STUDIES OF DNA MISMATCH REPAIR

工作概述：罕见的复制错误由细胞的拼写检查机制，复制后DNA错配修复（MMR）纠正。本项目的目的是了解正常和突变真核细胞中MMR的生物化学和遗传学。今年我们取得了三个主要成就。为了验证MSH3对MMR和降低癌症发病率很重要的假设，我们研究了单独缺乏MSH3或联合缺乏MSH6的小鼠。研究发现（-/-）小鼠的癌症发病率增加，从这些小鼠提取的细胞缺乏MMR活性。为了验证MSH2编码序列多态性可能具有重要功能的假设，我们研究了在1-8%等位基因频率上报道的三种不同的多态性。我们提供的证据表明，所有三个MSH2基因序列的变化都部分降低了MMR，这表明携带这些多态性的人患癌症的风险可能会增加。为了研究MMR中mh1opms1异源二聚体的功能，我们正在开发一种纯化该复合物的方法。作为这项工作的一部分，我们使用质谱法在部分纯化的蛋白质制备中鉴定蛋白质污染物。这允许重新设计纯化程序以去除污染物，从而为正在进行的生化表征提供高度纯化的MutL α异二聚体。这些研究对于理解DNA错配修复的遗传学和生物化学，错配修复基因的功能，癌症起始事件及其后续治疗的分子遗传学基础，以及暴露于DNA损伤剂对人群中个体造成的风险具有重要意义。

Summary of Work: Rare replication errors are corrected by the cell's spellchecking machinery, post-replication DNA mismatch repair (MMR). The goal of this project is to understand the biochemistry and genetics of MMR in normal and mutant eukaryotic cells. This year we had three main accomplishments. To test the hypothesis that MSH3 is important for MMR and for reducing cancer incidence, we studied mice deficient in MSH3 alone or in combination MSH6. The (-/-) mice were found to have an increased incidence of cancer and extracts of cells derived from these mice are deficient in MMR activity. To test the hypothesis that polymorphisms in the MSH2 coding sequence may be functionally important, we studied three different polymorphisms that are reported at 1-8% allele frequency. We provided evidence that all three MSH2 gene sequence changes partially reduced MMR, suggesting that people harboring these polymorphisms may be at increased risk of developing cancer. To investigate the function of the Mlh1oPms1 heterodimer in MMR, we are developing a procedure to purify this complex. As part of this effort, we used mass spectroscopy to identify protein contaminants in the partially purified protein preparation. This permitted redesign of the purification procedure to remove the contaminants, thus providing highly purified MutL alpha heterodimer for ongoing biochemical characterizations. These studies are important for understanding the genetics and biochemistry of DNA mismatch repair, the functions of mismatch repair genes, the molecular genetic basis for the initiating events in cancer and its subsequent treatment, and the risk posed to individuals in the population by exposure to DNA damaging agents.