MECHANISTIC STUDIES OF REPLICATIVE POLYMERASES

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DNA polymerase III holoenzyme is the major replicative polymerase of E. coli. Studies of it have revealed many of the salient features of replicative complexes common to all cells, eukaryotic and prokaryotic. These features include an acidic "sliding clamp" or processivity factor that encircles the primer-template and tethers the polymerase, a 5-protein ATPase that recognizes the primer terminus and transfers the sliding clamp onto DNA and a complex polymerase, capable of coordinating leading and lagging strand synthesis and the actions of the helicase/primase complex that function at the replication fork. The polymerase domain of most characterized DNA polymerases encompasses approximately 35,000 daltons, yet the catalytic subunits of replicative complexes are generally 4-5-times larger. This extra structure is involved in the protein-protein interactions and communications necessary for the polymerase to coordinate its actions with the other proteins at the replication fork. In this application, studies are proposed to focus on understanding the domains of the catalytic alpha subunit involved in (i) polymerization, (ii) interactions with tau, permitting dimerization of the replicative complex, (iii) interactions with beta, enabling high processivity, and (iv) interactions with epsilon, enabling proofreading. The experimental approach exploits the availability of a series of alpha fusion proteins we have created that contain a set of nested deletions. These deletion proteins will be characterized in terms of their catalytic activities, functional communication and their ability to bind other subunits. We will identify the key catalytic residues of the polymerase active site, and the key residues involved in protein-protein interactions.