DNA Mismatch Repair

DNA错配修复（MMR）的例子是E. coli甲基介导的MMR途径，靶向通过DNA复制错误、同源重组和DNA损伤引起的碱基对错配。MMR失活导致自发突变率大幅增加，并与散发性和遗传性癌症相关。除了其在复制后修复中的作用之外，MMR途径的组分还影响与亨廷顿病等综合征相关的三核苷酸重复序列的扩增，在确保减数分裂期间染色体的正确配对中发挥重要作用，调节涉及密切相关序列的同源重组，并参与免疫球蛋白基因座处抗体多样性的产生。 错配修复的关键步骤是MMR蛋白识别DNA错配并允许切除修复。 这在细胞中是一个关键问题，因为MMR的缺口DNA中间体如果不能有效修复，很容易转化为致命的双链断裂。 与Dorothy伊利博士合作，我们正在使用原子力显微镜（AFM）来检查MMR过程中形成的单个蛋白质-DNA复合物的构象。在不匹配的DNA上的特定位置处的分数占位符的测量允许我们确定MutS与不匹配的DNA结合的结合亲和力、特异性和化学计量。 MMR蛋白在错配结合位点中具有单个氨基酸变化的单分子研究正被用来定义错配识别的关键方面。 我们还研究了人类MMR蛋白MutSalpha（hMSH 2-MSH 6），MutSbeta（hMSH 2-hMSH 3）和MutLalpha（hMLH 1-hPMS 2）如何在DNA错配位点相互作用，以及核苷酸辅因子如何调节这种相互作用并许可下游切除步骤。 这些研究对于理解这种错配修复途径如何有助于基因组稳定性和癌症避免具有重要意义。

DNA mismatch repair (MMR) exemplified by the E. coli methyl-directed MMR pathway, targets base pair mismatches that arise through DNA replication errors, homologous recombination and DNA damage. Inactivation of MMR results in a large increase in the rate of spontaneous mutation and is associated with both sporadic and hereditary cancers. In addition to its role in post-replication repair, components of the MMR pathway also influence the expansion of trinucleotide repeats associated with syndromes such as Huntingtons disease, play an essential role in assuring the proper pairing of chromosomes during meiosis, modulate homologous recombination involving closely related sequences, and participate in the generation of antibody diversity at immunoglobulin gene loci. A key step in mismatch repair is the recognition of DNA mispairs by MMR proteins and the licensing of excision repair. This is a critical problem in cells because the gapped DNA intermediate of MMR is easily converted into lethal double-strand breaks if not efficiently repaired. In collaboration with Dr. Dorothy Erie, we are using atomic force microscopy (AFM) to examine the conformations of individual protein-DNA complexes that are formed during MMR. Measurements of fractional occupancies at specific locations on a mismatched DNA allow us to determine the binding affinity, specificity, and stoichiometry of MutS bound to mismatched DNAs. Single molecule studies of MMR proteins harboring single amino acid changes in the mismatch binding site are being exploited to define key aspects of mismatch recognition. We are also examining how human MMR proteins MutSalpha (hMSH2-MSH6), MutSbeta (hMSH2-hMSH3), and MutLalpha (hMLH1-hPMS2) interact with each other at the sites of DNA mismatches and how nucleotide cofactors modulate such interactions and license downstream excision steps. These studies have important implications for understanding how this mismatch repair pathway contributes to genome stability and cancer avoidance.