In this grant application we describe our plans to continue our in vitro studies on the molecular mechanisms, interactions, and regulation of the components of the DNA replication complex of bacteriophage T4. The T4 system has served as a paradigm for replication studies in higher organisms because, within its seven protein components, it includes a template-directed replication DNA polymerase (gp43), a single-stranded DNA binding protein (gp32), a trimeric sliding clamp (gp45) for controlling the processivity of the polymerases at the replication fork, an ATP-driven clamp-loading subassembly (gp44/62), and a primosome that contains the hexameric T4 replication helicase (gp41) that opens the DNA ahead of the replication fork and the primase (gp61) required for the RNA priming of lagging strand synthesis. During the next reporting period our Specific Aims are: (i) to complete the elucidation of the detailed reaction cycle involved in the ATP-driven loading of the gp45 clamp onto the primer- template DNA and subsequently onto the gp43 polymerase, as well as to determine how the stability of the loaded sliding clamp is controlled at various stages of the replication cycle; (ii) to continue our studies of the mechanisms whereby the T4 helicase (gp41) is able to open double-stranded DNA and expose the template sequences ahead of the moving replication fork (this study has general implications for the function of other hexameric helicases as well); (iii) to understand how the molecular activities and interactions within the primosome subassembly are coordinated with leading- and lagging-strand DNA synthesis; and (iv) to elucidate the molecular coupling mechanisms that synchronize and control the functions of the processive polymerases, primase, and helicase within the replication fork. In terms of their longer range significance for biomedical research, these studies will serve as molecular models for how the analogous DNA replication systems work in higher organisms and how the control of these systems may go awry in cancer and other diseases of inappropriate DNA replication and repair.
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