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

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.

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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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Involvement of POLA2 in Double Strand Break Repair and Genotoxic Stress

International Journal of Molecular Sciences ◽

10.3390/ijms21124245 ◽

2020 ◽

Vol 21 (12) ◽

pp. 4245

Author(s):

Tuyen T. Dang ◽

Julio C. Morales

Keyword(s):

Dna Damage ◽

Genomic Instability ◽

Dna Polymerase ◽

Genotoxic Stress ◽

Strand Break ◽

Double Strand Break ◽

Double Strand Break Repair ◽

Dsb Repair ◽

Dna Polymerase Alpha ◽

Break Repair

Cellular survival is dependent on the efficient replication and transmission of genomic information. DNA damage can be introduced into the genome by several different methods, one being the act of DNA replication. Replication is a potent source of DNA damage and genomic instability, especially through the formation of DNA double strand breaks (DSBs). DNA polymerase alpha is responsible for replication initiation. One subunit of the DNA polymerase alpha replication machinery is POLA2. Given the connection between replication and genomic instability, we decided to examine the role of POLA2 in DSB repair, as little is known about this topic. We found that loss of POLA2 leads to an increase in spontaneous DSB formation. Loss of POLA2 also slows DSB repair kinetics after treatment with etoposide and inhibits both of the major double strand break repair pathways: non-homologous end-joining and homologous recombination. In addition, loss of POLA2 leads to increased sensitivity to ionizing radiation and PARP1 inhibition. Lastly, POLA2 expression is elevated in glioblastoma multiforme tumors and correlates with poor overall patient survival. These data demonstrate a role for POLA2 in DSB repair and resistance to genotoxic stress.

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A Double-Strand Break Repair Component Is Essential for S Phase Completion in Fission Yeast Cell Cycling

Molecular Biology of the Cell ◽

10.1091/mbc.10.10.3331 ◽

1999 ◽

Vol 10 (10) ◽

pp. 3331-3343 ◽

Cited By ~ 28

Author(s):

Kimihiko Suto ◽

Akihisa Nagata ◽

Hiroshi Murakami ◽

Hiroto Okayama

Keyword(s):

Cell Division ◽

Fission Yeast ◽

Protein A ◽

Replication Protein A ◽

Strand Break ◽

Double Strand Break ◽

S Phase ◽

Double Strand Break Repair ◽

A Cell ◽

Break Repair

Fission yeast rad22 +, a homologue of budding yeast RAD52, encodes a double-strand break repair component, which is dispensable for proliferation. We, however, have recently obtained a cell division cycle mutant with a temperature-sensitive allele of rad22 +, designated rad22-H6, which resulted from a point mutation in the conserved coding sequence leading to one amino acid alteration. We have subsequently isolatedrad22 + and its novel homologuerti1 + as multicopy suppressors of this mutant. rti1 + suppresses all the defects of cells lacking rad22 +. Mating type switch-inactive heterothallic cells lacking eitherrad22 + or rti1 +are viable, but those lacking both genes are inviable and arrest proliferation with a cell division cycle phenotype. At the nonpermissive temperature, a synchronous culture ofrad22-H6 cells performs DNA synthesis without delay and arrests with chromosomes seemingly intact and replication completed and with a high level of tyrosine-phosphorylated Cdc2. However,rad22-H6 cells show a typical S phase arrest phenotype if combined with the rad1-1 checkpoint mutation.rad22 + genetically interacts withrad11 +, which encodes the large subunit of replication protein A. Deletion ofrad22 +/rti1 + or the presence of rad22-H6 mutation decreases the restriction temperature of rad11-A1 cells by 4–6°C and leads to cell cycle arrest with chromosomes incompletely replicated. Thus, in fission yeast a double-strand break repair component is required for a certain step of chromosome replication unlinked to repair, partly via interacting with replication protein A.

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MOF and Histone H4 Acetylation at Lysine 16 Are Critical for DNA Damage Response and Double-Strand Break Repair

Molecular and Cellular Biology ◽

10.1128/mcb.01476-09 ◽

2010 ◽

Vol 30 (14) ◽

pp. 3582-3595 ◽

Cited By ~ 188

Author(s):

Girdhar G. Sharma ◽

Sairei So ◽

Arun Gupta ◽

Rakesh Kumar ◽

Christelle Cayrou ◽

...

