MECHANISM AND FIDELITY OF DNA REPLICATION

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DNA replication occurs at fast rates and with a high degree of fidelity, both of which are necessary for the survival and propagation of organisms. The long term objective of this proposal is the structural, kinetic, and thermodynamic basis for high fidelity and efficiency of DNA Preliminary data by the principal investigator has established the pathway of T7 DNA polymerase by rapid chemical-quench-flow methods. In addition, the PI has constructed and fully char exonuclease deficient mutant of T7 DNA polymerase which has enabled quantitative measurement o the contributions of individual reactions to the fidelity of polymerization. These studies lay a strong foundation for this proposal. The specific aims are to: (1) Explore the structural basis for selectivity by examination of the kinetics and thermodynamics of incorporation as a function of variations in DNA sequence, through the use of nucleotide analogs, and by examination of the effect of the posi- tion of mismatches in the template/primer. (2) Relate the structure of the enzyme to the dynamics of catalysis by complete quantitation of the kinetic and thermodynamic consequences of single and multiple amino acid substitutions; initially, site-directed mutagenesis will be guided by the structure of DNA polymerase I, which is highly homologous to T7 DNA polymerase, but ultimately we hope to base our studies on the crystal structures of T7 DNA polymerase. (3) Begin studies to establish the structure of T7 DNA polymerase and gene 4 protein helicase/primase by x-ray crystallography in order to begin to address the problems of catalytic mechanism and fidelity of polymerization in molecular terms that can be related to the kinetics of reaction. (4) Establish the kinetics of the helicase and primase reactions of gene 4 protein, including definition of the pathway of coupling of ATP hydrolysis to DNA unwinding and examination of the elementary steps leading to RNA primer synthesis. (5) Characterize DNA polymerization at a replication fork by establishing the stoichiometries of DNA polymerase and the helicase/primase in the replication complex, by examination of the kinetics of leading and lagging strand synthesis by rapid chemical-quench flow methods, and by rapid photochemical crosslinking studies to establish the dynamics of protein-protein associations in the complex. These studies will provide fundamental mechanistic information describing DNA replication fidelity, with important implications for understanding eukaryotic polymerases and HIV reverse transcriptase.