ENZYMES AND REACTIONS FOR REPAIR OF DNA

DNA 修复的酶和反应

• 批准号: 2653940
• 负责人: THOMAS P BRENT
• 金额: $ 23.9万
• 依托单位: ST. JUDE CHILDREN'S RESEARCH HOSPITAL
• 依托单位国家: 美国
• 财政年份: 1977
• 资助国家: 美国
• 起止时间: 1977-08-01 至 2001-01-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/2653940
• 关键词: 5 methylcytosine; CpG islands; DNA footprinting; DNA repair; chemical binding; enzyme activity; enzyme induction /repression; genetic enhancer element; genetic promoter element; introns; methylguanidine; methyltransferase; reporter genes; site directed mutagenesis; southern blotting; transcription factor; western blottings

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.

几乎所有活着的生物都进化出DNA烷基转移酶来修复 DNA中鸟嘌呤O6-位诱发的致死和致突变加合物 通过烷基化试剂，如甲基亚硝脲。这些O6-烷基鸟嘌呤-DNA 烷基转移酶(ATase)也可以修复细胞毒DNA前体 抗癌氯乙基化药物(如BCNU)引起的交联。 鉴于所有正常人体组织和培养的细胞系(Mer+) 20-30%的肿瘤细胞系结构性表达ATase，称为MGMT (mer-)完全缺乏这种修复活动，对 简单甲基化试剂的细胞毒性和致突变作用以及 氯乙基化药物的治疗效果。MGMT似乎是 在特定组织或特定组织的特征水平上受结构性调节 细胞类型；然而，个体之间有相当大的变异性， 在恶性肿瘤中也有广泛的水平。这一行动的主要目标是 建议确定调节MGMT表达的机制 Mer+细胞，导致其在Mer-细胞中被完全抑制。 因为监管似乎是在转录层面上， 特定的目的主要集中在基因的启动子区域。我们 认为启动子区域的胞嘧啶甲基化决定 完全抑制Mer细胞中的MGMT转录(Aim 1)，并且 蛋白质与增强子或抑制子的结合决定 Mer+细胞中MGMT的组成水平(目标2和3)。转录 以甲基化依赖的方式结合这些DNA元件的因子将 被寻求(目标2)。最近在第一次发现的增强子元素 外显子/内含子边界将根据其特定的 结合蛋白和甲基化的影响(目标4)。高甲基化 MGMT基因体内的CpG岛将被定位，并水平 与MGMT表达相关的特定结合蛋白将是 决心(目标5)。这些研究将采用DNA条带移位和 鉴定和鉴定DNA结合蛋白的足迹技术， 报告基因结合序列的定点突变 构造以确立功能意义。对关键问题的澄清 MGMT基因中的调控元件，以及CpG甲基化和 调节结合蛋白在MGMT中的表达，将提供新的标志物 对环境致癌物的潜在易感性和潜在的 对癌症化疗的抗药性。所获得的信息也将 提出干预这些进程的新途径。