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Journal articles on the topic 'Double-Stranded DNA Breaks'

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Published: 4 June 2021
Last updated: 18 February 2022

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1

Goodsell, David S. "The Molecular Perspective: Double-Stranded DNA Breaks." Stem Cells 23, no. 7 (August 2005): 1021–22. http://dx.doi.org/10.1634/stemcells.fcm4.

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2

Goodsell, David S. "The Molecular Perspective: Double‐Stranded DNA Breaks." Oncologist 10, no. 5 (May 2005): 361–62. http://dx.doi.org/10.1634/theoncologist.10-5-361.

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3

Zee, Yeng Peng, Carmen López-Fernández, F. Arroyo, Stephen D. Johnston, William V. Holt, and Jaime Gosalvez. "Evidence that single-stranded DNA breaks are a normal feature of koala sperm chromatin, while double-stranded DNA breaks are indicative of DNA damage." REPRODUCTION 138, no. 2 (August 2009): 267–78. http://dx.doi.org/10.1530/rep-09-0021.

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In this study, we have used single and double comet assays to differentiate between single- and double-stranded DNA damage in an effort to refine the interpretation of DNA damage in mature koala spermatozoa. We have also investigated the likelihood that single-stranded DNA breakage is part of the natural spermiogenic process in koalas, where its function would be the generation of structural bends in the DNA molecule so that appropriate packaging and compaction can occur. Koala spermatozoa were examined using the sperm chromatin dispersion test (SCDt) and comet assays to investigate non-orthodox double-stranded DNA. Comet assays were conducted under 1) neutral conditions; and 2) neutral followed by alkaline conditions (double comet assay); the latter technique enabled simultaneous visualisation of both single-stranded and double-stranded DNA breaks. Following the SCDt, there was a continuum of nuclear morphotypes, ranging from no apparent DNA fragmentation to those with highly dispersed and degraded chromatin. Dispersion morphotypes were mirrored by a similar diversity of comet morphologies that could be further differentiated using the double comet assay. The majority of koala spermatozoa had nuclei with DNA abasic-like residues that produced single-tailed comets following the double comet assay. The ubiquity of these residues suggests that constitutive alkali-labile sites are part of the structural configuration of the koala sperm nucleus. Spermatozoa with ‘true’ DNA fragmentation exhibited a continuum of comet morphologies, ranging from a more severe form of alkaline-susceptible DNA with a diffuse single tail to nuclei that exhibited both single- and double-stranded breaks with two comet tails.
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4

Komor, Alexis C., Ahmed H. Badran, and David R. Liu. "Editing the Genome Without Double-Stranded DNA Breaks." ACS Chemical Biology 13, no. 2 (October 9, 2017): 383–88. http://dx.doi.org/10.1021/acschembio.7b00710.

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5

Hanada, Katsuhiro, Teruhito Yamashita, Yuko Shobuike, and Hideo Ikeda. "Role of DnaB Helicase in UV-Induced Illegitimate Recombination in Escherichia coli." Journal of Bacteriology 183, no. 17 (September 1, 2001): 4964–69. http://dx.doi.org/10.1128/jb.183.17.4964-4969.2001.

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ABSTRACT To study the involvement of DNA replication in UV-induced illegitimate recombination, we examined the effect of temperature-sensitive dnaB mutations on illegitimate recombination and found that the frequency of illegitimate recombination was reduced by an elongation-deficient mutation,dnaB14, but not by an initiation-deficient mutation,dnaB252. This result indicates that DNA replication is required for UV-induced illegitimate recombination. In addition, thednaB14 mutation also affected spontaneous or UV-induced illegitimate recombination enhanced by the recQmutation. Nucleotide sequence analyses of the recombination junctions showed that DnaB-mediated illegitimate recombination is short homology dependent. Previously, Michel et al. (B. Michel, S. Ehrlich, and M. Uzest, EMBO J. 16:430–438, 1997) showed that thermal treatment of the temperature-sensitive dnaB8 mutant induces double-stranded breaks, implying that induction of illegitimate recombination occurs. To explain the discrepancy between the observations, we propose a model for DnaB function, in which thednaB mutations may exhibit two types of responses, early and late responses, for double-stranded break formation. In the early response, replication forks stall at damaged DNA, resulting in the formation of double-stranded breaks, and the dnaB14mutation reduces the double-stranded breaks shortly after temperature shift-up. On the other hand, in the late response, the arrested replication forks mediated by the dnaB8 mutation may induce double-stranded breaks after prolonged incubation.
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6

Cowan, Richard, David Culpin, and David Gates. "Asymptotic results for a problem of DNA breakage." Journal of Applied Probability 27, no. 2 (June 1990): 433–39. http://dx.doi.org/10.2307/3214663.

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Double-stranded DNA molecules can be damaged by enzymic action or radiation, in a manner which creates randomly-located single-stranded breaks (nicks). The accumulation of these leads eventually to the double-stranded breakage of the molecule, because two opposite-strand nicks within a critical distance of each other establish conditions for breakage. We study the random variable N, defined as the number of nicks needed for double-stranded breakage to occur. We develop an asymptotic theory which is needed for practical computations.
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7

Cowan, Richard, David Culpin, and David Gates. "Asymptotic results for a problem of DNA breakage." Journal of Applied Probability 27, no. 02 (June 1990): 433–39. http://dx.doi.org/10.1017/s0021900200038894.

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Double-stranded DNA molecules can be damaged by enzymic action or radiation, in a manner which creates randomly-located single-stranded breaks (nicks). The accumulation of these leads eventually to the double-stranded breakage of the molecule, because two opposite-strand nicks within a critical distance of each other establish conditions for breakage. We study the random variable N, defined as the number of nicks needed for double-stranded breakage to occur. We develop an asymptotic theory which is needed for practical computations.
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8

Dillingham, Mark S., and Stephen C. Kowalczykowski. "RecBCD Enzyme and the Repair of Double-Stranded DNA Breaks." Microbiology and Molecular Biology Reviews 72, no. 4 (December 2008): 642–71. http://dx.doi.org/10.1128/mmbr.00020-08.

