Unrepaired DNA damage is a major source of mutations. In both bacterial and mammalian genomes many mutations have been found to occur at unusually high frequency at specific nucleotide sequences. A well defined mutagenic site in the human H-ras oncogene is at the second base pair of codon 12.
The specific aims of this proposal are to analyze mutational events resulting from insertion of specific DNA adducts at codon 12 in the human H-ras gene using a mammalian cell biological assay that we have recently developed. An autonomously replicating expression vector containing the human H-ras genomic sequence will be used for the insertion of specific nucleotide adducts at the second guanine of H-ras codon 12 or at nearby non activating sites with similar surrounding sequences for subsequent evaluation of mutational events after replication in a mammalian cell line. The frequency and type of mutations at the codon 12 site and at the non activating sites will be compared to evaluate in vivo processing of DNA lesions as a function of type of adduct and genomic location. The components of this assay have been purposefully designed to fill the current gap of knowledge between a known mutational site frequently found in mammalian tumors and the in vivo DNA replication/repair mechanisms contributing to mutational events at this location. The long-term goals of this project are to define cellular mechanisms of DNA damage and repair at tumor-specific locations in the mammalian genome.
