RecE/RecT and Redα/Redβ initiate double-stranded break repair by specifically interacting with their respective partners

The initial steps of double-stranded break (DSB) repair by homologous recombination mediated by the 5′–3′ exonuclease/annealing protein pairs, RecE/RecT and Redα/Redβ, were analyzed. Recombination was RecA-independent and required the expression of both components of an orthologous pair, even when the need for exonuclease activity was removed by use of preresected substrates. The required orthologous function correlated with a specific protein–protein interaction, and recombination was favored by overexpression of the annealing protein with respect to the exonuclease. The need for both components of an orthologous pair was observed regardless of whether recombination proceeded via a single-strand annealing or a putative strand invasion mechanism. The DSB repair reactions studied here are reminiscent of the RecBCD/RecA reaction and suggest a general mechanism that is likely to be relevant to other systems, including RAD52 mediated recombination.

Download Full-text

Conservative Repair of a Chromosomal Double-Strand Break by Single-Strand DNA through Two Steps of Annealing

Molecular and Cellular Biology ◽

10.1128/mcb.00672-06 ◽

2006 ◽

Vol 26 (20) ◽

pp. 7645-7657 ◽

Cited By ~ 75

Author(s):

Francesca Storici ◽

Joyce R. Snipe ◽

Godwin K. Chan ◽

Dmitry A. Gordenin ◽

Michael A. Resnick

Keyword(s):

Normal Cell ◽

Strand Break ◽

Double Strand Breaks ◽

Single Strand ◽

Dsb Repair ◽

Strand Breaks ◽

Single Strand Annealing ◽

Repair Mechanisms ◽

Strand Invasion ◽

Strand Annealing

ABSTRACT The repair of chromosomal double-strand breaks (DSBs) is essential to normal cell growth, and homologous recombination is a universal process for DSB repair. We explored DSB repair mechanisms in the yeast Saccharomyces cerevisiae using single-strand oligonucleotides with homology to both sides of a DSB. Oligonucleotide-directed repair occurred exclusively via Rad52- and Rad59-mediated single-strand annealing (SSA). Even the SSA domain of human Rad52 provided partial complementation for a null rad52 mutation. The repair did not involve Rad51-driven strand invasion, and moreover the suppression of strand invasion increased repair with oligonucleotides. A DSB was shown to activate targeting by oligonucleotides homologous to only one side of the break at large distances (at least 20 kb) from the break in a strand-biased manner, suggesting extensive 5′ to 3′ resection, followed by the restoration of resected DNA to the double-strand state. We conclude that long resected chromosomal DSB ends are repaired by a single-strand DNA oligonucleotide through two rounds of annealing. The repair by single-strand DNA can be conservative and may allow for accurate restoration of chromosomal DNAs with closely spaced DSBs.

Download Full-text

Yeastspt6-140Mutation, Affecting Chromatin and Transcription, Preferentially Increases Recombination in Which Rad51p-Mediated Strand Exchange Is Dispensable

Genetics ◽

10.1093/genetics/158.2.597 ◽

2001 ◽

Vol 158 (2) ◽

pp. 597-611 ◽

Cited By ~ 5

Author(s):

Francisco Malagón ◽

Andrés Aguilera

Keyword(s):

Strand Exchange ◽

Micrococcal Nuclease ◽

Sensitivity Analyses ◽

Inverted Repeats ◽

Major Mechanism ◽

Spontaneous Recombination ◽

Single Strand Annealing ◽

Strand Invasion ◽

Strand Annealing ◽

Break Induced Replication

AbstractWe have shown that the spt6-140 and spt4-3 mutations, affecting chromatin structure and transcription, stimulate recombination between inverted repeats by a RAD52-dependent mechanism that is very efficient in the absence of RAD51, RAD54, RAD55, and RAD57. Such a mechanism of recombination is RAD1-RAD59-dependent and yields gene conversions highly associated with the inversion of the repeat. The spt6-140 mutation alters transcription and chromatin in our inverted repeats, as determined by Northern and micrococcal nuclease sensitivity analyses, respectively. Hyper-recombination levels are diminished in the absence of transcription. We believe that the chromatin alteration, together with transcription impairment caused by spt6-140, increases the incidence of spontaneous recombination regardless of whether or not it is mediated by Rad51p-dependent strand exchange. Our results suggest that spt6, as well as spt4, primarily stimulates a mechanism of break-induced replication. We discuss the possibility that the chromatin alteration caused by spt6-140 facilitates a Rad52p-mediated one-ended strand invasion event, possibly inefficient in wild-type chromatin. Our results are consistent with the idea that the major mechanism leading to inversions might not be crossing over but break-induced replication followed by single-strand annealing.

