CELLULAR PROLIFERATION AND THE CDC7 PROTEIN KINASE

点击翻译按钮获取中文摘要

A central problem in cell biology is how the transitions between the various phases of the cell division cycle are controlled, ensuring orderly duplication and segregation of crucial cellular components. Results obtained using yeast genetics and animal cell biology and biochemistry have recently led to significant breakthroughs in our understanding of the mechanisms that regulate and promote entry into mitosis. By comparison, less is known about control of the G1/S transition and the initiation of DNA replication. In Saccharomyces cerevisiae, it appears that the same protein kinase is involved in regulating both the G1/S and the G2/M transitions, the cdc2/CDC28/MPF kinase. Our studies have been aimed at defining the events set in motion by activation of the Cdc28 protein in G1 in yeast. The overall gaol of our studies is to understand the role of the Cdc7 protein, also a protein kinase, in entry into S phase. The CDC7 gene functions late in G1. Our preliminary results suggest that Cdc7p is involved in a potential phosphorylation cascade during G1 that leads to initiation of DNA replication. We have found the Cdc28 immune complexes can phosphorylate Cdc7p in vitro and that Cdc7 immune complexes can phosphorylate the replication protein, RP-A, in vitro. If verified, these studies suggest that Cdc7 serves as a molecular link between the regulatory apparatus active in commitment to DNA synthesis and the catalytic apparatus involved in initiating DNA synthesis. We will further characterize the in vitro interactions of these proteins and try to show they reflect the cell cycle in vivo. First, we have found that Cdc7 protein purified from bacteria is inactive as a kinase. We will investigate the role of posttranslational modifications, including phosphorylation by Cdc28, and interactions with other yeast proteins in activating the Cdc7 kinase. The kinase activity of Cdc28 protein is regulated throughout the cell cycle by both covalent modifications and association with other proteins, including the cyclins, and Cdc7 may undergo similar modifications. Second, we will determine the amino acids in Cdc7 that are phosphorylated in vivo and in vitro. We will test their effect on the functions of Cdc7 by altering them by site directed mutagenesis and analysis of their ability to complement cdc7 mutants. Third, we will try to verify that RP-A is a substrate of Cdc7p in vivo and investigate other potential substrates.