The long-term objective of this proposal is to understand how a replisome responsible for DNA replication is assembled from its component proteins and how it functions in the coordination of leading and lagging strand DNA synthesis. The system being examined to gain such insights is the T4 phage replisome derived from 8 proteins including a polymerase, clamp loader complex, clamp, helicase, primase, helicase loader, and single-stranded DNA binding protein. These proteins can be grouped into subassemblies: the holoenzyme and the primosome reconstituted from the polymerase and clamp proteins, and from the helicase and primase proteins, respectively. We are specifically interested in how these subassemblies are formed, their composition and structure, the identity of their protein-protein contacts, their interactions with single-stranded DNA binding protein, their location relative to their DNA templates, their dynamic properties with respect to dissociation from the replisome and their movement at the replication fork. Answers to these complex questions will be sought with a wide assortment of techniques varying from crystallography and electron microscopy to single molecule and ensemble kinetics. The generality of the findings will be tested by extension of similar experiments to the yeast (S. cerevisae) replisome. Building on this understanding of normal replisome function, the proposed studies then will be expanded to investigate lesion bypass, and how a replisome copes with the problem of a damaged template base in creating a complementary strand. DNA replication is at the heart of a cell's ability to clonally expand; a deepened understanding of this fundamental process is essential for interpret ting the effects of changes in the fidelity and efficiency of replication in a variety of disease states, from viral infection to cancer and for the selection of specific replisomal proteins as potential therapeutic targets. ? ? ?
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