title: Yeast Rad17/Mec3/Ddc1: a sliding clamp for the DNA damage checkpoint.
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.
authors.affiliation: Department of Biochemistry and Molecular Biophysics, Washington University School of Medicine, St. Louis, MO 63110, USA.
authors.forename: Jerzy
authors.initials: J
authors.lastname: Majka
authors.forename: Peter M J
authors.initials: PM
authors.lastname: Burgers
mesh_terms: D000251:Adenosine Triphosphatases / Q000378:metabolism; D000255:Adenosine Triphosphate / Q000378:metabolism; D018797:Cell Cycle Proteins / Q000378:metabolism*; D002850:Chromatography, Gel; D004247:DNA / Q000378:metabolism; D004249:DNA Damage; D004268:DNA-Binding Proteins / Q000378:metabolism; D004591:Electrophoresis, Polyacrylamide Gel; D006868:Hydrolysis; D009687:Nuclear Proteins; D010750:Phosphoproteins / Q000378:metabolism*; D010957:Plasmids / Q000378:metabolism; D011485:Protein Binding; D051818:Replication Protein C; D012441:Saccharomyces cerevisiae / Q000378:metabolism*; D029701:Saccharomyces cerevisiae Proteins / Q000378:metabolism*; D020349:Surface Plasmon Resonance; D013997:Time Factors
chemical_list: D018797:Cell Cycle Proteins; D004268:DNA-Binding Proteins; C440013:Ddc1 protein, S cerevisiae; C099938:MEC3 protein, S cerevisiae; D009687:Nuclear Proteins; D010750:Phosphoproteins; C501395:RAD17 protein, S cerevisiae; D029701:Saccharomyces cerevisiae Proteins; D000255:Adenosine Triphosphate; D004247:DNA; D000251:Adenosine Triphosphatases; D051818:Replication Protein C