Investigating DNA Mismatch Repair Through Single-Molecule Approaches

智力优点：在活细胞中的DNA合成过程中，错误掺入核碱基的可能性（例如引入错配，如胸腺嘧啶而不是鸟嘌呤对面的胞嘧啶）高达每个核苷酸的10%。这些前致突变DNA改变需要通过复制后错配修复（MMR）系统去除。MMR的故障会带来灾难性的后果。尽管主要通过生物化学研究在阐明DNA错配修复方面取得了巨大进展，但DNA修复机制的许多至关重要的细节仍然模糊和有争议，即使是细菌（E. coli）MMR系统，这是最简单和最好的表征。这是因为MMR涉及许多蛋白质的协调活动，这些蛋白质形成难以真实的实时监测的瞬时复合物。在之前的项目中，原子力显微镜（AFM）用于直接可视化和检查MutS的行为，MutS是检测错配碱基的关键大肠杆菌MMR蛋白。这些AFM结果调和了以前相互排斥的模型的主要特征，为大肠杆菌MMR启动机制提供了新的见解。本研究的主要目的是进一步阐明E.通过应用一系列单分子技术（例如原子力显微镜、光镊（OT）和单分子荧光共振能量转移（SMFRET））进行大肠杆菌错配修复。具体而言，AFM成像和体积测量将用于检查MutS四聚体和MutS与MutL复合物的结构，以确定在腺嘌呤核苷酸存在和不存在的情况下，在与异源双链DNA的三元复合物中两种蛋白质的寡聚状态。AFM力谱将用于测量MutS二聚体内的相互作用以及腺嘌呤核苷酸对这些相互作用的影响，而光镊将用于测量MutS四聚体内以及MutS四聚体与DNA之间的类似相互作用。MutS控制的DNA循环机制和途径将使用光镊进行彻底检查。SM-FRET将用于真实的跟踪MMR蛋白相对于关键DNA位点（错配位点和链信号位点）的位置，并且在MMR反应期间，通过机械和光学（荧光）检测，通过所有三种技术探测这些关键DNA位点之间的距离和接触。这些单分子方法将测试一种新的MMR信号模型，其中错配位点和DNA切割位点通过两个MutS二聚体通过DNA环滑动机制的协同工作而接触。这些方法有望解开MMR机制的细节，到目前为止，已被证明是非常难以阐明的传统方法。新的结果将显着扩大知识基础的一个关键机制，负责维持基因组的稳定性，并将奠定基础，为未来的研究真核MMR，包括人类MMR。为这个模型系统开发的方法可能被证明是有用的研究其他DNA修复系统，更普遍地调查蛋白质-蛋白质和DNA-蛋白质相互作用。该项目将为两名研究生和一名本科生提供一个令人兴奋的教育和研究机会。由于该项目跨越多个学科（生物物理学，生物化学，分子生物学，力学，单分子仪器），它将为所有参与人员，初级和高级研究人员提供丰富的学习经验。它将使工程专业的学生接触到现代生物学和纳米科学之间的重要问题。其中一名研究生将访问韩国，接受用于这项研究的复杂单分子光学方法的培训。这种国际交流将有利于扩大美国和外国领先的研究和教育机构之间的国际合作。对社会的潜在好处是增加了对DNA中突变前损伤修复机制的基础知识。外联活动将涉及K-12学生，他们的教师和公众。

Intellectual Merit:During DNA synthesis in living cells, potential for erroneous incorporation of nucleobases (e.g. introduction of a mismatch such as a thymine instead of a cytosine across from a guanine) is as high as 10% per nucleotide. These pro-mutagenic DNA alterations need to be removed through post-replication mismatch repair (MMR) systems. Malfunction of MMR has disastrous consequences. Even though tremendous progress has been made in elucidating DNA mismatch repair, mainly through biochemical studies, many critically important details of DNA repair mechanisms remain obscure and controversial, even for bacterial (E. coli) MMR system, which is the simplest and best characterized. This is because MMR involves an orchestrated activity of many proteins, which form transient complexes that are difficult to monitor in real time. In the previous project, atomic force microscopy (AFM) was used to directly visualize and examine the behavior of MutS, the key E.coli MMR protein that detects mismatched bases. Those AFM results, which reconciled the main features of previous, mutually exclusive models, provided new insights into the mechanism by which E.coli MMR is initiated. The main goal of the current research is to further unravel the mechanism of E. coli mismatch repair through the application of a suite of single-molecule techniques such as AFM,Optical Tweezers (OT) and single-molecule fluorescence resonance energy transfer (SMFRET). Specifically, AFM imaging and volumetric measurements will be used to examine the structure of MutS tetramers and MutS complexes with MutL to determine the oligomeric status of both proteins in ternary complexes with heteroduplex DNA in the absence and presence of adenine nucleotides. AFM force spectroscopy will be used to measure the interactions within MutS dimers as well as the effect of adenine nucleotides on these interactions, while optical tweezers will be used to measure similar interactions within MutS tetramers as well as between MutS tetramers and DNA. MutS-controlled DNA looping mechanisms and pathways will be thoroughly examined using Optical Tweezers. SM-FRET will be used to follow, in real time, the position of the MMR proteins relative to key DNA sites (the mismatch site and strand signal site) and the distance and contact between these key DNA sites will be probed during the MMR reaction by all three techniques through mechanical and optical (fluorescence) detection. These single-molecule approaches will test a new MMR signaling model, in which the mismatch site and the site of DNA incision are brought into contact by the concerted work of two MutS dimers through a DNA looping-sliding mechanism. These approaches promise to unravel the details of the MMR mechanism that so far have proved very difficult to elucidate by traditional methods. The new results will significantly expand the knowledge base about one of the key mechanisms that is responsible for the maintenance of genomic stability and will lay the groundwork for future studies of eukaryotic MMR including human MMR. Methodologies developed for this model system may prove useful for studying other DNA repair systems and more generally to investigate protein-protein and DNA-protein interactions.Broader impacts. This project will provide an exciting education and research opportunity for two graduate students and one undergraduate student. Because the project crosses several disciplines (biophysics, biochemistry, molecular biology, mechanics, single-molecule instrumentation) it will provide a rich learning experience for all people involved, junior and senior researchers alike. It will expose engineering students to important problems at the interface between modern biology and nanoscience. One of the graduate students involved will visit South Korea to receive training in sophisticated single-molecule optical methods to be used in this research. This international exchange will be beneficial to expanding international collaborations between leading US and foreign research and educational institutions. The potential benefit to society is in the increase in fundamental knowledge of mechanisms by which pre-mutagenic lesions are repaired in DNA. Outreach activities will involve K-12 students, their teachers and the general public.