Keyword(s):

Dna Damage ◽

Dna Damage Response ◽

Strand Break ◽

Double Strand Break ◽

Double Strand Break Repair ◽

Histone H4 ◽

Dsb Repair ◽

Altered Expression ◽

Damage Response ◽

Break Repair

ABSTRACT The human MOF gene encodes a protein that specifically acetylates histone H4 at lysine 16 (H4K16ac). Here we show that reduced levels of H4K16ac correlate with a defective DNA damage response (DDR) and double-strand break (DSB) repair to ionizing radiation (IR). The defect, however, is not due to altered expression of proteins involved in DDR. Abrogation of IR-induced DDR by MOF depletion is inhibited by blocking H4K16ac deacetylation. MOF was found to be associated with the DNA-dependent protein kinase catalytic subunit (DNA-PKcs), a protein involved in nonhomologous end-joining (NHEJ) repair. ATM-dependent IR-induced phosphorylation of DNA-PKcs was also abrogated in MOF-depleted cells. Our data indicate that MOF depletion greatly decreased DNA double-strand break repair by both NHEJ and homologous recombination (HR). In addition, MOF activity was associated with general chromatin upon DNA damage and colocalized with the synaptonemal complex in male meiocytes. We propose that MOF, through H4K16ac (histone code), has a critical role at multiple stages in the cellular DNA damage response and DSB repair.

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DNA Replication Defects, Spontaneous DNA Damage, and ATM-dependent Checkpoint Activation in Replication Protein A-deficient Cells

Journal of Biological Chemistry ◽

10.1074/jbc.c400242200 ◽

2004 ◽

Vol 279 (32) ◽

pp. 34010-34014 ◽

Cited By ~ 63

Author(s):

Gerald E. Dodson ◽

Yuling Shi ◽

Randal S. Tibbetts

Keyword(s):

Dna Damage ◽

Dna Replication ◽

Protein A ◽

Replication Protein A ◽

Replication Protein ◽

Checkpoint Activation

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Faculty Opinions recommendation of p53 isoform Δ113p53/Δ133p53 promotes DNA double-strand break repair to protect cell from death and senescence in response to DNA damage.

Faculty Opinions – Post-Publication Peer Review of the Biomedical Literature ◽

10.3410/f.725365086.793539126 ◽

2017 ◽

Author(s):

Ygal Haupt ◽

Sue Haupt

Keyword(s):

Dna Damage ◽

Strand Break ◽

Double Strand Break ◽

Double Strand Break Repair ◽

Dna Double Strand Break ◽

Break Repair

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Watching a double strand break repair polymerase insert a pro-mutagenic oxidized nucleotide

Nature Communications ◽

10.1038/s41467-021-21354-6 ◽

2021 ◽

Vol 12 (1) ◽

Author(s):

Joonas A. Jamsen ◽

Akira Sassa ◽

David D. Shock ◽

William A. Beard ◽

Samuel H. Wilson

Keyword(s):

Active Site ◽

Dna Polymerases ◽

Time Lapse ◽

Strand Break ◽

Double Strand Break ◽

Double Strand Break Repair ◽

Dsb Repair ◽

X Ray Crystallography ◽

Polymerase Fidelity ◽

Break Repair

AbstractOxidized dGTP (8-oxo-7,8-dihydro-2´-deoxyguanosine triphosphate, 8-oxodGTP) insertion by DNA polymerases strongly promotes cancer and human disease. How DNA polymerases discriminate against oxidized and undamaged nucleotides, especially in error-prone double strand break (DSB) repair, is poorly understood. High-resolution time-lapse X-ray crystallography snapshots of DSB repair polymerase μ undergoing DNA synthesis reveal that a third active site metal promotes insertion of oxidized and undamaged dGTP in the canonical anti-conformation opposite template cytosine. The product metal bridged O8 with product oxygens, and was not observed in the syn-conformation opposite template adenine (At). Rotation of At into the syn-conformation enabled undamaged dGTP misinsertion. Exploiting metal and substrate dynamics in a rigid active site allows 8-oxodGTP to circumvent polymerase fidelity safeguards to promote pro-mutagenic double strand break repair.

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