Elucidating molecular interactions of MutL in mismatch repair using single molecule FRET

使用单分子 FRET 阐明错配修复中 MutL 的分子相互作用

• 批准号: 9327441
• 负责人: Sharonda LeBlanc
• 金额: $ 3.05万
• 依托单位: UNIV OF NORTH CAROLINA CHAPEL HILL
• 依托单位国家: 美国
• 财政年份: 2017
• 资助国家: 美国
• 起止时间: 2017-03-01 至 2017-08-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/9327441
• 关键词: ATP phosphohydrolase; Amino Acids; Atomic Force Microscopy; Base Pairing; Binding; Binding Proteins; Biochemical; Cancer Etiology; Cell physiology; Cells; Chemistry; Closure by clamp; Code; Colorectal Cancer; Complement; Complex; DNA; DNA Binding; DNA Ligases; DNA Repair; DNA Repair Gene; DNA biosynthesis; DNA-Directed DNA Polymerase; DNA-Protein Interaction; Data; Daughter; Detection; Disease; EXO1 gene; Exonuclease; Failure; Fluorescence Resonance Energy Transfer; Foundations; Future; Generations; Genes; Genome; Goals; Hereditary Nonpolyposis Colorectal Neoplasms; Human; Human Genome; Hydrolysis; Imagery; In Vitro; Inherited; Label; Lead; Left; Link; MLH1 gene; Malignant Neoplasms; Mismatch Repair; Molecular; Molecular Conformation; Molecular Models; Mutation; Nobel Prize; Nucleotides; Organism; Parents; Pathway interactions; Polymerase; Process; Property; Proteins; Recruitment Activity; Signal Transduction; Site; Slide; System; Techniques; Therapeutic; Tissues; Work; Yeasts; advanced disease; base; experimental study; in vivo; interfacial; molecular modeling; mutant; new therapeutic target; repaired; single molecule; single-molecule FRET; tumor; tumorigenesis

Project Summary 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. On the molecular level, we know that MutS initially recognizes a DNA mismatch, and undergoes ATP- dependent conformational changes to slide along the DNA. MutL is recruited to the site, and interacts with MutS on DNA to coordinate repair with PCNA, EXO1, DNA polymerase, RFC clamp loader, RPA single strand binding protein, and DNA ligase. We also know that MutL undergoes conformational changes upon ATP binding and hydrolysis, which likely functions to coordinate transient interactions with repair machinery. Previous studies show four distinct conformations of MutL that we believe must be regulated and functional in MMR. MutL is the central player in the middle of the pathway that directs multiple molecular interactions, but how it carries out its functions remains poorly understood. MutL mutations are associated with a spectrum of cancers, thus we need to understand its dynamic molecular interactions and MMR functions, which begin with the MutS-DNA recognition complex. 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. The molecular mechanism of mismatch repair is critical for further revealing how mutants fail to repair, and will provide a basis for identifying therapeutic strategies. We hypothesize that mutations in functional ATPase and interfacial regions of MutL are inadequate in their functional conformational changes and fail to advance repair. To explore these open questions, we propose the following specific aims: 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.

项目摘要 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 所有生物体的基因。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。 在分子水平上，我们知道MutS最初识别DNA错配，并经历ATP- 依赖于构象变化沿着DNA滑动。MutL被招募到该网站，并与 DNA上的MutS与PCNA、EXO 1、DNA聚合酶、RFC夹装载器、RPA单链协同修复 结合蛋白和DNA连接酶。我们还知道MutL在ATP作用下发生构象变化 结合和水解，这可能起到协调与修复机制的瞬时相互作用的作用。 以前的研究表明MutL有四种不同的构象，我们认为它们在细胞内必须受到调节和发挥功能。 MMR。MutL是指导多种分子相互作用的途径中间的中心参与者，但是 人们对它如何履行其职能仍然知之甚少。MutL突变与一系列 因此，我们需要了解它的动态分子相互作用和MMR功能，这开始与 MutS-DNA识别复合物。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- 蛋白质和蛋白质-DNA相互作用。错配修复的分子机制对于进一步研究错配修复的分子机制至关重要。 揭示突变体如何无法修复，并将为确定治疗策略提供基础。我们 假设MutL的功能性ATP酶和界面区的突变不足以使其 功能性构象变化，无法促进修复。为了探讨这些开放性问题，我们建议 具体目标如下： 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。