Duplication of the genetic material is crucial to all forms of life. Replication of a chromosome requires coordinated actions of numerous proteins to unwind the DNA, repeatedly prime synthesis on one antiparallel strand, and polymerize two daughter strands. In Escherichia coli, as in eukaryotes, the principle replicase is isolated as a multiprotein complex. The E. coli DNA polymerase III holoenzyme (polIII holoenzyme) is composed of a core polymerase and four accessory proteins. The accessory proteins confer special properties which distinguish the holoenzyme as a replicative polymerase. Thus polIII holoenzyme is extremely processive in synthesis, has high catalytic efficiency, and diffuses on duplex DNA in search of a primer terminus. By themselves the accessory proteins utilize ATP to form a tightly bound """"""""preinitiation complex"""""""" with a primer template. Upon completing a template to the last nucleotide, polIII holoenzyme rapidly cycles to a new primed template only if the new template is endowed with a preinitiation complex. Rapid cycling to new primers is a property anticipated of a replicative polymerase which must repeatedly cycle to multiple primers on the lagging strand of a moving replication fork. The proposed study aims for a clear view of polIII holoenzyme action in a growing chromosome. The dynamics of polIII holoenzyme in diffusion on a variety of DNA structures having single strand, duplex, and forks will be studied using precisely constructed DNA molecules. The mechanism by which polIII holoenzyme rapidly locates a shorter primer on a larger DNA substrate will be studied will by rapid reaction measurements. Individual subunits of polIII holoenzyme will be purified in large scale from overproducing strains of E. coli. Using pure subunits polIII holoenzyme and the preinitiation complex will be reconstituted on defined primed templates with the objective of a molecular description of how polymerase cycles to the preinitiation complex during replication (i.e. speed, polarity, energetics.) (3H)-subunits (prepared by reductive methylation) will be used in reconstitution studies to assess their stoichiometry and affinity in the holoenzyme particle. The long range goal is a firm grasp of the basic principles that underlie events at the replication fork of a growing chromosome.
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