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交易。MMR是由MutS和MutL同源物引发的，它们在原核生物和真核生物中高度保守。它们都是二聚体，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突变增加，重组不当，并对几种DNA损伤剂的细胞毒性作用产生抗性。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蛋白功能的多样性，了解这些不同过程背后的分子机制对于开发相关疾病和癌症的有效治疗至关重要。在真核生物中，MutS¿（MSH2-MSH6）和MutL¿（酵母中的MLH1-PMS2， Mlh1-Pms1）是负责MMR起始的主要MutS和MutL同源物。mut¿通过结合错配并经历atp依赖的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¿，将5‘和3’的子链切割到不匹配的位置。随后，MutS¿激活5‘-3’外切酶EXO1，以去除含有错误核苷酸的DNA。最后，DNA聚合酶催化再合成，DNA连接酶封闭缺口。结构和生化研究，包括我们实验室的一些研究，表明蛋白质和蛋白质- dna复合物的构象动力学和组装状态是MMR调控的核心。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。我们提出了一系列系统的实验，将我们灵敏的单分子荧光方法应用于以酿酒酵母为模型系统的活细胞。我们将利用现有的技术，如荧光蛋白，以及迅速兴起的技术，如非天然氨基酸标记。为了实现这个项目，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Thomas Kunkel博士的合作大大加强了我们执行项目以及测试结果模型的生物学意义的能力。我们的目标是：1)利用单分子荧光和超分辨率技术确定体内错配修复复合物的分子组成，包括蛋白质的化学计量和相对位置；2)利用单分子FRET检测错配修复起始蛋白MutS¿和MutL¿在修复过程中的体内构象动力学。提出的实验将回答关于MMR分子机制的许多悬而未决的问题，并将扩大检测多蛋白复合物的体内动力学和组成的技术范围。

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.