BASE EXCISION REPAIR AND MECHANISMS OF ALKYLATING AGENT INDUCED P53 DAMAGE

碱基切除修复途径负责DNA损伤的修复 由电离辐射、氧化损伤和烷基化损伤产生。 它还没有像核苷酸切除那样被彻底研究过 修复途径 用于化疗的DNA烷化剂产生 负责肿瘤杀伤效应的细胞毒性DNA损伤 这些代理人。 来自细菌的3-甲基腺嘌呤-DNA糖基化酶， 啮齿动物和哺乳动物来源催化第一个修复步骤，碱基 切除许多烷基化病变。 确定烷基化的范围 通过这三种酶修复底物，DNA将被一种 各种烷化剂，包括化学治疗剂， 氮芥、氯乙基亚硝基脲和环磷酰胺。 率 将确定每个碱基位置处的切割、体外修复， 细菌、大鼠和人3 meAdenine糖基化酶。 体内修复率 硫酸二甲酯和双氯乙基亚硝基脲的分布图将在 核苷酸水平的分辨率沿着PGK 1启动子和p53外显子， 用连接介导的聚合酶链法 反应（LMPCR）。 这些数据将提供有关序列背景的信息 以及碱基切除修复率的染色质依赖性。 基于pcr的 引物延伸试验，以取代用于修复的Bohr-Hanawalt试验 率，将制定一个人口筛选测定， 所有主要修复途径的活性。 本试验将用于检测 假设“许多脑肿瘤对 双氯乙基亚硝基脲和06苄基鸟嘌呤具有耐药性，因为它们 过表达3-甲基腺嘌呤DNA糖基化酶活性。"

The base excision repair pathway is responsible for repair of DNA lesions generated by ionizing radiation, oxidative damage, and alkylation damage. It has not been investigated as thoroughly as has the nucleotide excision repair pathway. DNA alkylating agents used in chemotherapy generate cytotoxic DNA lesions which are responsible for the tumor killing effect of these agents. The 3-methyladenine-DNA glycosylases from bacterial, rodent, and mammalian sources catalyze the first repair step, base excision, of many alkylated lesions. To identify the range of alkylated substrates repaired by these three enzymes, DNA will be modifies by a variety of alkylating agents including chemotherapeutic agents such as nitrogen mustards, chloroethyl-nitrosoureas, and cyclophosphamide. Rates of cleavage, in vitro repair, at each base position will be determined for bacterial, rat, and human 3meAdenine glycosylases. In vivo repair rates for dimethyl sulfate and bischloroethylnitrosourea will be mapped at the nucleotide level of resolution along the PGK1 promoter and p53 exons in normal human male fibroblasts using the ligation-mediated Polymerase Chain Reaction (LMPCR). These data will provide information on sequence context and chromatin dependence for base excision repair rates. A PCR-based primer extension assay to replace the Bohr-Hanawalt assay for repair rates, will be developed to produce a population screening assay for activity of all major repair pathways. This assay will be used to test the hypothesis "Many brain tumors resistant to a combination of bischloroethylnitrosourea and 06benzylguanine are resistant because they overexpress 3meadenine DNA glycosylase activity."