The T4 replication system is a model for DNA replication in general. The replication complex (replisome) is derived from eight proteins that constitute the holoenzyme and the primosome subassemblies which along with single strand binding protein acts at a DNA replication fork. The proposal describes a number of experiments to understand in molecular terms the dynamic and structural characteristics of an active replisome. Key objectives include: defining the composition, dynamics of assembly and disassembly of the primosome: and reconstituting the replisome at a replication fork in order to elucidate the kinetics of its movement with respect to both DNA strands as well as the protein-protein interactions within this multiprotein complex. The techniques to be used either independently or in collaboration span a variety of kinetic methods from rapid quench and stopped-flow fluorescent energy transfer to single molecule imaging as well as structural techniques from cross-linking to electron microscopy and X-ray crystallography. These studies will be extended to include DNA substrates containing lesions in order to gain insights into molecular events surrounding an active replisome attempting lesion bypass, an issue of considerable disease relevance.
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