This proposal addresses the mechanism by which cellular factors initiate DNA replication in yeast. One of the few cellular factors known to be required for this process is a multi-subunit protein complex called Replication Protein A (RPA). RPA is conserved throughout eukaryotic species, including the yeast S. cerevisiae. Biochemically, the remaining initiation factors will be isolated from yeast cells using an "origin unwinding" assay that mimics an early step in the initiation reaction. This assay is dependent upon RPA and a yeast origin of DNA replication but is relatively simple because it separates initiation from the elongation phase of DNA replication. Protein-affinity chromatography, using RPA as a specific ligand, will be used to enrich for rare proteins that interact with RPA at initiation. These factors will be purified to homogeneity and used to raise specific antisera. The purified proteins will be characterized to identify DNA-binding and DNA helicase activities. A crucial prediction about these initiation factors is that their activity will be regulated throughout the cell-cycle. The antisera will be used to study cell-cycle- dependent modifications to the protein and changes in their activity. The ultimate goal of this approach is to reconstitute complete DNA replication in-vitro and study its regulation. %%% It is known that DNA replication is necessary for a cell to divide, yet little is known about how the process actually begins. This study intends to identify the factors that initiate DNA replication using the well-characterized and highly conserved yeast system. Protein factors that initiate DNA replication will be purified using a DNA sequence that initiates replication in yeast. These proteins will be studied in order to understand how their activity is controlled. Since yeast cells use many of same proteins that human cells use to replicate their DNA, it is likely that understanding this simple model system will shed light on how DNA replication is controlled in human cells.
