Rad51-Independent Interchromosomal Double-Strand Break Repair by Gene Conversion Requires Rad52 but Not Rad55, Rad57, or Dmc1

ABSTRACT Homologous recombination (HR) is critical for DNA double-strand break (DSB) repair and genome stabilization. In yeast, HR is catalyzed by the Rad51 strand transferase and its “mediators,” including the Rad52 single-strand DNA-annealing protein, two Rad51 paralogs (Rad55 and Rad57), and Rad54. A Rad51 homolog, Dmc1, is important for meiotic HR. In wild-type cells, most DSB repair results in gene conversion, a conservative HR outcome. Because Rad51 plays a central role in the homology search and strand invasion steps, DSBs either are not repaired or are repaired by nonconservative single-strand annealing or break-induced replication mechanisms in rad51Δ mutants. Although DSB repair by gene conversion in the absence of Rad51 has been reported for ectopic HR events (e.g., inverted repeats or between plasmids), Rad51 has been thought to be essential for DSB repair by conservative interchromosomal (allelic) gene conversion. Here, we demonstrate that DSBs stimulate gene conversion between homologous chromosomes (allelic conversion) by >30-fold in a rad51Δ mutant. We show that Rad51-independent allelic conversion and break-induced replication occur independently of Rad55, Rad57, and Dmc1 but require Rad52. Unlike DSB-induced events, spontaneous allelic conversion was detected in both rad51Δ and rad52Δ mutants, but not in a rad51Δ rad52Δ double mutant. The frequencies of crossovers associated with DSB-induced gene conversion were similar in the wild type and the rad51Δ mutant, but discontinuous conversion tracts were fivefold more frequent and tract lengths were more widely distributed in the rad51Δ mutant, indicating that heteroduplex DNA has an altered structure, or is processed differently, in the absence of Rad51.

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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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Multiple Heterologies Increase Mitotic Double-Strand Break-Induced Allelic Gene Conversion Tract Lengths in Yeast

Genetics ◽

10.1093/genetics/153.2.665 ◽

1999 ◽

Vol 153 (2) ◽

pp. 665-679 ◽

Cited By ~ 3

Author(s):

Jac A Nickoloff ◽

Douglas B Sweetser ◽

Jennifer A Clikeman ◽

Guru Jot Khalsa ◽

Sarah L Wheeler

Keyword(s):

Gene Conversion ◽

Mismatch Repair ◽

Frameshift Mutation ◽

Strand Break ◽

Double Strand Break ◽

Chromosome Loss ◽

Allelic Gene ◽

Break Induced Replication ◽

Gene Conversion Tract ◽

Conversion Tract

Abstract Spontaneous and double-strand break (DSB)-induced allelic recombination in yeast was investigated in crosses between ura3 heteroalleles inactivated by an HO site and a +1 frameshift mutation, with flanking markers defining a 3.4-kbp interval. In some crosses, nine additional phenotypically silent RFLP mutations were present at ∼100-bp intervals. Increasing heterology from 0.2 to 1% in this interval reduced spontaneous, but not DSB-induced, recombination. For DSB-induced events, 75% were continuous tract gene conversions without a crossover in this interval; discontinuous tracts and conversions associated with a crossover each comprised ∼7% of events, and 10% also converted markers in unbroken alleles. Loss of heterozygosity was seen for all markers centromere distal to the HO site in 50% of products; such loss could reflect gene conversion, break-induced replication, chromosome loss, or G2 crossovers. Using telomere-marked strains we determined that nearly all allelic DSB repair occurs by gene conversion. We further show that most allelic conversion results from mismatch repair of heteroduplex DNA. Interestingly, markers shared between the sparsely and densely marked interval converted at higher rates in the densely marked interval. Thus, the extra markers increased gene conversion tract lengths, which may reflect mismatch repair-induced recombination, or a shift from restoration- to conversion-type repair.

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

Homologous Recombination - Methods in Molecular Biology ◽

10.1007/978-1-0716-0644-5_4 ◽

2020 ◽

pp. 47-57

Author(s):

Zhenxin Yan ◽

Sandeep Kumar ◽

Grzegorz Ira

Keyword(s):

Strand Break ◽

Double Strand Break ◽

Single Strand ◽

Dna Double Strand Break ◽

Single Strand Annealing ◽

Strand Annealing ◽

End Resection

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Spontaneous and double-strand break-induced recombination, and gene conversion tract lengths, are differentially affected by overexpression of wild-type or ATPase-defective yeast Rad54

Nucleic Acids Research ◽

10.1093/nar/gkf413 ◽

2002 ◽

Vol 30 (13) ◽

pp. 2727-2735 ◽

Cited By ~ 22

Author(s):

P. M. Kim

Keyword(s):

Gene Conversion ◽

Strand Break ◽

Double Strand Break ◽

Wild Type ◽

Gene Conversion Tract ◽

Conversion Tract

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The deinococcal DdrB protein is involved in an early step of DNA double strand break repair and in plasmid transformation through its single-strand annealing activity

DNA Repair ◽

10.1016/j.dnarep.2011.09.010 ◽

2011 ◽

Vol 10 (12) ◽

pp. 1223-1231 ◽

Cited By ~ 32

Author(s):

Claire Bouthier de la Tour ◽

Stéphanie Boisnard ◽

Cédric Norais ◽

Magali Toueille ◽

Esma Bentchikou ◽

...

