Analysis of the Mammalian DNA Damage Response

哺乳动物 DNA 损伤反应分析

• 批准号: 10319546
• 负责人: STEPHEN J ELLEDGE
• 金额: $ 40.13万
• 依托单位: BRIGHAM AND WOMEN'S HOSPITAL
• 依托单位国家: 美国
• 财政年份: 2019
• 资助国家: 美国
• 起止时间: 2019-01-01 至 2023-12-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/10319546
• 关键词: Aging; Autophagocytosis; Breast; CRISPR screen; Cancer Model; Canes; Cell Aging; Cell Cycle Arrest; Cell Cycle Progression; Cell physiology; Cisplatin; Clustered Regularly Interspaced Short Palindromic Repeats; DNA Damage; DNA Repair; DNA biosynthesis; Event; Excision; Fanconi Anemia pathway; Fanconi anemia protein; GATA4 gene; Genes; Genetic; Genetic Screening; Genotoxic Stress; Human; Immunologics; Inherited; Link; Maintenance; Malignant Neoplasms; Malignant neoplasm of lung; Mammals; Methods; Modification; Neurologic; Open Reading Frames; Pathway interactions; Phenotype; Phosphorylation; Phosphotransferases; Physiology; Play; Process; Proteins; Proteomics; RNA Interference; Regulation; Reporter; Resistance; Role; Set protein; Signal Transduction; Signaling Protein; Site; Stimulus; Structure; Syndrome; TP53 gene; Tumor Burden; Tumor Suppression; Tumor Suppressor Genes; WHSC1 gene; Yeasts; biological adaptation to stress; brca gene; cancer predisposition; candidate validation; chemotherapy; crosslink; developmental disease; exhaustion; follow-up; gain of function; genetic analysis; genetic regulatory protein; genome integrity; genotoxicity; homologous recombination; human DNA damage; human disease; inhibitor; innovation; loss of function; metabolomics; novel; overexpression; p53-binding protein 1; prevent; protein function; repaired; replication stress; response; screening; senescence; tool; tumor; tumorigenesis

Project Summary The DNA damage Response (DDR) is a regulatory network that coordinates cellular processed in response to DNA damage and replication stress. It also directly orchestrates DNA repair choices by sensing different DNA damage structures and transduces that information in cis to activate specific and appropriate repair options, thus optimizing repair. DDR's importance is highlighted by the many cancer predisposition syndromes resulting from its inactivation, including hereditary breast caner as well as its role in promoting sensitivity to genotoxic chemotherapy and synthetic lethality with the successful treatment of BRCA1/2-deficient tumors with PARP inhibitors. In the last 25 years we, and others, have investigated the composition of the central sensing and signaling apparati that detect and respond to genotoxic stress in yeast and now mammals. This revealed a conserved core of sensing and signaling proteins. Our analysis of substrates of this DDR kinase cascade has revealed an extremely diverse set of proteins and functions contacted by the DDR in mammals and the great majority of these activities are not conserved in yeast. This includes proteins like RHINO, Abraxas, SMARCAL1, RANZB3, WHSC1, NBA1/MERIT40, RAP80, the entire Fanconi anemia pathway including FANCI, FAN1, foci regulatory proteins Mdc1, p53BP1, RNF8, RNF168/RIDDLIN, RFWD3 not to mention other key DDR patheays like p53/ p21, USP28 and BDR7. Our analysis of ATM and ATR substrates and our and other's analyses of non-ATM/ATR regulated phosphorylation events have implicated over 1000 proteins in the DDR, the vast majority of which have no previous links to the DDR and have no yeast counterparts. Therefore, we think there are many, many new components of the DDR to be discovered in mammals and it is critical that we set out to identify these factors in order to generate a complete understanding of the DDR and its significance in cellular and organismal physiology. This includes a new effort in cellular senescence, a key response to DNA damage that prevents tumorigenesis and promotes aging. Toward this end, we have developed sophisticated genetic tools that allow us to employ RNAi, CRISPR and ORF expression to find new protein candidates involved in promoting survival in response to DNA damage. We have performed three preliminary screens for loss of function and gain of function to find new DDR candidate proteins that will serve as the basis for AIM1 and AIM2. In these AIMs we propose to carry out exhaustive validation of the candidates to identify bona fide new DDR proteins and will follow up on two candidate proteins already validated. In addition we have uncovered a key protein in regulation of the senescence secretory phenotype, GATA4, which responds to ATM and ATR to activate NFkB. We will further explore its role in senescence and a new protein required for senescence, ZNF292. These innovative methods will allow us to deeply probe the layers of the DDR including senescence to identify and prioritize new proteins and to then probe the functions of these proteins in the DDR using approaches we have pioneered for so many years.

