Though considerable progress has been made in elucidating the broad outline of how eukaryotic replication occurs, our knowledge of the details of this complicated macromolecular event is far from clear. Many of the enzymes that have been shown to be critical for DNA replication have been found to play roles in DNA repair and reconstruction. Both DNA repair and replication are important targets of many anticancer agents but we have little information about their mode of action. If we are to prevent aberrant growth, we must understand the critical signals governing DNA replication and its cell cycle regulation. Our goal is to study the function of key enzymatic components of the eukaryotic replication machinery. In particular, we have cloned and expressed replication factor C (RFC), a key accessory five subunit complex required to load proliferating cell nuclear antigen (PCNA) onto DNA which is required for processive DNA polymerase activities. We plan to a) determine the mechanism by which RFC opens the PCNA ring and topologically links PCNA to DNA; b) examine the role of RFC in its displacement of the pol alpha-primase complex from primed DNA templates (polymerase switching); c) study the influence of the interaction between Rad17 with RFC on the enzymatic properties of RFC. Rad17 is a component of the checkpoint regulation pathway; d) examine the mechanism of action of DNA polymerase delta from Schizosaccharomyces pombe which contains at least four distinct subunits, and has been found to be a dimeric polymerase. We have cloned and expressed all four subunits and plan to study and compare the properties of the holoenzyme and various subcomplexes. e) We have isolated the S. pombe origin recognition complex (ORC) and plan to clone and express all six subunits in order to carry out experiments on its interactions with origin sequences and with other key proteins that are involved in the formation of complexes critical for the initiation of DNA synthesis.
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