the message encoded in the DNA may be damaged when the DNA is in an unfriendly and hard environment. Therefore, it is particularly significant to improve the stability of DNA when it is exposed to a harsh environment during transmission. Here, we encoded the information into the DNA strands that were condensed for encryption to form a nanosphere covered with a shell of SiO2, which brings high-density messages and exhibits higher stability than separated DNA. The solid shell of SiO2 could prevent DNA from contacting the harsh environment, thereby protecting the DNA structure and maintaining the integrity of the information. At the same time, DNA nanospheres can achieve high throughput input and higher storage density per unit volume, which contribute to confusing the message strand (M-strand) with the interference strand in the stored information. Condensing DNA into the nanosphere that is used for DNA origami cryptography has the potential to be used in harsh conditions with higher confidentiality and integrity for the transmitted messages.
43
Mohamadi, Maryam, Ali Mostafavi, and Masoud Torkzadeh-Mahani. "Design of a Sensitive and Selective Electrochemical Aptasensor for the Determination of the Complementary cDNA of miRNA-145 Based on the Intercalation and Electrochemical Reduction of Doxorubicin." Journal of AOAC INTERNATIONAL 100, no. 6 (November 1, 2017): 1754–60. http://dx.doi.org/10.5740/jaoacint.16-0302.
Анотація:
Abstract The aim of this research was the determination of a microRNA (miRNA) using a DNA electrochemical aptasensor. In this biosensor, the complementary complementary DNA (cDNA) of miRNA-145 (a sense RNA transcript) was the target strand and the cDNA of miRNA-145 was the probe strand. Both cDNAs can be the product of the reverse transcriptase-polymerase chain reaction of miRNA. The proposed aptasensor’s function was based on the hybridization of target strands with probes immobilized on the surface of a working electrode and the subsequent intercalation of doxorubicin (DOX) molecules functioning as the electroactive indicators of any double strands that formed. Electrochemical transduction was performed by measuring the cathodic current resulting from the electrochemical reduction of the intercalated molecules at the electrode surface. In the experiment, because many DOX molecules accumulated on each target strand on the electrode surface, amplification was inherently easy, without a need for enzymatic or complicated amplification strategies. The proposed aptasensor also had the excellent ability to regenerate as a result of the melting of the DNA duplex. Moreover, the use of DNA probe strands obviated the challenges of working with an RNA probe, such as sensitivity to RNase enzyme. In addition to the linear relationship between the electrochemical signal and the concentration of the target strands that ranged from 2.0 to 80.0 nM with an LOD of 0.27 nM, the proposed biosensor was clearly capable of distinguishing between complementary (target strand) and noncomplementary sequences. The presented biosensor was successfully applied for the quantification of DNA strands corresponding to miRNA-145 in human serum samples.
44
Zhang, Jiahui, Ashkan Fakharzadeh, Feng Pan, Christopher Roland, and Celeste Sagui. "Atypical structures of GAA/TTC trinucleotide repeats underlying Friedreich’s ataxia: DNA triplexes and RNA/DNA hybrids." Nucleic Acids Research 48, no. 17 (August 21, 2020): 9899–917. http://dx.doi.org/10.1093/nar/gkaa665.
Анотація:
Abstract Expansion of the GAA/TTC repeats in the first intron of the FXN gene causes Friedreich’s ataxia. Non-canonical structures are linked to this expansion. DNA triplexes and R-loops are believed to arrest transcription, which results in frataxin deficiency and eventual neurodegeneration. We present a systematic in silico characterization of the possible DNA triplexes that could be assembled with GAA and TTC strands; the two hybrid duplexes [r(GAA):d(TTC) and d(GAA):r(UUC)] in an R-loop; and three hybrid triplexes that could form during bidirectional transcription when the non-template DNA strand bonds with the hybrid duplex (collapsed R-loops, where the two DNA strands remain antiparallel). For both Y·R:Y and R·R:Y DNA triplexes, the parallel third strand orientation is more stable; both parallel and antiparallel protonated d(GA+A)·d(GAA):d(TTC) triplexes are stable. Apparent contradictions in the literature about the R·R:Y triplex stability is probably due to lack of molecular resolution, since shifting the third strand by a single nucleotide alters the stability ranking. In the collapsed R-loops, antiparallel d(TTC+)·d(GAA):r(UUC) is unstable, while parallel d(GAA)·r(GAA):d(TTC) and d(GA+A)·r(GAA):d(TTC) are stable. In addition to providing new structural perspectives for specific therapeutic aims, our results contribute to a systematic structural basis for the emerging field of quantitative R-loop biology.
45
Sinden, Richard, R. "Slipped strand DNA structures." Frontiers in Bioscience 12, no. 12 (2007): 4788. http://dx.doi.org/10.2741/2427.
46
Bross, Linda, Masamichi Muramatsu, Kazuo Kinoshita, Tasuku Honjo, and Heinz Jacobs. "DNA Double-Strand Breaks." Journal of Experimental Medicine 195, no. 9 (May 6, 2002): 1187–92. http://dx.doi.org/10.1084/jem.20011749.
