Sequential and synergistic modification of human Replication Factor A stimulates chromosomal DNA repair.
Anantha RW, Vassin VM, Borowiec JA
Journal of Biological Chemistry (2007)
Category: DNA repair, OB-fold, SSB proteins ¤ Added: Dec 04, 2007 ¤ Rating: ◊◊
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-S29, a moiety formed by cyclin-Cdk, we observed that RPA2 was phosphorylated during mitosis in non-stressed cells. Robust phosphorylation of S29 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 S33 by ATR stimulates subsequent phosphorylation of Cdk sites S23 and S29. These events then facilitate modification of T21 and extreme N-terminal sites S4 and S8, likely 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 H2AX 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.