Analysis of DNA Double-Strand Break End Resection and Single-Strand Annealing in S. pombe

ABSTRACTMycobacteria encode three DNA double-strand break repair pathways: (i) RecA-dependent homologous recombination (HR), (ii) Ku-dependent nonhomologous end joining (NHEJ), and (iii) RecBCD-dependent single-strand annealing (SSA). Mycobacterial HR has two presynaptic pathway options that rely on the helicase-nuclease AdnAB and the strand annealing protein RecO, respectively. Ablation ofadnABorrecOindividually causes partial impairment of HR, but loss ofadnABandrecOin combination abolishes HR. RecO, which can accelerate annealing of single-stranded DNAin vitro, also participates in the SSA pathway. The functions of RecF and RecR, which, in other model bacteria, function in concert with RecO as mediators of RecA loading, have not been examined in mycobacteria. Here, we present a genetic analysis ofrecFandrecRin mycobacterial recombination. We find that RecF, like RecO, participates in the AdnAB-independent arm of the HR pathway and in SSA. In contrast, RecR is required for all HR in mycobacteria and for SSA. The essentiality of RecR as an agent of HR is yet another distinctive feature of mycobacterial DNA repair.IMPORTANCEThis study clarifies the molecular requirements for homologous recombination in mycobacteria. Specifically, we demonstrate that RecF and RecR play important roles in both the RecA-dependent homologous recombination and RecA-independent single-strand annealing pathways. Coupled with our previous findings (R. Gupta, M. Ryzhikov, O. Koroleva, M. Unciuleac, S. Shuman, S. Korolev, and M. S. Glickman, Nucleic Acids Res 41:2284–2295, 2013,http://dx.doi.org/10.1093/nar/gks1298), these results revise our view of mycobacterial recombination and place the RecFOR system in a central position in homology-dependent DNA repair.

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Analysis of BRCA1 Variants in Double-Strand Break Repair by Homologous Recombination and Single-Strand Annealing

Human Mutation ◽

10.1002/humu.22251 ◽

2013 ◽

Vol 34 (3) ◽

pp. 439-445 ◽

Cited By ~ 32

Author(s):

William I. Towler ◽

Jie Zhang ◽

Derek J. R. Ransburgh ◽

Amanda E. Toland ◽

Chikashi Ishioka ◽

...

Keyword(s):

Homologous Recombination ◽

Strand Break ◽

Double Strand Break ◽

Single Strand ◽

Double Strand Break Repair ◽

Single Strand Annealing ◽

Break Repair ◽

Strand Annealing

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Analysis of the Xenopus Werner syndrome protein in DNA double-strand break repair

The Journal of Cell Biology ◽

10.1083/jcb.200502077 ◽

2005 ◽

Vol 171 (2) ◽

pp. 217-227 ◽

Cited By ~ 20

Author(s):

Hong Yan ◽

Jill McCane ◽

Thomas Toczylowski ◽

Chinyi Chen

Keyword(s):

Werner Syndrome ◽

Strand Break ◽

Double Strand Break ◽

Single Strand ◽

Repair Pathway ◽

Dsb Repair ◽

Single Strand Annealing ◽

Werner Syndrome Protein ◽

Strand Annealing ◽

Syndrome Protein

Werner syndrome is associated with premature aging and increased risk of cancer. Werner syndrome protein (WRN) is a RecQ-type DNA helicase, which seems to participate in DNA replication, double-strand break (DSB) repair, and telomere maintenance; however, its exact function remains elusive. Using Xenopus egg extracts as the model system, we found that Xenopus WRN (xWRN) is recruited to discrete foci upon induction of DSBs. Depletion of xWRN has no significant effect on nonhomologous end-joining of DSB ends, but it causes a significant reduction in the homology-dependent single-strand annealing DSB repair pathway. These results provide the first direct biochemical evidence that links WRN to a specific DSB repair pathway. The assay for single-strand annealing that was developed in this study also provides a powerful biochemical system for mechanistic analysis of homology-dependent DSB repair.

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Replication protein A promotes 5′→3′ end processing during homology-dependent DNA double-strand break repair

The Journal of Cell Biology ◽

10.1083/jcb.201005110 ◽

2011 ◽

Vol 192 (2) ◽

pp. 251-261 ◽

Cited By ~ 29

Author(s):

Hong Yan ◽

Thomas Toczylowski ◽

Jill McCane ◽

Chinyi Chen ◽

Shuren Liao

Keyword(s):