Анотація:
The activation-induced cytidine deaminase (AID) is required for somatic hypermutation (SHM) and class-switch recombination (CSR) of immunoglobulin (Ig) genes, both of which are associated with DNA double-strand breaks (DSBs). As AID is capable of deaminating deoxy-cytidine (dC) to deoxy-uracil (dU), it might induce nicks (single strand DNA breaks) and also DNA DSBs via a U-DNA glycosylase-mediated base excision repair pathway (‘DNA-substrate model’). Alternatively, AID functions like its closest homologue Apobec1 as a catalytic subunit of a RNA editing holoenzyme (‘RNA-substrate model’). Although rearranged Vλ genes are preferred targets of SHM we found that germinal center (GC) B cells of AID-proficient and -deficient Vλ1-expressing GC B cells display a similar frequency, distribution, and sequence preference of DSBs in rearranged and also in germline Vλ1 genes. The possible roles of DSBs in relation to AID function and SHM are discussed.
47
Bolden, A. H., C. M. Nalin, C. A. Ward, M. S. Poonian, and A. Weissbach. "Primary DNA sequence determines sites of maintenance and de novo methylation by mammalian DNA methyltransferases." Molecular and Cellular Biology 6, no. 4 (April 1986): 1135–40. http://dx.doi.org/10.1128/mcb.6.4.1135-1140.1986.
Анотація:
Analysis of the enzymatic methylation of oligodeoxynucleotides containing multiple C-G groups showed that hemimethylated sites in duplex oligomers are not significantly methylated by human or murine DNA methyltransferase unless those sites are capable of being methylated de novo in the single- or double-stranded oligomers. Thus, the primary sequence of the target strand, rather than the methylation pattern of the complementary strand, determines maintenance methylation. This suggests that de novo and maintenance methylation are the same process catalyzed by the same enzyme. In addition, the study revealed that complementary strands of oligodeoxynucleotides are methylated at different rates and in different patterns. Both primary DNA sequence and the spacing between C-G groups seem important since in one case studied, maximal methylation required a specific spacing of 13 to 17 nucleotides between C-G pairs.
48
Bolden, A. H., C. M. Nalin, C. A. Ward, M. S. Poonian, and A. Weissbach. "Primary DNA sequence determines sites of maintenance and de novo methylation by mammalian DNA methyltransferases." Molecular and Cellular Biology 6, no. 4 (April 1986): 1135–40. http://dx.doi.org/10.1128/mcb.6.4.1135.
Анотація:
Analysis of the enzymatic methylation of oligodeoxynucleotides containing multiple C-G groups showed that hemimethylated sites in duplex oligomers are not significantly methylated by human or murine DNA methyltransferase unless those sites are capable of being methylated de novo in the single- or double-stranded oligomers. Thus, the primary sequence of the target strand, rather than the methylation pattern of the complementary strand, determines maintenance methylation. This suggests that de novo and maintenance methylation are the same process catalyzed by the same enzyme. In addition, the study revealed that complementary strands of oligodeoxynucleotides are methylated at different rates and in different patterns. Both primary DNA sequence and the spacing between C-G groups seem important since in one case studied, maximal methylation required a specific spacing of 13 to 17 nucleotides between C-G pairs.
49
Rogers, W. Benjamin, and Vinothan N. Manoharan. "Programming colloidal phase transitions with DNA strand displacement." Science 347, no. 6222 (February 5, 2015): 639–42. http://dx.doi.org/10.1126/science.1259762.
Анотація:
DNA-grafted nanoparticles have been called “programmable atom-equivalents”: Like atoms, they form three-dimensional crystals, but unlike atoms, the particles themselves carry information (the sequences of the grafted strands) that can be used to “program” the equilibrium crystal structures. We show that the programmability of these colloids can be generalized to the full temperature-dependent phase diagram, not just the crystal structures themselves. We add information to the buffer in the form of soluble DNA strands designed to compete with the grafted strands through strand displacement. Using only two displacement reactions, we program phase behavior not found in atomic systems or other DNA-grafted colloids, including arbitrarily wide gas-solid coexistence, reentrant melting, and even reversible transitions between distinct crystal phases.
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Giannattasio, Michele, and Dana Branzei. "DNA Replication Through Strand Displacement During Lagging Strand DNA Synthesis in Saccharomyces cerevisiae." Genes 10, no. 2 (February 21, 2019): 167. http://dx.doi.org/10.3390/genes10020167.
Анотація:
This review discusses a set of experimental results that support the existence of extended strand displacement events during budding yeast lagging strand DNA synthesis. Starting from introducing the mechanisms and factors involved in leading and lagging strand DNA synthesis and some aspects of the architecture of the eukaryotic replisome, we discuss studies on bacterial, bacteriophage and viral DNA polymerases with potent strand displacement activities. We describe proposed pathways of Okazaki fragment processing via short and long flaps, with a focus on experimental results obtained in Saccharomyces cerevisiae that suggest the existence of frequent and extended strand displacement events during eukaryotic lagging strand DNA synthesis, and comment on their implications for genome integrity.