The eukaryotic cell cycle is a cascade of highly complex processes that must occur with striking temporal and spatial precision. There exists regulatory networks capable of recognizing and correcting mistakes occurring during these processes. Cells respond to DNA damage and replication blocks in two ways: They arrest the cell cycle to allow time for repair and they induce the transcription of genes facilitating repair. The biochemical pathways ensuring this coordination are called checkpoints. The infrastructure of these checkpoint circuits will be probed in S. cerevisiae using the MEC, RAD, DPB, DRC, and DUN genes. DPB11 and DRC1 work together in the DNA polymerase epsilon pathway to control DNA synthesis and the S phase checkpoint. We will probe the relationship between the DNA replication and checkpoint functions of these genes to determine how it is sensing replication problems. We will determine what other proteins cooperate with these proteins to transmit cell cycle arrest signals using genetic and biochemical analysis. Cell cycle arrest when DNA replication is blocked depends upon the RAD53 protein kinase and 2 novel downstream genes ESH1 and ESH2. We propose to determine the mechanism of activation of Rad53 and to generate constitutively active mutants to identify downstream function. We will perform genetic and biochemical analyses of ESH1 and ESH2 to try to determine their role in preventing spindle elongation while replication is blocked. We will search for more essential genes in this checkpoint. The Dun1 and Chk1 kinase work together to prevent anaphase entry in response to DNA damage. Dun1 is thought to function by inhibiting the APC and Chk1 is thought to function by phosphorylating Pds1, an anaphase inhibitor. We propose to determine their roles in this checkpoint both biochemical and genetic analyses. We are interested in the target of Dun1 in the APC and the significance of Pds1 phosphorylation. Transcriptional activation of DNA damage inducible genes is a central response to DNA damage. We will determine the entire repertoire of damage-inducible genes in yeast using microarray analysis and the roles of checkpoint genes in the regulation of these transcripts to determine if there are layers of regulation. The proposed studies will contribute to our understanding of the mechanism of cell cycle control that is essential to the maintenance of genomic stability which prevents the accumulation of mutations leading to cancer.
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