title: Characterization of DNA damage-stimulated self-interaction of Saccharomyces cerevisiae checkpoint protein Rad17p.
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.
authors.affiliation: Department of Radiation Oncology and the Winship Cancer Institute, Emory University School of Medicine, Atlanta, Georgia 30322, USA.
authors.forename: H
authors.initials: H
authors.lastname: Zhang
authors.forename: Z
authors.initials: Z
authors.lastname: Zhu
authors.forename: G
authors.initials: G
authors.lastname: Vidanes
authors.forename: D
authors.initials: D
authors.lastname: Mbangkollo
authors.forename: Y
authors.initials: Y
authors.lastname: Liu
authors.forename: W
authors.initials: W
authors.lastname: Siede
mesh_terms: D000595:Amino Acid Sequence; D018797:Cell Cycle Proteins / Q000235:genetics*; D004249:DNA Damage; D004271:DNA, Fungal / Q000235:genetics; D004268:DNA-Binding Proteins; D015966:Gene Expression Regulation, Fungal; D008969:Molecular Sequence Data; D009687:Nuclear Proteins; D012441:Saccharomyces cerevisiae; D029701:Saccharomyces cerevisiae Proteins; D016415:Sequence Alignment
chemical_list: D018797:Cell Cycle Proteins; D004271:DNA, Fungal; D004268:DNA-Binding Proteins; D009687:Nuclear Proteins; C501395:RAD17 protein, S cerevisiae; D029701:Saccharomyces cerevisiae Proteins