This research proposal is a combined biochemical and clinical investigation on the fidelity of DNA synthesis. The overall objective is to determine the mechanism in animal cells that guarantees the accurate copying of the nucleotide sequence of DNA during each division cycle. We will also investigate depurination as a common mechanism for both spontaneous and induced mutagenesis by certain carcinogens. Our specific objectives are to: 1) Identify factors in animal cells that increase the fidelity by which purified DNA polymerase copies DNA templates; 2) Isolate and analyze mutations in the gene(s) coding for DNA polymerase-alpha; 3) Determine the mutation spectrum of metal mutagenesis; 4) Measure mutagenesis as a function of apurinic sites in animal cells; 5) Investigate the relationships between tumor progression and the fidelity by which cellular DNA sequences are replicated. To accomplich these objectives, we have designed a set of assays to measure the fidelity of DNA synthesis by purified DNA polymerases and crude cellular extracts and to examine the frequency of somatic mutations in normal and malignant human cells.
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