A Novel Carcinogen-Induced Cell Cycle Checkpoint

描述（由申请人提供）：在我们对DNA损伤被感知、处理和修复的机制的理解方面存在重大差距，并且这些事件与细胞生命周期整合的基础还没有完全理解。这些缺口的存在提出了一个根本性的问题，阻碍了我们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。本特定申请的目的是阐明称为“Rad 18”的E3泛素连接酶在通过跨损伤合成（TLS）耐受大体积DNA加合物（例如B[a]P-和UV-诱导的损伤）和修复S期期间出现的双链断裂（DSB）中的作用。基于强有力的初步数据，本申请的中心假设是细胞周期和DNA损伤响应性蛋白激酶对Rad 18的直接磷酸化对于激活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件整合以加强基因组维持是必需的。这项研究的基本原理是，阐明DNA修复的机制将直接导致更好地理解癌症的发病机制。本研究的具体目的是：（1）确定大体积DNA加合物的Rad 18依赖性TLS的启动机制。(2)确定Rad 18介导的DSB修复的TLS非依赖性机制。(3)阐明Rad 18在体内基因组维持和肿瘤抑制中的作用。对于目标1，Rad 18磷酸化和TLS蛋白的磷酸化依赖性调节将在体外和培养细胞中进行研究。目的2将研究Rad 18 E3连接酶依赖性的生化信号通路参与DSB传感和修复。在目标3中，将产生携带突变体rad 18等位基因的小鼠，并用于区分体内基因组维持和肿瘤发生中Rad 18的各种效应子途径。提出的想法和研究是创新的，因为它们代表了现有的范式的出发点，并寻求提供新的范式，其中不存在：磷酸化为基础的调节Rad 18是新的，与现有的范式相反，表明启动Rad 18介导的DNA修复是密切相关的检查点信号和细胞周期。Rad 18具有参与DSB信号传导的新底物的想法是创新的。拟议的小鼠研究是创新的，因为它们将首次测试Rad 18在基因组维持中的体内作用。这项工作意义重大，因为它将为Rad 18介导的基因组维持和肿瘤抑制提供新的范例，这些都与人类环境暴露有关。此外，由于TLS和DSB修复通常会使癌细胞对化疗和放疗产生抗性，因此对激活Rad 18依赖性修复的事件的更深入了解可以确定可药物化的靶点（例如Rad 18或其激活激酶），其抑制增强了现有癌症疗法的疗效。

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.