DDK phosphorylates checkpoint clamp component Rad9 and promotes its release from damaged chromatin

Mol Cell. 2010 Nov 24;40(4):606-18. doi: 10.1016/j.molcel.2010.10.026.

Abstract

When inappropriate DNA structures arise, they are sensed by DNA structure-dependent checkpoint pathways and subsequently repaired. Recruitment of checkpoint proteins to such structures precedes recruitment of proteins involved in DNA metabolism. Thus, checkpoints can regulate DNA metabolism. We show that fission yeast Rad9, a 9-1-1 heterotrimeric checkpoint-clamp component, is phosphorylated by Hsk1(Cdc7), the Schizosaccharomyces pombe Dbf4-dependent kinase (DDK) homolog, in response to replication-induced DNA damage. Phosphorylation of Rad9 disrupts its interaction with replication protein A (RPA) and is dependent on 9-1-1 chromatin loading, the Rad9-associated protein Rad4/Cut5(TopBP1), and prior phosphorylation by Rad3(ATR). rad9 mutants defective in DDK phosphorylation show wild-type checkpoint responses but abnormal DNA repair protein foci and decreased viability after replication stress. We propose that Rad9 phosphorylation by DDK releases Rad9 from DNA damage sites to facilitate DNA repair.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Amino Acid Sequence
  • Camptothecin / pharmacology
  • Cell Cycle Proteins / chemistry
  • Cell Cycle Proteins / metabolism*
  • Cell Cycle* / drug effects
  • Chromatin / drug effects
  • Chromatin / metabolism*
  • DNA Damage*
  • DNA Repair / drug effects
  • DNA Replication / drug effects
  • Enzyme Activation / drug effects
  • Kinetics
  • Models, Biological
  • Molecular Sequence Data
  • Mutant Proteins / chemistry
  • Mutant Proteins / metabolism
  • Phosphorylation / drug effects
  • Phosphoserine / metabolism
  • Phosphothreonine / metabolism
  • Protein Binding / drug effects
  • Protein Transport / drug effects
  • Protein-Serine-Threonine Kinases / metabolism*
  • Replication Protein A / metabolism
  • Schizosaccharomyces / cytology*
  • Schizosaccharomyces / drug effects
  • Schizosaccharomyces / enzymology*
  • Schizosaccharomyces pombe Proteins / metabolism*
  • Solubility / drug effects
  • Subcellular Fractions / drug effects
  • Subcellular Fractions / metabolism
  • Time Factors

Substances

  • Cell Cycle Proteins
  • Chromatin
  • Mutant Proteins
  • Replication Protein A
  • Schizosaccharomyces pombe Proteins
  • Phosphothreonine
  • rad9 protein
  • Phosphoserine
  • DDK protein, S pombe
  • Protein-Serine-Threonine Kinases
  • Camptothecin