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SUMMARY The RecBCD enzyme of Escherichia coli is a helicase-nuclease that initiates the repair of double-stranded DNA breaks by homologous recombination. It also degrades linear double-stranded DNA, protecting the bacteria from phages and extraneous chromosomal DNA. The RecBCD enzyme is, however, regulated by a cis-acting DNA sequence known as Chi (crossover hotspot instigator) that activates its recombination-promoting functions. Interaction with Chi causes an attenuation of the RecBCD enzyme's vigorous nuclease activity, switches the polarity of the attenuated nuclease activity to the 5′ strand, changes the operation of its motor subunits, and instructs the enzyme to begin loading the RecA protein onto the resultant Chi-containing single-stranded DNA. This enzyme is a prototypical example of a molecular machine: the protein architecture incorporates several autonomous functional domains that interact with each other to produce a complex, sequence-regulated, DNA-processing machine. In this review, we discuss the biochemical mechanism of the RecBCD enzyme with particular emphasis on new developments relating to the enzyme's structure and DNA translocation mechanism.
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9

Eid, Ayman, Sahar Alshareef, and Magdy M. Mahfouz. "CRISPR base editors: genome editing without double-stranded breaks." Biochemical Journal 475, no. 11 (June 11, 2018): 1955–64. http://dx.doi.org/10.1042/bcj20170793.

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The CRISPR (clustered regularly interspaced short palindromic repeat)/Cas9 adaptive immunity system has been harnessed for genome editing applications across eukaryotic species, but major drawbacks, such as the inefficiency of precise base editing and off-target activities, remain. A catalytically inactive Cas9 variant (dead Cas9, dCas9) has been fused to diverse functional domains for targeting genetic and epigenetic modifications, including base editing, to specific DNA sequences. As base editing does not require the generation of double-strand breaks, dCas9 and Cas9 nickase have been used to target deaminase domains to edit specific loci. Adenine and cytidine deaminases convert their respective nucleotides into other DNA bases, thereby offering many possibilities for DNA editing. Such base-editing enzymes hold great promise for applications in basic biology, trait development in crops, and treatment of genetic diseases. Here, we discuss recent advances in precise gene editing using different platforms as well as their potential applications in basic biology and biotechnology.
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10

Bhandoola, Avinash, Benjamin Dolnick, Nihal Fayad, Andre Nussenzweig, and Alfred Singer. "Immature Thymocytes Undergoing Receptor Rearrangements Are Resistant to an Atm-Dependent Death Pathway Activated in Mature T Cells by Double-Stranded DNA Breaks." Journal of Experimental Medicine 192, no. 6 (September 18, 2000): 891–98. http://dx.doi.org/10.1084/jem.192.6.891.

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Immature CD4+CD8+ thymocytes rearrange their T cell receptor (TCR)-α gene locus to generate clonotypic α/β TCR, after which a few cells expressing selectable TCR are signaled to further differentiate into mature T cells. Because of requirements for self-tolerance, immature CD4+CD8+ thymocytes are programmed to die in the thymus in response to a variety of stimuli that do not induce death of mature T cells. We now demonstrate that, in contrast to all previously described stimuli, immature CD4+CD8+ thymocytes are selectively more resistant than mature T cells to apoptotic death induced by DNA intercalating agents. Importantly, we demonstrate that DNA intercalating agents induce double-stranded DNA breaks in both immature thymocytes and mature T cells, but immature thymocytes tolerate these DNA breaks, whereas mature T cells are signaled to die by an Atm-dependent but p53-independent death mechanism. Thus, our results indicate that absence of an Atm-dependent but p53-independent pathway allows immature thymocytes to survive double-stranded DNA breaks. It is likely that the unique ability of immature thymocytes to survive DNA-damaging intercalating agents reflects their tolerance of double-stranded DNA breaks that occur normally during antigen receptor gene rearrangements.
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11

Eaton, Jonah, and Alexandra Zidovska. "Interphase Chromatin Dynamics in Response to Double Stranded DNA Breaks." Biophysical Journal 114, no. 3 (February 2018): 563a—564a. http://dx.doi.org/10.1016/j.bpj.2017.11.3082.

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12

Rackovsky, S., and W. A. Bernhard. "Electron trapping in double-stranded DNA: implications for formation of double-strand breaks." Journal of Physical Chemistry 93, no. 12 (June 1989): 5006–8. http://dx.doi.org/10.1021/j100349a066.

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13

Winters, M. K., D. W. Fairbairn, M. D. Standing, and K. L. O’Neill. "Determining DNA strand breaks using the laser scanning microscope." Proceedings, annual meeting, Electron Microscopy Society of America 51 (August 1, 1993): 272–73. http://dx.doi.org/10.1017/s042482010014720x.

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The single cell gel assay has been proven to be an effective, fast and accurate method of determining the amount of DNA single or double stranded breaks. The possible uses of this assay have already reached into some aspects of DNA damage caused by chemicals or other destructive agents. It has yet to reach even deeper into mutagenesis and carcinogenesis, both of which may show single or double stranded breaks to the DNA. Here we explore the effects that different concentrations of H2O2 have on the DNA of cells. The assay is performed by suspending cells in a low melt point agarose, spreading the agarose out on a frosted slide and letting it gel over ice. The cells are then treated with H2O2 to induce single strand DNA breakage. The cells are lysed in an alkaline environment, which separates the DNA into single strands and then electrophoresed. The gel is then stained with ethidium bromide.
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14

Szlachta, Karol, Arkadi Manukyan, Heather M. Raimer, Sandeep Singh, Anita Salamon, Wenying Guo, Kirill S. Lobachev, and Yuh-Hwa Wang. "Topoisomerase II contributes to DNA secondary structure-mediated double-stranded breaks." Nucleic Acids Research 48, no. 12 (June 5, 2020): 6654–71. http://dx.doi.org/10.1093/nar/gkaa483.

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Abstract DNA double-stranded breaks (DSBs) trigger human genome instability, therefore identifying what factors contribute to DSB induction is critical for our understanding of human disease etiology. Using an unbiased, genome-wide approach, we found that genomic regions with the ability to form highly stable DNA secondary structures are enriched for endogenous DSBs in human cells. Human genomic regions predicted to form non-B-form DNA induced gross chromosomal rearrangements in yeast and displayed high indel frequency in human genomes. The extent of instability in both analyses is in concordance with the structure forming ability of these regions. We also observed an enrichment of DNA secondary structure-prone sites overlapping transcription start sites (TSSs) and CCCTC-binding factor (CTCF) binding sites, and uncovered an increase in DSBs at highly stable DNA secondary structure regions, in response to etoposide, an inhibitor of topoisomerase II (TOP2) re-ligation activity. Importantly, we found that TOP2 deficiency in both yeast and human leads to a significant reduction in DSBs at structure-prone loci, and that sites of TOP2 cleavage have a greater ability to form highly stable DNA secondary structures. This study reveals a direct role for TOP2 in generating secondary structure-mediated DNA fragility, advancing our understanding of mechanisms underlying human genome instability.
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15

Torres-Machorro, Ana Lilia, John P. Aris, and Lorraine Pillus. "A moonlighting metabolic protein influences repair at DNA double-stranded breaks." Nucleic Acids Research 43, no. 3 (January 27, 2015): 1646–58. http://dx.doi.org/10.1093/nar/gku1405.

