Molecular mechanisms of DNA mismatch repair initiation

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 纠正在复制中逃避聚合酶校正的错配错误，但它也参与 细胞生理学的多个方面，包括双链DNA断裂修复，重组， 细胞对DNA损伤的反应人类DNA MMR的失败与几个直接相关 人类的癌症以及对化疗的抵抗力。 这个项目的目标是确定连接DNA错配的分子相互作用 识别下游修复事件。这种沟通是如何发生的仍然是最重要的问题之一。 MMR的神秘和争议方面。初始错配识别的多个群体 已经观察到蛋白质复合物的范围从从移动的夹钳滑离错配到 在失配处生长的静态复合物。我们将使用单分子方法对这些不同的 亚群，并确定哪些与复制持续性钳相互作用，这是必不可少的 用于随后的MMR信号传导，并将指示修复途径状态。在我们以前 根据这些出版物，我们确定了这些亚群的存在（Qiu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） 这些实验揭示了MMR蛋白质之间相互作用的细节以及对DNA的影响， 构象我们将开发这些检测的新组合（TPM+smFRET和smFRET 在活细胞内）以实现先前不可能的灵敏测量。此外，我们将直接 比较人类和水生栖热菌系统的结果，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。 目标1.确定MutS：MutL与β-钳的相互作用，其驱动下游MMR 我们将使用我们的smFRET试验，以确定是否滑动或静态Taq MMR复合物相互作用 这是MMR信号传导的下一个重要步骤。 目标二。用smFRET在体内测定MutS和MutL构象动力学 使用单分子FRET，我们将表征MMR蛋白在活细胞中的构象动力学。 目标3。确定哪些人和Taq MMR复合物激活下游切除 我们将使用对DNA弯曲、DNA压缩和DNA断裂敏感的系留粒子运动（TPM）测定。 单链切除，以检测哪些相互作用允许MMR级联反应继续， 最初的识别复合物，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。

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.