A Novel Carcinogen-Induced Cell Cycle Checkpoint

描述(由申请人提供)：我们对DNA损伤的感知、处理和修复机制的理解存在重大差距，这些事件与细胞生命周期相结合的基础还不完全清楚。这种差距的存在是一个根本性的问题，使我们无法理解细胞如何实现基因组维护和预防癌症，以应对环境诱导的DNA损伤。长期目标是阐明细胞协调细胞周期进程与DNA损伤的感知、耐受和修复的细胞机制。在这一特殊应用中的目的是阐明E3泛素连接酶‘RAD18’在通过跨损伤合成(TLS)耐受大体积DNA加合物(如B[a]P和紫外线诱导的损伤)和修复S期出现的双链断裂(DSB)中的作用。基于强大的初步数据，这一应用的中心假设是，细胞周期和DNA损伤反应蛋白激酶直接磷酸化Rad18是激活DNA修复、决定DNA修复途径的选择以及将DNA修复与细胞周期的其他元素整合以加强基因组维护所必需的。提出这项研究的理由是，阐明DNA修复的机制将直接导致对癌症发病机制的更好理解。这项提议的具体目的是：(1)确定大体积DNA加合物启动Rad18依赖的TLS的机制。(2)探讨RAD18介导DSB修复的TLS非依赖性机制(S)。(3)阐明Rad18在体内基因组维持和肿瘤抑制中的作用。对于目标1，将在体外和培养细胞中研究RAD18的磷酸化及其对TLS蛋白的磷酸化依赖的调节。目的2将研究参与DSB感知和修复的生化信号通路对Rad18 E3连接酶的依赖。在目标3中，将产生携带突变的rad18等位基因的小鼠，并用于区分Rad18在基因组维持和体内肿瘤发生中的不同效应途径。提出的想法和研究具有创新性，因为它们代表了对现有范式的背离，并试图提供不存在的新范式：基于磷酸化的Rad18调控是新颖的，与现有范式相反，表明Rad18介导的DNA修复的启动与检查点信号和细胞周期密切相关。Rad18有参与DSB信号转导的新底物的想法是创新的。拟议中的小鼠研究是创新的，因为他们将首次在体内测试Rad18在基因组维护中的作用。这项拟议的工作意义重大，因为它将为与人类环境暴露相关的Rad18介导的基因组维护和肿瘤抑制提供新的范例。此外，由于TLS和DSB修复经常导致癌细胞对化疗和放射治疗的耐药性，对激活Rad18依赖修复的事件的深入了解可以确定抑制其增强现有癌症治疗效果的药物靶点(例如Rad18或其激活的激酶)。

DESCRIPTION (provided by applicant): There are major gaps in our understanding of mechanisms by which DNA damage is sensed, processed and repaired, and the basis for integration of these events with the cellular life cycle is incompletely understood. Existence of such gaps presents a fundamental problem that precludes our understanding of how cells achieve genome maintenance and prevent cancer in response to environmentally-induced DNA damage. The long-term goal is to elucidate the cellular mechanisms whereby cells coordinate cell cycle progression with sensing, tolerance and repair of DNA damage. The objective in this particular application is to elucidate the roles of an E3 ubiquitin ligase termed 'Rad18' in tolerance of bulky DNA adducts (such as B[a]P- and UV-induced lesions) via Trans-Lesion Synthesis (TLS) and in repair of Double Strand Break (DSB) that arise during S-phase. Based on strong preliminary data, the central hypothesis of this application is that direct phosphorylation of Rad18 by cell cycle and DNA damage-responsive protein kinases is necessary for activating DNA repair, dictating choice of DNA repair pathway and integrating DNA repair with other elements of the cell cycle to enforce genome maintenance. The rationale for the proposed research is that elucidating mechanisms of DNA repair will lead directly to a better understanding of the pathogenesis of cancer. The specific aims of this proposal are: (1) To determine the mechanisms that initiate Rad18-dependent TLS of bulky DNA adducts. (2) To determine the TLS-independent mechanism(s) of Rad18-mediated DSB repair. (3) To elucidate roles of Rad18 in genome maintenance and tumor suppression in vivo. For Aim 1, Rad18 phosphorylation and its phosphorylation-dependent regulation of TLS proteins will be studied in vitro and in cultured cells. Aim 2 will examine the Rad18 E3 ligase-dependence of biochemical signaling pathways involved in DSB sensing and repair. In Aim 3, mice harboring mutant rad18 alleles will be generated and used to distinguish various effector pathways of Rad18 in genome maintenance and tumorigenesis in vivo. The proposed ideas and research are innovative because they represent a departure from existing paradigms, and seek to provide new paradigms where none exist: Phosphorylation-based regulation of Rad18 is novel and, contrary to existing paradigms, indicates that initiation of Rad18-mediated DNA repair is intimately connected with both checkpoint signaling and cell cycle. The idea that Rad18 has new substrates that are involved in DSB signaling is innovative. The proposed mouse studies are innovative because they will test in vivo roles of Rad18 in genome maintenance for the first time. The proposed work is significant because it will provide new paradigms for Rad18-mediated genome maintenance and tumor suppression that are relevant to human environmental exposures. Moreover, because TLS and DSB repair often confer resistance to chemotherapy and radiotherapy in cancer cells, a deeper understanding of events that activate Rad18-dependent repair could identify druggable targets (e.g. Rad18 or its activating kinases) whose inhibition enhances the efficacy of existing cancer therapies.