DNA-PK Phosphorylation of RPA32 Ser4/Ser8 Regulates Replication Stress Checkpoint Activation, Fork Restart, Homologous Recombination and Mitotic Catastrophe

Overview

Journal DNA Repair (Amst)

Publisher Elsevier

Specialties Biochemistry
Molecular Biology

Date 2014 May 14

PMID 24819595

Citations 75

Authors

Amanda K Ashley

Meena Shrivastav

Jingyi Nie

Courtney Amerin

Kyle Troksa

Jason G Glanzer

Shengqin Liu

Stephen O Opiyo

Diana D Dimitrova

Phuong Le

Brock Sishc

Susan M Bailey

Greg G Oakley

Jac A Nickoloff

Affiliations

Soon will be listed here.

Abstract

Genotoxins and other factors cause replication stress that activate the DNA damage response (DDR), comprising checkpoint and repair systems. The DDR suppresses cancer by promoting genome stability, and it regulates tumor resistance to chemo- and radiotherapy. Three members of the phosphatidylinositol 3-kinase-related kinase (PIKK) family, ATM, ATR, and DNA-PK, are important DDR proteins. A key PIKK target is replication protein A (RPA), which binds single-stranded DNA and functions in DNA replication, DNA repair, and checkpoint signaling. An early response to replication stress is ATR activation, which occurs when RPA accumulates on ssDNA. Activated ATR phosphorylates many targets, including the RPA32 subunit of RPA, leading to Chk1 activation and replication arrest. DNA-PK also phosphorylates RPA32 in response to replication stress, and we demonstrate that cells with DNA-PK defects, or lacking RPA32 Ser4/Ser8 targeted by DNA-PK, confer similar phenotypes, including defective replication checkpoint arrest, hyper-recombination, premature replication fork restart, failure to block late origin firing, and increased mitotic catastrophe. We present evidence that hyper-recombination in these mutants is ATM-dependent, but the other defects are ATM-independent. These results indicate that DNA-PK and ATR signaling through RPA32 plays a critical role in promoting genome stability and cell survival in response to replication stress.

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References

Vassin V, Anantha R, Sokolova E, Kanner S, Borowiec J . Human RPA phosphorylation by ATR stimulates DNA synthesis and prevents ssDNA accumulation during DNA-replication stress. J Cell Sci. 2009; 122(Pt 22):4070-80. PMC: 2776501. DOI: 10.1242/jcs.053702. View

Allen C, Ashley A, Hromas R, Nickoloff J . More forks on the road to replication stress recovery. J Mol Cell Biol. 2011; 3(1):4-12. PMC: 3030971. DOI: 10.1093/jmcb/mjq049. View

Brown E, Baltimore D . Essential and dispensable roles of ATR in cell cycle arrest and genome maintenance. Genes Dev. 2003; 17(5):615-28. PMC: 196009. DOI: 10.1101/gad.1067403. View

Shechter D, Costanzo V, Gautier J . ATR and ATM regulate the timing of DNA replication origin firing. Nat Cell Biol. 2004; 6(7):648-55. DOI: 10.1038/ncb1145. View

Shi W, Feng Z, Zhang J, Gonzalez-Suarez I, Vanderwaal R, Wu X . The role of RPA2 phosphorylation in homologous recombination in response to replication arrest. Carcinogenesis. 2010; 31(6):994-1002. PMC: 3916738. DOI: 10.1093/carcin/bgq035. View

Shao R, Cao C, Zhang H, Kohn K, Wold M, Pommier Y . Replication-mediated DNA damage by camptothecin induces phosphorylation of RPA by DNA-dependent protein kinase and dissociates RPA:DNA-PK complexes. EMBO J. 1999; 18(5):1397-406. PMC: 1171229. DOI: 10.1093/emboj/18.5.1397. View

