ENZYMES AND REACTIONS FOR REPAIR OF DNA

几乎所有生物体都进化出DNA烷基转移酶来修复 在DNA中鸟嘌呤的O 6-位诱导的致死和致突变加合物 通过烷基化剂如甲基亚硝基脲。 这些O 6-烷基鸟嘌呤-DNA 烷基转移酶（ATases）也可以修复细胞毒性DNA的前体 由抗癌氯乙基化药物诱导的交联（例如，BCNU）。 而所有正常人体组织和培养细胞系（Mer+） 组成型表达ATase，称为MGMT，20-30%的肿瘤细胞系 （Mer-）完全缺乏这种修复活性，对 简单甲基化剂的细胞毒性和致突变作用，以及 氯乙基化剂的治疗效果。 MGMT似乎是 在给定组织的特征性水平上组成性调节，或 细胞类型;然而，个体之间存在相当大的变异性， 并且在恶性肿瘤中有广泛的水平。 这个项目的主要目标是 一个建议是确定调节MGMT表达的机制， Mer+细胞，并导致其在Mer-细胞中的完全抑制。 因为调控似乎是在转录水平， 具体目标主要集中在基因的启动子区域。 我们 提出启动子区域的胞嘧啶甲基化决定了 完全抑制Mer-细胞中MGMT的转录（Aim 1）， 蛋白质与增强子或抑制子元件的结合决定了 Mer+细胞中MGMT的组成型水平（目的2和3）。 转录 以甲基化依赖方式结合这些DNA元件的因子将 目标2（Aim 2） 最近在第一次实验中发现了一种增强子元件， 外显子/内含子边界将根据其特异性 结合蛋白和甲基化的影响（目的4）。 甲基化 CpG岛在体内的MGMT基因将被定位， 与MGMT表达相关的特异性结合蛋白将被 目标5（Aim 5） 这些研究将采用DNA带移， 足迹技术用于鉴定和表征DNA结合蛋白， 以及报告基因中结合序列的定点突变 建立功能意义。 关键的解释 MGMT基因中的调控元件，以及CpG甲基化和 MGMT表达中的调节结合蛋白，将提供新的标记物 对环境致癌物的潜在易感性， 对癌症化疗的抵抗力。 获得的信息也将 提出了干预这些进程的新途径。

Almost all living organisms have evolved DNA alkyltransferases to repair lethal and mutagenic adducts induced at the O6-position of guanine in DNA by alkylating agents such as methylnitrosourea. These O6-alkyguanine-DNA alkyltransferases (ATases) can also repair precursors of cytotoxic DNA cross-links induced by anticancer chloroethylating drugs (e.g., BCNU). Whereas all normal human tissues and cultured cell lines (Mer+) constitutively express ATase, termed MGMT, 20-30% of tumor cell lines (Mer-) completely lack this repair activity and are hypersensitive to the cytotoxic and mutagenic effects of simple methylating agents as well as to the therapeutic effects of chloroethylating agents. MGMT appears to be constitutively regulated at levels characteristic for a given tissue or cell type; however, there is considerable variability among individuals, and a wide range of levels in malignant tumors. The major goal of this proposal is to determine the mechanisms that regulate MGMT expression in Mer+ cells and that result in its total suppression in Mer- cells. Because regulation appears to be at the transcriptional level, the specific aims focus primarily on the promoter region of the gene. We propose that cytosine methylation in the promoter region determines complete suppression of MGMT transcription in Mer- cells (Aim 1), and that binding of proteins to enhancer or suppressor elements determines constitutive levels of MGMT in Mer+ cells (Aims 2 and 3). Transcription factors that bind such DNA elements in a methylation-dependent manner will be sought (Aim 2). An enhancer element recently identified at the first exon/intron boundary will be characterized with respect to its specific binding protein and the effect of methylation (Aim 4). Hypermethylated CpG islands in the body of the MGMT gene will be located and the levels of specific binding proteins that correlate with MGMT expression will be determined (Aim 5). These studies will employ DNA band-shift and footprinting techniques to identify and characterize DNA binding proteins, and site-directed mutagenesis of binding sequences in reporter gene constructs to establish functional significance. Elucidation of critical regulatory elements in the MGMT gene, and the role of CpG methylation and regulatory binding proteins in MGMT expression, will provide new markers of potential susceptibility to environmental carcinogens and of potential resistance to cancer chemotherapy. The information gained will also suggest new avenues for intervention in these processes.