The physiological effects of exposure to trace quantities of metals in the workplace and in the environment are well documented. Several metals have been identified as mutagens as well as carcinogens. Notably, Mn, Co, Cd, Ag, Ba, Cr, Pb, Ni, and As have been found to increase base substitution errors by at least 30% in an in vitro DNA replication assay designed to detect misincorporation of an incorrectly paired nucleotide opposite a synthetic polynucleotide DNA template. In this proposal, a comprehensive analysis of the mutagenetic effects of metal salts on the fidelity of DNA synthesis will be undertaken using an in vitro fidelity assay. The assay allows detection of a variety of different errors occurring during synthesis of a 250 nucleotide sequence contained within a natural DNA template. Utilizing this system, we will systematically study the effects of a variety of metal salts on the fidelity of in vitro DNA synthesis. Specifically, we will measure dose effects of the selected metals on in vitro DNA synthesis and fidelity, determine mutation frequencies and error rates for specific types of mutations, and construct mutational spectra for the selected metals. The results of this work are expected to provide valuable information on mutagenic specificity and dose levels for the selected metals, and may provide further insight into the mechanism of metal-induced mutagenesis. This project will include students in every aspect. Students will learn fundamentals of DNA manipulations, plaque assays, in vitro DNA replication, microbiological techniques, DNA sequencing, data management, and safe use of hazardous metal salts and radionuclides.
