PHOSPHORYLATION OF DNA-POLYMERASE ALPHA-PRIMASE BY CYCLIN A-DEPENDENTKINASES REGULATES INITIATION OF DNA-REPLICATION IN-VITRO

DNA polymerase alpha-primase is the only known eukaryotic enzyme that can start DNA replication de novo. In this study, we investigated the regulation of DNA replication by phosphorylation of DNA polymerase alp ha-primase. The p180 and the p68 submits of DNA polymerase alpha-prima se were phosphorylated using Cyclin A-, B- and E- dependent kinases. T his phosphorylation did not influence its DNA polymerase activity on a ctivated DNA, but slightly stimulated primase activity using poly(dT) single-stranded DNA (ssDNA) without changing the product length of pri mers. In contrast, site-specific initiation of replication on plasmid DNA containing the SV40 origin is affected: Cyclin A-Cdk2 and Cyclin A -Cdc2 inhibited initiation of SV40 DNA replication in vitro, Cyclin B- Cdc2 had no effect and Cyclin E-Cdk2 stimulated the initation reaction . DNA polymerase alpha-primase that was pre-phosphorylated by Cyclin A -Cdk2 was completely unable to initiate the SV40 DNA replication in vi tro; Cyclin B-Cdc2-phosphorylated enzyme was moderately inhibited, whi le Cyclin E-Cdk2-treated DNA polymerase alpha-primase remained fully a ctive in the initiation reaction.