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.

项目摘要 DNA损伤响应（DDR）是一个调节网络，可协调细胞处理 对DNA损伤和复制应力的反应。它还通过 感知不同的DNA损伤结构，并传达CI中的信息以激活特定和 适当的维修选项，从而优化维修。 DDR的重要性由许多癌症强调 由于其失活而导致的易感性综合征，包括遗传性乳腺癌及其 成功地促进对遗传毒性化学疗法和合成致死性的敏感性 用PARP抑制剂治疗BRCA1/2缺陷肿瘤。在过去的25年中，我们和其他人有 研究了检测和响应的中央传感和信号设备的组成 酵母和哺乳动物的遗传毒性应激。这揭示了感应和信号传导的保守核心 蛋白质。我们对此DDR激酶级联的底物的分析显示了一组极其多样的 DDR在哺乳动物和其中绝大多数活动中联系的蛋白质和功能不是 在酵母中保守。这包括犀牛，Abraxas，smarcal1，ranzb3，whsc1，等蛋白质 NBA1/MERIT40，RAP80，整个Fanconi贫血途径，包括Fanci，Fan1，Foci调节性 蛋白质MDC1，P53BP1，RNF8，RNF168/RIDDLIN，RFWD3更不用说其他关键DDR PatheAys 例如p53/ p21，USP28和BDR7。我们对ATM和ATR底物以及我们和其他分析的分析 非ATM/ATR调节的磷酸化事件已与DDR中的1000多个蛋白质有关 绝大多数人以前没有与DDR的联系，也没有酵母菌。因此，我们 认为在哺乳动物中发现了很多新的DDR组件，这很关键 我们着手确定这些因素，以便对DDR及其ITS的完全了解 细胞和生物生理学的重要性。这包括在细胞衰老方面的新工作， 对DNA损伤的关键反应可防止肿瘤发生并促进衰老。为此，我们有 开发了复杂的遗传工具，使我们能够采用RNAi，CRISPR和ORF表达 新的蛋白质候选物涉及促进DNA损伤的生存。我们已经表演了 三个初步筛选，用于找到新的DDR候选蛋白的功能和功能增益 这将是AIM1和AIM2的基础。在这些目标中，我们建议进行详尽 验证候选人以识别真正的新DDR蛋白质，并将跟进两名候选人 蛋白质已经验证。此外，我们发现了衰老调节的关键蛋白 分泌表型GATA4，对ATM和ATR响应以激活NFKB。我们将进一步 探索其在衰老中的作用，并探索衰老所需的新蛋白质，Znf292。这些创新的 方法将使我们能够深入探测DDR的层，包括衰老以识别和 优先考虑新蛋白质，然后使用方法在DDR中探测这些蛋白质的功能 开创了这么多年。