DNA MISMATCH REPAIR AND CANCER IN MURINE MODELS

描述：本研究的目的是检查的作用， 错配修复蛋白MSH 3和MSH 6在癌症易感性中的作用 突变 hMSH 2基因突变导致HNPCC。 由于MSH 3和MSH 6被认为是 与MSH 2的功能复合物，预测是，敲除小鼠， 这些基因将是癌症的易感基因。 我们对宇宙的理解 哺乳动物DNA错配修复系统的特性来自于 大肠杆菌MutHLS系统的生化研究。 在细菌中， DNA错配的修复是DNA复制错误的结果， 由MutS蛋白与错配碱基的结合引发。 在 在真核生物中，DNA错配的识别更为复杂， 三种不同MutS同源物的子集：MSH 2、MSH 3和MSH 6。 研究 酵母已经表明DNA错配修复是由两种不同的 复合物：MSH 2-MSH 6之间的复合物，用于识别单个碱基 错配和MSH 2-MSH 3之间的复合物，用于识别两个 四个碱基对插入/缺失。 因为hMSH 2基因的种系突变 导致HNPCC可能是与hMSH 2相互作用的hMSH 3和hMSH 6， 也参与肿瘤的发展。 他们在其中的作用 然而，事态发展仍不明朗。 在本提案中，我们计划测试 假设酵母MSH 3和MSH 6的哺乳动物同源物具有相同的 在有丝分裂错配修复中的作用，并分析癌症易感性 携带这些基因的靶向突变的小鼠。 三个具体目标 这些建议是：1. 为了产生MSH 3和MSH 6缺陷的小鼠 并研究这些小鼠系的癌症易感性。 埃德尔曼医生 将研究MSH 3和MSH 6缺失对癌症的影响， 特别强调胃肠道致癌作用。 2. 为了验证哺乳动物msh 2，msh 3和msh 6基因是 基因上等同于它们的酵母同系物。 酵母实验 提示MSH 2-MSH 3和MSH 2-MSH 6参与了不同损伤的修复， DNA错配的类型 我们将使用msh 2、msh 3和msh 6突变小鼠系 和双突变msh 3/msh 6小鼠系作为小鼠胚胎发育的来源 成纤维细胞系，以分析微卫星不稳定性和测量 细胞提取物中异源双链体错配修复。 3. 检验…的影响 Apc肿瘤抑制基因的MSH 2、MSH 3和MSH 6缺陷。 结直肠 hMSH 2基因突变的癌细胞系积累突变， 肿瘤抑制基因APC。 我们将产生突变的小鼠品系 为msh 3或msh 6，为A1638等位基因杂合子。 的分析 肿瘤谱以及肿瘤形成的发生和进展将 说明MSH 2、MSH 3和MSH 6在胃肠功能中的意义。 肿瘤发生并提供突变谱的详细分析 这是由于这些修复基因中的每一个都存在缺陷。

DESCRIPTION: The purpose of this study is to examine the role of the mismatch repair proteins MSH3 and MSH6 in cancer susceptibility. Mutations in the hMSH2 gene lead to HNPCC. Since MSH3 and MSH6 are thought to form functional complexes with MSH2, the prediction is that knockout mice in these genes will be cancer prone. Most of our understanding of the properties of the mammalian DNA mismatch repair system is derived from biochemical studies of the Escherichia coli MutHLS system. In bacteria the repair of DNA mismatches, which are the result of DNA replication errors, is initiated by the binding of the MutS protein to the mismatched bases. In eukaryotes the recognition of DNA mismatches is more complex and requires subsets of three different MutS homologs: MSH2, MSH3 and MSH6. Studies in yeast have indicated the DNA mismatch repair is initiated by two different complexes: A complex between MSH2-MSH6 for the recognition of single base mismatches and a complex between MSH2-MSH3 for the recognition of two to four base pair insertion/deletions. Because germline mutations in hMSH2 lead to HNPCC it is likely that hMSH3 and hMSH6, which interact with hMSH2, are also involved in the development of neoplasms. Their role in these events, however, remains unclear. In this proposal we plan to test the hypothesis that the mammalian homologs of yeast MSH3 and MSH6 share the same function in mitotic mismatch repair and to analyze the cancer susceptibility of mice carrying targeted mutations in these genes. The 3 specific aims of this proposal are: 1. To generate mice that are deficient in MSH3 and MSH6 and to study the cancer susceptibility of these mouse lines. Dr. Edelmann will study the consequences of loss of MSH3 and MSH6 for cancer susceptibility with special emphasis on gastrointestinal carcinogenesis. 2. To test the hypothesis that the mammalian msh2, msh3 and msh 6 genes are genetically equivalent to their yeast homologs. Experiments in yeast suggest the MSH2-MSH3 and MSH2-MSH6 are involved in the repair of different types of DNA mismatches. We will use msh2, msh3 and msh6 mutant mouse lines and double mutant msh3/msh6 mouse lines as a source for mouse embryonic fibroblast cell lines to analyze microsatellite instability and to measure heteroduplex mismatch repair in cell extracts. 3. To examine the effect of MSH2, MSH3 and MSH6 deficiency of the Apc tumor suppressor gene. Colorectal cancer cell lines with mutations in the hMSH2 gene accumulate mutations in the tumor suppressor gene APC. We will generate mouse lines that are mutant for msh3 or msh6 and heterozygous for the A1638 allele. The analysis of the tumor spectrum and the onset and progression of tumor formation will indicate the significance of MSH2, MSH3 and MSH6 for gastrointestinal tumorigenesis and provide a detailed analysis of the spectrum of mutations that results from deficiency in each of these repair genes.