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16

Rogakou, Emmy P., Duane R. Pilch, Ann H. Orr, Vessela S. Ivanova, and William M. Bonner. "DNA Double-stranded Breaks Induce Histone H2AX Phosphorylation on Serine 139." Journal of Biological Chemistry 273, no. 10 (March 6, 1998): 5858–68. http://dx.doi.org/10.1074/jbc.273.10.5858.

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17

Li, Wuxing, and Hong Ma. "Double-stranded DNA breaks and gene functions in recombination and meiosis." Cell Research 16, no. 5 (May 2006): 402–12. http://dx.doi.org/10.1038/sj.cr.7310052.

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18

Keating, Derek, Alessandra Parrella, Zev Rosenwaks, and Gianpiero D. Palermo. "THE IMPACT OF DOUBLE-STRANDED SPERM DNA BREAKS ON ICSI OUTCOME." Fertility and Sterility 114, no. 3 (September 2020): e309-e310. http://dx.doi.org/10.1016/j.fertnstert.2020.08.843.

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19

Morimoto, Tsuda, Bunch, Sasanuma, Austin, and Takeda. "Type II DNA Topoisomerases Cause Spontaneous Double-Strand Breaks in Genomic DNA." Genes 10, no. 11 (October 30, 2019): 868. http://dx.doi.org/10.3390/genes10110868.

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Type II DNA topoisomerase enzymes (TOP2) catalyze topological changes by strand passage reactions. They involve passing one intact double stranded DNA duplex through a transient enzyme-bridged break in another (gated helix) followed by ligation of the break by TOP2. A TOP2 poison, etoposide blocks TOP2 catalysis at the ligation step of the enzyme-bridged break, increasing the number of stable TOP2 cleavage complexes (TOP2ccs). Remarkably, such pathological TOP2ccs are formed during the normal cell cycle as well as in postmitotic cells. Thus, this ‘abortive catalysis’ can be a major source of spontaneously arising DNA double-strand breaks (DSBs). TOP2-mediated DSBs are also formed upon stimulation with physiological concentrations of androgens and estrogens. The frequent occurrence of TOP2-mediated DSBs was previously not appreciated because they are efficiently repaired. This repair is performed in collaboration with BRCA1, BRCA2, MRE11 nuclease, and tyrosyl-DNA phosphodiesterase 2 (TDP2) with nonhomologous end joining (NHEJ) factors. This review first discusses spontaneously arising DSBs caused by the abortive catalysis of TOP2 and then summarizes proteins involved in repairing stalled TOP2ccs and discusses the genotoxicity of the sex hormones.
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20

Lal, Ashutosh, Amrita Bhagat, Wafa Atamna, Tal Offer, Elliott P. Vichinsky, Frans A. Kuypers, and Bruce N. Ames. "Increased Chromosomal Breaks in Sickle Cell Disease as Evidenced by the Presence of Micronuclei in Erythrocytes." Blood 106, no. 11 (November 16, 2005): 3807. http://dx.doi.org/10.1182/blood.v106.11.3807.3807.

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Abstract Double-stranded DNA breaks are serious lesions that contribute to the pathogenesis of cancer and chronic illnesses. Free radical injury is an important mechanism for the production of DNA modifications that eventually lead to chromosomal breaks. The generation of free radicals is increased in sickle cell disease (SCD) owing to the presence of the relatively unstable HbS and recurrent ischemia-reperfusion events. In the present study, a sensitive reticulocyte micronucleus assay was used to determine the frequency of chromosomal breaks in patients with SCD (Offer et al. FASEB J. 2005;19:485). A micronucleus is a piece of a chromosome left behind in a reticulocyte as a consequence of DNA double-stranded break after the nucleus is extruded. Blood samples were obtained from patients with SCD (n=33) and healthy volunteers (n=23). After immunomagnetic enrichment of CD71+ reticulocytes (Trf-Ret), RNA was removed with RNAse, and the cells were stained for miconuclei using the DNA dye 7-aminoactinomycin D (7-AAD) followed by flow cytometric evaluation. The appearance of reactive oxygen species in cytosol and membrane lipid oxidation in RBCs were measured by flow cytometry using the fluorescent markers 2, 7-dichlorofluorescin diacetate (DCF) and C11-BODIPY, respectively. The frequency of micronucleated Trf-Ret was significantly higher (p<.0001) in patients with SCD (mean 3.061/10,000 Trf-Ret, range 0.256 to 9.779) as compared with normal controls (mean 0.750/10,000 Trf-Ret, range 0.043 to 2.659). Mean DCF (125.1%) and C11-BODIPY (117.6%) fluorescence intensities were significantly greater than the mean control value (100%, p 0.0001). Our data indicate that patients with sickle cell disease have increased double stranded DNA-breaks compared with healthy controls, and suggest that elevated production of oxidants could contribute to the development of genetic damage in these patients.
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21

Merrill, Bradley J., and Connie Holm. "A Requirement for Recombinational Repair in Saccharomyces cerevisiae Is Caused by DNA Replication Defects of mec1 Mutants." Genetics 153, no. 2 (October 1, 1999): 595–605. http://dx.doi.org/10.1093/genetics/153.2.595.

