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

• 批准号: 9323473
• 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
• 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/9323473
• 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; 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; Recruitment Activity; 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; repaired; sensor; single molecule; temporal measurement; tool; tumor growth

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.

项目摘要 我们的基因组编码每个细胞，组织和组织健康功能所需的关键信息 器官。然而，基因组DNA是在正常细胞期间连续积累的毒性损伤 过程，或者是由阳光和化学致癌物等环境条件引起的。双倍的- 滞留的DNA断裂（DSB）是最危险的病变。当DNA的两个链时发生DSB 双螺旋在彼此近距离上被打破，将染色体分成两个不同的碎片。 如果未修复，即使是单个DSB也可以引起细胞功能障碍，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来修复DSB：易于错误的非同源末端或高保真 同源重组。值得注意的是，确定DNA修复途径的主要分子步骤 仍然不完全知道。因此，迫切需要了解健康的细胞如何修复其 碎片DNA以及这些过程中的破坏如何导致癌症。 我们的长期目标是了解专门的DNA修复蛋白如何用作分子 基因组的看护人。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 图像多种酶，并在实时修复DNA时记录其生化活性。使用此 技术，该提案的目的将研究一组人酶如何协调第一步 DSB修复。首先，我们将确定MRE11/RAD50/NBS1（MRN）络合物如何充当分子 DSB的传感器在染色质的背景下。其次，我们将研究MRN如何募集其他酶 到DSB，以及这些酶如何处理核小体涂层的DNA轨道。第三，我们将确定如何 MRN将修复指向同源重组途径。总而言之，我们的研究将阐明第一个 DSB修复的关键步骤并回答了这些酶如何定义这些酶的长期问题 DSB维修途径。最终，开发新诊断和 专门针对失去正确修复其基因组能力的癌细胞的治疗剂。