Because cancer cells display abnormal chromosome numbers and high mutation rates, knowledge of the mechanism of chromosomal DNA replication and repair can be used in clinical diagnosis, prognosis and in the design of therapies. This proposal continues an ongoing study of the regulation of the initiation of eukaryotic DNA replication by protein phosphorylation using budding yeast cells as a model system. The roles of both Cdc7/Dbf4 and Cdc28/Clb protein kinases as important regulators of DNA replication are the main focus of this study. Both protein kinases are hypothesized to phosphorylate the Mcm-complex at origins of DNA replication. In this way, the kinases may remodel chromatin structure at origins in G1 phase of the cell cycle to permit access to origins by the DNA replication machinery. Because all these proteins are conserved in evolution from yeast to humans, this regulatory mechanism may be universal. The major hypothesis in the first specific aim is that these two kinases are involved in removing the inhibition to DNA replication by phosphorylation of the Mcm-complex. A combined genetic and biochemical approaches will be used to test the hypotheses in each Specific Aim. Mutations will be isolated that remove the inhibition thereby allowing for bypass of the phosphorylation step. Sites of phosphorylation in the Mcm substrates will be identified by electrospray mass spectrometry. Another aim will focus on the regulation of this kinase during the cell cycle by phosphorylation by other kinases and the modulation of Dbf4 protein levels. Other kinases will be identified by protein purification. Dbf4 protein synthesis and degradation will be analyzed using a tagged Dbf4 protein. The Rad53 checkpoint protein kinase as a possible regulatory of Cdc7/Dbf4 kinase will also be studied. The next two aims are focused on the role of Cdc7/Dbf4 kinase in meiosis and in error-probe DNA repair (induced- mutagenesis), which are two processes that have not been well-studied. The hypothesis is that upstream regulatory events such as the binding of Dbf4 are also used in these two cellular processes, but downstream events such as substrate phosphorylation are different. Because Cdc7/Dbf4 kinase is not used for meiotic replication, meiotic replication may be regulated differently even though it uses similar replication proteins and origins.
The final aim will examine the role of the Pakl protein kinase in DNA replication. Similar genetic and biochemical method will be employed in these latter aims as well.
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