Mechanisms of PARP-1 interaction with chromatin

项目总结/摘要 这项合作提案解决了人类多聚腺苷二磷酸（ADP）- 核糖）聚合酶1（PARP-1）。PARP-1是细胞代谢中的多功能参与者。它涉及恶性 在许多不同的侵袭性癌症中过表达;它是公认的 作为抗癌药物和治疗靶点。蛋白质的聚腺苷二磷酸核糖化是一种翻译后修饰 与DNA修复、转录调节和凋亡相关。不同的模型解释了 已经提出了PARP-1在这些过程中的染色质重组中的作用;然而，它们需要进一步的研究。 系统评价，因为对基本机制仍然知之甚少。 我们各个小组先前进行的研究已经导致了定义的，结构上的发展。 概括PARP-1作用的重要方面的易于处理的实验模型：与核小体的结合 以及它们自发的和转录依赖的重组。我们以前的研究主要集中在 不同的，但相关的方面PARP-1的行动（结合染色质和促进转录），使用先进的 实验方法（时间分辨生物化学和单粒子福斯特共振能量转移 （spFRET））。因此，我们启动了合作研究，利用我们独特的，但 互补的，个人的力量，以检查PARP-1对自发和转录的影响- 核小体的依赖性重组。我们已经证明，PARP-1与染色质的结合会导致 核小体结构发生了相当大的变化，并且通过染色质的转录被强烈地促进 PARP-1，可能涉及PARP-1诱导的中间体结构的变化， 转录。鉴于这些初步合作努力取得的成功（见下文），我们现在建议扩大 这些研究旨在分析PARP-1依赖性自发和转录的机制， 核小体的依赖性重组，如本申请中所概述的。 我们提出了四个主要问题：（1）PARP-1结合诱导的结构变化是什么 到核小体(2)PARP-1如何影响连接组蛋白与核小体的结合？(3)如何 PARP-1影响转录延长中间体的结构和稳定性？(4)不同的PARP-1 相互之间有机械联系的活动？这些问题将在体外使用高度纯化的 使用生物化学， 时间分辨和单分子技术以及基因组方法。整合我们的努力， 研究RNA聚合酶II或调节蛋白因子 遇到核小体屏障，以及PARP-1如何帮助消除和重建这种染色质屏障。 对PARP-1与染色质相互作用的机制的理解将导致新的潜在靶点的鉴定 用于开发更有效和更具体的抗癌药物。

Project Summary/Abstract This collaborative proposal addresses mechanisms of chromatin reorganization by human poly(ADP- ribose)polymerase 1 (PARP-1). PARP-1 is a multifunctional player in cell metabolism. It is involved in malignant transformation and tumor survival, and is overexpressed in many different aggressive cancers; it is a recognized target for anticancer drugs and therapies. Poly(ADP-ribosyl)ation of proteins is a posttranslational modification associated with DNA repair, transcriptional regulation and apoptosis. Different models explaining the role of PARP-1 in chromatin reorganization during these processes have been proposed; however, they require further systematic evaluation, since underlying mechanisms remain poorly understood. Previous studies performed by our individual groups have resulted in the development of defined, structurally tractable experimental models that recapitulate the important aspects of PARP-1 action: binding to nucleosomes and their spontaneous and transcription-dependent reorganization. Our previous studies have been focused on distinct, but related aspects of PARP-1 action (binding to chromatin and facilitating transcription) using advanced experimental approaches (time-resolved biochemistry and single-particle Förster resonance energy transfer (spFRET)). We have therefore initiated collaborative studies that take advantage of our distinct, but complementary, individual strengths to examine the effects of PARP-1 on spontaneous and transcription- dependent reorganization of nucleosomes. We have shown that PARP-1 binding to chromatin results in a considerable change in the nucleosome structure and that transcription through chromatin is strongly facilitated by PARP-1, likely involving PARP-1-induced changes in the structures of the intermediates formed during transcription. Given the success of these initial collaborative efforts (described below), we now propose to extend these studies towards analysis of the mechanisms of PARP-1-dependent spontaneous and transcription- dependent reorganization of nucleosomes, as outlined in this application. We propose to address four primary questions: (1) What are the structural changes induced by PARP-1 binding to nucleosomes? (2) How is the binding of linker histones to nucleosome affected by PARP-1? (3) How does PARP-1 affect the structure and stability of transcript elongation intermediates? (4) Are the different PARP-1 activities mechanistically related to each other? These questions will be addressed in vitro using highly purified proteins with well-defined mono- and polynucleosomal chromatin templates using a combination of biochemical, time-resolved and single-molecule techniques and genomic approaches. Integrating our efforts will allow us to examine the currently mysterious processes that occur when RNA polymerase II or regulatory protein factors encounters a nucleosomal barrier, and how PARP-1 helps to remove and rebuild this chromatin barrier. Mechanistic understanding of PARP-1 interaction with chromatin will lead to identification of new potential targets for development of more potent and specific anti-cancer drugs.