The general objective of this proposal are to define the kinetic and structural characteristics of the polymerization process catalyzed by repair and replicative DNA enzymes with emphasis on how replicative fidelity is achieved. Specifically, the minimal reaction sequence for incorporation and misincorporation of deoxynucleotide 5'-triphosphates into template-primers by repair polymerases such as Po1 I and on the other hand to evaluate the influence of the accessory proteins of the T4 replication complex on leading strand synthesis and ultimately at a replication fork. In the case of the HIV reverse transcriptase such a scheme will permit the quantitation of the effects and mode of action of various nucleotide analogs on HIV reverse transcriptase catalyzed replication. The experimental approach is based on rapid quench methods that examine the initial turnover. To compliment studies on the dynamics of these polymerization processes structural studies employing fluorescently labelled DNA, inter strand cross-linked DNA, and photoaffinity labelled DNA will be used to map protein DNA interactions in order to identify specific domains and residues important in both the binding and catalytic processes. The expansion of these methods to the T4 replicative proteins will aid in identification of those proteins that interact with the DNA or with other protein surfaces. The ultimate objective of this work is to elucidate the relationship between polymerase structure and function that results in the enzymes high catalytic efficiency and fidelity.
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