We are investigating the structures and catalytic mechanisms of the enzymes from bacteriophage T7 that catalyze DNA replication. The experiments described in this application will focus on the interactions of these replication proteins with one another and with DNA in the context of a replication fork. The T7 replisome is a model system for understanding the dynamic commerce of DNA replication-the unwinding of two DNA strands, the continuous synthesis of DNA on the leading strand of the replication fork, the priming of Okazaki fragments and discontinuous synthesis of DNA on the lagging strand. These features are universal for all DNA-based life forms, and they are most easily examined at the molecular level in a simple model replication system like that encoded by phage T7. In spite of the cartoons of replication forks that are shown in college textbooks, very little is actually known about the overall organization and molecular architecture of any replication system. Our goal is to identify and characterize the transient and stable protein-protein and protein-DNA interactions that underlie the coupled synthesis of two antiparallel DNA strands. These processes are fundamental for the growth and development of humans and other organisms. Unique features of the replication process in prokaryotes are attractive targets for the development of selective antimicrobial agents to treat infectious diseases. ? ? ?
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