Keyword(s):

Strand Break ◽

Double Strand Break ◽

Single Strand ◽

Double Strand Break Repair ◽

Early Step ◽

Plasmid Transformation ◽

Dna Double Strand Break ◽

Single Strand Annealing ◽

Break Repair ◽

Strand Annealing

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Genetic Requirements for RAD51- andRAD54-Independent Break-Induced Replication Repair of a Chromosomal Double-Strand Break

Molecular and Cellular Biology ◽

10.1128/mcb.21.6.2048-2056.2001 ◽

2001 ◽

Vol 21 (6) ◽

pp. 2048-2056 ◽

Cited By ~ 104

Author(s):

Laurence Signon ◽

Anna Malkova ◽

Maria L. Naylor ◽

Hannah Klein ◽

James E. Haber

Keyword(s):

Saccharomyces Cerevisiae ◽

Homologous Recombination ◽

Gene Conversion ◽

Strand Break ◽

Double Strand Break ◽

Telomere Maintenance ◽

Diploid Cells ◽

Break Induced Replication ◽

In Cells

ABSTRACT Broken chromosomes can be repaired by several homologous recombination mechanisms, including gene conversion and break-induced replication (BIR). In Saccharomyces cerevisiae, an HO endonuclease-induced double-strand break (DSB) is normally repaired by gene conversion. Previously, we have shown that in the absence ofRAD52, repair is nearly absent and diploid cells lose the broken chromosome; however, in cells lacking RAD51, gene conversion is absent but cells can repair the DSB by BIR. We now report that gene conversion is also abolished when RAD54, RAD55, and RAD57 are deleted but BIR occurs, as withrad51Δ cells. DSB-induced gene conversion is not significantly affected when RAD50, RAD59, TID1(RDH54), SRS2, or SGS1 is deleted. Various double mutations largely eliminate both gene conversion and BIR, including rad51Δ rad50Δ, rad51Δ rad59Δ, andrad54Δ tid1Δ. These results demonstrate that there is aRAD51- and RAD54-independent BIR pathway that requires RAD59, TID1, RAD50, and presumablyMRE11 and XRS2. The similar genetic requirements for BIR and telomere maintenance in the absence of telomerase also suggest that these two processes proceed by similar mechanisms.

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Double-strand break repair deficiency in NONO knockout murine embryonic fibroblasts and compensation by spontaneous upregulation of the PSPC1 paralog

Nucleic Acids Research ◽

10.1093/nar/gku650 ◽

2014 ◽

Vol 42 (15) ◽

pp. 9771-9780 ◽

Cited By ~ 27

Author(s):

Shuyi Li ◽

Zhentian Li ◽

Feng-Jue Shu ◽

Hairong Xiong ◽

Andrew C. Phillips ◽

...

Keyword(s):

Genotoxic Stress ◽

Strand Break ◽

Double Strand Break ◽

Stable Complex ◽

Mrna Levels ◽

Wild Type ◽

Long Term Effects ◽

Dsb Repair ◽

Embryonic Fibroblasts ◽

Compensatory Response

Abstract NONO, SFPQ and PSPC1 make up a family of proteins with diverse roles in transcription, RNA processing and DNA double-strand break (DSB) repair. To understand long-term effects of loss of NONO, we characterized murine embryonic fibroblasts (MEFs) from knockout mice. In the absence of genotoxic stress, wild-type and mutant MEFs showed similar growth rates and cell cycle distributions, and the mutants were only mildly radiosensitive. Further investigation showed that NONO deficiency led to upregulation of PSPC1, which replaced NONO in a stable complex with SFPQ. Knockdown of PSPC1 in a NONO-deficient background led to severe radiosensitivity and delayed resolution of DSB repair foci. The DNA-dependent protein kinase (DNA-PK) inhibitor, NU7741, sensitized wild-type and singly deficient MEFs, but had no additional effect on doubly deficient cells, suggesting that NONO/PSPC1 and DNA-PK function in the same pathway. We tested whether NONO and PSPC1 might also affect repair indirectly by influencing mRNA levels for other DSB repair genes. Of 12 genes tested, none were downregulated, and several were upregulated. Thus, NONO or related proteins are critical for DSB repair, NONO and PSPC1 are functional homologs with partially interchangeable functions and a compensatory response involving PSPC1 blunts the effect of NONO deficiency.

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