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

通过研究关键 DNA 修复途径阐明癌症发展的分子机制

基本信息

批准号：
10264134
负责人：
Sharonda LeBlanc
金额：
$ 13.16万
依托单位：
NORTH CAROLINA STATE UNIVERSITY RALEIGH
依托单位国家：
美国
项目类别：
财政年份：
2020
资助国家：
美国
起止时间：
2020-09-01 至 2023-08-31
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/10264134
关键词：
ATP phosphohydrolase Amino Acids Atomic Force Microscopy Base Pairing Binding Biochemical Biochemistry Biological Sciences Biophysics Cancer Etiology Cell physiology Cells Chemistry Code Colorectal Cancer Complement Complex Confocal Microscopy Coupled DNA DNA Binding DNA Repair DNA Repair Gene DNA Repair Pathway DNA biosynthesis DNA-Protein Interaction Data Daughter Detection Development Disease Doctor of Philosophy Exonuclease Failure Film Fluorescence Fluorescence Resonance Energy Transfer Foundations Future Generations Genes Genome Goals Hereditary Nonpolyposis Colorectal Neoplasms Human Human Genome Hydrolysis In Vitro Inherited Institution Knowledge Label Lead Left Link MLH1 gene Malignant Neoplasms Mismatch Repair Mismatch Repair Deficiency Molecular Molecular Conformation Mutation Nobel Prize Nucleotides Organism Parents Pathway interactions Physical Chemistry Physics Polymerase Positioning Attribute Preparation Process Property Proteins Quantum Dots Research Research Personnel Research Training Science Signal Transduction Spectrum Analysis Structure System Techniques Testing Time Tissues Visualization Work Yeasts advanced disease base career design electric field experience experimental study in vivo interfacial molecular modeling mutant nanoscale nanoscience new therapeutic target plasmonics repaired single molecule single-molecule FRET skills tenure track therapeutic target tumor tumorigenesis

项目摘要

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.

项目概要！ Sharonda LeBlanc 博士获得了纳米科学博士学位，研究电场对使用共焦显微镜与时间分辨结合的单量子点 (SQD) 荧光发射光谱学。她发现了适度应用场和等离子体薄膜的有趣光物理效应在 SQD 上。 LeBlanc 博士目前正在从基础物理化学转向生物化学/生物物理学同时仍然利用单分子技术。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）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。这该提案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。具体目标1和2如下：具体目标 1：表征 MutL 构象的核苷酸依赖性动力学使用单分子 FRET 体外检测不存在错配 DNA。具体目标 2：研究野生型和突变体 MutL 构象的动态使用 smFRET 在体外用核苷酸、MutS 和错配 DNA 启动错配修复。 DNA 错配修复 (MMR) 是一种蛋白质复制后系统，可纠正基因组中罕见的错误所有生物体。在 60 亿个碱基的人类基因组中，每轮复制大约有 600 个错误，每个细胞。如果不加以纠正，错误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 同源物的任务是识别 107 个正确配对中的不匹配碱基，区分母链和子链，然后开始修复。单氨基酸突变 MutS 和 MutL 蛋白与遗传性和散发性结直肠癌有关，这是第三大全世界常见的癌症。尽管这些突变主要与 MutL 相关，已在在癌症病例中，目前尚不清楚 MMR 缺陷如何引发和进展该疾病。不匹配导致的失败修复途径可能引发肿瘤发生，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) 具有独特的研究能力 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。这些实验可以为确定治疗靶点提供基础。该提案的最终目标是旨在促进 LeBlanc 博士过渡到独立职业，研究策略中概述了：具体目标 3：提出想法、设计实验并测试与杰出研究相关的新假设 DNA 修复问题，为独立研究生涯做准备。