Control of chromosomal replication is crucial for the growth and development of mammalian cells. During each cell cycle, a dividing cell accurately duplicates its entire complement chromosomes, composed of several billion basepairs. Perturbations of this fundamental process lead to abnormal growth and are associated with various disease states. The long-term objective of this work is to increase our understanding of the molecular mechanisms of chromosomal DNA replication. Because the process of chromosomal replication is complex, simpler viral model systems such as simian virus 40 (SV40) are essential for identifying and purifying replication proteins. By dissecting such systems and studying the properties of individual proteins in detail, it is possible to understand how these proteins function in concert to replicate chromosomal DNA. This proposal focuses on a novel multi-subunit, human, single-stranded DNA- binding protein, Replication Protein-A (RP-A). This protein is absolutely required for SV40 DNA replication and is probably involved in chromosomal replication. The principal goal of these studies is to elucidate the physiological role of RP-A during DNA replication. A SV40-based cell-free replication system will be used as a functional assay. There will be three major experimental components of this research: (1) Both biochemical properties and functional activities, such as single- stranded DNA-binding activity, will be studied to determine how they influence the role of RP-A in DNA replication. (2) The affect of RP-A on the enzymatic activities of other replication proteins will be determined Specific interactions will be analyzed for in immunoprecipitation and cross-linking studies. The mechanistic consequences of such interactions will be examined. (3) Protein affinity chromatography will be used to isolate novel replication proteins. The functional significance of such proteins will be analyzed in the SV40 cell-free system. Previously unidentified proteins will be purified and characterized. In the future, these studies will be expanded to examine the roles of other DNA replication proteins. One future direction will be to establish non- viral model reactions for chromosomal DNA replication.
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