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Abstract To examine the role of the RAD52 recombinational repair pathway in compensating for DNA replication defects in Saccharomyces cerevisiae, we performed a genetic screen to identify mutants that require Rad52p for viability. We isolated 10 mec1 mutations that display synthetic lethality with rad52. These mutations (designated mec1-srf for synthetic lethality with rad-fifty-two) simultaneously cause two types of phenotypes: defects in the checkpoint function of Mec1p and defects in the essential function of Mec1p. Velocity sedimentation in alkaline sucrose gradients revealed that mec1-srf mutants accumulate small single-stranded DNA synthesis intermediates, suggesting that Mec1p is required for the normal progression of DNA synthesis. sml1 suppressor mutations suppress both the accumulation of DNA synthesis intermediates and the requirement for Rad52p in mec1-srf mutants, but they do not suppress the checkpoint defect in mec1-srf mutants. Thus, it appears to be the DNA replication defects in mec1-srf mutants that cause the requirement for Rad52p. By using hydroxyurea to introduce similar DNA replication defects, we found that single-stranded DNA breaks frequently lead to double-stranded DNA breaks that are not rapidly repaired in rad52 mutants. Taken together, these data suggest that the RAD52 recombinational repair pathway is required to prevent or repair double-stranded DNA breaks caused by defective DNA replication in mec1-srf mutants.
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22

Bilang, R., A. Peterhans, A. Bogucki, and J. Paszkowski. "Single-stranded DNA as a recombination substrate in plants as assessed by stable and transient recombination assays." Molecular and Cellular Biology 12, no. 1 (January 1992): 329–36. http://dx.doi.org/10.1128/mcb.12.1.329.

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Two separate assays, one that requires stable integration of recombination products and one that does not, were employed to elucidate the role of single-stranded DNA in extrachromosomal homologous recombination in Nicotiana tabacum. Both assays revealed that single-stranded DNA in linear and in circular forms was an efficient substrate for recombination, provided that the cotransformed recombination substrates were of complementary sequence, so that direct annealing was possible. Recombination was inefficient when both single-stranded recombination partners contained homologous regions of identical sequence and generation of a double-stranded DNA was required prior to heteroduplex formation. These results indicate that direct annealing of single strands is an important initial step for intermolecular recombination in tobacco cells. Annealed cotransformed single-stranded molecules yielded intermediates that could be further processed by either continuous or discontinuous second-strand synthesis. The type of intermediate had no influence on the recombination efficiency. Double-stranded circles were unable to recombine efficiently either with each other or with single-stranded DNA. Our results suggest that a helicase activity is involved in the initial steps of double-stranded DNA recombination which unwinds duplex molecules at the site of double-strand breaks.
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23

Bilang, R., A. Peterhans, A. Bogucki, and J. Paszkowski. "Single-stranded DNA as a recombination substrate in plants as assessed by stable and transient recombination assays." Molecular and Cellular Biology 12, no. 1 (January 1992): 329–36. http://dx.doi.org/10.1128/mcb.12.1.329-336.1992.

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Two separate assays, one that requires stable integration of recombination products and one that does not, were employed to elucidate the role of single-stranded DNA in extrachromosomal homologous recombination in Nicotiana tabacum. Both assays revealed that single-stranded DNA in linear and in circular forms was an efficient substrate for recombination, provided that the cotransformed recombination substrates were of complementary sequence, so that direct annealing was possible. Recombination was inefficient when both single-stranded recombination partners contained homologous regions of identical sequence and generation of a double-stranded DNA was required prior to heteroduplex formation. These results indicate that direct annealing of single strands is an important initial step for intermolecular recombination in tobacco cells. Annealed cotransformed single-stranded molecules yielded intermediates that could be further processed by either continuous or discontinuous second-strand synthesis. The type of intermediate had no influence on the recombination efficiency. Double-stranded circles were unable to recombine efficiently either with each other or with single-stranded DNA. Our results suggest that a helicase activity is involved in the initial steps of double-stranded DNA recombination which unwinds duplex molecules at the site of double-strand breaks.
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24

Do, To Uyen, Bay Ho, Shyh-Jen Shih, and Andrew Vaughan. "Zinc Finger Nuclease induced DNA double stranded breaks and rearrangements in MLL." Mutation Research/Fundamental and Molecular Mechanisms of Mutagenesis 740, no. 1-2 (December 2012): 34–42. http://dx.doi.org/10.1016/j.mrfmmm.2012.12.006.

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25

Frock, Richard L., Jiazhi Hu, Robin M. Meyers, Yu-Jui Ho, Erina Kii, and Frederick W. Alt. "Genome-wide detection of DNA double-stranded breaks induced by engineered nucleases." Nature Biotechnology 33, no. 2 (December 15, 2014): 179–86. http://dx.doi.org/10.1038/nbt.3101.

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26

Puc, Janusz, and Ramon Parsons. "PTEN Loss Inhibits CHK1 to Cause Double Stranded-DNA Breaks in Cells." Cell Cycle 4, no. 7 (May 2, 2005): 927–29. http://dx.doi.org/10.4161/cc.4.7.1795.

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27

Jayaram, M. "Mating type-like conversion promoted by the 2 micrograms circle site-specific recombinase: implications for the double-strand-gap repair model." Molecular and Cellular Biology 6, no. 11 (November 1986): 3831–37. http://dx.doi.org/10.1128/mcb.6.11.3831.

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Double-strand breaks in DNA are known to promote recombination in Saccharomyces cerevisiae. Yeast mating type switching, which is a highly efficient gene conversion event, is apparently initiated by a site-specific double-strand break. The 2 micrograms circle site-specific recombinase, FLP, has been shown to make double-strand breaks in its substrate DNA. By using a hybrid 2 micrograms circle::Tn5 plasmid, a portion of which resembles, in its DNA organization, the active (MAT) and the silent (HML) yeast mating type loci, it is shown that FLP mediates a conversion event analogous to mating type switching. Whereas the FLP site-specific recombination is not dependent on the RAD52 gene product, the FLP-induced conversion is abolished in a rad52 background. The FLP-promoted conversion in vivo can be faithfully reproduced by making a double-stranded gap in vitro in the vicinity of the FLP site and allowing the gap to be repaired in vivo.
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Jayaram, M. "Mating type-like conversion promoted by the 2 micrograms circle site-specific recombinase: implications for the double-strand-gap repair model." Molecular and Cellular Biology 6, no. 11 (November 1986): 3831–37. http://dx.doi.org/10.1128/mcb.6.11.3831-3837.1986.