Download Full-text

Repairing a double–strand chromosome break by homologous recombination: revisiting Robin Holliday's model

Philosophical Transactions of the Royal Society B Biological Sciences ◽

10.1098/rstb.2003.1367 ◽

2004 ◽

Vol 359 (1441) ◽

pp. 79-86 ◽

Cited By ~ 53

Author(s):

James E. Haber ◽

Gregorz Ira ◽

Anna Malkova ◽

Neal Sugawara

Keyword(s):

Homologous Recombination ◽

Repair Process ◽

Strand Break ◽

Holliday Junctions ◽

Chromosome Break ◽

Invasion Mechanism ◽

Strand Invasion ◽

Strand Annealing ◽

Break Induced Replication ◽

Mutant Cells

Since the pioneering model for homologous recombination proposed by Robin Holliday in 1964, there has been great progress in understanding how recombination occurs at a molecular level. In the budding yeast Saccharomyces cerevisiae , one can follow recombination by physically monitoring DNA after the synchronous induction of a double–strand break (DSB) in both wild–type and mutant cells. A particularly well–studied system has been the switching of yeast mating–type ( MAT ) genes, where a DSB can be induced synchronously by expression of the site–specific HO endonuclease. Similar studies can be performed in meiotic cells, where DSBs are created by the Spo11 nuclease. There appear to be at least two competing mechanisms of homologous recombination: a synthesis–dependent strand annealing pathway leading to noncrossovers and a two–end strand invasion mechanism leading to formation and resolution of Holliday junctions (HJs), leading to crossovers. The establishment of a modified replication fork during DSB repair links gene conversion to another important repair process, break–induced replication. Despite recent revelations, almost 40 years after Holliday's model was published, the essential ideas he proposed of strand invasion and heteroduplex DNA formation, the formation and resolution of HJs, and mismatch repair, remain the basis of our thinking.

Download Full-text

RNF169 limits 53BP1 deposition at DSBs to stimulate single-strand annealing repair

Proceedings of the National Academy of Sciences ◽

10.1073/pnas.1804823115 ◽

2018 ◽

Vol 115 (35) ◽

pp. E8286-E8295 ◽

Cited By ~ 13

Author(s):

Liwei An ◽

Chao Dong ◽

Junshi Li ◽

Jie Chen ◽

Jingsong Yuan ◽

...

Keyword(s):

Single Strand ◽

Fine Tuning ◽

Dna Double Strand Breaks ◽

Dsb Repair ◽

Strand Breaks ◽

Single Strand Annealing ◽

Molecular Rheostat ◽

Dna End Resection ◽

Strand Annealing ◽

End Resection

Unrestrained 53BP1 activity at DNA double-strand breaks (DSBs) hampers DNA end resection and upsets DSB repair pathway choice. RNF169 acts as a molecular rheostat to limit 53BP1 deposition at DSBs, but how this fine balance translates to DSB repair control remains undefined. In striking contrast to 53BP1, ChIP analyses of AsiSI-induced DSBs unveiled that RNF169 exhibits robust accumulation at DNA end-proximal regions and preferentially targets resected, RPA-bound DSBs. Accordingly, we found that RNF169 promotes CtIP-dependent DSB resection and favors homology-mediated DSB repair, and further showed that RNF169 dose-dependently stimulates single-strand annealing repair, in part, by alleviating the 53BP1-imposed barrier to DSB end resection. Our results highlight the interplay of RNF169 with 53BP1 in fine-tuning choice of DSB repair pathways.

Download Full-text

FKBP25 participates in DNA double-strand break repair

Biochemistry and Cell Biology ◽

10.1139/bcb-2018-0328 ◽

2020 ◽

Vol 98 (1) ◽

pp. 42-49 ◽

Cited By ~ 2

Author(s):

David Dilworth ◽

Fade Gong ◽

Kyle Miller ◽

Christopher J. Nelson

Keyword(s):

Homologous Recombination ◽

Strand Break ◽

Repair Pathway ◽

Dna Double Strand Breaks ◽

Dsb Repair ◽

Peptide Bonds ◽

Single Strand Annealing ◽

Strand Annealing ◽

Fk506 Binding Proteins

FK506-binding proteins (FKBPs) alter the conformation of proteins via cis–trans isomerization of prolyl-peptide bonds. While this activity can be demonstrated in vitro, the intractability of detecting prolyl isomerization events in cells has limited our understanding of the biological processes regulated by FKBPs. Here we report that FKBP25 is an active participant in the repair of DNA double-strand breaks (DSBs). FKBP25 influences DSB repair pathway choice by promoting homologous recombination (HR) and suppressing single-strand annealing (SSA). Consistent with this observation, cells depleted of FKBP25 form fewer Rad51 repair foci in response to etoposide and ionizing radiation, and they are reliant on the SSA repair factor Rad52 for viability. We find that FKBP25’s catalytic activity is required for promoting DNA repair, which is the first description of a biological function for this enzyme activity. Consistent with the importance of the FKBP catalytic site in HR, rapamycin treatment also impairs homologous recombination, and this effect is at least in part independent of mTor. Taken together these results identify FKBP25 as a component of the DNA DSB repair pathway.

