The objective of this proposal is to use DNA polymerase-beta (pol-beta) from rat brain, which is the smallest and simplest DNA polymerase known (39 kDa), to study important issues related to DNA and RNA polymerases. There are three major goals: (i) to understand structure-function relationships in this enzyme at the highest resolution possible, particularly in relation to catalytic mechanism and fidelity; (ii) to obtain mutants of pol-beta with """"""""useful"""""""" properties, e.g., improved fidelity and reversed stereoselectivity; and (iii) to use pol-beat-beta as a model system to understand the drug resistance for possible application to HIV reverse transcriptase (RT).
In Specific Aim 1, the complete kinetic mechanism will be determined by pre-steady-state kinetics using both normal template-primer DNA and single-gapped DNA substrates. Stopped-flow fluorescence will be used to identify possible substrate-induced conformational changes (in collaboration with Dr. Smita Patel). Resonance assignment and conformational analysis by NMR will be pursued to the farthest extent possible.
In Specific Aim 2, the residues interacting with dNTP or its partner on the template will be mutated and the mutants analyzed by the procedures developed in Specific Aim 1. Various substrate analogs will also be used to test the changes in the substrate specificity of the mutants.
In Specific Aim 3, mutants with improved fidelity will be pursued in three ways: changing the active site topology to constrict the base-pair binding pocket; reducing pol-beta triphosphate interactions; and random mutagenesis.
Specific Aim 4 proposes to change the stereoselectivity of pol-beta toward diastereomers of dNTP-alphaS using four approaches: a combination of site-directed mutagenesis and metal ion exchange, ligand-assisted catalysis, introducing new interactions with alpha-phosphate, and random mutagenesis.
In Specific Aim 5, pol-beta will be used to test a hypothesis that """"""""one of the consequences of drug resistance is an improvement in substrate specificity."""""""" The result will enhance understanding of the drug resistance of HIV RT. Mutants displaying significant functional properties will be subjected to structural determination by x-ray crystallography by Dr. B.C. Wang at the University of Georgia.
