FKBP25 participates in DNA double-strand break repair

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.

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Mouse RAD54 Affects DNA Double-Strand Break Repair and Sister Chromatid Exchange

Molecular and Cellular Biology ◽

10.1128/mcb.20.9.3147-3156.2000 ◽

2000 ◽

Vol 20 (9) ◽

pp. 3147-3156 ◽

Cited By ~ 102

Author(s):

Mies L. G. Dronkert ◽

H. Berna Beverloo ◽

Roger D. Johnson ◽

Jan H. J. Hoeijmakers ◽

Maria Jasin ◽

...

Keyword(s):

Homologous Recombination ◽

Gene Conversion ◽

Sister Chromatid ◽

Sister Chromatid Exchange ◽

Embryonic Stem ◽

Strand Break ◽

Dna Double Strand Breaks ◽

Dsb Repair ◽

Single Strand Annealing ◽

Strand Annealing

ABSTRACT Cells can achieve error-free repair of DNA double-strand breaks (DSBs) by homologous recombination through gene conversion with or without crossover. In contrast, an alternative homology-dependent DSB repair pathway, single-strand annealing (SSA), results in deletions. In this study, we analyzed the effect of mRAD54, a gene involved in homologous recombination, on the repair of a site-specific I-SceI-induced DSB located in a repeated DNA sequence in the genome of mouse embryonic stem cells. We used six isogenic cell lines differing solely in the orientation of the repeats. The combination of the three recombination-test substrates used discriminated among SSA, intrachromatid gene conversion, and sister chromatid gene conversion. DSB repair was most efficient for the substrate that allowed recovery of SSA events. Gene conversion with crossover, indistinguishable from long tract gene conversion, preferentially involved the sister chromatid rather than the repeat on the same chromatid. Comparing DSB repair in mRAD54wild-type and knockout cells revealed direct evidence for a role ofmRAD54 in DSB repair. The substrate measuring SSA showed an increased efficiency of DSB repair in the absence ofmRAD54. The substrate measuring sister chromatid gene conversion showed a decrease in gene conversion with and without crossover. Consistent with this observation, DNA damage-induced sister chromatid exchange was reduced in mRAD54-deficient cells. Our results suggest that mRAD54 promotes gene conversion with predominant use of the sister chromatid as the repair template at the expense of error-prone SSA.

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RecF and RecR Play Critical Roles in the Homologous Recombination and Single-Strand Annealing Pathways of Mycobacteria

Journal of Bacteriology ◽

10.1128/jb.00290-15 ◽

2015 ◽

Vol 197 (19) ◽

pp. 3121-3132 ◽

Cited By ~ 11

Author(s):

Richa Gupta ◽

Stewart Shuman ◽

Michael S. Glickman

Keyword(s):

Dna Repair ◽

Homologous Recombination ◽

Strand Break ◽

Single Strand ◽

Central Position ◽

End Joining ◽

Dna Double Strand Break ◽

Single Strand Annealing ◽

Strand Annealing

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 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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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.

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Multiple Pathways for Repair of DNA Double-Strand Breaks in Mammalian Chromosomes

Molecular and Cellular Biology ◽

10.1128/mcb.19.12.8353 ◽

1999 ◽

Vol 19 (12) ◽

pp. 8353-8360 ◽

Cited By ~ 52

Author(s):

Yunfu Lin ◽

Tamas Lukacsovich ◽

Alan S. Waldman

Keyword(s):

Homologous Recombination ◽

Double Strand Breaks ◽

Nonhomologous End Joining ◽

Dna Double Strand Breaks ◽

End Joining ◽

Base Pairs ◽

Dsb Repair ◽

Strand Breaks ◽

Single Strand Annealing ◽

Strand Annealing

ABSTRACT To study repair of DNA double-strand breaks (DSBs) in mammalian chromosomes, we designed DNA substrates containing a thymidine kinase (TK) gene disrupted by the 18-bp recognition site for yeast endonuclease I-SceI. Some substrates also contained a second defective TK gene sequence to serve as a genetic donor in recombinational repair. A genomic DSB was induced by introducing endonuclease I-SceI into cells containing a stably integrated DNA substrate. DSB repair was monitored by selection for TK-positive segregants. We observed that intrachromosomal DSB repair is accomplished with nearly equal efficiencies in either the presence or absence of a homologous donor sequence. DSB repair is achieved by nonhomologous end-joining or homologous recombination, but rarely by nonconservative single-strand annealing. Repair of a chromosomal DSB by homologous recombination occurs mainly by gene conversion and appears to require a donor sequence greater than a few hundred base pairs in length. Nonhomologous end-joining events typically involve loss of very few nucleotides, and some events are associated with gene amplification at the repaired locus. Additional studies revealed that precise religation of DNA ends with no other concomitant sequence alteration is a viable mode for repair of DSBs in a mammalian genome.

