Multiple phosphorylation sites of DNA polymerase alpha-primase cooperate to regulate the initiation of DNA replication in vitro

J Biol Chem. 2001 Oct 12;276(41):38076-83. doi: 10.1074/jbc.M104975200. Epub 2001 Aug 13.


DNA polymerase alpha-primase (pol-prim) is the only enzyme that can start DNA replication de novo. The 180-kDa (p180) and 68-kDa (p68) subunits of the human four-subunit enzyme are phosphorylated by Cyclin-dependent kinases (Cdks) in a cell cycle-dependent manner. Cyclin A-Cdk2 physically interacts with pol-prim and phosphorylates N-terminal amino acids of the p180 and the p68 subunits, leading to an inhibition of pol-prim in initiating cell-free SV40 DNA replication. Mutation of conserved putative Cdk phosphorylation sites in the N terminus of human p180 and p68 reduced their phosphorylation by Cyclin A-Cdk2 in vitro. In contrast to wild-type pol-prim these mutants were no longer inhibited by Cyclin A-Cdk2 in the initiation of viral DNA replication. Importantly, rather than inhibiting it, Cyclin A-Cdk2 stimulated the initiation activity of pol-prim containing a triple N-terminal alanine mutant of the p180 subunit. Together these results suggest that Cyclin A-Cdk2 executes both stimulatory and inhibitory effects on the activity of pol-prim in initiating DNA replication.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Animals
  • CDC2-CDC28 Kinases*
  • Cells, Cultured
  • Cyclin A / metabolism
  • Cyclin-Dependent Kinase 2
  • Cyclin-Dependent Kinases / metabolism
  • DNA Polymerase I / chemistry
  • DNA Polymerase I / metabolism*
  • DNA Primase / chemistry
  • DNA Primase / metabolism*
  • DNA Replication*
  • Humans
  • Insecta
  • Phosphorylation
  • Precipitin Tests
  • Protein Serine-Threonine Kinases / metabolism


  • Cyclin A
  • Protein Serine-Threonine Kinases
  • CDC2-CDC28 Kinases
  • CDK2 protein, human
  • Cyclin-Dependent Kinase 2
  • Cyclin-Dependent Kinases
  • DNA Primase
  • DNA polymerase alpha-primase
  • DNA Polymerase I