项目概要 DNA 损伤反应 (DDR) 是一个调节网络，协调细胞内的加工过程 对 DNA 损伤和复制应激的反应。它还通过以下方式直接协调 DNA 修复选择： 感知不同的 DNA 损伤结构并以顺式转换该信息以激活特定的和 适当的修复选项，从而优化修复。许多癌症凸显了 DDR 的重要性 因其失活而导致的易感综合症，包括遗传性乳腺癌及其 成功地促进了对基因毒性化疗和合成致死的敏感性的作用 用 PARP 抑制剂治疗 BRCA1/2 缺陷型肿瘤。在过去 25 年里，我们和其他人已经 研究了检测和响应的中央传感和信号装置的组成 酵母和现在哺乳动物的基因毒性应激。这揭示了传感和信号传导的保守核心 蛋白质。我们对 DDR 激酶级联底物的分析揭示了一组极其多样化的底物 哺乳动物中 DDR 所接触的蛋白质和功能，并且这些活动中的绝大多数不是 保存在酵母中。这包括 RHINO、Abraxas、SMARCAL1、RANZB3、WHSC1 等蛋白质， NBA1/MERIT40、RAP80、整个范可尼贫血途径，包括 FANCI、FAN1、病灶调节 蛋白质 Mdc1、p53BP1、RNF8、RNF168/RIDDLIN、RFWD3 更不用说其他关键 DDR 通路 如 p53/p21、USP28 和 BDR7。我们对 ATM 和 ATR 底物的分析以及我们和其他人的分析 非 ATM/ATR 调节的磷酸化事件涉及 DDR 中的 1000 多种蛋白质， 其中绝大多数以前与 DDR 没有联系，也没有酵母对应物。因此，我们 认为在哺乳动物中发现了很多很多新的 DDR 成分，这一点至关重要 我们着手确定这些因素，以便全面了解 DDR 及其 在细胞和有机体生理学中具有重要意义。这包括在细胞衰老方面的一项新努力， 对 DNA 损伤的关键反应，可预防肿瘤发生并促进衰老。为此，我们有 开发了复杂的遗传工具，使我们能够利用 RNAi、CRISPR 和 ORF 表达来寻找 新的候选蛋白质参与促进 DNA 损伤的生存。我们表演过 针对功能丧失和功能获得的三个初步筛选，以寻找新的 DDR 候选蛋白 这将作为 AIM1 和 AIM2 的基础。在这些目标中，我们建议进行详尽的 验证候选者以确定真正的新 DDR 蛋白，并将对两个候选者进行跟进 蛋白质已经被验证。此外，我们还发现了调节衰老的关键蛋白质 分泌表型 GATA4，响应 ATM 和 ATR 激活 NFkB。我们将进一步 探索其在衰老中的作用以及衰老所需的新蛋白质 ZNF292。这些创新的 这些方法将使我们能够深入探测 DDR 的各层，包括衰老，以识别和 优先考虑新蛋白质，然后使用我们的方法探测这些蛋白质在 DDR 中的功能 已经开拓了这么多年。