Download Full-text

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.

Download Full-text

Genetic Steps of Mammalian Homologous Repair with Distinct Mutagenic Consequences

Molecular and Cellular Biology ◽

10.1128/mcb.24.21.9305-9316.2004 ◽

2004 ◽

Vol 24 (21) ◽

pp. 9305-9316 ◽

Cited By ~ 313

Author(s):

Jeremy M. Stark ◽

Andrew J. Pierce ◽

Jin Oh ◽

Albert Pastink ◽

Maria Jasin

Keyword(s):

Mammalian Cells ◽

Atp Hydrolysis ◽

Chromosomal Rearrangements ◽

Strand Break ◽

Dsb Repair ◽

Mutagenic Potential ◽

Gross Chromosomal Rearrangements ◽

Single Strand Annealing ◽

Chromosomal Breaks ◽

Strand Annealing

ABSTRACT Repair of chromosomal breaks is essential for cellular viability, but misrepair generates mutations and gross chromosomal rearrangements. We investigated the interrelationship between two homologous-repair pathways, i.e., mutagenic single-strand annealing (SSA) and precise homology-directed repair (HDR). For this, we analyzed the efficiency of repair in mammalian cells in which double-strand break (DSB) repair components were disrupted. We observed an inverse relationship between HDR and SSA when RAD51 or BRCA2 was impaired, i.e., HDR was reduced but SSA was increased. In particular, expression of an ATP-binding mutant of RAD51 led to a >90-fold shift to mutagenic SSA repair. Additionally, we found that expression of an ATP hydrolysis mutant of RAD51 resulted in more extensive gene conversion, which increases genetic loss during HDR. Disruption of two other DSB repair components affected both SSA and HDR, but in opposite directions: SSA and HDR were reduced by mutation of Brca1, which, like Brca2, predisposes to breast cancer, whereas SSA and HDR were increased by Ku70 mutation, which affects nonhomologous end joining. Disruption of the BRCA1-associated protein BARD1 had effects similar to those of mutation of BRCA1. Thus, BRCA1/BARD1 has a role in homologous repair before the branch point of HDR and SSA. Interestingly, we found that Ku70 mutation partially suppresses the homologous-repair defects of BARD1 disruption. We also examined the role of RAD52 in homologous repair. In contrast to yeast, Rad52 − / − mouse cells had no detectable HDR defect, although SSA was decreased. These results imply that the proper genetic interplay of repair factors is essential to limit the mutagenic potential of DSB repair.

Download Full-text

HELQ is a dual-function DSB repair enzyme modulated by RPA and RAD51

Nature ◽

10.1038/s41586-021-04261-0 ◽

2021 ◽

Author(s):

Roopesh Anand ◽

Erika Buechelmaier ◽

Ondrej Belan ◽

Matthew Newton ◽

Aleksandra Vancevska ◽

...

Keyword(s):

Single Molecule ◽

Cellular Level ◽

Dual Function ◽

Dna Unwinding ◽

End Joining ◽

Dsb Repair ◽

Single Strand Annealing ◽

Dna Strands ◽

Strand Annealing ◽

Dna Strand

AbstractDNA double-stranded breaks (DSBs) are deleterious lesions, and their incorrect repair can drive cancer development1. HELQ is a superfamily 2 helicase with 3′ to 5′ polarity, and its disruption in mice confers germ cells loss, infertility and increased predisposition to ovarian and pituitary tumours2–4. At the cellular level, defects in HELQ result in hypersensitivity to cisplatin and mitomycin C, and persistence of RAD51 foci after DNA damage3,5. Notably, HELQ binds to RPA and the RAD51-paralogue BCDX2 complex, but the relevance of these interactions and how HELQ functions in DSB repair remains unclear3,5,6. Here we show that HELQ helicase activity and a previously unappreciated DNA strand annealing function are differentially regulated by RPA and RAD51. Using biochemistry analyses and single-molecule imaging, we establish that RAD51 forms a complex with and strongly stimulates HELQ as it translocates during DNA unwinding. By contrast, RPA inhibits DNA unwinding by HELQ but strongly stimulates DNA strand annealing. Mechanistically, we show that HELQ possesses an intrinsic ability to capture RPA-bound DNA strands and then displace RPA to facilitate annealing of complementary sequences. Finally, we show that HELQ deficiency in cells compromises single-strand annealing and microhomology-mediated end-joining pathways and leads to bias towards long-tract gene conversion tracts during homologous recombination. Thus, our results implicate HELQ in multiple arms of DSB repair through co-factor-dependent modulation of intrinsic translocase and DNA strand annealing activities.

