EXCEED THE SPACE PROVIDED. The T4 replication s5 stem is a model for DNA replication in general. The replication complex treplisome) 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 cornpositmn, 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 nmltiprotein 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 alq active replisome attempting lesion bypass, an issue of considerable disease relevance. PERFORMANCE SITE ========================================Section End===========================================
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