The proposed research addresses the enzymology of DNA replication and related nucleotide metabolism, with especial emphasis on mechanisms employed by the cell to synthesize DNA molecules relatively free of errors. These processes may be divided into two categories, error prevention mechanisms, and error correction mechanisms. Since errors in replicating DNA faithfully are manifested as mutations, mutants with abnormally high, or low spontaneous mutation rates are excellent experimental systems to study. Biochemical characterization of the defective agent responsible for the abnormal mutation rate in these mutants almost invariably leads to the discovery of proteins (enzymes) responsible for preventing or correcting errors during replication.
The specific aims of this proposal include: a - characterize a protein coded for by the mutator gene, mutT, which prevents specific transversions during replication b - determine the basis for the unusual specificity of an enzyme that phosphorylates deoxynucleotides c - investigate the relationship of DNA helix stability to error correction by a polymerase proofreading enzyme d - investigate the metabolic role of a unique dGTPase, found only in the enteric bacteria e - attempt to isolate mutator gene mutants of yeast. The study of factors involved in the fidelity of nucleic acid synthesis are essential to our understanding of the basis of normal and abnormal development, genetic diseases, viral immunity, and in general, any health processes based on gene expression.
