Mechanistic Characterization of the First Steps of Human DNA Break Repair

项目摘要 我们的基因组编码的关键信息是每个细胞，组织， 器官.然而，基因组DNA在正常细胞生长过程中不断积累毒性损伤， 过程，或由环境条件如阳光和化学致癌物引起。双倍- DNA链断裂（DSB）是最危险的损伤。当DNA的两条链 双螺旋在彼此非常接近的地方断裂，将染色体分成两个不同的片段。 如果未修复，即使是单个DSB也可以引发细胞功能障碍、恶性转化和肿瘤生长。 我们的细胞可以通过两种不同的途径修复DSB：易错的非同源末端连接或高保真 同源重组值得注意的是，决定DNA修复途径的主要分子步骤 仍然不完全清楚。因此，迫切需要了解健康细胞如何修复它们的功能。 片段化的DNA以及这些过程的中断如何导致癌症。 我们的长期目标是了解专门的DNA修复蛋白如何作为分子 基因组的守护者为了实现这一目标，我们开创了一种独特的体外显微镜技术， 对多种酶进行成像并记录它们在真实的修复DNA时的生化活动。使用此 这项技术的目的是研究一组人类酶如何协调第一步 DSB修复首先，我们将确定Mre 11/Rad 50/Nbs 1（MRN）复合物如何作为分子 在染色质的背景下的DSB传感器。其次，我们将研究MRN如何招募额外的酶 到DSB，以及这些酶如何处理核小体包裹的DNA轨道。第三，我们将确定如何 MRN引导修复朝向同源重组途径。总之，我们的研究将阐明第一个 DSB修复的关键步骤，并回答这些酶如何生化定义的长期问题 DSB修复途径。最终，开发新的诊断方法需要这些知识， 这些治疗剂特异性靶向已经失去正确修复其基因组能力的癌细胞。

Project Summary Our genome encodes critical information that is required for the healthy function of every cell, tissue, and organ. However, genomic DNA is continuously accumulating toxic damage that arises during normal cellular processes, or is caused by environmental conditions such as sunlight and chemical carcinogens. Double- stranded DNA breaks (DSBs) are the most dangerous lesions. DSBs occur when both strands of the DNA double helix are broken in close proximity to each other, fragmenting the chromosome into two distinct pieces. If unrepaired, even a single DSB can initiate cellular dysfunction, malignant transformation, and tumor growth. Our cells can repair DSBs via two distinct pathways: error-prone non-homologous end joining or high-fidelity homologous recombination. Remarkably, the primary molecular steps that determine the DNA repair pathway are still not completely known. Thus, there is a critical need to understand how healthy cells repair their fragmented DNA and how disruptions in these processes can lead to cancer. Our long-term goal is to understand how specialized DNA repair proteins serve as the molecular caretakers of the genome. To achieve this goal, we pioneered a unique in vitro microscopy technique that can image multiple enzymes and record their biochemical activities as they repair DNA in real time. Using this technique, the Aims in this proposal will investigate how a group of human enzymes coordinate the first steps of DSB repair. First, we will determine how the Mre11/Rad50/Nbs1 (MRN) complex acts as the molecular sensor for DSBs in the context of chromatin. Second, we will investigate how MRN recruits additional enzymes to the DSB, and how these enzymes process a nucleosome-coated DNA track. Third, we will determine how MRN directs repair towards the homologous recombination pathway. In sum, our studies will elucidate the first critical steps of DSB repair and answer the long-standing question of how these enzymes biochemically define the DSB repair pathway. Ultimately, this knowledge will be required for developing new diagnostics and therapeutics that specifically target cancer cells that have lost the ability to correctly repair their genomes.