Checkpoint-dependent inhibition of DNA replication initiation by Sld3 and Dbf4 phosphorylation

Nature. 2010 Sep 23;467(7314):474-8. doi: 10.1038/nature09373. Epub 2010 Sep 12.

Abstract

The initiation of eukaryotic DNA replication is regulated by three protein kinase classes: cyclin-dependent kinases (CDK), Dbf4-dependent kinase (DDK) and the DNA damage checkpoint kinases. CDK phosphorylation of two key initiation factors, Sld2 and Sld3, promotes essential interactions with Dpb11 (refs 2-4), whereas DDK acts by phosphorylating subunits of the Mcm2-7 helicase. CDK has an additional role in replication by preventing the re-loading of Mcm2-7 during the S, G2 and M phases, thus preventing origin re-firing and re-replication. During the G1 phase, both CDK and DDK are downregulated, which allows origin licensing and prevents premature replication initiation. Origin firing is also inhibited during the S phase when DNA damage or replication fork stalling activates the checkpoint kinases. Here we show that, analogous to the situation in the G1 phase, the Saccharomyces cerevisiae checkpoint kinase Rad53 inhibits both CDK- and DDK-dependent pathways, which acts redundantly to block further origin firing. Rad53 acts on DDK directly by phosphorylating Dbf4, whereas the CDK pathway is blocked by Rad53-mediated phosphorylation of the downstream CDK substrate, Sld3. This allows CDK to remain active during the S phase in the presence of DNA damage, which is crucial to prevent re-loading of Mcm2-7 onto origins that have already fired. Our results explain how checkpoints regulate origin firing and demonstrate that the slowing of S phase by the 'intra-S checkpoint' is primarily due to the inhibition of origin firing.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Cell Cycle Proteins / antagonists & inhibitors
  • Cell Cycle Proteins / genetics
  • Cell Cycle Proteins / metabolism*
  • Checkpoint Kinase 2
  • Cyclin-Dependent Kinases / metabolism
  • DNA Damage
  • DNA Replication / physiology*
  • DNA-Binding Proteins / antagonists & inhibitors
  • DNA-Binding Proteins / genetics
  • DNA-Binding Proteins / metabolism*
  • Phosphorylation
  • Protein-Serine-Threonine Kinases / genetics
  • Protein-Serine-Threonine Kinases / metabolism
  • Replication Origin / physiology
  • S Phase / physiology*
  • Saccharomyces cerevisiae / cytology*
  • Saccharomyces cerevisiae / genetics
  • Saccharomyces cerevisiae / metabolism*
  • Saccharomyces cerevisiae Proteins / antagonists & inhibitors
  • Saccharomyces cerevisiae Proteins / genetics
  • Saccharomyces cerevisiae Proteins / metabolism*
  • Substrate Specificity

Substances

  • Cell Cycle Proteins
  • DNA-Binding Proteins
  • DPB11 protein, S cerevisiae
  • Dbf4 protein, S cerevisiae
  • Saccharomyces cerevisiae Proteins
  • Sld3 protein, S cerevisiae
  • Checkpoint Kinase 2
  • Protein-Serine-Threonine Kinases
  • Cyclin-Dependent Kinases
  • RAD53 protein, S cerevisiae