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Double-strand breaks in DNA are known to promote recombination in Saccharomyces cerevisiae. Yeast mating type switching, which is a highly efficient gene conversion event, is apparently initiated by a site-specific double-strand break. The 2 micrograms circle site-specific recombinase, FLP, has been shown to make double-strand breaks in its substrate DNA. By using a hybrid 2 micrograms circle::Tn5 plasmid, a portion of which resembles, in its DNA organization, the active (MAT) and the silent (HML) yeast mating type loci, it is shown that FLP mediates a conversion event analogous to mating type switching. Whereas the FLP site-specific recombination is not dependent on the RAD52 gene product, the FLP-induced conversion is abolished in a rad52 background. The FLP-promoted conversion in vivo can be faithfully reproduced by making a double-stranded gap in vitro in the vicinity of the FLP site and allowing the gap to be repaired in vivo.
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29

Sauvageau, Synthia, Alicja Z. Stasiak, Isabelle Banville, Mickaël Ploquin, Andrzej Stasiak, and Jean-Yves Masson. "Fission Yeast Rad51 and Dmc1, Two Efficient DNA Recombinases Forming Helical Nucleoprotein Filaments." Molecular and Cellular Biology 25, no. 11 (June 1, 2005): 4377–87. http://dx.doi.org/10.1128/mcb.25.11.4377-4387.2005.

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ABSTRACT Homologous recombination is important for the repair of double-strand breaks during meiosis. Eukaryotic cells require two homologs of Escherichia coli RecA protein, Rad51 and Dmc1, for meiotic recombination. To date, it is not clear, at the biochemical level, why two homologs of RecA are necessary during meiosis. To gain insight into this, we purified Schizosaccharomyces pombe Rad51 and Dmc1 to homogeneity. Purified Rad51 and Dmc1 form homo-oligomers, bind single-stranded DNA preferentially, and exhibit DNA-stimulated ATPase activity. Both Rad51 and Dmc1 promote the renaturation of complementary single-stranded DNA. Importantly, Rad51 and Dmc1 proteins catalyze ATP-dependent strand exchange reactions with homologous duplex DNA. Electron microscopy reveals that both S. pombe Rad51 and Dmc1 form nucleoprotein filaments. Rad51 formed helical nucleoprotein filaments on single-stranded DNA, whereas Dmc1 was found in two forms, as helical filaments and also as stacked rings. These results demonstrate that Rad51 and Dmc1 are both efficient recombinases in lower eukaryotes and reveal closer functional and structural similarities between the meiotic recombinase Dmc1 and Rad51. The DNA strand exchange activity of both Rad51 and Dmc1 is most likely critical for proper meiotic DNA double-strand break repair in lower eukaryotes.
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30

Paix, Alexandre, Andrew Folkmann, Daniel H. Goldman, Heather Kulaga, Michael J. Grzelak, Dominique Rasoloson, Supriya Paidemarry, Rachel Green, Randall R. Reed, and Geraldine Seydoux. "Precision genome editing using synthesis-dependent repair of Cas9-induced DNA breaks." Proceedings of the National Academy of Sciences 114, no. 50 (November 28, 2017): E10745—E10754. http://dx.doi.org/10.1073/pnas.1711979114.

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The RNA-guided DNA endonuclease Cas9 has emerged as a powerful tool for genome engineering. Cas9 creates targeted double-stranded breaks (DSBs) in the genome. Knockin of specific mutations (precision genome editing) requires homology-directed repair (HDR) of the DSB by synthetic donor DNAs containing the desired edits, but HDR has been reported to be variably efficient. Here, we report that linear DNAs (single and double stranded) engage in a high-efficiency HDR mechanism that requires only ∼35 nucleotides of homology with the targeted locus to introduce edits ranging from 1 to 1,000 nucleotides. We demonstrate the utility of linear donors by introducing fluorescent protein tags in human cells and mouse embryos using PCR fragments. We find that repair is local, polarity sensitive, and prone to template switching, characteristics that are consistent with gene conversion by synthesis-dependent strand annealing. Our findings enable rational design of synthetic donor DNAs for efficient genome editing.
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31

Vidal-Eychenié, Sophie, Chantal Décaillet, Jihane Basbous, and Angelos Constantinou. "DNA structure-specific priming of ATR activation by DNA-PKcs." Journal of Cell Biology 202, no. 3 (July 29, 2013): 421–29. http://dx.doi.org/10.1083/jcb.201304139.

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Three phosphatidylinositol-3-kinase–related protein kinases implement cellular responses to DNA damage. DNA-dependent protein kinase catalytic subunit (DNA-PKcs) and ataxia-telangiectasia mutated respond primarily to DNA double-strand breaks (DSBs). Ataxia-telangiectasia and RAD3-related (ATR) signals the accumulation of replication protein A (RPA)–covered single-stranded DNA (ssDNA), which is caused by replication obstacles. Stalled replication intermediates can further degenerate and yield replication-associated DSBs. In this paper, we show that the juxtaposition of a double-stranded DNA end and a short ssDNA gap triggered robust activation of endogenous ATR and Chk1 in human cell-free extracts. This DNA damage signal depended on DNA-PKcs and ATR, which congregated onto gapped linear duplex DNA. DNA-PKcs primed ATR/Chk1 activation through DNA structure-specific phosphorylation of RPA32 and TopBP1. The synergistic activation of DNA-PKcs and ATR suggests that the two kinases combine to mount a prompt and specific response to replication-born DSBs.
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32

S'yakste, N. I., T. G. S'yakste, and N. D. Zaleskaya. "Reversible accumulation of double- and single-stranded DNA breaks in DNA in growth-arrested cells." Bulletin of Experimental Biology and Medicine 102, no. 2 (August 1986): 1059–61. http://dx.doi.org/10.1007/bf00836195.

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33

Wang, Jianlei, and Douglas A. Julin. "DNA Helicase Activity of the RecD Protein fromDeinococcus radiodurans." Journal of Biological Chemistry 279, no. 50 (October 4, 2004): 52024–32. http://dx.doi.org/10.1074/jbc.m408645200.

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The bacteriumDeinococcus radioduransis extremely resistant to high levels of DNA-damaging agents, including gamma rays and ultraviolet light that can lead to double-stranded DNA breaks. Surprisingly, the organism does not appear to have a RecBCD enzyme, an enzyme that is critical for double-strand break repair in many other bacteria. TheD. radioduransgenome does encode a protein whose closest characterized homologues are RecD subunits of RecBCD enzymes in other bacteria. We have purified this novelD. radioduransRecD protein and characterized its biochemical activities. TheD. radioduransRecD protein is a DNA helicase that unwinds short (20 base pairs) DNA duplexes with either a 5′-single-stranded tail or a forked end, but not blunt-ended or 3′-tailed duplexes. Duplexes with 10–12 nucleotide (nt) 5′-tails are good unwinding substrates and are bound tightly, while DNA with shorter tails (4–8 nt) are poor unwinding substrates and are bound much less tightly. The RecD protein is much less efficient at unwinding slightly longer substrates (52 or 76 base pairs, with 12 nt 5′-tails). Unwinding of the longer substrates is stimulated somewhat (4–5-fold) by the single-stranded DNA-binding protein fromD. radiodurans. These results show that theD. radioduransRecD protein is a DNA helicase with 5′-3′ polarity and low processivity.
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34

Rathmell, W. K., and G. Chu. "A DNA end-binding factor involved in double-strand break repair and V(D)J recombination." Molecular and Cellular Biology 14, no. 7 (July 1994): 4741–48. http://dx.doi.org/10.1128/mcb.14.7.4741.

