More than twenty proteins cooperate at the replication fork in E. Coli to synthesize the nascent leading and lagging strands of DNA. The rapid and accurate duplication of the chromosome requires a high level of coordination between the proteins in the replisome to ensure coupling of leading and lagging strand synthesis and the proper temporal sequence of events during each and every cycle of Okazaki fragment synthesis. The goal of this application is to understand the biochemical basis underlying the synthetic and regulatory mechanisms at the replication fork. In the previous grant period the investigators demonstrated that an interaction between the tau subunit of the DNA polymerase lll holoenzyme (HE) and the replication fork helicase. DnaB was required for replication fork progression at high rates. They will investigate how this contact tolerates the relative rotations of the proteins involved as the replication fork moves forward by isolating mutants of DnaB that can no longer interact with tau and studying their action during rolling circle DNA synthesis. This will serve as an entry into understanding the dynamics of the protein-protein interactions at the replication fork. Okazaki fragment synthesis occurs once every one or two seconds, thus the cycling of the lagging strand polymerase from the just-completed fragment to the next primer terminus must be coordinated precisely to the cycle of primase action. The investigators have demonstrated that the distributive interaction between the primase and DnaB at the replication fork was the primary regulator of Okazaki fragment size. They will investigate the degree of interdependence between the primase and lagging-strand polymerase cycles by using the rolling circle replication fork system to define: i) the signal for primer synthesis, ii) the trigger for lagging-strand polymerase cycling, and iii) whether coupling of the leading and lagging-strand polymerases is passive or informational. The phiX174-type primosome which has been thought to provide the helicase and primase activities at the replication fork, has recently been suggested to be involved in the resolution of homologous recombination and double-strand break repair intermediates. To illuminate the role of the primosome in E. Coli the investigators will: i) study the phenotype of temperature-sensitive priA alleles, ii) determine the activities of the DnaC809 and PriA301 proteins that are encoded by alleles that suppress the priA recombination defect and lethality of recF overexpression, respectively, and iii) model double-strand break repair in vitro with purified proteins.
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