GENETIC AND MOLECULAR ANALYSIS OF YEAST DNA REPLICATION

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Knowledge of the regulation of cellular proliferation is important for understanding the molecular mechanisms of diseases in which the regulation is perturbed. This knowledge will be useful in both early diagnosis and treatment. Most likely, cancer is the result of an unregulated or poorly regulated cell cycle that leads to uncontrolled proliferation. Furthermore, carcinogenesis is a multistep process that involves an accumulation of mutations in a number of oncogenes and anti-oncogenes. The Cdc7 protein of Saccharomyces cerevisiae is a ser/thr protein kinase that regulates both cellular proliferation and mutagenesis and therefore, will be used as a model system for investigating these two important processes. Cdc7 catalyzed-phosphorylations may activate replication proteins and enzymes or may alter chromatin structure to permit access of these proteins to the DNA. The mechanism of Cdc7 kinase regulation during the cell cycle is the major focus of this study. Two possible mechanisms, phosphorylation by another kinase and interaction with other proteins, most notably, the Dbf4 protein, will be studied. Genetic experiments will correlate changes in putative Cdc7 phosphorylation sites with Cdc7 functions. Biochemical experiments will test if Cdc7-phosphorylation is activating in vitro and will correlate the phosphorylation of specific residues of Cdc7 with kinase activity throughout the cell cycle. Cdc7- substrates and regulators will be identified by analysis of the BOB1 and DBF4 gene products, respectively. The role and regulation of the Dbf4 protein will be investigated in detail because we have shown it to be an important regulator of Cdc7 activity. Both genetic techniques, including the "two hybrid" system to show direct interactions, and biochemical techniques, including "tagged" proteins to facilitate protein purification and the production of specific antibodies as reagents, will be utilized. The long term objective of this combined biochemical and genetic approach is to delineate the regulatory mechanisms of DNA replication and replication-associated fidelity.