Regulation of the replication initiator protein p65(cdc18) by CDK phosphorylation

Prasad V. Jallepalli, Grant W. Brown, Marco Muzi-Falconi, Deborah Tien, Thomas J. Kelly

Research output: Contribution to journalArticlepeer-review

Abstract

Cyclin-dependent kinases (CDKs) promote the initiation of DNA replication and prevent reinitiation before mitosis, presumably through phosphorylation of key substrates at origins of replication. In fission yeast, the p65(cdc18) protein is required to initiate DNA replication and interacts with the origin recognition complex (ORC) and the p34(cdc2) CDK. Here we report that p65(cdc18) becomes highly phosphorylated as cells undergo the G1 → S phase transition. This modification is dependent on p34(cdc18) protein kinase activity, as well as six consensus CDK phosphorylation sites within the p65(cdc18) polypeptide. Genetic interactions between cdc18+ and the S-phase cyclin cig2+ suggest that CDK-dependent phosphorylation antagonizes cdc18+ function in vivo. Using site-directed mutagenesis, we show that phosphorylation at CDK consensus sites directly targets p65(cdc18) for rapid degradation and inhibits its replication activity, as strong expression of a constitutively hypophosphorylated mutant form of p65(cdc18) results in large amounts of DNA over-replication in vivo. Furthermore, the over-replication phenotype produced by this mutant p65(cdc18) is resistant to increased mitotic cyclin/CDK activity, a known inhibitor of over-replication. Therefore, p65(cdc18) is the first example of a cellular initiation factor directly regulated in vivo by CDK-dependent phosphorylation and proteolysis. Regulation of p65(cdc18) by CDK phosphorylation is likely to contribute to the CDK-driven 'replication switch' that restricts initiation at eukaryotic origins to once per cell cycle.

Original languageEnglish (US)
Pages (from-to)2767-2779
Number of pages13
JournalGenes and Development
Volume11
Issue number21
DOIs
StatePublished - Nov 1 1997

Keywords

  • Cell cycle
  • Cyclin-dependent kinase
  • Fission yeast
  • Over- replication
  • S phase

ASJC Scopus subject areas

  • Genetics
  • Developmental Biology

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