Werner Syndrome ◽

Protein A ◽

Replication Protein A ◽

Strand Break ◽

Double Strand Break ◽

Single Strand ◽

Replication Protein ◽

Dsb Repair ◽

Dna Double Strand Break ◽

Single Strand Annealing

Replication protein A (RPA), the eukaryotic single-strand deoxyribonucleic acid (DNA [ss-DNA])–binding protein, is involved in DNA replication, nucleotide damage repair, mismatch repair, and DNA damage checkpoint response, but its function in DNA double-strand break (DSB) repair is poorly understood. We investigated the function of RPA in homology-dependent DSB repair using Xenopus laevis nucleoplasmic extracts as a model system. We found that RPA is required for single-strand annealing, one of the homology-dependent DSB repair pathways. Furthermore, RPA promotes the generation of 3′ single-strand tails (ss-tails) by stimulating both the Xenopus Werner syndrome protein (xWRN)–mediated unwinding of DNA ends and the subsequent Xenopus DNA2 (xDNA2)–mediated degradation of the 5′ ss-tail. Purified xWRN, xDNA2, and RPA are sufficient to carry out the 5′-strand resection of DNA that carries a 3′ ss-tail. These results provide strong biochemical evidence to link RPA to a specific DSB repair pathway and reveal a novel function of RPA in the generation of 3′ ss-DNA for homology-dependent DSB repair.

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DNA Length Dependence of the Single-Strand Annealing Pathway and the Role of Saccharomyces cerevisiae RAD59 in Double-Strand Break Repair

Molecular and Cellular Biology ◽

10.1128/mcb.20.14.5300-5309.2000 ◽

2000 ◽

Vol 20 (14) ◽

pp. 5300-5309 ◽

Cited By ~ 169

Author(s):

Neal Sugawara ◽

Grzegorz Ira ◽

James E. Haber

Keyword(s):

Saccharomyces Cerevisiae ◽

Gene Conversion ◽

Strand Break ◽

Double Strand Break ◽

Single Strand ◽

Sequence Length ◽

Homologous Sequence ◽

Single Strand Annealing ◽

Strand Annealing ◽

Almost All

ABSTRACT A DNA double-strand break (DSB) created by the HO endonuclease inSaccharomyces cerevisiae will stimulate recombination between flanking repeats by the single-strand annealing (SSA) pathway, producing a deletion. Previously the efficiency of SSA, using homologous sequences of different lengths, was measured in competition with that of a larger repeat further from the DSB, which ensured that nearly all cells would survive the DSB if the smaller region was not used (N. Sugawara and J. E. Haber, Mol. Cell. Biol. 12:563–575, 1992). Without competition, the efficiency with which homologous segments of 63 to 205 bp engaged in SSA was significantly increased. A sequence as small as 29 bp was used 0.2% of the time, and homology dependence was approximately linear up to 415 bp, at which size almost all cells survived. A mutant with a deletion of RAD59, a homologue of RAD52, was defective for SSA, especially when the homologous-sequence length was short; however, even with 1.17-kb substrates, SSA was reduced fourfold. DSB-induced gene conversion also showed a partial dependence on Rad59p, again being greatest when the homologous-sequence length was short. We found that Rad59p plays a role in removing nonhomologous sequences from the ends of single-stranded DNA when it invades a homologous DNA template, in a manner similar to that previously seen with srs2 mutants. Δrad59 affected DSB-induced gene conversion differently from msh3 and msh2, which are also defective in removing nonhomologous ends in both DSB-induced gene conversion and SSA. A msh3 rad59 double mutant was more severely defective in SSA than either single mutant.

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A Simulated Shift Work Schedule Does Not Increase DNA Double-Strand Break Repair by NHEJ in the Drosophila Rr3 System

Genes ◽

10.3390/genes13010150 ◽

2022 ◽

Vol 13 (1) ◽

pp. 150

Author(s):

Lydia Bergerson ◽

Caleb Fitzmaurice ◽

Tyler Knudtson ◽

Halle McCormick ◽

Alder M. Yu

Keyword(s):

Shift Work ◽

Strand Break ◽

Double Strand Break ◽

Dsb Repair ◽

Dna Double Strand Break ◽

Significant Difference ◽

Nhej Repair ◽

Single Strand Annealing ◽

Strand Annealing ◽

Shift Work Schedule

Long-term shift work is widely believed to increase the risk of certain cancers, but conflicting findings between studies render this association unclear. Evidence of interplay between the circadian clock, cell cycle regulation, and DNA damage detection machinery suggests the possibility that circadian rhythm disruption consequent to shift work could alter the DNA double-strand break (DSB) repair pathway usage to favor mutagenic non-homologous end-joining (NHEJ) repair. To test this hypothesis, we compared relative usage of NHEJ and single-strand annealing (SSA) repair of a complementary ended chromosomal double-stranded break using the Repair Reporter 3 (Rr3) system in Drosophila between flies reared on 12:12 and 8:8 (simulated shift work) light:dark schedules. Actimetric analysis showed that the 8:8 light:dark schedule effectively disrupted the rhythms in locomotor output. Inaccurate NHEJ repair was not a frequent outcome in this system overall, and no significant difference was seen in the usage of NHEJ or SSA repair between the control and simulated shift work schedules. We conclude that this circadian disruption regimen does not alter the usage of mutagenic NHEJ DSB repair in the Drosophila male pre-meiotic germline, in the context of the Rr3 system.

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