DNA REPLICATION INITIATION--PROTEIN VISUALIZATION

California State Poly U Pomona, Pomona CA

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Abstract

Our objective is to understand the sequence of molecular events that regulate the initiation of DNA synthesis. Regulation of DNA synthesis is essential for maintaining genomic and cellular integrity. When the process malfunctions, DNA fidelity is not maintained and a cell can divide without controls to stop unwanted cell proliferation. Improper cell proliferation and DNA mutations can lead to the formation and progression of tumors. The budding yeast, Saccharomyces cerevisiae, is a useful model organism to study the initiation of DNA synthesis. It is a simple eukaryote amenable to genetic and molecular biology manipulations. In budding yeast, a serine/threonine kinase, Cdc7, interacts with proteins that bind to regions in DNA required for initiation, called origins, and through these interactions, Cdc7 is thought to trigger DNA replication. Cdc7 kinase activity varies during the cell cycle with a peak in activity during the G1 phase and early S phase of the cell cycle when DNA synthesis begins. Cdc7 interacts with another protein, Dbf4. Our hypothesis that Dbf4 regulates Cdc7 kinase activity through direct interaction with Cdc7 only during the G1-S transition and that changes in the protein stability and expression of Dbf4 during the cell cycle modulate the kinase activity of Cdc7. Additional regulation may be imparted by the subcellular localization of Cdc7 and Dbf4, which must coalesce at the origins during the G1-S transition to instigate DNA synthesis. Recent development of the green fluorescent protein (GFP) has made possible the direct visualization of proteins involved in dynamic cell processes. Our specific aims are: 1) the production and characterization of fluorescent tagged fusion proteins, cGFP-Cdc7 and vGFP-Dbf4; 2) determination of the protein expression and subcellular localization of Cdc7 and Dbf4 during the cell cycle; 3) characterization of Cdc/Dbf4 complex formation at different stages of the cell cycle; and 4) examination of the dynamics of Cdc7 and Dbf4 interactions with origins of DNA replication. These studies will reveal the molecular events, dynamic processes and precise timing required for the initiation of DNA synthesis.