Elucidating Molecular Mechanisms of Cancer Development by Investigating Key DNA Repair Pathways

项目摘要 ! 博士Sharonda LeBlanc获得了纳米科学博士学位，研究电场对 单量子点（SQD）荧光发射使用共焦显微镜耦合时间分辨 谱她发现了适度应用场和等离子体薄膜的有趣物理效应 关于SQD LeBlanc博士目前正在从基础物理化学过渡到生物化学/生物物理学 同时仍然使用单分子技术。目前，她是美国查佩尔山的博士后研究员 研究错配修复蛋白的分子相互作用。她在两个实验室之间工作， （化学）和NC状态（物理）完成她的研究。她的职业目标是获得终身教职 在一家研究机构工作。她想把她过去在纳米科学方面的研究经验与联合收割机结合起来， 生物化学/生物物理学，以最终研究体内癌症发展的分子机制。的 本提案和研究培训计划的具体目标旨在提高LeBlanc博士的技能， 生物科学知识，特别是DNA修复。具体目标1和2如下： 具体目标1：表征在细胞中MutL构象的核苷酸依赖性动力学 使用单分子FRET在体外不存在错配DNA。 具体目标2：研究野生型和突变型MutL构象的动力学， 用核苷酸、MutS和错配DNA在体外用smFRET启动错配修复。 DNA错配修复（MMR）是一种蛋白质复制后系统，可纠正基因组中的罕见错误 所有有机体。在60亿个碱基的人类基因组中，每轮复制大约有600个错误， cell.如果不加以纠正，错误就会积累成基因组中的永久性突变，并可能导致疾病 在有机体中。MutS和MutL同源物的任务是识别107个正确配对的错配 碱基，区分亲本和子链，然后启动修复。单个氨基酸突变 MutS和MutL蛋白与遗传性和散发性结直肠癌有关，这是第三大 全球常见癌症虽然这些突变，主要与MutL相关，已被确定在 在癌症病例中，目前还不清楚MMR缺陷如何启动和促进疾病。不匹配故障 修复途径可能启动肿瘤发生，但我们缺乏对MMR过程的基本了解。 单分子荧光共振能量转移（smFRET）是唯一能够研究 MMR的分子机制涉及多种瞬时蛋白质-蛋白质和蛋白质-DNA相互作用。 这些实验可能为确定治疗靶点提供基础。本提案的最终目的是 旨在促进LeBlanc博士向独立职业的过渡，如研究策略所述： 具体目标3：提出想法，设计实验，并测试与杰出的 DNA修复的问题，为独立的研究生涯做准备。

Project Summary ! Dr. Sharonda LeBlanc earned her PhD in Nanoscale Science, investigating the effects of electric fields on single quantum dot (SQD) fluorescence emission using confocal microscopy coupled with time-resolved spectroscopy. She discovered interesting photophysical effects of moderate applied fields and plasmonic films on SQDs. Dr. LeBlanc is currently transitioning from fundamental physical chemistry to biochemistry/biophysics while still utilizing single molecule techniques. Currently, she is a postdoctoral researcher at UNC Chapel Hill investigating molecular interactions of mismatch repair proteins. She works between two labs at UNC (Chemistry) and NC State (Physics) to complete her research. Her career goal is to obtain a tenure-track position at a research institution. She would like to combine her past research experience in nanoscience with biochemistry/biophysics to ultimately investigate molecular mechanisms of cancer development in vivo. The specific aims of this proposal and research training plan are designed to enhance Dr. LeBlanc's skills and knowledge in the biological sciences, specifically DNA repair. Specific Aims 1 and 2 are as follows: Specific Aim 1: Characterize the nucleotide-dependent dynamics of MutL conformations in the absence of mismatch DNA in vitro using single molecule FRET. Specific Aim 2: Investigate the dynamics of wild-type and mutant MutL conformations in the context of mismatch repair initiation with nucleotides, MutS, and mismatch DNA in vitro with smFRET. DNA mismatch repair (MMR) is a post-replicative system of proteins that corrects rare mistakes in the genome of all organisms. In the human genome of 6 billion bases, there are ~ 600 errors per round of replication, per cell. If left uncorrected, errors accumulate as permanent mutations in a genome, and can lead to a disease state in the organism. MutS and MutL homologs are tasked with recognizing a mismatch in 107 correctly paired bases, discriminating between parent and daughter strand, then initiating repair. Single amino acid mutations in MutS and MutL proteins have been linked to hereditary and sporadic colorectal cancer, the third most common cancer worldwide. Although these mutations, mostly associated with MutL, have been identified in cancer cases, it is unclear how MMR deficiencies initiate and advance the disease. Failures in the mismatch repair pathway likely initiate tumorigenesis, but we lack a fundamental understanding of the MMR process. Single molecule fluorescence resonance energy transfer (smFRET) is uniquely capable of investigating the molecular mechanism of MMR that involves multiple transient protein-protein and protein-DNA interactions. These experiments may provide a basis for identifying therapeutic targets. The final aim of this proposal is designed to facilitate Dr. LeBlanc's transition to an independent career, outlined in the Research Strategy: Specific Aim 3: Develop ideas, design experiments, and test new hypotheses related to outstanding questions in DNA repair in preparation for an independent research career.