MECHANISMS OF CHEMICAL CARCINOGEN INDUCED P53 MUTATIONS

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Damage of DNA by environmental carcinogens is assumed to be a principle factor in the induction of mutations and the development of cancer in humans. The p53 tumor suppressor gene is the most frequently mutated gene in human malignancies. One popular hypothesis is that the spectrum of mutations found in different cancers may bear the signature of the mutagenesis process. To test this hypothesis, we will undertake a detailed analysis of the mutagenesis process at the p53 locus using in vitro systems. As a first step in this direction, we will investigate in different cell types the chromatin structure at the critical p53 exons including an analysis of nucleosome association and DNA binding proteins. To understand the mechanisms of mutagenesis, we will analyze DNA damage and DNA repair events at the DNA sequence level in human cells treated with several different mutagens and carcinogens. We will apply the ligation-mediated polymerase chain reaction (LMPCR), a uniquely sensitive technique for in vivo mapping of DNA adducts, to determine the frequency of a number of different mutagenic DNA adducts at each nucleotide position of the p53 gene's exons five through nine. Repair rates for selected DNA adducts will analyzed to determine if repair is sequence-specific. An increased adduct frequency and/or decreased repair rates at specific nucleotide positions may create mutation hot spots. Data from analysis of chromatin structure, sequence-dependent adduct frequency maps, and repair rates will be compared with published frequencies of mutants found in a variety of human cancers including those for with involvement of an environmental carcinogen is suspected . Thus we will attempt to provide in vitro model systems to study the basic molecular mechanism that give rise to mutations in the p53 gene.