STUDIES OF DNA MISMATCH REPAIR

工作总结：罕见的复制错误是由细胞的拼写检查机制，复制后DNA错配修复（MMR）纠正。 本项目的目标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，我们取得了三大成就。 为了验证MSH 3对MMR和降低癌症发病率很重要的假设，我们研究了MSH 3单独或联合MSH 6缺陷的小鼠。 发现（-/-）小鼠具有增加的癌症发病率，并且源自这些小鼠的细胞提取物缺乏MMR活性。 为了验证MSH 2编码序列多态性可能在功能上重要的假设，我们研究了三种不同的多态性，报告在1-8%的等位基因频率。 我们提供的证据表明，所有三个MSH 2基因序列的变化部分降低了MMR，这表明携带这些多态性的人可能会增加患癌症的风险。 为了研究Mlh 1 oPms 1异二聚体在MMR中的功能，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，我们使用质谱法来鉴定部分纯化的蛋白质制剂中的蛋白质污染物。 这允许重新设计纯化程序以去除污染物，从而提供高度纯化的MutL α异二聚体用于正在进行的生物化学表征。 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构成的风险非常重要。

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.