Analysis of the Mammalian DNA Damage Response

项目摘要 DNA损伤反应(DDR)是一个调节网络，协调细胞在 对DNA损伤和复制压力的反应。它还通过以下方式直接协调DNA修复选择 感知不同的DNA损伤结构和转导信息，以激活特定的和 适当的维修选项，从而优化维修。许多癌症突显了DDR的重要性 由其失活引起的易感综合征，包括遗传性乳腺癌及其 在提高对基因毒性化疗的敏感性和合成致死率方面的作用 PARP抑制剂治疗BRCA1/2缺陷肿瘤在过去的25年里，我们和其他人 调查了检测和响应的中央传感和信号设备的组成 酵母和现在的哺乳动物的基因毒性应激。这揭示了感知和信号传递的保守核心 蛋白质。我们对这种DDR激酶级联反应底物的分析揭示了一组极其不同的 在哺乳动物中与DDR接触的蛋白质和功能，而这些活动中的绝大多数不是 在酵母中保存。这包括犀牛，Abraxas，SMARCAL1，RANZB3，WHSC1， NBA1/MERIT40，RAP80，整个Fanconi贫血途径，包括FANCI，FAN1，Foci调节 Mdc1、p53BP1、RNF8、RNF168/RIDDLIN、RFWD3蛋白，更不用说其他关键的DDR途径了 如p53/p21、USP28和BDR7。我们对ATM和ATR基板的分析以及OUR和其他人的分析 在非ATM/ATR调控的磷酸化事件中，已有1000多个蛋白质参与了DDR， 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 我认为在哺乳动物身上发现了很多很多新的DDR成分，这是至关重要的 我们着手确定这些因素，以便对复员方案及其 在细胞和生物体生理学中的意义。这包括在细胞衰老方面的一项新努力，一种 对DNA损伤的关键反应，防止肿瘤发生和促进衰老。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存活以应对DNA损伤。我们已经表演了 三种功能丧失和功能获得的初步筛选以寻找新的DDR候选蛋白 这将成为AIM1和AIM2的基础。在这些目标中，我们建议进行详尽的 验证候选以确定真正的新DDR蛋白，并将跟踪两个候选 蛋白质已经过验证了。此外，我们还发现了一种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 探索它在衰老中的作用以及衰老所需的一种新蛋白质ZNF292。这些创新之处 这些方法将使我们能够深入探索DDR的各个层面，包括衰老，以识别和 确定新蛋白质的优先顺序，然后使用我们的方法探索这些蛋白质在DDR中的功能 已经开拓了这么多年。

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.