INITIATION OF LAGGING-STRAND DNA SYNTHESIS

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A major part of the effort of this laboratory is directed at developing an understanding of how the replication machinery gains access to the DNA template and subsequently remains bound over tremendous distances while actively synthesizing both the leading- and lagging-strands of DNA. To this end, detailed investigations of the activities of enzymes present at the replication fork are presented. Particular attention is given to the protein-protein and protein-DNA interactions that characterize the replication fork. Studies will be continued that are designed to characterize the interaction of the primosome (a mobile priming apparatus that requires seven E. coli-encoded proteins for assembly and which can assemble at a specific site on the DNA (primosome assembly sites), migrate processively 5' to 3' and occasionally synthesize a primer) with primosome assembly sites. The protein responsible for primosome movement will be established and the three genetically undefined primosomal proteins (replication factor Y (protein n') and proteins n and n") will be molecularly cloned and their genes characterized. The structure and movement of the replication fork will be investigated using novel primer-templates that sustain rolling- circle DNA replication in which synthesis of the leading- and lagging-strands of DNA are coupled. The protein-protein interactions required and the role of the DNA polymerase III holoenzyme in coupling the replication machinery at the fork will be established. Rates of replication fork movement will be measured and factors that influence progress of the replication fork will be identified.