DNA Mismatch Repair

以大肠杆菌甲基导向的MMR途径为代表的DNA错配修复(MMR)，针对通过DNA复制错误、同源重组和DNA损伤而产生的碱基对错配。MMR失活会导致自发突变率的大幅增加，并与散发性和遗传性癌症有关。除了在复制后修复中的作用外，MMR途径的组成部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大，在确保减数分裂过程中染色体的正确配对、调节涉及密切相关序列的同源重组以及参与免疫球蛋白基因座抗体多样性的产生方面发挥重要作用。 错配修复的一个关键步骤是MMR蛋白识别DNA错配和切除修复的许可。这在细胞中是一个关键问题，因为如果不有效修复，MMR的缺口DNA中间体很容易转化为致命的双链断裂。与多萝西·伊利博士合作，我们正在使用原子力显微镜(AFM)来研究在MMR过程中形成的单个蛋白质-DNA复合体的构象。通过测量错配DNA上特定位置的占有率，我们可以确定错配DNA结合的MUT的亲和力、特异性和化学计量比。对错配结合部位含有单一氨基酸变化的MMR蛋白质的单分子研究正在被用来定义错配识别的关键方面。我们还在研究人类MMR蛋白MutSalpha(hMSH2-MSH6)、MutSbeta(hMSH2-hMSH3)和MutLpha(hMLH1-hPMS2)如何在DNA错配位置相互作用，以及核苷酸辅助因子如何调节这种相互作用并许可下游的切除步骤。这些研究对于理解错配修复途径如何有助于基因组稳定和避免癌症具有重要意义。

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.