The goal of this research is to understand the molecular mechanisms of DNA replication and mutagenesis in mammalian cells, using the DNA polymerase of herpes simplex virus type 1 as a model. Genetic, biochemical and computer analysis will be used to define mechanisms of enzyme action, understand modifications of these mechanisms in mutant enzymes and model possible structures on the polymerase molecule. The first objective is to understand the mechanism of substrate recognition by analyzing the binding of nucleoside triphosphates to mutant and normal DNA polymerases. Amino acid changes in polymerases with altered substrate interactions will be identified and used to suggest interactions between residues in the polymerase and nucleoside triphosphates. Directed mutations will be made to test these predictions. A second objective is to understand the interactions between DNA polymerase and other proteins in the replication complex. Replication in vitro by DNA polymerase and other replication proteins (e.g. DNA binding protein) will be studied. Interactions between the DNA binding protein and DNA polymerase will also be investigated by isolating mutations within the binding protein gene which suppress polymerase defects. The third objective is to understand the role of DNA polymerase in viral mutagenesis by studying polymerases which produce mutator or antimutator phenotypes and analyzing the types of mutations made in each case. Finally, computer analysis of the DNA sequence will be used to search for similarities between the herpes simplex and other DNA polymerases and to predict secondary structures within the herpes simplex gene. Included in this analysis will be the mutant data above, possible structures of the related Epstein-Barr polymerases and possible similarities to the structure proposed for polymerase I of Escherichia coli.
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