Characterization of DNA damage-stimulated self-interaction of Saccharomyces cerevisiae checkpoint protein Rad17p

J Biol Chem. 2001 Jul 13;276(28):26715-23. doi: 10.1074/jbc.M103682200. Epub 2001 May 16.

Abstract

Saccharomyces cerevisiae Rad17p is necessary for cell cycle checkpoint arrests in response to DNA damage. Its known interactions with the checkpoint proteins Mec3p and Ddc1p in a PCNA-like complex indicate a sensor role in damage recognition. In a novel application of the yeast two-hybrid system and by immunoprecipitation, we show here that Rad17p is capable of increased self-interaction following DNA damage introduced by 4-nitroquinoline-N-oxide, camptothecin or partial inactivation of DNA ligase I. Despite overlap of regions required for Rad17p interactions with Rad17p or Mec3p, single amino acid substitutions revealed that Rad17p x Rad17p complex formation is independent of Mec3p. E128K (rad17-1) was found to inhibit Rad17p interaction with Mec3p but not with Rad17p. On the other hand, Phe-121 is essential for Rad17p self-interaction, and its function in checkpoint arrest but not for Mec3p interaction. These differential effects indicate that Rad17p-Rad17p interaction plays a role that is independent of the Rad17p x Mec3p x Ddc1p complex, although our results are also compatible with Rad17p-mediated supercomplex formation of the Rad17p x Mec3p x Ddc1p heterotrimer in response to DNA damage.

Publication types

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

MeSH terms

  • Amino Acid Sequence
  • Cell Cycle Proteins / genetics*
  • DNA Damage*
  • DNA, Fungal / genetics
  • DNA-Binding Proteins
  • Gene Expression Regulation, Fungal
  • Molecular Sequence Data
  • Nuclear Proteins
  • Saccharomyces cerevisiae
  • Saccharomyces cerevisiae Proteins
  • Sequence Alignment

Substances

  • Cell Cycle Proteins
  • DNA, Fungal
  • DNA-Binding Proteins
  • Nuclear Proteins
  • RAD17 protein, S cerevisiae
  • Saccharomyces cerevisiae Proteins