DNA REPAIR IN NORMAL AND HYPERMUTABLE CANCER CELL LINES

错配修复(MMR)中的缺陷已被证明与 遗传性非息肉病性结肠癌和散发性结肠癌 癌症。这些癌症具有肿瘤特有的微卫星突变。 重复序列。这些重复序列被认为很容易 复制过程中出现链错配。如果不改正，这将是 导致复制错误和插入/删除的累积 (I/D)突变。这种表型被称为MIN(微卫星 不稳定)。获得超可变表型会导致 基因组的不稳定，似乎是癌症的早期事件 发展。高度可变的癌细胞系分成几个不同的 类与它们的突变谱有关。其中大部分都是 是最小的，并且错配修复不足。MMR功能有缺陷 导致高度可变的癌症表型。 一些高度可变的癌细胞株具有微卫星不稳定性和 精通错配校正。也就是说，他们认识到并 纠正所有碱基错配和单链DNA环 核苷酸。这一发现表明，其他DNA修复途径可能是 这些细胞有缺陷。 初步证据表明，修复大型DNA环的一条途径 存在，并且它不同于MMR。假设这一点 矫正路径也可能预置到MIN将直接测试 在使用DNA环底物的高度可变的癌细胞系中。 我将探索这些细胞中的大环修复是否有缺陷 以及它是否对Min表型有贡献。要做到这一点 我的计划是：1.筛选修复大DNA的超变MIN细胞系 圈数。2.确定大的DNA环修复是否有别于 失配修复。3.确定此次维修的功能需求 系统。4.分离这一新型修复系统的组件。这 最初的努力将集中在DNA环的特异性识别上 蛋白。 第二个项目涉及筛选第二类超可变 精通MMR的癌细胞株。这些都是超可变的 在一种检测转换/颠换突变的体外试验中 HPRT基因座，但不显示最小。的突变谱 这些细胞系与修复系统的缺陷是一致的，例如 碱基切除修复，纠正特定的错误结合的碱基或 由水解物自发产生的那些。我会检查是否有瑕疵 在几种糖基酶中使用了一种简单的测定方法，并进一步研究了缺陷 细胞系。

Defects in mismatch repair (MMR) have been shown to be associated with both Hereditary Non-Polyposis Colon Cancers (HNPCC) and sporadic cancers. These cancers have tumor specific mutations in microsatellite repeat sequences. These repeat sequences are thought to be prone to slipped strand mispairing during replication. If not corrected this will lead to replication errors and an accumulation of insertion/deletion (I/D) mutations. This phenotype has been termed MIN (Microsatellite Instability). Acquisition of the hypermutable phenotype leads to destabilization of the genome and appears to be an early event in cancer development. Hypermutable cancer cell lines fall into several distinct classes with regard to their mutation spectrum. The majority of these are MIN + and are mismatch repair deficient. Defective MMR function contributes to the hypermutable cancer phenotype. Some hypermutable cancer cell lines have microsatellite instability and are proficient in mismatch correction. That is, they recognize and correct all base-base mispairs and single stranded DNA loops up to nucleotides. This finding suggests other DNA repair pathways may be defective in these cells. Preliminary evidence shows that a pathway which repairs large DNA loops exists and that it is distinct from MMR. The hypothesis that this corrective pathway may also predispose to MIN will be tested directly in hypermutable cancer cell lines using DNA loop substrates. I will explore whether large loop repair is defective in these cell lines and whether it contributes to the MIN+ phenotype. To achieve this I plan to: 1. Screen hypermutable MIN cell lines for repair of large DNA laps. 2. Determine whether large DNA loop repair is distinct from mismatch repair. 3. Determine the functional requirements of this repair system. 4. Isolate the components of this novel repair system. This effort will initially focus on the DNA loop-specific recognition protein. A second project involves screening of a second class of hypermutable cancer cell lines which are proficient in MMR. These are hypermutable in an in-vitro assay which detects transition/transversion mutations at the HPRT locus, but do not demonstrate MIN. The mutation spectra of these cell lines is consistent with defects in repair systems, such as base excision repair, which corrects specific misincorporated bases or those produced spontaneously by hydrolysis. I will screen for defects in several glycosylases using a simple assay and further study defective cell lines.