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

2011 ◽  
Vol 192 (2) ◽  
pp. 251-261 ◽  
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.

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

2005 ◽  
Vol 171 (2) ◽  
pp. 217-227 ◽  
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.

scholarly journals Preferential localization of hyperphosphorylated replication protein A to double-strand break repair and checkpoint complexes upon DNA damage

2005 ◽  
Vol 391 (3) ◽  
pp. 473-480 ◽  
Author(s):  
Xiaoming Wu ◽  
Zhengguan Yang ◽  
Yiyong Liu ◽  
Yue Zou
Keyword(s):  
Dna Damage ◽  
Protein A ◽  
Replication Protein A ◽  
Strand Break ◽  
Double Strand Break ◽  
Replication Protein ◽  
Double Strand Break Repair ◽  
Dsb Repair ◽  
Checkpoint Activation ◽  
In Cells

RPA (replication protein A) is an essential factor for DNA DSB (double-strand break) repair and cell cycle checkpoint activation. The 32 kDa subunit of RPA undergoes hyperphosphorylation in response to cellular genotoxic insults. However, the potential involvement of hyperphosphorylated RPA in DSB repair and checkpoint activation remains unclear. Using co-immunoprecipitation assays, we showed that cellular interaction of RPA with two DSB repair factors, Rad51 and Rad52, was predominantly mediated by the hyperphosphorylated species of RPA in cells after UV and camptothecin treatment. Moreover, Rad51 and Rad52 displayed higher affinity for the hyperphosphorylated RPA than native RPA in an in vitro binding assay. Checkpoint kinase ATR (ataxia telangiectasia mutated and Rad3-related) also interacted more efficiently with the hyperphosphorylated RPA than with native RPA following DNA damage. Consistently, immunofluorescence microscopy demonstrated that the hyperphosphorylated RPA was able to co-localize with Rad52 and ATR to form significant nuclear foci in cells. Our results suggest that hyperphosphorylated RPA is preferentially localized to DSB repair and the DNA damage checkpoint complexes in response to DNA damage.

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

2007 ◽  
Vol 28 (3) ◽  
pp. 897-906 ◽  
Author(s):  
Thomas J. Pohl ◽  
Jac A. Nickoloff
Keyword(s):  
Gene Conversion ◽  
Strand Break ◽  
Double Strand Break ◽  
Single Strand ◽  
Homology Search ◽  
Wild Type ◽  
Dsb Repair ◽  
Single Strand Annealing ◽  
In The Wild ◽  
Break Induced Replication

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.

scholarly journals FANCA Promotes DNA Double-Strand Break Repair by Catalyzing Single-Strand Annealing and Strand Exchange

2018 ◽  
Vol 71 (4) ◽  
pp. 621-628.e4 ◽  
Author(s):  
Anaid Benitez ◽  
Wenjun Liu ◽  
Anna Palovcak ◽  
Guanying Wang ◽  
Jaewon Moon ◽  
...  
Keyword(s):  
Strand Break ◽  
Double Strand Break ◽  
Single Strand ◽  
Strand Exchange ◽  
Double Strand Break Repair ◽  
Dna Double Strand Break ◽  
Single Strand Annealing ◽  
Break Repair ◽  
Strand Annealing

scholarly journals Human RecQ Helicases in DNA Double-Strand Break Repair

2021 ◽  
Vol 9 ◽  
Author(s):  
Huiming Lu ◽  
Anthony J. Davis
Keyword(s):  
Werner Syndrome ◽  
Genetic Disorders ◽  
Strand Break ◽  
Bloom Syndrome ◽  
Double Strand Break ◽  
Premature Aging ◽  
Telomere Maintenance ◽  
Dsb Repair ◽  
Recq Helicases ◽  
Dna Double Strand Break

RecQ DNA helicases are a conserved protein family found in bacteria, fungus, plants, and animals. These helicases play important roles in multiple cellular functions, including DNA replication, transcription, DNA repair, and telomere maintenance. Humans have five RecQ helicases: RECQL1, Bloom syndrome protein (BLM), Werner syndrome helicase (WRN), RECQL4, and RECQL5. Defects in BLM and WRN cause autosomal disorders: Bloom syndrome (BS) and Werner syndrome (WS), respectively. Mutations in RECQL4 are associated with three genetic disorders, Rothmund–Thomson syndrome (RTS), Baller–Gerold syndrome (BGS), and RAPADILINO syndrome. Although no genetic disorders have been reported due to loss of RECQL1 or RECQL5, dysfunction of either gene is associated with tumorigenesis. Multiple genetically independent pathways have evolved that mediate the repair of DNA double-strand break (DSB), and RecQ helicases play pivotal roles in each of them. The importance of DSB repair is supported by the observations that defective DSB repair can cause chromosomal aberrations, genomic instability, senescence, or cell death, which ultimately can lead to premature aging, neurodegeneration, or tumorigenesis. In this review, we will introduce the human RecQ helicase family, describe in detail their roles in DSB repair, and provide relevance between the dysfunction of RecQ helicases and human diseases.

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

Genes ◽  
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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