Human RPA phosphorylation by ATR stimulates DNA synthesis and prevents ssDNA accumulation during DNA-replication stress.
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.
Department of Biochemistry, New York University School of Medicine, New York, NY 10016, USA.
Vitaly M
VM
Vassin
Rachel William
RW
Anantha
Elena
E
Sokolova
Shlomo
S
Kanner
James A
JA
Borowiec
D017209:Apoptosis / Q000235:genetics; D064007:Ataxia Telangiectasia Mutated Proteins; D018797:Cell Cycle Proteins / Q000378:metabolism*; D002460:Cell Line; D002843:Chromatin / Q000378:metabolism; D004247:DNA / Q000096:biosynthesis*; D004249:DNA Damage; D004261:DNA Replication; D004277:DNA, Single-Stranded / Q000235:genetics / Q000378:metabolism*; D006801:Humans; D007399:Interphase / Q000235:genetics; D010766:Phosphorylation; D017346:Protein Serine-Threonine Kinases / Q000378:metabolism*; D011994:Recombinant Proteins / Q000235:genetics / Q000378:metabolism; D051716:Replication Protein A / Q000235:genetics / Q000378:metabolism*
D018797:Cell Cycle Proteins; D002843:Chromatin; D004277:DNA, Single-Stranded; D011994:Recombinant Proteins; D051716:Replication Protein A; D004247:DNA; C576597:ATR protein, human; D064007:Ataxia Telangiectasia Mutated Proteins; D017346:Protein Serine-Threonine Kinases; C516794:RPA2 protein, human