Single-Molecule Studies of Human DNA Double Strand Break Repair

项目总结/摘要 我们的基因组DNA编码的关键信息是每个细胞，组织， 器官.然而，DNA在正常细胞过程中不断积累毒性损伤， 或由环境条件如阳光和化学致癌物引起。双链DNA 破裂（DSB）是最危险的病变。当DNA双螺旋的两条链都 彼此靠近断裂，将染色体分裂成两个不同的片段。如果不修理， 即使是单一的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：快速，易错的反应或通过第二个过程， 无任何错误值得注意的是，决定DNA修复途径的主要分子步骤仍然没有 完全知道。因此，迫切需要了解健康细胞如何修复其碎片化的DNA 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 基因组的守护者在我的研究生工作中，我将研究一组人类酶如何协调 DSB修复的第一步我将首先研究Mre 11/Rad 50/Nbs 1（MRN）复合体如何作为 DSBs的分子传感器。我还将探讨如何MRN利用其多种生化活动开始 处理游离DNA末端。接下来，我将确定MRN如何招募额外的酶，以及这是如何实现的。 这些蛋白质的空间和时间有序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，我将描述这些如何 DNA修复蛋白识别并阻断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 无法直接观察同一DNA上多种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， 单个的DNA分子，并记录多个酶在真实的时间内修复DNA的电影。使用此 在荧光显微镜下，我将直接观察关键的人类酶如何协调它们的行动， 无差错的DNA修复这些研究的预期结果将回答一个长期存在的问题， 人的DNA被修复。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/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.