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

项目摘要 好了！ Sharonda LeBlanc博士获得了纳米科学博士学位，研究电场对 时间分辨共聚焦显微镜用于单量子点(SQD)的荧光发射 光谱学。她发现了中等电场和等离子体薄膜的有趣的光物理效应。 在SQDS上。勒布朗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 同时仍在使用单分子技术。目前，她是北卡罗来纳大学教堂山分校的博士后研究员 研究错配修复蛋白的分子相互作用。她在北卡罗来纳大学的两个实验室之间工作 (化学)和北卡罗来纳州立大学(物理学)完成她的研究。她的职业目标是获得终身教职。 在研究机构的职位。她希望将她过去在纳米科学方面的研究经验与 生物化学/生物物理学，最终研究体内癌症发展的分子机制。这个 这项建议和研究培训计划的具体目标旨在提高勒布朗博士的技能和 生物科学知识，特别是DNA修复方面的知识。具体目标1和2如下： 具体目标1：表征MutL构象的核苷酸依赖的动力学 用单分子FRET检测体外无错配DNA。 具体目标2：研究野生型和突变型MutL构象的动态 错配修复启动与核苷酸，MutS，和错配DNA与smFRET。 DNA错配修复(MMR)是一种蛋白质复制后系统，可以纠正基因组中罕见的错误 在所有有机体中。在人类基因组的60亿个碱基中，每一轮复制都有大约600个错误 手机。如果不加以纠正，错误就会累积为基因组中的永久性突变，并可能导致疾病 在生物体中的状态。MutS和MutL同源基因的任务是识别107对正确配对的错配 碱基，区分父母和女儿链，然后启动修复。单一氨基酸突变 在MutS和MutL蛋白中，MutS和MutL蛋白与遗传性和散发性结直肠癌有关，在 全世界常见的癌症。虽然这些突变大多与MutL有关，但已在 在癌症病例中，目前尚不清楚MMR缺陷是如何引发和推进这种疾病的。不匹配中的失败 修复途径可能会启动肿瘤的发生，但我们对MMR过程缺乏基本的了解。 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的蛋白质-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依据。这项提议的最终目的是 旨在促进勒布朗博士过渡到独立的职业生涯，在研究战略中概述： 具体目标3：开发想法，设计实验，并测试与突出问题相关的新假设 DNA修复中的问题，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.