Analysis of the Mammalian DNA Damage Response

哺乳动物 DNA 损伤反应分析

• 批准号: 10568991
• 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 2024-12-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/10568991
• 关键词: Aging; Autophagocytosis; Breast; CRISPR screen; Cancer Model; 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; Hereditary Breast Carcinoma; Human; Immunologics; Inherited; Link; Maintenance; Malignant Neoplasms; Malignant neoplasm of lung; Mammals; Methods; Modification; NF-kappa B; Neurologic; Open Reading Frames; Pathway interactions; Phenotype; Phosphorylation; Phosphotransferases; Physiology; Play; Poly(ADP-ribose) Polymerase Inhibitor; Process; Production; 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; follow-up; gain of function; genetic analysis; genetic regulatory protein; genome integrity; genotoxicity; homologous recombination; human DNA damage; human disease; 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损伤反应（DDR）是一个调节网络，它协调细胞内的DNA损伤。 对DNA损伤和复制应激的反应。它还直接协调DNA修复选择， 检测不同的DNA损伤结构并顺式转导该信息以激活特异性和 适当的维修选项，从而优化维修。DDR的重要性是由许多癌症 基因失活导致的易感综合征，包括遗传性乳腺癌及其 促进对遗传毒性化疗的敏感性和合成致死性的作用， 用PARP抑制剂治疗BRCA 1/2缺陷型肿瘤。在过去的25年里，我们和其他人， 研究了中央传感和信号装置的组成， 酵母和哺乳动物的遗传毒性压力。这揭示了一个保守的传感和信号核心 proteins.我们对这种DDR激酶级联反应底物的分析揭示了一组极其多样的 蛋白质和功能接触的DDR在哺乳动物和绝大多数这些活动不是 保存在酵母中。这包括蛋白质如RHINO，Abraxas，SMARCAL 1，RANZB 3，WHSC 1， NBA 1/MERIT 40、RAP 80、整个范可尼贫血通路，包括FANCI、FAN 1、病灶调节 蛋白质Mdc 1、p53 BP 1、RNF 8、RNF 168/RIDDLIN、RFWD 3，更不用说其他关键DDR通路 如p53/ p21、USP 28和BDR 7。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中超过1000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。所以我们 我认为，在哺乳动物中，有许多新的DDR成分有待发现，这是至关重要的 我们着手确定这些因素，以便全面了解复员方案及其 在细胞和生物生理学中的重要性。这包括在细胞衰老方面的新努力， 对DNA损伤的关键反应，防止肿瘤发生并促进衰老。为此，我们 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对DNA损伤的反应。我们已执行 对功能丧失和功能获得进行三次初步筛选，以发现新的DDR候选蛋白 这将作为AIM 1和AIM 2的基础。在这些目标中，我们建议进行详尽的 验证候选物以鉴定真正的新DDR蛋白质，并将跟踪两个候选物 蛋白质已经验证。此外，我们还发现了一个调控衰老的关键蛋白 分泌表型，GATA 4，其响应ATM和ATR以激活NF κ B。我们将进一步 探讨其在衰老中的作用以及一种新的衰老所需蛋白ZNF 292。这些创新 方法将使我们能够深入探测DDR的层，包括衰老，以识别和 优先考虑新的蛋白质，然后使用我们的方法探测DDR中这些蛋白质的功能， 已经开创了这么多年。