Structural Biochemistry of PARP-1

描述（由申请人提供）：DNA结构的断裂是对基因组完整性的持续压力，并且它们构成染色体重排和基因突变的重大风险，这可能挑战生物体的健康并促进癌症的发展。有几种细胞机制可以监测基因组的状态，并快速启动修复机制以应对DNA损伤，从而将健康的基因组传递给下一代。聚（ADP-核糖）聚合酶-1，或PARP-1，是DNA结构断裂的主要反应者。PARP-1具有独特的催化活性，可合成ADP-核糖聚合物作为靶蛋白的翻译后修饰，主要是PARP-1本身（自修饰）。在与DNA断裂结合后，PARP-1活性被“打开”以调节DNA损伤修复途径，从而促进细胞存活。相反，过度的DNA损伤导致PARP-1活性水平升高，从而导致细胞死亡。因此，PARP-1活性的调节是决定细胞命运的关键因素。此外，PARP-1的抑制剂最近已经成为治疗癌症和炎症的有前景的治疗剂。尽管对PARP-1抑制剂的兴趣越来越大，并且发现PARP-1活性在DNA修复、转录调节和凋亡信号传导中的作用扩大，但对PARP-1活性和调节的机制的了解很少。这项研究计划的长期目标是在分子水平上建立控制PARP-1活性的机制。DNA损伤是PARP-1最有效的激活剂;因此我们选择首先关注PARP-1的DNA依赖性激活机制。使用X射线晶体学和生物化学分析的组合，拟议的研究将推进我们对PARP-1识别DNA损伤以及PARP-1与染色质相互作用（特异性目的1）的理解。这些研究将提供与DNA结合的PARP-1的第一个观点，因此将提供机制的见解，这将大大推进PARP研究领域，并对DNA修复和染色质生物学领域产生更广泛的影响。拟议的工作将展示PARP-1的多个结构域如何合作，将聚（ADP-核糖）合成与结构特异性DNA结合（特异性目的2）偶联。PARP-1活性和调节的详细结构分析将改进PARP-1生物学功能的当前模型，并可能揭示特异性抑制PARP-1活性的新策略。 公共卫生相关性：聚ADP核糖聚合酶1（Poly（ADP-ribose）Polymerase 1，PARP-1）在DNA损伤修复、基因表达调控和细胞死亡信号转导中具有重要作用。PARP-1的抑制剂已经成为治疗癌症和炎症的有前景的治疗剂。该提案中的研究将促进我们对PARP-1作用和调节机制的理解，以改善PARP-1生物学功能的现有模型，并揭示抑制PARP-1的新策略。

DESCRIPTION (provided by applicant): Breaks in the structure of DNA are a persistent stress on the integrity of the genome, and they pose a substantial risk of chromosomal rearrangement and genetic mutation that can challenge the well-being of an organism and promote the development of cancer. There are several cellular mechanisms that monitor the state of the genome and rapidly initiate repair mechanisms in response to DNA damage so that a healthy genome is passed on to the next generation. Poly(ADP-ribose) Polymerase-1, or PARP-1, is a primary responder to breaks in the structure of DNA. PARP-1 has a unique catalytic activity that synthesizes polymers of ADP-ribose as a posttranslational modification on target proteins, primarily on PARP-1 itself (automodification). Upon binding to DNA breaks, PARP-1 activity is "turned on" to modulate DNA damage repair pathways and thereby promote cell survival. In contrast, excessive DNA damage leads to an elevated level of PARP-1 activity that results in cell death. Regulation of PARP-1 activity is therefore a critical factor in determining the fate of a cell. Furthermore, inhibitors of PARP-1 have recently emerged as promising therapeutic agents for the treatment of cancer and inflammation. Despite a growing interest in PARP-1 inhibitors and the discovery of expanded roles for PARP-1 activity in DNA repair, transcriptional regulation, and apoptotic signaling, there are few insights into the mechanism of PARP-1 activity and regulation. The long-term objective of this research program is to establish at the molecular level the mechanisms that control PARP-1 activity. DNA damage is the most potent activator of PARP-1; therefore we have chosen to first focus on the mechanism of DNA-dependent activation of PARP-1. Using a combination of x-ray crystallography and biochemical analysis, the proposed research will advance our understanding of PARP-1 recognition of DNA damage, and PARP-1 interaction with chromatin (Specific Aim 1). These studies will provide the first views of PARP-1 bound to DNA and will therefore provide mechanistic insights that will significantly advance the PARP field of research, as well as having a more broad impact on the field of DNA repair and chromatin biology. The proposed work will demonstrate how multiple domains of PARP-1 collaborate to couple poly(ADP-ribose) synthesis to structure-specific DNA binding (Specific Aim 2). The detailed structural analysis of PARP-1 activity and regulation will improve current models of PARP-1 biological functions and potentially reveal novel strategies for specifically inhibiting PARP-1 activity. PUBLIC HEALTH RELEVANCE: Poly(ADP-ribose) Polymerase 1 (PARP-1) has important roles in DNA damage repair, regulation of gene expression, and cell death signaling. Inhibitors of PARP-1 have emerged as promising therapeutic agents for the treatment of cancer and inflammation. The studies in this proposal will advance our understanding of PARP-1 mechanism of action and regulation to improve current models of PARP-1 biological functions and to reveal novel strategies for inhibiting PARP-1.