Cell Cycle-Dependent Regulation of Human DNA Polymerase α-Primase Activity by Phosphorylation

Abstract

DNA polymerase α-primase is known to be phosphorylated in human and yeast cells in a cell cycle-dependent manner on the p180 and p68 subunits. Here we show that phosphorylation of purified human DNA polymerase α-primase by purified cyclin A/cdk2 in vitro reduced its ability to initiate simian 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 and poorly by cyclin E/cdk2. The p180 phosphopeptides were identical with both 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 displayed a phosphorylation-dependent shift to slower electrophoretic mobility. Mutation of the four putative phosphorylation sites within p68 peptide residues 141 to 160 prevented its phosphorylation by cyclin A/cdk2 and the inhibition of replication activity. Phosphopeptide maps of the p68 subunit of DNA polymerase α-primase from human cells, synchronized and labeled in G₁/S and in G₂, revealed a cyclin E/cdk2-like pattern in G₁/S and a cyclin A/cdk2-like pattern in G₂. The slower-electrophoretic-mobility form of p68 was absent in human cells in G₁/S and appeared as the cells entered G₂/M. Consistent with this, the ability of DNA polymerase α-primase isolated from synchronized human cells to initiate SV40 replication was maximal in G₁/S, decreased as the cells completed S phase, and reached a minimum in G₂/M. These results suggest that the replication activity of DNA polymerase α-primase in human cells is regulated by phosphorylation in a cell cycle-dependent manner.

ACKNOWLEDGMENTS

We thank K. Gould, C. Prives, D. Reese, M. Becker, M. Westfall, D. Lane, D. Morgan, T. Wang, E. Harlow, and the members of the Fanning lab for sharing reagents and advice with us. We thank V. Podust and A. Altman for helpful criticism of the manuscript.

We gratefully acknowledge the financial support of the NIH (RO1 GM 52948 to E.F. and RR00480 to the MSU-NIH Mass Spectrometry Facility), Vanderbilt University, a Boehringer Ingelheim Fonds predoctoral scholarship for C. Rehfuess, and the NSF (Shared Instrumentation Grant BIR-941667). Mutagenesis of the p68 cDNA was supported in part by the DFG (Fa138/6-1 and Na190/6-3) and the European Community (CHRX-CT93-0248 DG 12).