Integrative single molecule studies: DNA repair and technology development

项目摘要 这项提议的总体目标是开发和应用单分子技术来获得机械 对DNA修复过程中发生的关键过程的洞察。DNA修复过程，这是守护者 基因组，涉及多个顺序的酶步骤，需要协调组装和作用 DNA上有很多蛋白质。这些交互的瞬变性质在以下方面提出了重大挑战 用传统的生化方法阐明DNA修复的分子机制。单分子 方法非常适合于克服这些困难；然而，它们提出了自己的挑战，需要 创新解决方案。我实验室的研究重点是阐明DNA的分子机制 错配修复(MMR)和单分子工具的开发将使我们能够访问以前 无法获得的信息和/或极大地促进了实施或分析的吞吐量。MMR扮演着 在避免突变方面发挥重要作用，包括纠正DNA复制错误，调节细胞反应 DNA损伤剂，并防止不同序列之间的重组。突变 失活的MMR蛋白会导致林奇综合征，这是最常见的遗传性癌症。此外，它们还会导致 对几种DNA损伤剂的细胞毒性作用的耐药性，这些药物经常用于治疗白血病 癌症。因此，了解这些不同过程背后的分子机制是至关重要的。 为相关癌症开发有效的治疗方法。MUTSα通过绑定到不匹配启动修复 以及经历依赖于三磷酸腺苷的构象变化，促进其与一个或多个MutLα的相互作用 蛋白质。随后，增殖细胞核抗原激活MutLα，以依赖于ATP的方式切割子链。 一旦MutLα将DNA5‘端错配，MutSα可激活5’-3‘外切酶EXO1 切除含有错误的δ/ε或促进POL DNA启动链置换合成。最后， DNA聚合酶δ或ε催化再合成，DNA连接酶封闭缺口。单分子、结构和 生化研究，包括我们实验室的几项研究表明，构象动力学和 蛋白质和蛋白质-DNA复合体的组装状态是MMR调控的中心。我们会 扩大我们正在进行的研究，以破译MMR的关键分子机制。我们要参加一场 一种综合方法，我们利用一系列单分子技术来检测多个 体外和体内的生物体。我们将重点研究MMR的时间和空间组装 在MMR启动过程中DNA上的蛋白质。为了进一步提高我们(和其他人)探索这些机制的能力，我们 将继续开发新的单分子工具，重点是：1)开发高通量平台 用于原子力显微镜样品的制备和成像；2)优化我们新发明的静电力 一种名为DREEM的成像技术，使我们能够“看到”蛋白质-DNA复合体中的DNA。除了……之外 扩展单分子工具箱，这项技术将直接有益于我们对MMR的研究。

Project Summary The overall objective of this proposal is to develop and apply single-molecule techniques to gain mechanistic insights into the critical processes occurring during DNA repair. DNA repair processes, which are the guardian of the genome, involve multiple sequential enzymatic steps that require the coordinated assembly and action of many proteins on the DNA. The transient nature of these interactions presents significant challenges in elucidating the molecular mechanisms of DNA repair using traditional biochemical methods. Single molecule approaches are well suited to overcome these difficulties; however, they present their own challenges, requiring innovative solutions. Research in my laboratory focuses on elucidating the molecular mechanisms of DNA mismatch repair (MMR) and on the development of single-molecule tools that will give us access to previously unattainable information and/or greatly facilitate throughput of implementation or analysis. MMR plays a major role in mutation avoidance, including correcting DNA replication errors, modulating cellular responses to DNA damaging agents, and preventing recombination between diverged sequences. Mutations that inactivate MMR proteins cause Lynch syndrome, the most common hereditary cancer. In addition, they cause resistance to the cytotoxic effects of several DNA damaging agents that are often used in the treatment of cancer. As such, understanding the molecular mechanisms that underlie these different processes is essential for developing effective treatments for the associated cancers. MutSα initiates repair by binding to a mismatch and undergoing ATP-dependent conformational changes that promotes its interaction with one or more MutLα proteins. Subsequently, PCNA activates MutLα to incise the daughter strand in an ATP-dependent manner. Once MutLα nicks the DNA 5' to the mismatch, MutSα can activate the 5'-3' exonuclease EXO1 to processively excise the DNA containing the error or promote POLδ/ε to initiate strand-displacement synthesis. Finally, DNA polymerase δ or ε catalyzes resynthesis, and DNA ligase seals the nick. Single-molecule, structural, and biochemical studies, including several from our laboratory, indicate that the conformational dynamics and assembly states of the proteins and protein-DNA complexes are central to the regulation of MMR. We will extend our ongoing studies to decipher the molecular mechanisms critical to MMR. We are taking an integrative approach in which we utilize an array of single-molecule techniques to examine MMR in multiple organisms in vitro and in vivo. We will focus on examining the temporal and spatial assembly of MMR proteins on the DNA during MMR initiation. To further our (and others) ability probe these mechanisms, we will continue to develop new single-molecule tools, focusing on: 1) development of a high-throughput platform for preparation and imaging of AFM samples and 2) optimization of our newly invented electrostatic force imaging technique, called DREEM, that allows us to “see” DNA inside protein-DNA complexes. In addition to expanding the single-molecule toolbox, the this technology will directly benefit our studies of MMR.