HUMAN MISMATCH REPAIR IN CHEMICAL CARCINOGENESIS

描述：李博士的长期目标是了解分子 错配修复(MMR)机制及其在避免癌症中的作用MMR IS 一个突变避免系统，并在维持 遗传稳定性。已经证明，人类系统中的缺陷 有很强的癌症倾向，包括遗传性息肉病 结直肠癌。环境致癌物共价修饰DNA形成 致癌物-DNA加合物，它诱导突变，启动致癌作用。 最近，MMR组件已被证明可以识别某些形式的 致癌物-DNA加合物，以前被认为只被 核苷酸切除修复(NER)。此外，还记录了MMR-Experent 细胞对病毒的细胞毒作用更加敏感 N-甲基-N‘-硝基-亚硝胺(MNNG)与MMR缺陷细胞的比较。 根据这些发现，我们假设MMR组件或者直接 参与致癌物-DNA加合物的修复或作为传感器发挥作用 启动程序性细胞死亡。为了验证这一假设，有四条工作路线 是在本申请中提出的。首先，使用电泳迁移率 Shift分析，纯化的hMutSa，一种人类错配识别蛋白，将被 测试其结合含有以下物质的寡核苷酸双链的能力 乙酰氨基荧酮、苯并[a]芘二醇环氧化物、 和MNNG，这三种最关键的化学致癌物。第二，单元格 从不同MMR背景的肿瘤细胞中提取的提取物将 被检查以处理含有位点特异性加合物的环状质粒DNA 上面列出的致癌物质。第三，调查研究生理学 识别和处理致癌物-DNA加合物的重要性 MMR、MMR-正常和突变细胞将用列出的致癌物进行处理 以SV40为基础的致癌物修饰的pZ189质粒以上或转染 穿梭载体，并进行细胞凋亡分析。由于MNNG诱导的细胞死亡 精通MMR的细胞被归因于他们徒劳的尝试 去除模板DNA链中的MMNG加合物，预计 致癌物修饰的基因组或质粒DNA的复制将导致 精通MMR的细胞中的凋亡。最后，要确定致癌物是否 MMR可在体内去除加合物，从MMR-Experent中去除基因组DNA 用致癌物治疗的MMR缺陷细胞将被消化 转化为单核苷，然后进行加合物检测和定量。

DESCRIPTION: Dr. Li's long term goal is to understand the molecular mechanism of mismatch repair (MMR) and its role in cancer avoidance. MMR is a mutation avoidance system and plays an important role in maintaining genetic stability. It has been shown that defects in the human system confer a strong cancer predisposition including hereditary polyposis colorectal cancer. Environmental carcinogens covalently modify DNA to form carcinogen-DNA adducts, which induce mutations that initiate carcinogenesis. Recently, MMR components have been shown to recognize certain forms of carcinogen-DNA adducts that are previously thought to be only processed by nucleotide excision repair (NER). It is also documented that MMR-proficient cells are much more sensitive to the cytotoxic effects of N-methyl-N'-nitro-nitrosoguanidine (MNNG) compared to MMR-deficient cells. Given these findings, we hypothesize that MMR components either directly participate in repair of carcinogen-DNA adducts or function as a sensor to activate programmed cell death. To test this hypothesis, four lines of work are proposed in this application. First, using an electrophoretic mobility shift assay, purified hMutSa, a human mismatch recognition protein, will be tested for its ability to bind oligonucleotide duplexes containing site-specific adducts of acetylaminofluorene, benzo[a]pyrene diol epoxide, and MNNG, the three most critical chemical carcinogens. Second, cell extracts derived from tumor cells with different MMR backgrounds will be examined to process circular plasmid DNA containing site-specific adducts of the carcinogens listed above. Third, to investigate the physiological importance of the recognition and processing of carcinogen-DNA adducts by MMR, MMR-normal and mutant cells will be treated with carcinogens listed above or transfected with carcinogen-modified pZ189 plasmid, an SV40-based shuttle vector, and analyzed for apoptosis. Since MNNG-induced cell death of MMR-proficient cells has been attributed to their futile attempts to remove MMNG adducts in the template DNA strand, it is anticipated that replication of carcinogen-modified genomic or plasmid DNA will induce apoptosis in MMR-proficient cells. Finally, to determine if carcinogen adducts can be remove in vivo by MMRcriti, genomic DNA from MMR-proficient and MMR-deficient cells that are treated with carcinogens will be digested into mononucleosides, and followed by adduct detection and quantitation.