The focus of this proposal is to understand in molecular terms the structural and dynamic characteristics of the DNA replication process carried out by the T4 bacteriophage. The T4 replication system is a multiprotein complex comprised of eight proteins that constitute the holoenzyme, the primosome and single strand binding units which act at a DNA replication fork. This proposal describes a series of experiments to define the protein-protein and protein-DNA contacts within and between the above three replicative complex components as well as the dynamics of their assembly, rate of travel, and disassembly as individual and coupled units at in vitro replication forks. A key objective is to reconstitute leading and lagging strand synthesis under conditions of minimal turnover in order to avoid the kinetic complexities introduced by multiple cycles of synthesis. The main objectives and techniques to be used involve: measurement of the opening/closing of the holoenzyme clamp protein (gp 45) by the clamp loader protein complex (gp 44/62) through time-dependent fluorescent energy transfer; detection of specific protein-protein contacts within the holoenzyme (gp 45, gp 44/62, polymerase) and the primosome (the helicase, gp 41; the primase, gp 61) as well as between the two units by photoactivatible crosslinkers; determination of the rate of primosome travel during leading strand synthesis at a replicative fork by a combination of rapid quench and fluorescence stopped-flow methods; and elucidation of the composition and stoichiometry of the replication complex by protein mutagenesis, two hybrid system analysis, immunoprecipitation and surface plasmon resonance. Novel DNA substrates will be generated for protein-DNA crosslinking and monitoring the position and travel of various assemblies of the units of the replicative complex. These studies should greatly deepen our knowledge of how the T4 replicative system functions and serves as a paradigm for DNA replication in general.
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