Mechanistic Characterization of the First Steps of Human DNA Break Repair

项目摘要 我们的基因组编码每个细胞、组织和组织的健康功能所需的关键信息 管风琴。然而，基因组dna不断积累着在正常细胞中产生的毒性损伤。 过程，或者是由阳光和化学致癌物等环境条件引起的。双倍- DNA断裂(DSB)是最危险的损伤。当DNA的两条链都发生DSB时 双螺旋结构紧密相连地断裂，将染色体分割成两个截然不同的片段。 如果不加以修复，即使是一个DSB也会引发细胞功能障碍、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：容易出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 仍然不是完全清楚的。因此，迫切需要了解健康的细胞是如何修复其 以及这些过程中的干扰如何导致癌症。 我们的长期目标是了解特殊的dna修复蛋白如何作为分子。 基因组的保管者。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，可以 对多种酶进行成像，并记录它们在实时修复DNA时的生化活动。使用这个 技术，这个提案的目的是研究一组人类酶是如何协调第一步的 DSB修理部。首先，我们将确定Mre11/Rad50/Nbs1(MRN)复合体作为分子的作用 染色质背景下的DSB传感器。其次，我们将调查MRN如何招募更多的酶 DSB，以及这些酶是如何处理核小体包裹的DNA轨迹的。第三，我们将决定如何 MRN引导修复朝向同源重组途径。总而言之，我们的研究将阐明第一个 DSB修复的关键步骤和回答长期存在的问题，即这些酶如何在生物化学上定义 DSB修复途径。最终，这些知识将被用于开发新的诊断和 专门针对已经失去正确修复基因组能力的癌细胞的治疗技术。

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.