Quantitative analysis of transient DNA repair processes in vivo

描述（由申请人提供）：DNA错配修复（MMR）系统纠正复制过程中发生的DNA合成错误，也参与其他几种DNA处理。MMR由MutS和MutL同源物启动，其在原核生物和真核生物中高度保守。它们都是二聚体，并含有DNA结合和ATP酶活性，这是体内MMR所必需的。这些蛋白质的失活导致增加的诱变、不适当的重组和对几种DNA损伤剂的细胞毒性作用的抗性。在人类中，错配修复基因的突变与遗传性非息肉病性结直肠癌（HNPCC）直接相关，并与几种散发性癌症相关。由于MMR蛋白所执行的功能的多样性，理解这些不同过程的分子机制对于开发相关疾病和癌症的有效治疗是至关重要的。在真核生物中，MutS ²（MSH 2-MSH 6）和MutL ²（酵母中的MLH 1-PMS 2、Mlh 1-Pms 1）是负责启动MMR的主要MutS和MutL同源物。MutS通过与错配结合并经历ATP依赖性构象变化来启动修复，从而促进其与MutL的相互作用。然后，PCNA激活MutL？切割子链5'和3'到错配。随后，MutS？激活5 '-3'核酸外切酶EXO 1，以将含有不正确核苷酸的DNA前切除。最后，DNA聚合酶催化再合成，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，表明蛋白质和蛋白质-DNA复合物的构象动力学和组装状态对MMR的调节至关重要。本提案的总体目标是阐明控制体内MMR启动的结构/功能关系。我们提出了一个系统的一系列实验，使我们敏感的单分子荧光方法活细胞使用S。酿酒酵母作为模型系统。我们将利用现有的技术，如荧光蛋白，以及迅速新兴的技术，如非天然氨基酸标记。为了使这个项目取得成果，我们组建了一个团队，他们在项目的各个方面都有很强的专业知识，并且对协作工作有很强的兴趣。此外，我们与托马斯昆克尔博士的合作大大加强了我们开展该项目的能力，以及测试所产生的模型的生物学意义。我们的目标是1）确定体内错配修复复合物的分子组成，包括化学计量和蛋白质的相对位置，使用单分子荧光结合超分辨率技术，和2）检查在体内的错配修复起始蛋白，MutS <$和MutL <$，在修复过程中使用单分子FRET构象动力学。拟议的实验将回答许多悬而未决的问题，MMR的分子机制，他们将扩大范围的技术，检查在体内的动力学和组合物的多蛋白复合物。

DESCRIPTION (provided by applicant): The DNA mismatch repair (MMR) system corrects DNA synthesis errors that occur during replication and is also 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, MutS¿ (MSH2-MSH6) and MutL¿ (MLH1-PMS2, Mlh1-Pms1 in yeast) are the primary MutS and MutL homologs responsible for initiation of MMR. MutS¿ initiates repair by binding to a mismatch and undergoing an ATP-dependent conformational change that promotes its interaction with MutL¿. PCNA then activates MutL¿ to incise the daughter strand both 5' and 3' to the mismatch. Subsequently, MutS¿ activates the 5'-3' exonuclease EXO1 to processively excise the DNA containing the incorrect nucleotide. Finally, DNA polymerase ¿ or ¿ catalyzes resynthesis, and DNA ligase seals the nick. Structural and biochemical studies, including several from our labs, 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 of MMR in vivo. We propose a systematic series of experiments that bring our sensitive single-molecule fluorescence methods to live cells using S. cerevisiae as a model system. We will take advantage of both established technologies, such as fluorescent proteins, as well as rapidly emerging technologies, such as unnatural amino acid labeling. To bring this project to fruition, we have assembled a team with both strong expertise in every aspect of the project and a strong penchant for collaborative work. In addition, our collaboration with Dr. Thomas Kunkel significantly strengthens our ability to carry out the project as well as to test the biological significance of the models that result. ur goals are 1) to determine the molecular compositions of in vivo mismatch repair complexes, including stoichiometries and relative locations of proteins, using single-molecule fluorescence coupled with super-resolution techniques, and 2) examine the in vivo conformational dynamics of the mismatch repair initiation proteins, MutS¿ and MutL¿, during repair using single-molecule FRET. The proposed experiments will answer many outstanding questions about the molecular mechanisms of MMR, and they will expand the range of techniques for examining the in vivo dynamics and compositions of multiprotein complexes.