Download Full-text

Examining a DNA Replication Requirement for Bacteriophage λ Red- and Rac Prophage RecET-Promoted Recombination inEscherichia coli

mBio ◽

10.1128/mbio.01443-16 ◽

2016 ◽

Vol 7 (5) ◽

Cited By ~ 7

Author(s):

Lynn C. Thomason ◽

Nina Costantino ◽

Donald L. Court

Keyword(s):

Dna Replication ◽

Homologous Recombination ◽

Genetic Engineering ◽

Single Strand ◽

Genetic Modifications ◽

Single Strand Annealing ◽

Strand Invasion ◽

Strand Annealing ◽

Λ Red

ABSTRACTRecombineering,in vivogenetic engineering with bacteriophage homologous recombination systems, is a powerful technique for making genetic modifications in bacteria. Two systems widely used inEscherichia coliare the Red system from phage λ and RecET from the defective Rac prophage. We investigated thein vivodependence of recombineering on DNA replication of the recombining substrate using plasmid targets. For λ Red recombination, when DNA replication of a circular target plasmid is prevented, recombination with single-stranded DNA oligonucleotides is greatly reduced compared to that under replicating conditions. For RecET recombination, when DNA replication of the targeted plasmid is prevented, the recombination frequency is also reduced, to a level identical to that seen for the Red system in the absence of replication. The very low level of oligonucleotide recombination observed in the absence of any phage recombination functions is the same in the presence or absence of DNA replication. In contrast, both the Red and RecET systems recombine a nonreplicating linear dimer plasmid with high efficiency to yield a circular monomer. Therefore, the DNA replication requirement is substrate dependent. Our data are consistent with recombination by both the Red and RecET systems occurring predominately by single-strand annealing rather than by strand invasion.IMPORTANCEBacteriophage homologous recombination systems are widely used forin vivogenetic engineering in bacteria. Single- or double-stranded linear DNA substrates containing short flanking homologies to chromosome targets are used to generate precise and accurate genetic modifications when introduced into bacteria expressing phage recombinases. Understanding the molecular mechanism of these recombination systems will facilitate improvements in the technology. Here, two phage-specific systems are shown to require exposure of complementary single-strand homologous targets for efficient recombination; these single-strand regions may be created during DNA replication or by single-strand exonuclease digestion of linear duplex DNA. Previously,in vitrostudies reported that these recombinases promote the single-strand annealing of two complementary DNAs and also strand invasion of a single DNA strand into duplex DNA to create a three-stranded region. Here,in vivoexperiments show that recombinase-mediated annealing of complementary single-stranded DNA is the predominant recombination pathway inE. coli.

Download Full-text

Multi-Pathway DNA Double-Strand Break Repair Reporters Reveal Extensive Cross-Talk Between End-Joining, Single Strand Annealing, and Homologous Recombination

10.1101/2021.12.22.473809 ◽

2021 ◽

Author(s):

Bert van de Kooij ◽

Alex Kruswick ◽

Haico van Attikum ◽

Michael B. Yaffe

Keyword(s):

Homologous Recombination ◽

Cross Talk ◽

Single Strand ◽

Repair Pathway ◽

End Joining ◽

Dsb Repair ◽

Alu Elements ◽

Single Strand Annealing ◽

Strand Annealing ◽

End Resection

DNA double-strand breaks (DSB) are repaired by multiple distinct pathways, with outcomes ranging from error-free repair to extensive mutagenesis and genomic loss. Repair pathway cross-talk and compensation within the DSB-repair network is incompletely understood, despite its importance for genomic stability, oncogenesis, and the outcome of genome editing by CRISPR/Cas9. To address this, we constructed and validated three fluorescent Cas9-based reporters, named DSB-Spectrum, that simultaneously quantify the contribution of multiple distinct pathways to repair of a DSB. These reporters distinguish between DSB-repair by error-free canonical non-homologous end-joining (c-NHEJ) versus homologous recombination (HR; reporter 1), mutagenic repair versus HR (reporter 2), and mutagenic end-joining versus single strand annealing (SSA) versus HR (reporter 3). Using these reporters, we show that inhibition of the essential c-NHEJ factor DNA-PKcs not only increases repair by HR, but also results in a substantial increase in mutagenic repair by SSA. We show that SSA-mediated repair of Cas9-generated DSBs can occur between Alu elements at endogenous genomic loci, and is enhanced by inhibition of DNA-PKcs. Finally, we demonstrate that the short-range end-resection factors CtIP and Mre11 promote both SSA and HR, whereas the long-range end-resection factors DNA2 and Exo1 promote SSA, but reduce HR, when both pathways compete for the same substrate. These new Cas9-based DSB-Spectrum reporters facilitate the rapid and comprehensive analysis of repair pathway crosstalk and DSB-repair outcome.

Download Full-text