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

Christian Voitenleitner, Christoph Rehfuess, Melissa Hilmes, Lynda O'Rear, Pao Chi Liao, Douglas A. Gage, Robert Ott, Heinz Peter Nasheuer, Ellen Fanning

Research output: Contribution to journalArticlepeer-review

70 Citations (Scopus)


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.

Original languageEnglish
Pages (from-to)646-656
Number of pages11
JournalMolecular and Cellular Biology
Issue number1
Publication statusPublished - 1999 Jan

All Science Journal Classification (ASJC) codes

  • Molecular Biology
  • Cell Biology


Dive into the research topics of 'Cell cycle-dependent regulation of human DNA polymerase α-primase activity by phosphorylation'. Together they form a unique fingerprint.

Cite this