Integrative single molecule studies: DNA repair and technology development

项目摘要 这项提案的总体目标是利用现有的和开发新的单分子技术， 对DNA修复过程中发生的关键过程的机械见解。DNA修复过程， 基因组的守护者，涉及多个连续的酶促步骤，需要协调组装 以及许多蛋白质对DNA的作用。这些相互作用的短暂性质带来了重大挑战 使用传统的生物化学方法阐明DNA修复的分子机制。单分子 这些方法非常适合克服这些困难;然而，它们也提出了自己的挑战，需要 创新的解决方案。我的实验室致力于阐明DNA错配修复的分子机制 (MMR)以及开发单分子工具，让我们获得以前无法获得的信息。 MMR在减少基因组突变方面起着重要作用，包括纠正DNA复制错误， 调节细胞对DNA损伤剂的反应，并防止分化的 序列的MMR蛋白的突变导致Lynch综合征，这是最常见的遗传性 癌症，以及对用于治疗癌症的几种DNA损伤剂的抗性。令人惊讶的是，在三核苷酸 重复序列（TNR）扩增，导致一些神经退行性疾病，MMR蛋白引起突变， 促进重复扩张和疾病。MutSa/MutSb通过结合错配/插入启动MMR 缺失环，并经历ATP依赖性构象变化，促进其与一个或多个 更多MutLa蛋白随后，PCNA和ATP激活MutLa切割子链，MutSa 激活EXO 1以将含有错误的DNA原切掉，然后再合成和连接。 类似地，在TRE中，MutSb结合环出的DNA三核苷酸重复序列并募集MutLa和/或MutLg，但 这一过程不是导致修复，而是促进扩张。了解分子机制， 这些不同过程的基础对于开发相关癌症的有效治疗至关重要， 神经退行性疾病单分子、结构和生物化学研究，包括我们的一些研究。 实验室，表明蛋白质和蛋白质-DNA的构象动力学和组装状态， 复合物是调节MMR和TRN扩增的中心。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办法， 一系列单分子技术，包括AFM和单分子荧光，以检查MMR在 在体外和体内的多种生物体，以及启动TNR扩增的研究。我们将专注于 检查MMR和TNR启动期间DNA上蛋白质的时间和空间组装 扩张.最后，我们将继续开发单分子工具，例如DREEM，它可以让我们“看到”。 蛋白质-DNA复合物内的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，并直接有利于我们的MMR研究。

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.