The aim of this project is to understand, at the molecular level, the mechanism of initiation and termination of replication of a linear double-stranded DNA molecule: the 72 kb pair genome of coliphage N4. N4 DNA has at its ends short non-complementary 3' single-stranded tails. Preliminary evidence suggests that in vivo replication starts at or near the ends. We have recently developed an in vitro soluble replication system specific for N4 DNA where replication also starts at the ends of the DNA. Presently, available information, however, does not allow us to postulate a unique model for N4 DNA replication. In order to determine the mechanism of N4 DNA replication we will use biochemical and genetic approaches to: a) determine the exact structure at the ends of N4 DNA. b) investigate the role of the 3' single-stranded ends in DNA replication in vivo and in the in vitro system. c) determine the structure of in vivo and in vitro replication intermediates. d) determine the role of the terminally redundant sequences in replication. Several strategies are proposed in this application to explain how N4 DNA replicates. We expect that accomplishment of the above goals will allow us to formulate, test and confirm a unique model for N4 DNA replication. To understand the reactions and components involved in N4 DNA replication we propose to a) fractionate the in vitro replication system in order to identify additional host and phage components required for replication with specific emphasis on the role of the virion-associated RNA polymerase. b) clone and overproduce the proteins required in order to aid in their purification and characterization.
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