RPA2 is a direct downstream target for ATR to regulate the S-phase checkpoint.
Upon DNA damage, replication is inhibited by the S-phase checkpoint. ATR (ataxia telangiectasia mutated- and Rad3-related) is specifically involved in the inhibition of replicon initiation when cells are treated with DNA damage-inducing agents that stall replication forks, but the mechanism by which it acts to prevent replication is not yet fully understood. We observed that RPA2 is phosphorylated on chromatin in an ATR-dependent manner when replication forks are stalled. Mutation of the ATR-dependent phosphorylation sites in RPA2 leads to a defect in the down-regulation of DNA synthesis following treatment with UV radiation, although ATR activation is not affected. Threonine 21 and serine 33, two residues among several phosphorylation sites in the amino terminus of RPA2, are specifically required for the UV-induced, ATR-mediated inhibition of DNA replication. RPA2 mutant alleles containing phospho-mimetic mutations at ATR-dependent phosphorylation sites have an impaired ability to associate with replication centers, indicating that ATR phosphorylation of RPA2 directly affects the replication function of RPA. Our studies suggest that in response to UV-induced DNA damage, ATR rapidly phosphorylates RPA2, disrupting its association with replication centers in the S-phase and contributing to the inhibition of DNA replication.
Olson
Erin
E
Department of Molecular and Experimental Medicine, The Scripps Research Institute, La, Jolla, California 92037, USA.
Nievera
Christian J
CJ
Klimovich
Vitaly
V
Fanning
Ellen
E
Wu
Xiaohua
X
D064007:Ataxia Telangiectasia Mutated Proteins; D001665:Binding Sites; D018797:Cell Cycle Proteins / Q000737:chemistry / Q000502:physiology*; D002460:Cell Line; D045744:Cell Line, Tumor; D002843:Chromatin / Q000737:chemistry; D004249:DNA Damage; D004259:DNA-Directed DNA Polymerase / Q000096:biosynthesis / Q000502:physiology*; D006801:Humans; D006863:Hydrogen-Ion Concentration; D009154:Mutation; D010766:Phosphorylation; D011485:Protein Binding; D017346:Protein Serine-Threonine Kinases / Q000737:chemistry / Q000502:physiology*; D051716:Replication Protein A; D016196:S Phase; D012694:Serine / Q000737:chemistry; D013912:Threonine / Q000737:chemistry; D014466:Ultraviolet Rays
D018797:Cell Cycle Proteins; D002843:Chromatin; D051716:Replication Protein A; D013912:Threonine; D012694:Serine; C576597:ATR protein, human; D064007:Ataxia Telangiectasia Mutated Proteins; D017346:Protein Serine-Threonine Kinases; D004259:DNA-Directed DNA Polymerase; C516794:RPA2 protein, human