Sequential and synergistic modification of human RPA stimulates chromosomal DNA repair.
The activity of human replication protein A (RPA) in DNA replication and repair is regulated by phosphorylation of the middle RPA2 subunit. It has previously been shown that up to nine different N-terminal residues are modified in vivo and in response to genotoxic stress. Using a novel antibody against phospho-Ser(29), a moiety formed by cyclin-Cdk, we observed that RPA2 was phosphorylated during mitosis in nonstressed cells. Robust phosphorylation of Ser(29) was also seen in interphase cells following treatment with the DNA-damaging agent camptothecin, a rare example of stress stimulating the modification of a repair factor by cyclin-Cdk. RPA2 phosphorylation is regulated both in cis and trans. Cis-phosphorylation follows a preferred pathway. (That is, the initial modification of Ser(33) by ATR stimulates subsequent phosphorylation of Cdk sites Ser(23) and Ser(29)). These events then facilitate modification of Thr(21) and extreme N-terminal sites Ser(4) and Ser(8), probably by DNA-PK. Our data also indicate that the phosphorylation of one RPA molecule can influence the phosphorylation of other RPA molecules in trans. Cells in which endogenous RPA2 was "replaced" with a double S23A/S29A-RPA2 mutant were seen to have an abnormal cell cycle distribution both in normal and in stressed cells. Such cells also showed aberrant DNA damage-dependent RPA foci and had persistent staining of gammaH2AX following DNA damage. Our data indicate that RPA phosphorylation facilitates chromosomal DNA repair. We postulate that the RPA phosphorylation pattern provides a means to regulate the DNA repair pathway utilized.
Anantha
Rachel W
RW
Department of Biochemistry and New York University Cancer Institute, New York University School of Medicine, New York, New York 10016, USA.
Vassin
Vitaly M
VM
Borowiec
James A
JA
D019943:Amino Acid Substitution; D000906:Antibodies / Q000737:chemistry; D000972:Antineoplastic Agents, Phytogenic / Q000493:pharmacokinetics; D064007:Ataxia Telangiectasia Mutated Proteins; D002166:Camptothecin / Q000494:pharmacology; D018797:Cell Cycle Proteins / Q000235:genetics / Q000378:metabolism; D002460:Cell Line; D016213:Cyclins / Q000378:metabolism; D004249:DNA Damage / Q000187:drug effects / Q000502:physiology*; D004260:DNA Repair / Q000187:drug effects / Q000502:physiology*; D004261:DNA Replication / Q000187:drug effects / Q000502:physiology; D006657:Histones / Q000235:genetics / Q000378:metabolism; D006801:Humans; D007399:Interphase / Q000187:drug effects / Q000502:physiology; D008938:Mitosis / Q000187:drug effects / Q000502:physiology; D020125:Mutation, Missense; D010766:Phosphorylation / Q000187:drug effects; D017346:Protein Serine-Threonine Kinases / Q000235:genetics / Q000378:metabolism; D021122:Protein Subunits / Q000235:genetics / Q000378:metabolism; D051716:Replication Protein A / Q000235:genetics / Q000378:metabolism*
D000906:Antibodies; D000972:Antineoplastic Agents, Phytogenic; D018797:Cell Cycle Proteins; D016213:Cyclins; C468783:H2AX protein, human; D006657:Histones; D021122:Protein Subunits; D051716:Replication Protein A; C576597:ATR protein, human; D064007:Ataxia Telangiectasia Mutated Proteins; D017346:Protein Serine-Threonine Kinases; C516794:RPA2 protein, human; D002166:Camptothecin