Oakley G, Loberg L, Yao J, Risinger M, Yunker R, Khanna K . UV-induced hyperphosphorylation of replication protein a depends on DNA replication and expression of ATM protein. Mol Biol Cell. 2001; 12(5):1199-213. PMC: 34578. DOI: 10.1091/mbc.12.5.1199. View

Morrison C, Sonoda E, Takao N, Shinohara A, Yamamoto K, Takeda S . The controlling role of ATM in homologous recombinational repair of DNA damage. EMBO J. 2000; 19(3):463-71. PMC: 305583. DOI: 10.1093/emboj/19.3.463. View

Hastings P, Lupski J, Rosenberg S, Ira G . Mechanisms of change in gene copy number. Nat Rev Genet. 2009; 10(8):551-64. PMC: 2864001. DOI: 10.1038/nrg2593. View

10.

Lieber M . The mechanism of double-strand DNA break repair by the nonhomologous DNA end-joining pathway. Annu Rev Biochem. 2010; 79:181-211. PMC: 3079308. DOI: 10.1146/annurev.biochem.052308.093131. View

11.

Shrivastav M, De Haro L, Nickoloff J . Regulation of DNA double-strand break repair pathway choice. Cell Res. 2007; 18(1):134-47. DOI: 10.1038/cr.2007.111. View

12.

Moynahan M, Jasin M . Mitotic homologous recombination maintains genomic stability and suppresses tumorigenesis. Nat Rev Mol Cell Biol. 2010; 11(3):196-207. PMC: 3261768. DOI: 10.1038/nrm2851. View

13.

Zhou Y, Paull T . DNA-dependent protein kinase regulates DNA end resection in concert with Mre11-Rad50-Nbs1 (MRN) and ataxia telangiectasia-mutated (ATM). J Biol Chem. 2013; 288(52):37112-25. PMC: 3873567. DOI: 10.1074/jbc.M113.514398. View

14.

Wu X, Yang Z, Liu Y, Zou Y . Preferential localization of hyperphosphorylated replication protein A to double-strand break repair and checkpoint complexes upon DNA damage. Biochem J. 2005; 391(Pt 3):473-80. PMC: 1276948. DOI: 10.1042/BJ20050379. View

15.

Block W, Yu Y, Lees-Miller S . Phosphatidyl inositol 3-kinase-like serine/threonine protein kinases (PIKKs) are required for DNA damage-induced phosphorylation of the 32 kDa subunit of replication protein A at threonine 21. Nucleic Acids Res. 2004; 32(3):997-1005. PMC: 373400. DOI: 10.1093/nar/gkh265. View

16.

Wilson 3rd D, Thompson L . Molecular mechanisms of sister-chromatid exchange. Mutat Res. 2006; 616(1-2):11-23. DOI: 10.1016/j.mrfmmm.2006.11.017. View

17.

Romanova L, Willers H, Blagosklonny M, Powell S . The interaction of p53 with replication protein A mediates suppression of homologous recombination. Oncogene. 2004; 23(56):9025-33. DOI: 10.1038/sj.onc.1207982. View

18.

Matsuoka S, Ballif B, Smogorzewska A, McDonald 3rd E, Hurov K, Luo J . ATM and ATR substrate analysis reveals extensive protein networks responsive to DNA damage. Science. 2007; 316(5828):1160-6. DOI: 10.1126/science.1140321. View

19.

Peng Y, Woods R, Beamish H, Ye R, Lees-Miller S, Lavin M . Deficiency in the catalytic subunit of DNA-dependent protein kinase causes down-regulation of ATM. Cancer Res. 2005; 65(5):1670-7. DOI: 10.1158/0008-5472.CAN-04-3451. View

20.

Hagelstrom R, Askin K, Williams A, Ramaiah L, Desaintes C, Goodwin E . DNA-PKcs and ATM influence generation of ionizing radiation-induced bystander signals. Oncogene. 2008; 27(53):6761-9. DOI: 10.1038/onc.2008.276. View