Phosphorylation of the PCNA binding domain of the large subunit of replication factor C by Ca2+/calmodulin-dependent protein kinase II inhibits DNA synthesis.
Replication factor C (RF-C) is a heteropentameric protein essential for DNA replication and DNA repair. It is a molecular matchmaker required 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 [Fotedar, R., Mossi, R., Fitzgerald, P., Rousselle, T., Maga, G., Brickner, H., Messier, H., Khastilba. S., Hübscher, 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 (CaMKII), an enzyme required for cell cycle progression in eukaryotic cells. The DNA binding domain, on the other hand, is not phosphorylated. Phosphorylation by CaMKII reduces the binding of PCNA to RF-C and consequently inhibits RF-C-dependent DNA synthesis by DNA polymerases delta1 and epsilon. Once bound to PCNA and DNA, RF-C is protected from phosphorylation by CaMKII, suggesting a possible role of CaMKII in regulating the dynamics of interaction between PCNA and RF-C and thus interfering in the formation of an active sliding clamp by DNA polymerases delta and epsilon.
Maga
G
G
Department of Veterinary Biochemistry, University Zürich-Irchel, Switzerland.
Mossi
R
R
Fischer
R
R
Berchtold
M W
MW
Hübscher
U
U
D000818:Animals; D001665:Binding Sites; D017871:Calcium-Calmodulin-Dependent Protein Kinases / Q000378:metabolism* / Q000494:pharmacology; D002417:Cattle; D004257:DNA Polymerase II; D004258:DNA Polymerase III; D004261:DNA Replication; D004268:DNA-Binding Proteins / Q000378:metabolism*; D004259:DNA-Directed DNA Polymerase / Q000378:metabolism; D018398:Homeodomain Proteins; D006801:Humans; D015778:Minor Histocompatibility Antigens; D019384:Nucleic Acid Synthesis Inhibitors; D010766:Phosphorylation; D018809:Proliferating Cell Nuclear Antigen / Q000378:metabolism*; D019253:Proto-Oncogene Proteins c-bcl-2; D051818:Replication Protein C; D012097:Repressor Proteins; D029701:Saccharomyces cerevisiae Proteins
C096816:BCL2-related protein A1; D004268:DNA-Binding Proteins; D018398:Homeodomain Proteins; C096063:MATA1 protein, S cerevisiae; D015778:Minor Histocompatibility Antigens; D019384:Nucleic Acid Synthesis Inhibitors; D018809:Proliferating Cell Nuclear Antigen; D019253:Proto-Oncogene Proteins c-bcl-2; D012097:Repressor Proteins; D029701:Saccharomyces cerevisiae Proteins; D017871:Calcium-Calmodulin-Dependent Protein Kinases; D004257:DNA Polymerase II; D004258:DNA Polymerase III; D004259:DNA-Directed DNA Polymerase; D051818:Replication Protein C