Structure Function Studies of DNA Mismatch Repair

DNA错配修复（MMR）系统纠正复制过程中发生的DNA合成错误， 在其他几个DNA交易中MMR由MutS和MutL同源物启动，它们在整个MMR中高度保守。 原核生物和真核生物。它们都是二聚体，含有DNA结合和ATP酶活性，这些活性对于 体内MMR。这些蛋白质的失活导致增加的诱变、不适当的重组和对大肠杆菌的抗性。 几种DNA损伤剂的细胞毒性作用。在人类中，错配修复基因的突变直接 与遗传性非息肉病性结直肠癌（HNPCC）有关，并与几种散发性癌症有关。因为 由于MMR蛋白具有多种多样的功能，因此了解其分子机制是至关重要的。 这些不同的过程的基础，以开发有效的治疗相关疾病和癌症。在 在真核生物中，MutSα（MSH 2-MSH 6）和MutLα（MLH 1-PMS 2）是主要的MutS和MutL同源物， MMR的启动。MutSα通过与错配结合并经历ATP依赖性构象变化来启动修复 促进其与MutLα相互作用的变化。然后PCNA激活MutLα切割子链的5'和3'端， 到不匹配。随后，MutSα激活5 '-3'核酸外切酶EXO 1，以将含有DNA的 不正确的核苷酸最后，DNA聚合酶δ或ε催化再合成，DNA连接酶密封切口。结构 和生物化学研究，包括我们实验室的几项研究，表明构象动力学和组装状态 蛋白质和蛋白质-DNA复合物是MMR调节的中心。本提案的总体目标是 阐明了MMR起始步骤的结构-功能关系。我们提出了一系列系统的 实验中，我们表征的结构，构象，和动态特性的MMR复合物形成 与MutSα使用原子力显微镜（AFM），我们新开发的双共振频率增强 静电力显微镜（DREEM），它允许通过蛋白质的DNA的路径可视化，和 单分子荧光。这些研究将辅之以对生物化学和 我们的合作者在Paul Modrich和Peggy Hsieh的实验室完成了功能特性。的这种组合 技术将使我们能够充分表征复合物的结合，动力学，构象和功能特性 控制MMR和基因组稳定性的基因。我们的目标是：1）解剖的分子机制， MutSα的错配识别; 2）确定MutSα-MutLα-错配复合物的构象性质 控制着修复的开始

The DNA mismatch repair (MMR) system corrects DNA synthesis errors that occur during replication and also is involved in several other DNA transactions. MMR is initiated by MutS and MutL homologs, which are highly conserved throughout prokaryotes and eukaryotes. They are both dimers and contain DNA binding and ATPase activities that are essential for MMR in vivo. Inactivation of these proteins leads to increased mutagenesis, improper recombination, and resistance to the cytotoxic effects of several DNA damaging agents. In humans, mutations in the mismatch repair genes are directly linked to hereditary non-polyposis colorectal cancer (HNPCC) and are associated with several sporadic cancers. Because of the diversity of functions carried out by the MMR proteins, it will be essential to understand the molecular mechanisms that underlie these different processes to develop effective treatment for the associated diseases and cancers. In eukaryotes, MutSα (MSH2-MSH6) and MutLα (MLH1-PMS2) are the primary MutS and MutL homologs responsible for initiation of MMR. MutSα initiates repair by binding to a mismatch and undergoing an ATP-dependent conformational change that promotes its interaction with MutLα. PCNA then activates MutLα to incise the daughter strand both 5' and 3' to the mismatch. Subsequently, MutSα activates the 5'-3' exonuclease EXO1 to processively excise the DNA containing the incorrect nucleotide. Finally, DNA polymerase δ or ε catalyzes resynthesis, and DNA ligase seals the nick. Structural and biochemical studies, including several from our lab, indicate that the conformational dynamics and assembly states of the proteins and protein-DNA complexes are central to the regulation of MMR. The overall goal of this proposal is to elucidate the structure-function relationships that govern the initiation steps of MMR. We propose a systematic series of experiments, in which we characterize the structural, conformational, and dynamic properties of MMR complexes formed with MutSα using atomic force microscopy (AFM), our newly developed Dual Resonance frequency Enhanced Electrostatic force Microscopy (DREEM), which allows visualization of the path of the DNA through the proteins, and single-molecule fluorescence. These studies will be complemented with a thorough examination of the biochemical and functional properties done by our collaborators in Paul Modrich's and Peggy Hsieh's laboratories. This combination of techniques will allow us to fully characterize the binding, dynamic, conformational, and functional properties of complexes that govern MMR and the preservation of genomic stability. Our goals are to: 1) dissect the molecular mechanisms of mismatch recognition by MutSα, and 2) determine the conformational properties of MutSα-MutLα-mismatch complexes that govern the initiation of repair.