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Molecular and Cellular Biology, November 2009, p. 5696-5709, Vol. 29, No. 21
0270-7306/09/$08.00+0     doi:10.1128/MCB.00191-09
Copyright © 2009, American Society for Microbiology. All Rights Reserved.

Protein Phosphatase 2A-Dependent Dephosphorylation of Replication Protein A Is Required for the Repair of DNA Breaks Induced by Replication Stress{triangledown} ,{dagger}

Junjie Feng,1 Timothy Wakeman,1 Sheila Yong,1 Xiaohua Wu,2 Sally Kornbluth,1 and Xiao-Fan Wang1*

Department of Pharmacology and Cancer Biology, Duke University Medical Center, Durham, North Carolina 27710,1 Department of Molecular and Experimental Medicine, The Scripps Research Institute, La Jolla, California 920372

Received 10 February 2009/ Returned for modification 13 March 2009/ Accepted 14 August 2009

Eukaryotic genomic integrity is safeguarded by cell cycle checkpoints and DNA repair pathways, collectively known as the DNA damage response, wherein replication protein A (RPA) is a key regulator playing multiple critical roles. The genotoxic insult-induced phosphorylation of the 32-kDa subunit of human RPA (RPA32), most notably the ATM/ATR-dependent phosphorylation at T21 and S33, acts to suppress DNA replication and recruit other checkpoint/repair proteins to the DNA lesions. It is not clear, however, how the DNA damage-responsive function of phosphorylated RPA is attenuated and how the replication-associated activity of the unphosphorylated form of RPA is restored when cells start to resume the normal cell cycle. We report here that in cells recovering from hydroxyurea (HU)-induced genotoxic stress, RPA32 is dephosphorylated by the serine/threonine protein phosphatase 2A (PP2A). Interference with PP2A catalytic activity causes persistent RPA32 phosphorylation and increased HU sensitivity. The PP2A catalytic subunit binds to RPA following DNA damage and can dephosphorylate RPA32 in vitro. Cells expressing a RPA32 persistent phosphorylation mimetic exhibit normal checkpoint activation and reenter the cell cycle normally after recovery but display a pronounced defect in the repair of DNA breaks. These data indicate that PP2A-mediated RPA32 dephosphorylation is required for the efficient DNA damage repair.

* Corresponding author. Mailing address: Department of Pharmacology and Cancer Biology, Duke University Medical Center, C218 LSRC Bldg., La Salle Street Extension, Durham, NC 27710. Phone: (919) 681-4861. Fax: (919) 681-7152. E-mail: wang0011{at}mc.duke.edu

{triangledown} Published ahead of print on 24 August 2009.

{dagger} Supplemental material for this article may be found at http://mcb.asm.org/.

Molecular and Cellular Biology, November 2009, p. 5696-5709, Vol. 29, No. 21
0270-7306/09/$08.00+0     doi:10.1128/MCB.00191-09
Copyright © 2009, American Society for Microbiology. All Rights Reserved.





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