MECHANISM AND FIDELITY OF DNA REPLICATION

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The goal of this proposal is to establish the structural, kinetic and thermodynamic basis for the fidelity and efficiency of DNA replication. Analysis of HIV reverse transcriptase will allow definition of the structural constraints that ultimately may limit the ability of the virus to avoid an appropriate combination of nucleoside analogs in the treatment of AIDS and other viral infections. Analysis of the mitochondrial DNA polymerase will define the origins of the toxicity of nucleoside analogs thought to be due to incorporation of the analogs into mitochondrial DNA. Studies on T7 DNA polymerase and HIV RT have shown that the selectivity of the polymerase is a function of a two-step nucleotide binding reaction involving a nucleotide-induced change in conformation from an "open" to a "cloned" state of the ternary E.DNA.dNTP complex. Based upon the crystal structure of HIV RT complexed with DNA, a working model has been formulated proposing the movement of the "fingers" domain of the polymerase into the major groove of DNA in response to the binding of a correct base pair. The specific aims of this proposal are to: (1.) Identify the protein structural domains involved in the conformational change leading to tight nucleotide binding and rapid polymerization; specific photo-affinity labeling will be used to localize amino acids which are brought into close contact with the DNA major groove following the conformational change. (2.) Transient state kinetic analysis of site- directed mutants will be employed to further define the contacts made in the "closed" conformational state and to quantify the contributions of individual amino acids. The kinetics of the conformational change will be examined by stopped-flow methods using fluorescently labeled nucleotides, DNA and protein. (3.) The effects of DNA structure on the conformational change and the kinetics of polymerization will be examined. In particular, the role of DNA bends will be investigated, and the mechanisms of frameshift mutagenesis will be examined by measuring the kinetics of extension over premutational frameshift intermediates. (4.) The effect of RNA secondary structure on the kinetics of polymerization catalyzed by RT will be investigated and the effect of the nucleocapsid protein on the ability of the polymerase to read through hairpins will be assessed. (5.) Genes for the mitochondrial polymerase will be cloned and conditions will be optimized for the overexpression and purification of active protein. The mechanism and fidelity of the mitochondrial polymerase will be established by kinetic measurements.