In this proposal we outline a detailed physico-chemical study of the structure and interactions of the various protein and nucleic acid components of the bacteriophage T4 DNA replication complex. Building on the results of Alberts and Nossal and their coworkers, who have defined this in vitro system, as well as on our own earlier studies, we will use chemical and photochemical cross-linking experiments to determine structural and topological relationships and to localize protein-protein and protein-nucleic acid contacts. Studies of binding equilibria between various components, and the dependence of these binding constants on salt concentration, pH and other environmental variables will be carried out to determine the nature and the free energy contributions to complex formation and stability of various types of functional group interactions. Rates of DNA synthesis and replication fork movement, as well as the processivity and fidelity of the synthesis catalyzed by various partial replication systems will be examined in order to attempt to understand, on a molecular basis, the role each protein plays in the coupled (leading and lagging strand) DNA elongation phase of the integrated replication process, and the protein-protein and protein-nucleic acid interaction mechanisms involved in the partial reactions of the overall system.
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