Molecular mechanisms of DNA mismatch repair initiation

DNA错配修复启动的分子机制

• 批准号: 10155520
• 负责人: Keith R Weninger
• 金额: $ 27.3万
• 依托单位: NORTH CAROLINA STATE UNIVERSITY RALEIGH
• 依托单位国家: 美国
• 财政年份: 2019
• 资助国家: 美国
• 起止时间: 2019-07-25 至 2023-05-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/10155520
• 关键词: Bacteria; Base Pairing; Behavior; Binding; Biological; Biological Assay; Cell physiology; Cells; Closure by clamp; Communication; Complex; DNA; DNA Damage; DNA Double Strand Break; DNA replication fork; Data; Daughter; Detection; Disease; Event; Excision; Failure; Fluorescence Resonance Energy Transfer; Genetic; Genetic Recombination; Genome Stability; Genomics; Goals; Homologous Gene; Human; In Vitro; Individual; Knowledge; Link; Malignant Neoplasms; Measurement; Methods; Mismatch Repair; Molecular; Molecular Conformation; Motion; Outcome; Pathway interactions; Phenotype; Polymerase; Population; Positioning Attribute; Process; Protein Conformation; Proteins; Publications; Repair Complex; Replication Error; Resistance; Side; Signal Transduction; Slide; Structure; System; Tertiary Protein Structure; Thermus; Work; base; cancer therapy; chemotherapy; design; experimental study; human DNA; in vivo; mutant; novel; particle; protein complex; recruit; repaired; response; single molecule; single-molecule FRET; therapy design

Project Summary: Molecular Mechanisms of DNA mismatch repair initiation DNA mismatch repair (MMR) is fundamental to genetic stability. In humans, DNA MMR not only corrects mismatch errors that escape polymerase proofreading in replication, but also it is involved in multiple aspects of cellular physiology including double-strand DNA break repair, recombination, and cellular responses to DNA damage. Failure of DNA MMR in humans is directly linked to several cancers in humans as well as contributing to resistance to chemotherapy. The goal of this project is to determine the molecular interactions that connect DNA mismatch recognition to downstream repair events. How this communication occurs remains one of the most mysterious and controversial aspects of MMR. Multiple populations of the initial mismatch recognition protein complex have been observed ranging from mobile clamps sliding away from the mismatch to static complexes growing at the mismatch. We will use single molecule methods to sort these various subpopulations and determine which interacts with replication processivity clamp, which is essential for subsequent MMR signaling, and will indicate on-repair-pathway states. In our previous publications, we established the existence of these subpopulations (Qiu et al., PNAS 2015). Our project will apply single molecule FRET (smFRET) and tethered particle motion (TPM) experiments to reveal details of the interactions among MMR proteins as well as the impact on DNA conformation. We will develop novel combinations of these assays (TPM+smFRET and smFRET inside live cells) to enable sensitive measurements not previously possible. In addition, we will directly compare results from human and Thermus aquaticus systems to establish conserved features. Guided by strong preliminary data, we designed 3 aims to achieve these goals. Aim 1. Determine MutS:MutL interactions with β-clamp that drive downstream MMR We will use our smFRET assay to determine whether sliding or static Taq MMR complexes interact with β-clamp, which is the next, essential step in MMR signaling. Aim 2. In vivo determination of MutS and MutL conformational dynamics with smFRET Using single molecule FRET, we will characterize MMR protein conformational dynamics in live cells. Aim 3. Determine which human and Taq MMR complexes activate downstream excision We will use a tethered particle motion (TPM) assay sensitive to DNA bending, DNA compaction and single strand excision to detect which interactions permit continuation of the MMR cascade beyond the initial recognition complex, to the point of DNA excision. These studies will reveal the basic mechanisms that underlie mismatch repair, which will be important for designing treatment of cancers involving malfunction of DNA mismatch repair.

DNA错配修复启动的分子机制 DNA错配修复(MMR)是遗传稳定性的基础。在人类中，DNA MMR不仅 纠正在复制过程中逃过聚合酶校对的错配错误，但它也参与了 细胞生理学的多个方面，包括双链DNA断裂修复、重组和 细胞对DNA损伤的反应。人类DNA MMR的失败直接与几个 对人类癌症的危害以及对化疗的抗药性。 这个项目的目标是确定连接DNA错配的分子相互作用 对下游维修事件的识别。这种交流是如何发生的，仍然是最多的 MMR的神秘和有争议的方面。多种群初始失配识别 观察到了蛋白质复合体，从可移动的钳子从错配滑动到 静态复合体在错配处生长。我们将使用单分子方法来对这些不同的 亚群，并确定哪些与复制过程钳相互作用，这是必不可少的 用于随后的MMR信号，并将指示修复途径状态。在我们之前的 在发表的文章中，我们确定了这些亚群的存在(邱等人，PNAS 2015)。 我们的项目将应用单分子FRET(SmFRET)和拴系粒子运动(TPM) 揭示MMR蛋白之间相互作用及其对DNA影响的实验细节 构象。我们将开发这些分析的新组合(TPM+smFRET和smFRET 在活细胞内)，以实现以前不可能进行的灵敏测量。此外，我们还将直接 比较人类和水生热水鱼系统的结果，以建立保守的特征。 在强大的初步数据的指导下，我们设计了3个目标来实现这些目标。 目的1.确定MutS：MutL与驱动下游MMR的β钳的相互作用 我们将使用我们的smFRET分析来确定滑动或静态Taq MMR复合体是否相互作用 使用β钳位，这是MMR信号的下一个关键步骤。 目的2.SmFRET测定MutS和MutL的体内构象动力学 利用单分子FRET，我们将表征活细胞中MMR蛋白的构象动力学。 目的3.确定哪些人和Taq MMR复合体激活了下游切除 我们将使用拴系粒子运动(TPM)分析对DNA弯曲、DNA紧凑和 单链切除以检测哪些相互作用允许MMR级联的延续 最初的识别复合体，到DNA切除的程度。 这些研究将揭示错配修复的基本机制，这将是重要的 用于设计涉及DNA错配修复故障的癌症的治疗方法。