Structure and Mechanism of Chromatin-Bound PARP1

项目总结 聚(ADP-核糖)聚合酶1(PARP1)是DNA修复受损的肿瘤治疗的一个有前途的靶点 肿瘤。一种PARP1抑制剂(奥拉帕利)用于临床治疗卵巢癌，~20例Ⅲ期临床 PARP1抑制剂的试验目前正在进行中。PARP1是一种高度丰富的核蛋白，在 结合受损的DNA会在自身和许多其他核蛋白上增加长链的聚(ADP-核糖)。这 巴洛克式的翻译后改造招募了DNA修复机器的关键部件。现有的缓蚀剂 PARP1的需要高有效剂量(100-400 mg/天)，并且与 抑制PARP1活性，可能是因为它们还针对与丰富结合的非活性PARP1 未受损的染色质。该PARP1群体具有许多超出维护范围的核功能 基因组完整性，如染色质紧凑，以及转录和DNA甲基化的调节。 我们的目标是使用严格的定量、结构和机械方法来研究PARP1如何 在染色质的背景下，与受损的DNA和完整的DNA的相互作用不同。我们的目标是了解是否有选择性 抑制PARP1的DNA结合活性构象是提高药物疗效的潜在途径 和专一性。使用新的酶分析方法，我们将测试不同类型的激活的不同结果 DNA损伤的可能性。我们还建议研究PARP1与不太活跃的和 PARP2含量较低。我们的研究将揭示PARP1和PARP2的构象多样性，并展示如何 这影响了复杂的反应机制，导致不同的下游信号事件。我们的机械师 研究将为如何设计更好的筛查和分析方法提供关键见解，以开发更多 选择性的、基于机理的PARP抑制剂。总之，我们建议的研究将有助于 用于癌症治疗的下一代PARP抑制剂。

PROJECT SUMMARY Poly(ADP-ribose) polymerase 1 (PARP1) is a promising target for cancer therapy in DNA repair-compromised tumors. One PARP1 inhibitor (Olaparib) is used in the clinic to treat ovarian cancer, and ~20 phase III clinical trials with inhibitors of PARP1 are currently ongoing. PARP1 is a highly abundant nuclear protein that upon binding damaged DNA adds long chains of poly(ADP-ribose) onto itself and many other nuclear proteins. This baroque post-translational modification recruits key components of the DNA repair machinery. Existing inhibitors of PARP1 require a high effective dose (100-400 mg/day) and show poor correlation of drug efficacy with inhibition of PARP1 activity, possibly because they also target inactive PARP1 that is abundantly bound to undamaged chromatin. This PARP1 population has many nuclear functions that extend beyond the maintenance of genome integrity, such as chromatin compaction, and the regulation of transcription and DNA methylation. Our goal is to use rigorous quantitative, structural, and mechanistic approaches to investigate how PARP1 interacts differently with damaged vs. intact DNA in the context of chromatin. We aim to understand if selective inhibition of the DNA-bound active conformation of PARP1 is a potential approach for increasing drug efficacy and specificity. Using novel enzymatic assays, we will test the varied outcomes of activation by different types of DNA damage. We also propose to investigate the interplay between PARP1 and the much less active and less abundant PARP2. Our studies will reveal the conformational diversity of PARP1 and PARP2, and show how this affects the complex reaction mechanism, resulting in different downstream signaling events. Our mechanistic studies will provide key insights into how to design better screens and assays for the development of more selective, mechanism-based PARP inhibitors. Altogether our proposed research will aid the development of the next generation of PARP inhibitors for use in cancer therapy.