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

项目摘要 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。 在分子水平上，我们知道MutS最初识别DNA错配，并经历ATP- 依赖于沿着DNA滑动的构象变化。MutL被招募到网站，并与 DNA上突变体与增殖细胞核抗原、EXO1、DNA聚合酶、RFC钳夹加载器、RPA单链协同修复 结合蛋白和DNA连接酶。我们还知道MutL在ATP上经历了构象变化 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。 以前的研究显示了MutL的四种不同的构象，我们认为它们必须在 MMR。MutL是引导多个分子相互作用的途径中间的中心参与者，但 它是如何履行其职能的，人们仍然知之甚少。MutL突变与一系列 因此，我们需要了解其动态的分子相互作用和MMR功能，这始于 MutS-DNA识别复合体。单分子荧光共振能量转移(SmFRET)是 独一无二地能够研究涉及多个瞬时蛋白的MMR的分子机制 蛋白质和蛋白质-DNA的相互作用。错配修复的分子机制是进一步研究错配修复的关键 揭示突变是如何无法修复的，并将为确定治疗策略提供基础。我们 假设MutL的功能ATPase和界面区的突变在他们的 功能构象改变，未能提前修复。为了探索这些悬而未决的问题，我们建议 以下是具体目标： 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。

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.