ROLE OF MISMATCH REPAIR IN SPORADIC TUMOR SUPPRESSION

描述(申请人摘要)：本项目的长期目标 是为了了解人类错配修复的分子机制及其 对人类癌症的影响。DNA错配修复在维持 通过校正DNA生物合成产生的错配来实现基因组稳定性 错误和DNA重组。人类错配修复的缺陷是主要的 遗传性结直肠癌和散发性结直肠癌的病因 显示微卫星不稳定。微卫星不稳定，这 与错配修复缺陷相关，在一项 许多类型的散发性癌症的相当一部分，包括膀胱癌 癌症。最近，散发性膀胱癌表现出较高的 微卫星不稳定性的发生率高于其他散发性癌症。的目标 此应用程序用于确定错配修复缺陷是否与 并分离和鉴定新的错配 修复组件/基因。实验将在以下三个阶段进行 特定领域。1)膀胱癌细胞株的错配修复能力 微卫星的不稳定性将使用体外生化来确定。 错配修复试验。2)新的错配修复活动将是第一个 以使用已知错配修复的互补实验为特征的 蛋白质和/或突变细胞系，然后从HeLa核中纯化 萃取物凭借其恢复小说错配修复的能力 突变的细胞系。3)编码新蛋白(S)的基因(S)将被克隆 通过“反向遗传”的方法。多肽序列将从 新蛋白(S)及其用于设计简并引物扩增DNA 感兴趣的片段，这些片段依次用于识别全长的cDNA。 这项研究不仅将为了解散发性膀胱的病因提供帮助 癌症，但也导致识别新的错配修复 组件。

DESCRIPTION (Applicant's abstract): The long-term objectives of this project are to understand the molecular mechanism of human mismatch repair and its impact on human cancer. DNA mismatch repair plays a crucial role in maintaining genomic stability by correcting mismatches generated from DNA biosynthetic errors and DNA recombination. Defects in human mismatch repair are the primary cause of both hereditary colorectal cancer and sporadic colorectal cancers that display microsatellite instability. Microsatellite instability, which correlates with mismatch repair deficiency, has also been demonstrated in a substantial fraction of many types of sporadic cancer, including bladder cancers. Recently, sporadic bladder cancers have been shown to display a higher rate of microsatellite instability than other sporadic cancers. The goals of this application are to determine if mismatch repair deficiency is associated with sporadic bladder cancers and to isolate and characterize novel mismatch repair components/genes. Experiments will be developed in the following three specific areas. 1) The mismatch repair proficiency of bladder cell lines with microsatellite instability will be determined using an in vitro biochemical mismatch repair assay. 2) Novel mismatch repair activities will be first characterized by complementation experiments using the known mismatch repair proteins and/or mutant cell lines, and then be purified from HeLa nuclear extracts by virtue of their ability to restore mismatch repair to the novel mutant cell lines. 3) The gene(s) encoding the novel protein(s) will be cloned by the "reverse genetic" approach. Peptide sequences will be obtained from the novel protein(s) and used for designing degenerate primers to amplify DNA fragments of interest, which are in turn used to identify full length cDNAs. This study will not only provide insight into the etiology of sporadic bladder cancers, but also lead to the identification of novel mismatch repair components.