The long-term objective of this project is to understand the underlying mechanisms of mutagenesis and define the mutational pathways and repair systems operating in living cells. Because of the relationship between mutagenesis and carcinogenesis, it is the contention of Dr. Miller that this project has a direct bearing on human health.
The specific aims of the application involve detecting and characterizing different mutator genes in bacteria, and finding and characterizing their homologs in human cDNA and human genomic libraries. Initially, the work will focus on four mutators which were discovered during the previous funding period. The mutY and mutM genes encode proteins that counteract the effects of the oxidation product 8-oxodG. Work is already ongoing with a fragment of the mutY homolog from a human cDNA library, and this fragment will be used to determine the sequence of the entire gene, detect the genomic segment it is carried on, and to map the gene on the human chromosome. Ultimately, it is planned to screen human tumor lines that are mutators for defects in this gene, and others that can be detected which are homologous to bacterial mutators. Additional studies will focus on the mutA and mutC genes which Dr. Miller has found to create mutations by a novel pathway. A missense suppressor is created that inserts glycine at the aspartic acid codon and, in doing so, probably creates a mutator polymerase by substituting in a fraction of the population, a glycine for an essential aspartic acid in the epsilon subunit of polymerase III. It is contended that this pathway of mutagenesis in bacteria has possible implications for understanding contributions to aging and possibly cancer susceptibility in humans.
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