The long term goals of this proposal are to define the structural, kinetic, thermodynamic and mechanistic basis for nucleotide selectivity during DNA replication by a high fidelity DNA polymerase. Elementary steps in the reactions governing nucleotide selectivity will be defined by comprehensive and rigorous kinetic analysis using transient-state kinetic methods (stopped-flow and chemical-quench-flow), combined with site-directed mutagenesis and site-specific labeling. The T7 DNA polymerase will be used as a model system, because it represents the best available high fidelity polymerase with a high resolution crystal structure of the E-DNA-nucleotide ternary complex, poised to carry out catalysis. The kinetics of nucleotide-induced changes in protein structure, and the role of this important rearrangement of the active site residues in the specificity and efficiency of DNA replication, will be established. The kinetic partitioning between the DNA polymerase active site and the proofreading exonuclease active site will be examined, including analysis of the role of structural elements in the protein that sense mismatches at the polymerase site and those that stabilize the binding and catalysis of single-stranded DNA at the exonuclease site. This work will provide a comprehensive picture of the mechanisms by which DNA polymerases achieve such extraordinary fidelity in replicating DNA. The work is important, in that it will provide the standard for critical events responsible for nucleotide recognition and discrimination against mismatches or nucleotide analogs. The results will be applied to improve our understanding of processes underlying the origins of some cancers, aging and hereditary disorders related to mitochondria! DNA replication errors, the selective incorporation of nucleoside analogs by viral polymerases, and the toxic side effects of nucleoside analogs used to treat viral infections. The fundamental molecular details provided by this work will provide the basis for understanding nucleotide selectivity in systems that may not be amenable to such detailed analysis and are causally related to these significant human health issues.
