Cell cycle-dependent regulation of human DNA polymerase alpha-primase activity by phosphorylation

DNA polymerase alpha-primase is known to be phosphorylated in human and yea st cells in a cell cycle-dependent manner on the p180 and p68 subunits. Her e we show that phosphorylation of purified human DNA polymerase alpha-prima se by purified cyclin A/cdk2 in vitro reduced its ability to initiate simia n virus 40 (SV40) DNA replication in vitro, while phosphorylation by cyclin E/cdk2 stimulated its initiation activity. Tryptic phosphopeptide mapping revealed a family of p68 peptides that was modified well by cyclin A/cdk2 a nd poorly by cyclin E/cdk2. The p180 phosphopeptides were identical with bo th kinases. By mass spectrometry, the p68 peptide family was identified as residues 141 to 160. Cyclin A/cdk2- and cyclin A/cdc2-modified p68 also dis played a phosphorylation-dependent shift to slower electrophoretic mobility . Mutation of the four putative phosphorylation sites within p68 peptide re sidues 141 to 160 prevented its phosphorylation by cyclin A/cdk2 and the in hibition of replication activity. Phosphopeptide maps of the p68 subunit of DNA polymerase alpha-primase from human cells, synchronized and labeled in G(1)/S and in G(2), revealed a cyclin E/cdk2-like pattern in G(1)/S and a cyclin A/cdk2-like pattern in G(2). The slower-electrophoretic-mobility for m of p68 was absent in human cells in G(1)/S and appeared as the cells ente red G(2)/M. Consistent with this, the ability of DNA polymerase alpha-prima se isolated from synchronized human cells to initiate SV40 replication was maximal in G(1)/S, decreased as the cells completed S phase, and reached a minimum in G(2)/M. These results suggest that the replication activity of D NA polymerase alpha-primase in human cells is regulated by phosphorylation in a cell cycle-dependent manner.