GL CYCLINS AND THE REGULATION OF DNA REPLICATION

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The regulation of cell division and growth control is critical for all organisms. Cyclin-dependent kinases (CDKs) are essential regulators of cell cycle division in all eukaryotic organisms. Many growth-stimulatory and inhibitory signals affect the activity of CDKs; furthermore, mutations which deregulate expression of CDKs or affect the function of CDK inhibitors are associated with alterations in growth control and cancers. The CDKs are known to be protein kinases, but few substrates of CDKs have been identified. This proposal utilizes a combination of genetic, molecular and biochemical approaches to identify and characterize substrates and regulators of Cdc28, the CDK in Saccharomyces cerevisiae, focusing on those required for the regulation and fidelity of DNA synthesis. DNA damage in G1 causes a "checkpoint" response; the cell delays transit through the cell cycle until damage is repaired. The role of the Cdc28 kinase in this response will be determined by examining the effects of both overexpression and deletion of the G1 cyclins (CLNs). Regulators of the CLNs and genes required downstream of CLN function for promotion of DNA synthesis after START will be identified and characterized, utilizing three different approaches. The FIRST approach follows from the observation that a mutation in a checkpoint gene, mec1-1, and GAL-CLN1 are synthetically lethal. This suggests that high, constitutive levels of CLN1 may override a G1 checkpoint for DNA damage, or that GAL-CLN1 may promote partially defective DNA synthesis. Suppressors of this synthetic lethality will be selected and characterized genetically, molecularly and biochemically. The SECOND approach continues the analysis of ERC11, mutations in which affect DNA synthesis and repair. The defects in DNA synthesis are suppressed by the expression of CLN1 or CLN2, but not by CLN3, demonstrating that CLN1/CLN2 promote transit through the cell cycle differently than CLN3. To characterize this ability of CLN1/CLN2, we will characterize two suppressors of erc11, SEL1 and CDC9, which may encode components of a CLN1/CLN2-activated pathway for DNA replication. FINALLY, new cln2 mutant alleles unable to promote DNA synthesis in erc11-2 mutants will be isolated. These mutant cln2 alleles will be characterized biochemically and genetically to determine whether they affect a subset of cln2's functions.