PHOSPHORYLATION OF THE PCNA BINDING DOMAIN OF THE LARGE SUBUNIT OF REPLICATION FACTOR-C BY CA2+ CALMODULIN-DEPENDENT PROTEIN-KINASE INHIBITS DNA-SYNTHESIS/

Replication factor C (RF-C) is a heteropentameric protein essential fo r DNA replication and DNA repair. It is a molecular matchmaker require d for loading of the proliferating cell nuclear antigen (PCNA) sliding clamp onto double-strand DNA and for PCNA-dependent DNA synthesis by DNA polymerases delta and epsilon. The DNA and PCNA binding domains of the large 140 kDa subunit of human RF-C have been recently cloned [Fo tedar, R., Mossi, R., Fitzgerald, P., Rousselle, T., Maga, G., Brickne r, H., Messier, H., Khastilba, S., Hiibscher, U., & Fotedar, A. (1996) EMBO J. 15, 4423-4433]. Here we show that the PCNA binding domain is phosphorylated by the Ca2+/calmodulin-dependent protein kinase II (CaM KII), an enzyme required for cell cycle progression in eukaryotic cell s. The DNA binding domain, on the other hand, is not phosphorylated. P hosphorylation by CaMKII reduces the binding of PCNA to RF-C and conse quently inhibits RF-C-dependent DNA synthesis by DNA polymerases delta and epsilon. Once bound to PCNA and DNA, RF-C is protected from phosp horylation by CaMKII, suggesting a possible role of CaMKII in regulati ng the dynamics of interaction between PCNA and RF-C and thus interfer ing in the formation of an active sliding clamp by DNA polymerases del ta and epsilon.