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We have identified a nuclear factor that binds to double-stranded DNA ends, independently of the structure of the ends. It had equivalent affinities for DNA ends created by sonication or by restriction enzymes leaving 5', 3', or blunt ends but had no detectable affinity for single-stranded DNA ends. Since X rays induce DNA double-strand breaks, extracts from several complementation groups of X-ray-sensitive mammalian cells were tested for this DNA end-binding (DEB) activity. DEB activity was deficient in three independently derived cell lines from complementation group 5. Furthermore, when the cell lines reverted to X-ray resistance, expression of the DEB factor was restored to normal levels. Previous studies had shown that group 5 cells are defective for both double-strand break repair and V(D)J recombination. The residual V(D)J recombination activity in these cells produces abnormally large deletions at the sites of DNA joining (F. Pergola, M. Z. Zdzienicka, and M. R. Lieber, Mol. Cell. Biol. 13:3464-3471, 1993, and G. Taccioli, G. Rathbun, E. Oltz, T. Stamato, P. Jeggo, and F. Alt, Science 260:207-210, 1993), consistent with deficiency of a factor that protects DNA ends from degradation. Therefore, DEB factor may be involved in a biochemical pathway common to both double-strand break repair and V(D)J recombination.
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35

Rathmell, W. K., and G. Chu. "A DNA end-binding factor involved in double-strand break repair and V(D)J recombination." Molecular and Cellular Biology 14, no. 7 (July 1994): 4741–48. http://dx.doi.org/10.1128/mcb.14.7.4741-4748.1994.

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We have identified a nuclear factor that binds to double-stranded DNA ends, independently of the structure of the ends. It had equivalent affinities for DNA ends created by sonication or by restriction enzymes leaving 5', 3', or blunt ends but had no detectable affinity for single-stranded DNA ends. Since X rays induce DNA double-strand breaks, extracts from several complementation groups of X-ray-sensitive mammalian cells were tested for this DNA end-binding (DEB) activity. DEB activity was deficient in three independently derived cell lines from complementation group 5. Furthermore, when the cell lines reverted to X-ray resistance, expression of the DEB factor was restored to normal levels. Previous studies had shown that group 5 cells are defective for both double-strand break repair and V(D)J recombination. The residual V(D)J recombination activity in these cells produces abnormally large deletions at the sites of DNA joining (F. Pergola, M. Z. Zdzienicka, and M. R. Lieber, Mol. Cell. Biol. 13:3464-3471, 1993, and G. Taccioli, G. Rathbun, E. Oltz, T. Stamato, P. Jeggo, and F. Alt, Science 260:207-210, 1993), consistent with deficiency of a factor that protects DNA ends from degradation. Therefore, DEB factor may be involved in a biochemical pathway common to both double-strand break repair and V(D)J recombination.
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36

Xu, Yixi, and Dongyi Xu. "Repair pathway choice for double-strand breaks." Essays in Biochemistry 64, no. 5 (July 10, 2020): 765–77. http://dx.doi.org/10.1042/ebc20200007.

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Abstract Deoxyribonucleic acid (DNA) is at a constant risk of damage from endogenous substances, environmental radiation, and chemical stressors. DNA double-strand breaks (DSBs) pose a significant threat to genomic integrity and cell survival. There are two major pathways for DSB repair: nonhomologous end-joining (NHEJ) and homologous recombination (HR). The extent of DNA end resection, which determines the length of the 3′ single-stranded DNA (ssDNA) overhang, is the primary factor that determines whether repair is carried out via NHEJ or HR. NHEJ, which does not require a 3′ ssDNA tail, occurs throughout the cell cycle. 53BP1 and the cofactors PTIP or RIF1-shieldin protect the broken DNA end, inhibit long-range end resection and thus promote NHEJ. In contrast, HR mainly occurs during the S/G2 phase and requires DNA end processing to create a 3′ tail that can invade a homologous region, ensuring faithful gene repair. BRCA1 and the cofactors CtIP, EXO1, BLM/DNA2, and the MRE11–RAD50–NBS1 (MRN) complex promote DNA end resection and thus HR. DNA resection is influenced by the cell cycle, the chromatin environment, and the complexity of the DNA end break. Herein, we summarize the key factors involved in repair pathway selection for DSBs and discuss recent related publications.
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37

Traver, B. E., M. A. E. Anderson, and Z. N. Adelman. "Homing endonucleases catalyze double-stranded DNA breaks and somatic transgene excision inAedes aegypti." Insect Molecular Biology 18, no. 5 (October 2009): 623–33. http://dx.doi.org/10.1111/j.1365-2583.2009.00905.x.

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38

García-Medel, Paola L., Noe Baruch-Torres, Antolín Peralta-Castro, Carlos H. Trasviña-Arenas, Alfredo Torres-Larios, and Luis G. Brieba. "Plant organellar DNA polymerases repair double-stranded breaks by microhomology-mediated end-joining." Nucleic Acids Research 47, no. 6 (January 30, 2019): 3028–44. http://dx.doi.org/10.1093/nar/gkz039.

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39

Kloosterman, Wigard P., Masoumeh Tavakoli-Yaraki, Markus J. van Roosmalen, Ellen van Binsbergen, Ivo Renkens, Karen Duran, Lucia Ballarati, et al. "Constitutional Chromothripsis Rearrangements Involve Clustered Double-Stranded DNA Breaks and Nonhomologous Repair Mechanisms." Cell Reports 1, no. 6 (June 2012): 648–55. http://dx.doi.org/10.1016/j.celrep.2012.05.009.

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40

Guikema, Jeroen E. J., Erin K. Linehan, Daisuke Tsuchimoto, Yusaku Nakabeppu, Phyllis R. Strauss, Janet Stavnezer, and Carol E. Schrader. "APE1- and APE2-dependent DNA breaks in immunoglobulin class switch recombination." Journal of Experimental Medicine 204, no. 12 (November 19, 2007): 3017–26. http://dx.doi.org/10.1084/jem.20071289.

