The investigator's long-term goal is to understand how DNA replication is regulated during the eukaryotic cell cycle. A central question about this regulation is how replication origins are prevented from re-initiating DNA replication within a single cell cycle. Strict enforcement of this block at the hundreds to thousands of origins in an eukaryotic genome is required for the faithful propagation and stable transmission of genetic information. Loss of this control could contribute to the genomic instability associated with tumorigenesis. The investigator is studying the block to re-initiation in the budding yeast Saccharomyces cerevisiae, where combined genetic and biochemical analysts have identified many of the molecular components involved in regulating the cell cycle and initiating DNA replication. Work from their lab and that of others has shown that cyclin dependent kinases (CDKs; particularly Clb/Cdc28 in budding yeast) play a pivotal role in preventing re-initiation. In this proposal, he wishes to identify the critical targets of these kinases and determine how phosphorylation of these targets inhibits re-initiation. The investigator's preliminary studies raise the possibility that Clb kinases use multiple overlapping mechanisms to target three components of the initiation complex: the origin recognition complex (ORC); Cdc6; and the Mcm2-7 family of proteins. To test this hypothesis he will: (1) Determine whether simultaneous deregulation of these three initiation components leads to re-initiation of DNA replication; (2) Determine whether ORC and Mcm proteins are substrates of Clb kinases in vivo ; (3) Investigate the molecular mechanisms underlying the block to re-initiation; and (4) Test additional mechanisms that may contribute to the block to re-initiation. Because the components of the initiation machinery are conserved among eukaryotes, he anticipates that an understanding of how this machinery is regulated during the budding yeast cell cycle will serve as a paradigm for understanding this regulation in other eukaryotes.
