Single-Molecule Studies of Human DNA Double Strand Break Repair

项目摘要/摘要 我们的基因组DNA编码了每个细胞、组织和组织的健康功能所需的关键信息 管风琴。然而，DNA正在不断积累在正常细胞过程中产生的毒性损伤， 或者是由阳光和化学致癌物等环境条件引起的。双链DNA 破损(DSB)是最危险的损伤。当DNA双螺旋的两条链都是 彼此紧密相连断裂，将染色体分割成两个截然不同的片段。如果没有修复， 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：快速、容易出错的反应或通过第二个过程 没有错误。值得注意的是，决定DNA修复途径的主要分子步骤仍然不是 完全知道。因此，迫切需要了解健康细胞是如何修复其碎片dna的。 以及这些过程的中断如何导致癌症。 我的长期目标是了解专门的dna修复蛋白如何作为分子。 基因组的保管者。在我的研究生工作中，我将研究一组人类酶是如何协调的 DSB修复的第一步。我将首先研究Mre11/Rad50/Nbs1(MRN)复合体是如何发挥作用的 DSB分子传感器。我还将探索MRN如何利用其多种生化活动开始 处理自由的DNA结束了。接下来，我将确定MRN如何招募额外的酶，以及如何 这些蛋白质在空间和时间上有序的组装催化了第一个生化步骤， 确定DSB修复途径。当我过渡到博士后职位时，我将描述这些 DNA修复蛋白识别并被阻止在DNA的正常末端，端粒。 破译这些关键的分子事件仍然具有挑战性，因为传统的方法 无法直接观察到同一DNA上多个修复蛋白的复杂分子编排 分子。为了实现我的目标，我开创了一种独特的、超灵敏的显微镜技术，可以成像 当它们实时修复DNA时，可以记录单个DNA分子和多个酶的电影。使用这个 在荧光显微镜下，我将直接观察关键的人体酶是如何协调它们的活动来启动的 无错误的DNA修复。这些研究的预期结果将回答一个长期存在的问题，即如何 人类的DNA被修复了。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/Abstract Our genomic DNA encodes critical information that is required for the healthy function of every cell, tissue, and organ. However, 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: a rapid, error-prone reaction or via a second process that is largely error-free. 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. My long-tern goal is to understand how specialized DNA repair proteins serve as the molecular caretakers of the genome. In my graduate work, I will investigate how a group of human enzymes coordinate the first steps of DSB repair. I will first investigate how the Mre11/Rad50/Nbs1 (MRN) complex acts as the molecular sensor for DSBs. I will also explore how MRN harnesses its multiple biochemical activities to begin processing the free DNA ends. Next, I will determine how MRN recruits additional enzymes, and how this spatially and temporally ordered assembly of these proteins catalyzes the first biochemical steps that determine the DSB repair pathway. As I transition into a postdoctoral position, I will characterize how these DNA repair proteins recognize and are blocked at the normal ends of DNA, telomeres. Deciphering these critical molecular events remains challenging because traditional approaches are unable to directly observe the intricate molecular choreography of multiple repair proteins on the same DNA molecule. To achieve my aims, I have pioneered a unique, ultra-sensitive microscopy technique that can image individual molecules of DNA and record movies of multiple enzymes as they repair DNA in real time. Using this fluorescence microscope, I will directly observe how critical human enzymes coordinate their actions to initiate error-free DNA repair. The anticipated results of these studies will answer a long-standing question of how human DNA is repaired. 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.