The comprehension of the initiation of chromosomal DNA replication in eukaryotes depends upon the identification of key regulatory molecules. This knowledge is important for understanding the control o( cellular proliferation tn both somatic and term line tissues. Genetic, molecular and physiological experiments have indicated that the CDC7 protein of Saccharomyces cerevisiae is a putative protein kinase which represents a regulatory molecule. lt is hypothesized that different target molecules are phosphorylated at a specific time in the cell cycle to accomplish the regulation. Direct biochemical evidence for the hypothesis will be obtained by overexpression of the protein in yeast or bacterial cells, protein purification and by the use of CDC7- specific polyclonal antibodies which we have produced. Antibodies will also be used to determine the subcellular location of the CDC7 protein. Both protein kinase assays and localization studies will be performed on cells at different stages of the cell cycle. Identification of the target molecules in vitro will rely upon a CDC7 protein kinase assay and antibodies. A combined biochemical (PK assay, protein purification and reverse genetics) and genetic (cdc7 extragenic suppressors, other DNA initiation mutants) approach will be used to identify the target protein(s) of CDC7 phosphorylations in the cell. The long term objective is to delineate the molecular mechanisms of this regulatory process.
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