The replication fork is a complex structure. Upward of 20 different protomers are likely to operate together in an enzyme machine that has been termed the replisome. In Escherichia coli, this protein conglomerate, which is made up of primosomal proteins and the DNA polymerase III holoenzyme (itself composed of 10 subunits), moves on the parental DNA at 1000 nt/sec, simultaneously unwinding the template and synthesizing the nascent leading- and lagging-strands in a coordinated fashion. It is our goal to understand how these replication proteins assemble on the DNA, to illuminate the interactions that hold the replication fork together, and to determine how each protein at the replication fork contributes to the orderly process of semi-conservative DNA replication. To do so, we study: i) The independent activities of individual replication proteins, ii) the nature of various partial reactions catalyzed by subsets of the complete complement of replication fork proteins, iii) the control circuits and parameters that affect Okazaki fragment synthesis catalyzed by replication forks reconstituted in vitro with purified primosomal proteins and the DNA polymerase III holoenzyme, and iv) the phenotypes of E. coli mutant strains deficient in the PriA, PriB, and PriC replication proteins. Our studies on replication fork action have allowed us to develop a model describing control of Okazaki fragment synthesis. A key feature of this model is the interaction between DnaB (the replication fork helicase) and DnaG (the primase), and between DnaB and DnaC (a primosomal protein). These aspects of the model will be tested by isolating new mutant proteins that are defective in their interactions. These will be examined, along with existing mutant proteins, in the reconstituted replication fork system for their affect on Okazaki fragment synthesis. Studies on the action of the subassemblies of the DNA polymerase III holoenzyme have suggested differential participation of the subunits in: i) nascent strand synthesis, ii) the interaction between the leading- and lagging-strand polymerase complex, iii) preinitiation and initiation complex formation on the new primer terminus on the lagging strand, and iv) in mediating the disassembly of the lagging-strand polymerase after termination of Okazaki fragment synthesis and in its transit to the new primer terminus. The roles of the Pol III HE subunits at the fork will be defined by reconstituting replication forks with individually purified subunits. The precise coordination required between proteins and DNA strands at the replication fork, and the important role of specific protein-protein interactions in replication fork action, implies a defined molecular architecture. We will study the protein-DNA structures present at the replication fork using replication proteins labelled metabolically to high specific activity, gel shift analysis, electron microscopy and immunoelectron microscopy. The validity of our models on replication fork action will be tested in vivo by studying Okazaki fragment synthesis in strains where DnaG production can be controlled. The role of the PriA, PriB, and PriC primosomal proteins in cellular replication, and the reason that PriA inactivation induces the SOS response, will be probed by studying Okazaki fragment synthesis and replication fork movement in priA, priB, and priC strains.
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