The catalytic and non-catalytic functions of PARP1 in cancer biology

癌症放射治疗会产生DNA损伤和活性氧，从而激活Poly ADP-核糖聚合酶1（PARP 1）及其相关家族成员PARP 2。激活的PARP 1/2转移ADP- 核糖对自身和组蛋白。由此产生的聚（ADP-核糖）（PAR）链进一步招募其他修复 蛋白质（例如XRCC 1-Lig 3复合物）。PARP 1和PARP 2的特异性抑制剂（称为PARPi）优先 靶向BRCA 1/2缺陷癌细胞，并与其他遗传毒性癌症疗法（包括放射）协同作用。 虽然这些效应最初归因于缺乏XRCC 1的PAR依赖性募集，但XRCC 1的缺失导致了XRCC 1的缺失。 XRCC 1不具有与PARPi相同的细胞毒性。最近，PARPi被发现在DNA上捕获PARP 1， 断裂，将短暂的PARP 1灶（<10 min）转化为持久的（>30 min）。如果不去除，PARP 1-DNA 加合物可以阻止DNA修复和停止复制。因此，PARP 1缺失使癌细胞对 PARPi，揭示了PARP 1在癌症治疗中的结构功能。在初步研究中，我们发现 出乎意料的是，PARP 1从DNA中的释放受到PARP 2和单ADP核糖基化的调节 （MARylation），但不是PARylation。为了了解PARP 1在体内的结构功能，我们生成了 表达组成型（Parp 1A）或诱导型（Parp 1AN）PAR化缺陷型PARP 1（E988 A）的小鼠模型 和条件性PARP 1小鼠模型（Parp 1C）。与正常的发育和性别相比， Parp 1-/-小鼠的分布，Parp 1 +/A小鼠显示雌性特异性胚胎致死性。雄性Parp 1 +/A细胞 显示出对DNA损伤剂的严重超敏反应，超过了在Parp 1-/-ceptin中发现的。鉴于 PARP抑制剂在几乎只影响女性（乳腺癌和卵巢癌）的癌症类型中的应用， 性别偏见是有趣的，可能是由PARP 1在染色质上的断裂独立结合引起的 以及PARP 1蛋白在X失活和其他表观遗传调节中的作用。女性Parp 1 +/-Parp 2-/-（但不 Parp 1-/-Parp 2 +/-）小鼠也显示出雌性特异性致死性，伴有X染色体特异性不稳定性。 基于这些发现，我们假设PARP 1在染色质生物学和DNA中具有结构功能 对癌症治疗至关重要的修复。为了验证这一点，我们将使用小鼠遗传学，结构生物学和细胞 生物学方法1）确定在DNA损伤位点调节PARP 1动力学的机制， 揭示PARP 1捕获的生物学后果，2）表征PARP 1 E988 A小鼠模型， 确定催化失活的PARP 1对DNA修复、X-失活、体内肿瘤发生的影响。的 这项研究的完成将阐明PARP 1以前未被认识的结构功能，揭示 PARP 1和PARP 2之间的功能相互作用以及PARP 1在女性中的性别特异性作用。在 在不久的将来，我们的研究过程中产生的机制和新的动物模型也将 促进PARP抑制剂用于癌症治疗的开发和使用。

Cancer radiation therapy generates DNA lesions and reactive oxygen species, which activate Poly ADP-ribose polymerase 1 (PARP1) and its related family member PARP2. Activated PARP1/2 transfers ADP- Ribose to themselves and histones. The resulted poly (ADP-ribose) (PAR) chains further recruit others repair proteins (e.g. XRCC1-Lig3 complex). Specific inhibitors for both PARP1 and 2 (refer to as PARPi) preferentially target BRCA1/2 deficient cancer cells and synergize with other genotoxic cancer therapies, including radiation. While these effects were originally attributed to the lack of PAR dependent recruitment of XRCC1, deletion of XRCC1 does not have the same cytotoxicity as PARPi. More recently PARPi was found to trap PARP1 at DNA breaks, converting transient PARP1 foci (<10min) to durable ones (>30 min). If not removed, the PARP1-DNA adduct can prevent DNA repair and stall replication. Accordingly PARP1 deletion “desensitizes” cancer cells to PARPi, revealing a structural function of PARP1 in cancer therapy. In preliminary studies, we found unexpectedly the release of PARP1 from DNA is regulated by PARP2 and mono-ADP-ribosylation (MARylation), but not PARylation. To understand this structural function of PARP1 in vivo, we generated mouse models expressing constitutive (Parp1A) or inducible (Parp1AN) PARylation defective PARP1 (E988A) and a conditional PARP1 mouse model (Parp1C). In contrast to the normal development and gender distribution of Parp1-/- mice, Parp1+/A mice display female specific embryonic lethality. And male Parp1+/A cells display severe hypersensitivity to DNA damage agents beyond what is found in Parp1-/- ctrl. Given the application of PARP inhibitor in cancer types almost exclusively affecting women (breast and ovarian), this gender bias is intriguing and might be caused by the breaks independent binding of PARP1 on the chromatin and an role of PARP1 protein in X-inactivation and other epigenetic regulation. Female Parp1+/-Parp2-/- (but not Parp1-/-Parp2+/-) mice also display female specific lethality accompanied by X-chromosome specific instability. Based on these findings, we hypothesize that PARP1 has structural function in chromatin biology and DNA repair that are critical for cancer therapy. To test this, we will use mouse genetics, structural biology and cell biology approaches to 1) determine the mechanism that regulates PARP1 dynamics at DNA damage sites, reveal the biological consequence of PARP1 trapping and 2) characterize PARP1 E988A mouse models to determine the impact of catalytic inactive PARP1 on DNA repair, X-inactivation, oncogenesis in vivo. The completion of this study will elucidate the previously un-recognized structural function of PARP1, reveal functional interactions between PARP1 and PARP2 and the gender specific role of PARP1 in female. In the near future, the mechanism and the novel animal models generated in the course of our study will also facilitate the development and use of PARP inhibitors for cancer therapy.