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Antibody class switch recombination (CSR) occurs by an intrachromosomal deletion requiring generation of double-stranded breaks (DSBs) in switch-region DNA. The initial steps in DSB formation have been elucidated, involving cytosine deamination by activation-induced cytidine deaminase and generation of abasic sites by uracil DNA glycosylase. However, it is not known how abasic sites are converted into single-stranded breaks and, subsequently, DSBs. Apurinic/apyrimidinic endonuclease (APE) efficiently nicks DNA at abasic sites, but it is unknown whether APE participates in CSR. We address the roles of the two major mammalian APEs, APE1 and APE2, in CSR. APE1 deficiency causes embryonic lethality in mice; we therefore examined CSR and DSBs in mice deficient in APE2 and haploinsufficient for APE1. We show that both APE1 and APE2 function in CSR, resulting in the DSBs necessary for CSR and thereby describing a novel in vivo function for APE2.
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41

Meek, Katheryn. "Activation of DNA-PK by hairpinned DNA ends reveals a stepwise mechanism of kinase activation." Nucleic Acids Research 48, no. 16 (July 27, 2020): 9098–108. http://dx.doi.org/10.1093/nar/gkaa614.

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Abstract As its name implies, the DNA dependent protein kinase (DNA-PK) requires DNA double-stranded ends for enzymatic activation. Here, I demonstrate that hairpinned DNA ends are ineffective for activating the kinase toward many of its well-studied substrates (p53, XRCC4, XLF, HSP90). However, hairpinned DNA ends robustly stimulate certain DNA-PK autophosphorylations. Specifically, autophosphorylation sites within the ABCDE cluster are robustly phosphorylated when DNA-PK is activated by hairpinned DNA ends. Of note, phosphorylation of the ABCDE sites is requisite for activation of the Artemis nuclease that associates with DNA-PK to mediate hairpin opening. This finding suggests a multi-step mechanism of kinase activation. Finally, I find that all non-homologous end joining (NHEJ) defective cells (whether deficient in components of the DNA-PK complex or components of the ligase complex) are similarly deficient in joining DNA double-stranded breaks (DSBs) with hairpinned termini.
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42

Williamson, Adele, Ulli Rothweiler, and Hanna-Kirsti Schrøder Leiros. "Enzyme–adenylate structure of a bacterial ATP-dependent DNA ligase with a minimized DNA-binding surface." Acta Crystallographica Section D Biological Crystallography 70, no. 11 (October 29, 2014): 3043–56. http://dx.doi.org/10.1107/s1399004714021099.

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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 DNAviaan adenylated intermediate. Here, the structure and activity of a recombinantly produced ATP-dependent DNA ligase from the bacteriumPsychromonassp. 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. ThePsychromonassp. 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.
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43

Blackwood, John K., Neil J. Rzechorzek, Sian M. Bray, Joseph D. Maman, Luca Pellegrini, and Nicholas P. Robinson. "End-resection at DNA double-strand breaks in the three domains of life." Biochemical Society Transactions 41, no. 1 (January 29, 2013): 314–20. http://dx.doi.org/10.1042/bst20120307.

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During DNA repair by HR (homologous recombination), the ends of a DNA DSB (double-strand break) must be resected to generate single-stranded tails, which are required for strand invasion and exchange with homologous chromosomes. This 5′–3′ end-resection of the DNA duplex is an essential process, conserved across all three domains of life: the bacteria, eukaryota and archaea. In the present review, we examine the numerous and redundant helicase and nuclease systems that function as the enzymatic analogues for this crucial process in the three major phylogenetic divisions.
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44

Lin, F. L., K. Sperle, and N. Sternberg. "Repair of double-stranded DNA breaks by homologous DNA fragments during transfer of DNA into mouse L cells." Molecular and Cellular Biology 10, no. 1 (January 1990): 113–19. http://dx.doi.org/10.1128/mcb.10.1.113.

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To test the validity of various models for recombination between extrachromosomal DNAs in mammalian cells, we measured recombination between a plasmid containing a herpesvirus thymidine kinase (tk) gene with an internal BamHI linker insertion mutation (ptkB8) and a tk gene deleted at both ends (tk delta 3' delta 5'). The two DNAs shared 885 base pairs of perfect tk homology except for the interruption at the linker insertion site. Recombination events that restored the mutated insertion site to wild type were monitored by the generation of hypoxanthine-aminopterine-thymidine-resistant colonies after cotransformation of Ltk- cells with the two DNAs. We found that cleavage of the ptkB8 DNA at the linker insertion site was essential for gene restoration. If the tk delta 3' delta 5' DNA was ligated into mp10 vector DNA, then recombination with the cleaved ptkB8 DNA was inefficient. In contrast, if it was excised from that vector by cleavage at flanking restriction sites, then recombination was stimulated about 150-fold. Using restriction site polymorphisms, we showed that most of the recombination events leading to restoration of the tk gene with the excised tk delta 3' delta 5' fragment involved three double-strand duplexes: two ptkB8 DNAs and one tk delta 3' delta 5' fragment. These results are much more readily explained by the single-strand annealing model of recombination than by the double-strand break repair model, and they suggest that the deficiency of the latter pathway for extrachromosomal mammalian recombination may be due, at least in part, to the obligate tripartite nature of the reaction. Finally, we measured the effect of DNA homology on the efficiency of the ptkB8-tk delta 3' delta 5' reaction. Our results showed a near-linear relationship between the efficiency of recombination and the amount of homology flanking either side of the linker insertion site. Moreover, we could detect thymidine kinase-positive transformants with as little as 10 base pairs of homology.
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45

Lin, F. L., K. Sperle, and N. Sternberg. "Repair of double-stranded DNA breaks by homologous DNA fragments during transfer of DNA into mouse L cells." Molecular and Cellular Biology 10, no. 1 (January 1990): 113–19. http://dx.doi.org/10.1128/mcb.10.1.113-119.1990.