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The Yeast Recombinational Repair Protein Rad59 Interacts With Rad52 and Stimulates Single-Strand Annealing

Genetics ◽

10.1093/genetics/159.2.515 ◽

2001 ◽

Vol 159 (2) ◽

pp. 515-525 ◽

Cited By ~ 2

Author(s):

Allison P Davis ◽

Lorraine S Symington

Keyword(s):

Protein A ◽

Single Strand ◽

Physical Interaction ◽

Dna Double Strand Breaks ◽

Single Stranded Dna ◽

Single Strand Annealing ◽

Strand Annealing ◽

Recombination Pathway

Abstract The yeast RAD52 gene is essential for homology-dependent repair of DNA double-strand breaks. In vitro, Rad52 binds to single- and double-stranded DNA and promotes annealing of complementary single-stranded DNA. Genetic studies indicate that the Rad52 and Rad59 proteins act in the same recombination pathway either as a complex or through overlapping functions. Here we demonstrate physical interaction between Rad52 and Rad59 using the yeast two-hybrid system and co-immunoprecipitation from yeast extracts. Purified Rad59 efficiently anneals complementary oligonucleotides and is able to overcome the inhibition to annealing imposed by replication protein A (RPA). Although Rad59 has strand-annealing activity by itself in vitro, this activity is insufficient to promote strand annealing in vivo in the absence of Rad52. The rfa1-D288Y allele partially suppresses the in vivo strand-annealing defect of rad52 mutants, but this is independent of RAD59. These results suggest that in vivo Rad59 is unable to compete with RPA for single-stranded DNA and therefore is unable to promote single-strand annealing. Instead, Rad59 appears to augment the activity of Rad52 in strand annealing.

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DNA double-strand break repair: a theoretical framework and its application

Journal of The Royal Society Interface ◽

10.1098/rsif.2015.0679 ◽

2016 ◽

Vol 13 (114) ◽

pp. 20150679 ◽

Cited By ~ 9

Author(s):

Philip J. Murray ◽

Bart Cornelissen ◽

Katherine A. Vallis ◽

S. Jon Chapman

Keyword(s):

Dna Damage ◽

Auger Electron ◽

Strand Break ◽

Dna Double Strand Breaks ◽

Dsb Repair ◽

Histone H2ax ◽

Dna Double Strand Break ◽

A Cell

DNA double-strand breaks (DSBs) are formed as a result of genotoxic insults, such as exogenous ionizing radiation, and are among the most serious types of DNA damage. One of the earliest molecular responses following DSB formation is the phosphorylation of the histone H2AX, giving rise to γ H2AX. Many copies of γ H2AX are generated at DSBs and can be detected in vitro as foci using well-established immuno-histochemical methods. It has previously been shown that anti- γ H2AX antibodies, modified by the addition of the cell-penetrating peptide TAT and a fluorescent or radionuclide label, can be used to visualize and quantify DSBs in vivo . Moreover, when labelled with a high amount of the short-range, Auger electron-emitting radioisotope, 111 In, the amount of DNA damage within a cell can be increased, leading to cell death. In this report, we develop a mathematical model that describes how molecular processes at individual sites of DNA damage give rise to quantifiable foci. Equations that describe stochastic mean behaviours at individual DSB sites are derived and parametrized using population-scale, time-series measurements from two different cancer cell lines. The model is used to examine two case studies in which the introduction of an antibody (anti- γ H2AX-TAT) that targets a key component in the DSB repair pathway influences system behaviour. We investigate: (i) how the interaction between anti- γ H2AX-TAT and γ H2AX effects the kinetics of H2AX phosphorylation and DSB repair and (ii) model behaviour when the anti- γ H2AX antibody is labelled with Auger electron-emitting 111 In and can thus instigate additional DNA damage. This work supports the conclusion that DSB kinetics are largely unaffected by the introduction of the anti- γ H2AX antibody, a result that has been validated experimentally, and hence the hypothesis that the use of anti- γ H2AX antibody to quantify DSBs does not violate the image tracer principle. Moreover, it provides a novel model of DNA damage accumulation in the presence of Auger electron-emitting 111 In that is supported qualitatively by the available experimental data.

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Double-strand-break-induced homologous recombination in mammalian cells

Biochemical Society Transactions ◽

10.1042/bst0290196 ◽

2001 ◽

Vol 29 (2) ◽

pp. 196-201 ◽

Cited By ~ 186

Author(s):

R. D. Johnson ◽

M. Jasin

Keyword(s):

Homologous Recombination ◽

Sister Chromatid ◽

Mammalian Cells ◽

Indirect Evidence ◽

Strand Break ◽

Double Strand Breaks ◽

Crossing Over ◽

Dna Double Strand Breaks ◽

Dsb Repair ◽

Strand Breaks

In mammalian cells, the repair of DNA double-strand breaks (DSBs) occurs by both homologous and non-homologous mechanisms. Indirect evidence, including that from gene targeting and random integration experiments, had suggested that non-homologous mechanisms were significantly more frequent than homologous ones. However, more recent experiments indicate that homologous recombination is also a prominent DSB repair pathway. These experiments show that mammalian cells use homologous sequences located at multiple positions throughout the genome to repair a DSB. However, template preference appears to be biased, with the sister chromatid being preferred by 2–3 orders of magnitude over a homologous or heterologous chromosome. The outcome of homologous recombination in mammalian cells is predominantly gene conversion that is not associated with crossing-over. The preference for the sister chromatid and the bias against crossing-over seen in mitotic mammalian cells may have developed in order to reduce the potential for genome alterations that could occur when other homologous repair templates are utilized. In attempts to understand further the mechanism of homologous recombination, the proteins that promote this process are beginning to be identified. To date, four mammalian proteins have been demonstrated conclusively to be involved in DSB repair by homologous recombination: Rad54, XRCC2, XRCC3 and BRCAI. This paper summarizes results from a number of recent studies.

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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.

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