Integrative single molecule studies: DNA repair and technology development

项目摘要 这项提议的总体目标是利用现有的和开发新的单分子技术来获得 对DNA修复过程中发生的关键过程的机械性见解。DNA修复过程，这是 基因组的守护者，涉及需要协调组装的多个顺序的酶步骤 以及多种蛋白质对DNA的作用。这些相互作用的暂时性带来了巨大的挑战 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。我的实验室致力于阐明DNA错配修复的分子机制 (MMR)和单分子工具的开发，使我们能够获得以前无法获得的信息。 MMR在减少基因组突变方面发挥着重要作用，包括纠正DNA复制错误， 调节细胞对DNA损伤剂的反应，防止不同分支之间的重组 序列。MMR蛋白失活的突变会导致林奇综合征，这是最常见的遗传性疾病 癌症，以及对几种用于治疗癌症的DNA损伤剂的耐药性。令人惊讶的是，在三核苷酸中 重复(TNR)扩张，导致一些神经退行性疾病，MMR蛋白导致突变 促进重复扩张和疾病。Mutsa/MutSb通过绑定到不匹配/插入来启动MMR 缺失环，并经历依赖于ATP的构象变化，促进其与一个或一个或多个 更多的Mutla蛋白质。随后，增殖细胞核抗原和三磷酸腺苷激活MUTRA切割子链，MUTSA 激活EXO1，连续切除含有错误的DNA，然后重新合成和连接。 同样，在Tre中，MutSb结合循环的DNA三核苷酸重复并招募Mutla和/或MutLg，但 这一过程不仅没有导致修复，反而促进了扩张。了解分子机制， 这些不同的过程对于开发有效的治疗相关癌症和 神经退行性疾病。单分子、结构和生化研究，包括我们的几个 实验室，表明蛋白质和蛋白质-DNA的构象动力学和组装状态 复合体是MMR和TRN扩张调控的中心。我们将继续我们的机械研究 并将其扩展到TNR扩展。我们正在采取一种综合的方法，其中我们利用 一系列单分子技术，包括原子力显微镜和单分子荧光法，以检测MMR在 在体外和体内的多种生物，以及开始研究TNR的扩张。我们将重点关注 MMR和TNR启动过程中蛋白质在DNA上的时空组装 扩张。最后，我们将继续开发单分子工具，如DREEM，它可以让我们“看到” 蛋白质-DNA复合体中的DNA和高通量AFM。这些技术扩展了单一的- 分子工具箱，丰富了原子力显微镜的结构信息，为我们的MMR研究提供了直接的帮助。

Project Summary The overall objective of this proposal is to use existing and develop new 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 DNA. The transient nature of these interactions presents significant challenges to 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. My laboratory focuses on elucidating the molecular mechanisms of DNA mismatch repair (MMR) and on development of single-molecule tools that give us access to previously unattainable information. MMR plays a major role in reducing genomic mutations, 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, as well as resistance to several DNA damaging agents used to treat cancer. Surprisingly, in trinucleotide repeat (TNR) expansion, which causes some neurodegenerative diseases, MMR proteins cause mutations that promote repeat expansion and disease. MutSa/MutSb initiates MMR by binding to a mismatch/insertion deletion loop and undergoing ATP-dependent conformational changes that promote its interaction with one or more MutLa proteins. Subsequently, PCNA and ATP activate MutLa to incise the daughter strand, and MutSa activates EXO1 to processively excise the DNA containing the error, followed by resynthesis and ligation. Similarly, in TRE, MutSb binds looped out DNA trinucleotide repeats and recruits MutLa and/or MutLg, but instead of leading to repair, this process promotes expansions. Understanding the molecular mechanisms that underlie these different processes is essential for developing effective treatments for the associated cancers and neurodegenerative diseases. 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 and TRN expansion. We will continue our mechanistic studies of MMR and extend them to TNR expansion. We are taking an integrative approach in which we utilize an array of single-molecule techniques, including AFM and single-molecule fluorescence, to examine MMR in multiple organisms in vitro and in vivo, as well as initiating studies on TNR expansion. We will focus on examining the temporal and spatial assembly of proteins on DNA during initiation of MMR and TNR expansion. Finally, we will continue to develop single-molecule tools, such as DREEM that allows us to “see” DNA inside protein-DNA complexes and “high-throughput” AFM. These technologies expand the single- molecule toolbox, enrich the structural information from AFM, and directly benefit our MMR studies.