Quantitative analysis of transient DNA repair processes in vivo

体内瞬时 DNA 修复过程的定量分析

基本信息

批准号：
8667200
负责人：
Eric Michael Brustad
金额：
$ 26.89万
依托单位：
UNIV OF NORTH CAROLINA CHAPEL HILL
依托单位国家：
美国
项目类别：
财政年份：
2014
资助国家：
美国
起止时间：
2014-06-01 至 2018-05-31
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/8667200
关键词：
ATP phosphohydrolase Amino Acids Apoptosis Bacteria Base-Base Mismatch Binding Biochemical Biological Models Biological Testing Cell Survival Cell physiology Cells Collaborations Complex Coupled DNA DNA Binding DNA Damage DNA Ligases DNA Repair DNA biosynthesis DNA-Directed DNA Polymerase DNA-Protein Interaction Data Daughter Development Disease Double Strand Break Repair EXO1 gene Emerging Technologies Eukaryota Fluorescence Fluorescence Resonance Energy Transfer Frequencies Future Genes Genetic Recombination Goals Grant Hereditary Nonpolyposis Colorectal Neoplasms Homologous Gene Human Image In Vitro Label Laboratories Licensing Life Link Location MLH1 gene MSH2 gene MSH3 gene MSH6 gene Malignant Neoplasms Methods Mismatch Repair Mismatch Repair Gene Inactivation Modeling Molecular Monitor Multiprotein Complexes Mutagenesis Mutation Nucleotides PMS2 gene Process Prokaryotic Cells Property Proteins Published Comment Regulation Relative (related person)Repair Complex Resistance Resolution Saccharomyces cerevisiae Sampling Series Signal Transduction Site Staging Structure-Activity Relationship System Techniques Technology Treatment-Related Cancer Work Yeasts cancer therapy chemotherapy chromatin immunoprecipitation cytotoxic dimer effective therapy in vivo insertion/deletion mutation insight protein protein interaction public health relevance repaired research study seal single molecule single-molecule FRET spleen exonuclease stoichiometry therapy design yeast genetics

项目摘要

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.

描述（由申请人提供）：DNA 错配修复（MMR）系统可纠正复制过程中发生的 DNA 合成错误，并且还参与其他几种 DNA 处理，MMR 由 MutS 和 MutL 同源物启动，它们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，这些活性对于体内 MMR 至关重要。这些蛋白质的失活会导致诱变增加、重组不当和耐药性。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，并且由于其功能的多样性而与多种散发性癌症相关。 MMR 蛋白，了解这些不同过程背后的分子机制对于开发针对真核生物中相关疾病和癌症的有效治疗方法至关重要。 (MSH2-MSH6) 和 MutL¿ （酵母中的 MLH1-PMS2、Mlh1-Pms1）是负责 MMR 启动的主要 MutS 和 MutL 同源物。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¿将子链 5' 和 3' 切割至不匹配的位置，随后，MutS¿激活 5'-3' 核酸外切酶 EXO1 以持续切除含有错误核苷酸的 DNA 最后，DNA 聚合酶 ¿或者催化再合成，DNA 连接酶密封切口结构和生化研究（包括我们实验室的多项研究）表明，蛋白质和蛋白质-DNA 复合物的构象动力学和组装状态是 MMR 调节的核心。我们的建议是阐明控制体内 MMR 启动的结构/功能关系，我们提出了一系列系统实验，将我们敏感的单分子荧光方法应用到活细胞中。我们将利用荧光蛋白等成熟技术以及非天然氨基酸标记等快速新兴技术，以实现该项目，我们组建了一支拥有强大专业知识的团队。此外，我们与 Thomas Kunkel 博士的合作极大地增强了我们执行该项目以及测试所产生的模型的生物学意义的能力。 1）确定使用单分子荧光结合超分辨率技术，研究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，以及 2) 检查错配修复起始蛋白 MutS¿和 MutL¿ ，在使用单分子 FRET 进行修复期间，所提出的实验将回答有关 MMR 分子机制的许多悬而未决的问题，并且它们将扩大用于检查多蛋白复合物的体内动力学和组成的技术范围。