MECHANISMS OF CHEMICAL CARCINOGEN INDUCED P53 MUTATIONS

环境致癌物对DNA的损伤被认为是一种原理 在诱发突变和癌症发展中的因素 人类。P53抑癌基因是最常发生突变的基因 在人类的恶性肿瘤中。一种流行的假设是， 在不同癌症中发现的突变可能带有 诱变过程。为了验证这一假设，我们将进行一项 P53基因座突变过程的详细分析 体外系统。 作为这个方向的第一步，我们将在不同的细胞中进行调查 在关键的p53外显子上键入染色质结构，包括 核小体结合和DNA结合蛋白的分析。 为了了解突变的机制，我们将分析dna损伤。 和DNA序列水平上的DNA修复事件 几种不同的诱变剂和致癌物。我们将应用 连接介导的聚合酶链式反应(LMPCR)，一种唯一敏感的 DNA加合物的体内定位技术，以确定频率 在每个核苷酸位置有多个不同的致突变DNA加合物 P53基因的外显子5到9。选定DNA的修复率 将对加合物进行分析，以确定修复是否具有序列特异性。一个 在特定情况下增加加合物频率和/或降低修复率 核苷酸位置可能会产生突变热点。 来自染色质结构、序列依赖加合物分析的数据 频率图和维修率将与公布的 在多种人类癌症中发现的突变频率，包括 因为涉嫌与环境致癌物质有关。因此， 我们将尝试提供体外模型系统来研究基础 导致P53基因突变的分子机制。

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.