Considerable progress has been made in elucidating the mechanism of DNA replication in prokaryotes and eukaryotes. These studies have emphasized its complexity and fundamental similarities. Even with this progress, we are far from understanding its intricacies and we have only recently begun to appreciate its regulation and relationship to DNA repair and cell growth. DNA replication has been the target of anticancer therapies for years with little appreciation of how these agents work. Thus, if we are to understand aberrant growth, we must have fundamental knowledge of how DNA replication occurs. During the previous grant period, we have studied the biochemical mechanism by which simian virus (SV) 40 DNA is replicated. We have reconstituted the replication pathway with purified proteins. We have investigated how SV4O T antigen, the only viral protein required, activates the SV4O origin, and through its intrinsic DNA helicase participates in the replication reaction. The identification, isolation and mechanism of action of the other proteins, supplied by the host, have yielded important information about their role in chromosomal replication. During the tenure of this grant, we will investigate the mode of action of a number of the host proteins involved in SV4O DNA replication. These include l) the mechanism of assembly of the accessory proteins needed to confer high processivity onto the catalytic polymerases. The clamp loader, activator 1 (A1, also called RFC), contains five different subunits all of which have been cloned. We will investigate the role of each subunit in the loading of the processivity factor proliferating cell nuclear antigen (PCNA) onto DNA which acts as a sliding clamp tethering the polymerase to DNA. We will investigate the mechanism by which A1 tracks along DNA until it finds primer ends. 2) The role of PCNA will be examined with particular emphasis on the influence of various growth regulators (p21, Gadd45) which interact with PCNA and influence its action. 3) The synthesis of the trimeric DNA binding protein, called human single stranded binding protein (or RPA) will be investigated. HSSB is the only binding protein that supports SV40 replication. All of the subunits have been cloned and expressed. We will examine its synthesis and the role of each subunit in the SV4O pathway. 3) We plan to investigate the replication of Schizosaccharomyces pombe DNA. The availability of an origin sequence that acts as an autonomous replication sequence (ARS) in plasmids, coupled with the identification of the origin replication complex (ORC) which interacts with the origin of Saccharomyces cerevisiae has focused our attention to this system. We plan to isolate the ORC polypeptides from S. pombe and to further characterize two distinct protein isolated from S. pombe that interact with the S. pombe conserved core sequence.
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