Yeast Rad17/Mec3/Ddc1: a sliding clamp for the DNA damage checkpoint

Proc Natl Acad Sci U S A. 2003 Mar 4;100(5):2249-54. doi: 10.1073/pnas.0437148100. Epub 2003 Feb 25.

Abstract

The Saccharomyces cerevisiae Rad24 and Rad17 checkpoint proteins are part of an early response to DNA damage in a signal transduction pathway leading to cell cycle arrest. Rad24 interacts with the four small subunits of replication factor C (RFC) to form the RFC-Rad24 complex. Rad17 forms a complex with Mec3 and Ddc1 (Rad1731) and shows structural similarities with the replication clamp PCNA. This parallelism with a clamp-clamp loader system that functions in DNA replication has led to the hypothesis that a similar clamp-clamp loader relationship exists for the DNA damage response system. We have purified the putative checkpoint clamp loader RFC-Rad24 and the putative clamp Rad1731 from a yeast overexpression system. Here, we provide experimental evidence that, indeed, the RFC-Rad24 clamp loader loads the Rad1731 clamp around partial duplex DNA in an ATP-dependent process. Furthermore, upon ATP hydrolysis, the Rad1731 clamp is released from the clamp loader and can slide across more than 1 kb of duplex DNA, a process which may be well suited for a search for damage. Rad1731 showed no detectable exonuclease activity.

Publication types

  • Research Support, U.S. Gov't, P.H.S.

MeSH terms

  • Adenosine Triphosphatases / metabolism
  • Adenosine Triphosphate / metabolism
  • Cell Cycle Proteins / metabolism*
  • Chromatography, Gel
  • DNA / metabolism
  • DNA Damage*
  • DNA-Binding Proteins / metabolism
  • Electrophoresis, Polyacrylamide Gel
  • Hydrolysis
  • Nuclear Proteins
  • Phosphoproteins / metabolism*
  • Plasmids / metabolism
  • Protein Binding
  • Replication Protein C
  • Saccharomyces cerevisiae / metabolism*
  • Saccharomyces cerevisiae Proteins / metabolism*
  • Surface Plasmon Resonance
  • Time Factors

Substances

  • Cell Cycle Proteins
  • DNA-Binding Proteins
  • Ddc1 protein, S cerevisiae
  • MEC3 protein, S cerevisiae
  • Nuclear Proteins
  • Phosphoproteins
  • RAD17 protein, S cerevisiae
  • Saccharomyces cerevisiae Proteins
  • Adenosine Triphosphate
  • DNA
  • Adenosine Triphosphatases
  • Replication Protein C