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Mechanistic Characterization of the First Steps of Human DNA Break Repair

人类 DNA 断裂修复第一步的机制表征

基本信息

批准号：
9752585
负责人：
ILYA J FINKELSTEIN
金额：
$ 29.93万
依托单位：
UNIVERSITY OF TEXAS AT AUSTIN
依托单位国家：
美国
项目类别：
财政年份：
2016
资助国家：
美国
起止时间：
2016-08-01 至 2021-07-31
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/9752585
关键词：
ATP phosphohydrolase Address Affinity Binding Biochemical Biochemical Reaction Biophysics Cell Death Cell physiology Cells Chromatin Chromosomes Complex DNA DNA Binding DNA Damage DNA Double Strand Break DNA Repair DNA Repair Gene DNA Repair Pathway DNA lesion Dangerousness Diffusion Dimensions Double Strand Break Repair Engineering Enzymes Excision Family Functional disorder Future G22P1 gene Genetic Genome Genome Scan Genomic DNA Goals Histone H2A Human Image In Vitro Individual Knowledge Label Lead Lesion Life Malignant - descriptor Malignant Neoplasms Metabolism Microscopy Modeling Molecular Molecular Biology Multienzyme Complexes Nonhomologous DNA End Joining Nucleosomes Organ Pathway interactions Process Proteins Repair Complex Scanning Single-Stranded DNA Sum Sunlight Techniques Testing Therapeutic Time Tissues XRCC5 gene base cancer cell chemical carcinogen design ds-DNA helicase homologous recombination human DNA imaging platform interdisciplinary approach nanomolar nanoscale novel diagnostics novel therapeutics nuclease recruit repaired sensor single molecule temporal measurement tool tumor growth

项目摘要

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.

项目概要我们的基因组编码每个细胞、组织和健康功能所需的关键信息。器官。然而，基因组 DNA 不断积累正常细胞过程中产生的毒性损伤。过程，或由环境条件（例如阳光和化学致癌物质）引起。双倍的- 链状 DNA 断裂 (DSB) 是最危险的损伤。当 DNA 的两条链双螺旋彼此靠近地断裂，将染色体分裂成两个不同的片段。如果不修复，即使是单个 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度同源重组。值得注意的是，决定 DNA 修复途径的主要分子步骤仍不完全为人所知。因此，迫切需要了解健康细胞如何修复它们的 DNA 碎片以及这些过程的破坏如何导致癌症。我们的长期目标是了解专门的 DNA 修复蛋白如何充当分子基因组的看护者。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，可以对多种酶进行成像并实时记录它们修复 DNA 时的生化活动。使用这个技术，该提案的目标将研究一组人类酶如何协调第一步 DSB修复。首先，我们将确定 Mre11/Rad50/Nbs1 (MRN) 复合物如何充当分子染色质背景下的 DSB 传感器。其次，我们将研究 MRN 如何招募额外的酶 DSB，以及这些酶如何处理核小体包被的 DNA 轨迹。第三，我们将确定如何 MRN 将修复引导至同源重组途径。总之，我们的研究将阐明第一个 DSB 修复的关键步骤并回答了这些酶如何在生化上定义这一长期存在的问题 DSB 修复途径。最终，开发新的诊断方法和方法将需要这些知识。专门针对失去正确修复基因组能力的癌细胞的疗法。