Phosphorylation of the PCNA binding domain of the large subunit of replication factor C on Thr506 by cyclin‐dependent kinases regulates binding to PCNA

Replication factor C (RF‐C) complex binds to DNA primers and loads PCNA onto DNA, thereby increasing the processivity of DNA polymerases. We have previously identified a distinct region, domain B, in the large subunit of human RF‐C (RF‐Cp145) which binds to PCNA. We show here that the functional interaction of RF‐Cp145 with PCNA is regulated by cdk‐cyclin kinases. Phosphorylation of either RF‐Cp145 as a part of the RF‐C complex or RF‐Cp145 domain B by cdk‐cyclin kinases inhibits their ability to bind PCNA. A cdk‐cyclin phosphorylation site, Thr506 in RF‐Cp145, identified by mass spectrometry, is also phosphorylated in vivo. A Thr506→Ala RF‐Cp145 domain B mutant is a poor in vitro substrate for cdk‐cyclin kinase and, consequently, the ability of this mutant to bind PCNA was not suppressed by phosphorylation. By generating an antibody directed against phospho‐Thr506 in RF‐Cp145, we demonstrate that phosphorylation of endogenous RF‐Cp145 at Thr506 is mediated by CDKs since it is abolished by treatment of cells with the cdk‐cyclin inhibitor roscovitine. We have thus mapped an in vivo cdk‐cyclin phosphorylation site within the PCNA binding domain of RF‐Cp145.