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.