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First published online 20 October 2009
doi: 10.1242/jcs.053702

Journal of Cell Science 122, 4070-4080 (2009)
Published by The Company of Biologists 2009
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Research Article

Human RPA phosphorylation by ATR stimulates DNA synthesis and prevents ssDNA accumulation during DNA-replication stress

Vitaly M. Vassin, Rachel William Anantha, Elena Sokolova, Shlomo Kanner and James A. Borowiec*

Department of Biochemistry and New York University Cancer Institute, New York University School of Medicine, New York, NY 10016, USA

* Author for correspondence (james.borowiec{at}med.nyu.edu)

Accepted 30 August 2009

ATR is an essential kinase activated in response to DNA-replication stress, with a known target being the RPA2 subunit of human replication protein A (RPA). We find that S33-RPA2 phosphorylation by ATR occurs primarily in the late-S and G2 phases, probably at sites of residual stalled DNA-replication forks, with S33-P-RPA2 contained within nuclear repair centers. Although cells in which endogenous RPA2 was `replaced' with an RPA2 protein with mutations T21A and S33A (T21A/S33A-RPA) had normal levels of DNA replication under non-stress conditions, the mutant cells were severely deficient in the amount of DNA synthesis occurring during replication stress. These cells also had abnormally high levels of chromatin-bound RPA, indicative of increased amounts of single-stranded DNA (ssDNA) and showed defective recovery from stress. Cells replaced with the mutant RPA2 also generated G1 cells with a broader DNA distribution and high levels of apoptosis following stress, compared with cells expressing wild-type RPA2. Surprisingly, cells expressing the wild-type RPA2 subunit had increased levels of stress-dependent DNA breaks. Our data demonstrate that RPA phosphorylation at the T21 and S33 sites facilitates adaptation of a DNA-replication fork to replication stress.

Key words: ATR, DNA damage, DNA replication, RPA, Replication stress


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