Investigating DNA Mismatch Repair Through Single-Molecule Approaches

智力优势：在活细胞的DNA合成过程中，核苷酸碱基错误结合的可能性高达每个核苷酸10%(例如，引入不匹配的胸腺嘧啶而不是鸟嘌呤的胞嘧啶)。这些促突变的DNA改变需要通过复制后错配修复(MMR)系统来移除。MMR的故障会带来灾难性的后果。尽管在阐明DNA错配修复方面已经取得了巨大的进展，主要是通过生物化学研究，但DNA修复机制的许多关键细节仍然不清楚和有争议，即使对于细菌(E.Coli)MMR系统也是如此，这是最简单和最具特征的系统。这是因为MMR涉及许多蛋白质的精心编排的活动，这些蛋白质形成了难以实时监测的瞬时复合体。在之前的项目中，原子力显微镜(AFM)被用来直接可视化和检查MutS的行为，MutS是大肠杆菌MMR的关键蛋白质，可以检测不匹配的碱基。这些AFM结果与以前相互排斥的模型的主要特征相一致，为启动E.Coli MMR的机制提供了新的见解。本研究的主要目的是通过应用原子力显微镜、光钳(OT)和单分子荧光共振能量转移(SmFRET)等一系列单分子技术，进一步揭示大肠杆菌错配修复的机制。具体地说，AFM成像和体积测量将用于研究MutS四聚体和MutS与MutL的复合体的结构，以确定在没有和存在腺嘌呤核苷酸的情况下，这两种蛋白质在与异源双链DNA的三元复合体中的寡聚状态。AFM力光谱将被用来测量MutS二聚体内部的相互作用以及腺嘌呤核苷酸对这些相互作用的影响，而光学镊子将被用来测量MutS四聚体内部以及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钳进行彻底检查。SM-FRET将被用来实时跟踪MMR蛋白质相对于关键DNA位点(错配位点和链信号位点)的位置，以及这些关键DNA位点之间的距离和接触将通过所有三种技术通过机械和光学(荧光)检测来探测。这些单分子方法将测试一种新的MMR信号模型，在该模型中，错配位置和DNA切割位置通过DNA环-滑动机制由两个MutS二聚体协同工作而接触。这些方法有望揭开MMR机制的细节，到目前为止，这些细节很难用传统方法来阐明。这些新的结果将极大地扩展关于负责维持基因组稳定性的关键机制之一的知识库，并将为未来包括人类MMR在内的真核MMR的研究奠定基础。为这个模型系统开发的方法可能被证明对研究其他DNA修复系统以及更广泛地研究蛋白质-蛋白质和DNA-蛋白质相互作用有用。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。由于该项目跨越了几个学科(生物物理学、生物化学、分子生物学、力学、单分子仪器)，它将为所有参与其中的人提供丰富的学习体验，无论是初级还是高级研究人员。它将使工程学学生接触到现代生物学和纳米科学之间的重要问题。其中一名参与研究的研究生将访问韩国，接受这项研究中使用的尖端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中前突变损伤修复机制的基础知识。外展活动将涉及K-12学生、他们的老师和普通公众。

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.