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To test the validity of various models for recombination between extrachromosomal DNAs in mammalian cells, we measured recombination between a plasmid containing a herpesvirus thymidine kinase (tk) gene with an internal BamHI linker insertion mutation (ptkB8) and a tk gene deleted at both ends (tk delta 3' delta 5'). The two DNAs shared 885 base pairs of perfect tk homology except for the interruption at the linker insertion site. Recombination events that restored the mutated insertion site to wild type were monitored by the generation of hypoxanthine-aminopterine-thymidine-resistant colonies after cotransformation of Ltk- cells with the two DNAs. We found that cleavage of the ptkB8 DNA at the linker insertion site was essential for gene restoration. If the tk delta 3' delta 5' DNA was ligated into mp10 vector DNA, then recombination with the cleaved ptkB8 DNA was inefficient. In contrast, if it was excised from that vector by cleavage at flanking restriction sites, then recombination was stimulated about 150-fold. Using restriction site polymorphisms, we showed that most of the recombination events leading to restoration of the tk gene with the excised tk delta 3' delta 5' fragment involved three double-strand duplexes: two ptkB8 DNAs and one tk delta 3' delta 5' fragment. These results are much more readily explained by the single-strand annealing model of recombination than by the double-strand break repair model, and they suggest that the deficiency of the latter pathway for extrachromosomal mammalian recombination may be due, at least in part, to the obligate tripartite nature of the reaction. Finally, we measured the effect of DNA homology on the efficiency of the ptkB8-tk delta 3' delta 5' reaction. Our results showed a near-linear relationship between the efficiency of recombination and the amount of homology flanking either side of the linker insertion site. Moreover, we could detect thymidine kinase-positive transformants with as little as 10 base pairs of homology.
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46

Kara, Neesha, Felix Krueger, Peter Rugg-Gunn, and Jonathan Houseley. "Genome-wide analysis of DNA replication and DNA double-strand breaks using TrAEL-seq." PLOS Biology 19, no. 3 (March 24, 2021): e3000886. http://dx.doi.org/10.1371/journal.pbio.3000886.

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Faithful replication of the entire genome requires replication forks to copy large contiguous tracts of DNA, and sites of persistent replication fork stalling present a major threat to genome stability. Understanding the distribution of sites at which replication forks stall, and the ensuing fork processing events, requires genome-wide methods that profile replication fork position and the formation of recombinogenic DNA ends. Here, we describe Transferase-Activated End Ligation sequencing (TrAEL-seq), a method that captures single-stranded DNA 3′ ends genome-wide and with base pair resolution. TrAEL-seq labels both DNA breaks and replication forks, providing genome-wide maps of replication fork progression and fork stalling sites in yeast and mammalian cells. Replication maps are similar to those obtained by Okazaki fragment sequencing; however, TrAEL-seq is performed on asynchronous populations of wild-type cells without incorporation of labels, cell sorting, or biochemical purification of replication intermediates, rendering TrAEL-seq far simpler and more widely applicable than existing replication fork direction profiling methods. The specificity of TrAEL-seq for DNA 3′ ends also allows accurate detection of double-strand break sites after the initiation of DNA end resection, which we demonstrate by genome-wide mapping of meiotic double-strand break hotspots in a dmc1Δ mutant that is competent for end resection but not strand invasion. Overall, TrAEL-seq provides a flexible and robust methodology with high sensitivity and resolution for studying DNA replication and repair, which will be of significant use in determining mechanisms of genome instability.
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47

Wang, Hailong, Zhengping Shao, Linda Z. Shi, Patty Yi-Hwa Hwang, Lan N. Truong, Michael W. Berns, David J. Chen, and Xiaohua Wu. "CtIP Protein Dimerization Is Critical for Its Recruitment to Chromosomal DNA Double-stranded Breaks." Journal of Biological Chemistry 287, no. 25 (April 27, 2012): 21471–80. http://dx.doi.org/10.1074/jbc.m112.355354.

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48

Yeeles, Joseph T. P., and Mark S. Dillingham. "The processing of double-stranded DNA breaks for recombinational repair by helicase–nuclease complexes." DNA Repair 9, no. 3 (March 2010): 276–85. http://dx.doi.org/10.1016/j.dnarep.2009.12.016.

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49

Burger, Kaspar, Margarita Schlackow, Martin Potts, Svenja Hester, Shabaz Mohammed, and Monika Gullerova. "Nuclear phosphorylated Dicer processes double-stranded RNA in response to DNA damage." Journal of Cell Biology 216, no. 8 (June 22, 2017): 2373–89. http://dx.doi.org/10.1083/jcb.201612131.

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The endoribonuclease Dicer is a key component of the human RNA interference pathway and is known for its role in cytoplasmic microRNA production. Recent findings suggest that noncanonical Dicer generates small noncoding RNA to mediate the DNA damage response (DDR). Here, we show that human Dicer is phosphorylated in the platform–Piwi/Argonaute/Zwille–connector helix cassette (S1016) upon induction of DNA damage. Phosphorylated Dicer (p-Dicer) accumulates in the nucleus and is recruited to DNA double-strand breaks. We further demonstrate that turnover of damage-induced nuclear, double-stranded (ds) RNA requires additional phosphorylation of carboxy-terminal Dicer residues (S1728 and S1852). DNA damage-induced nuclear Dicer accumulation is conserved in mammals. Dicer depletion causes endogenous DNA damage and delays the DDR by impaired recruitment of repair factors MDC1 and 53BP1. Collectively, we place Dicer within the context of the DDR by demonstrating a DNA damage-inducible phosphoswitch that causes localized processing of nuclear dsRNA by p-Dicer to promote DNA repair.
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50

Raymond, Amy C., Bart L. Staker, and Alex B. Burgin. "Substrate Specificity of Tyrosyl-DNA Phosphodiesterase I (Tdp1)." Journal of Biological Chemistry 280, no. 23 (April 4, 2005): 22029–35. http://dx.doi.org/10.1074/jbc.m502148200.

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Tyrosyl-DNA phosphodiesterase I (Tdp1) hydrolyzes 3′-phosphotyrosyl bonds to generate 3′-phosphate DNA and tyrosine in vitro. Tdp1 is involved in the repair of DNA lesions created by topoisomerase I, although the in vivo substrate is not known. Here we study the kinetic and binding properties of human Tdp1 (hTdp1) to identify appropriate 3′-phosphotyrosyl DNA substrates. Genetic studies argue that Tdp1 is involved in double and single strand break repair pathways; however, x-ray crystal structures suggest that Tdp1 can only bind single strand DNA. Separate kinetic and binding experiments show that hTdp1 has a preference for single-stranded and blunt-ended duplex substrates over nicked and tailed duplex substrate conformations. Based on these results, we present a new model to explain Tdp1/DNA binding properties. These results suggest that Tdp1 only acts upon double strand breaks in vivo, and the roles of Tdp1 in yeast and mammalian cells are discussed.
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