Koczor Christopher A, Haider Aaron J, Saville Kate M, Li Jianfeng, Andrews Joel F, Beiser Alison V, Sobol Robert W
Department of Pharmacology, College of Medicine, University of South Alabama, Mobile, AL 36688, USA.
Mitchell Cancer Institute, University of South Alabama, Mobile, AL 36604, USA.
Cancers (Basel). 2022 Jul 28;14(15):3676. doi: 10.3390/cancers14153676.
Poly(ADP-ribose) (PAR) is a molecular scaffold that aids in the formation of DNA repair protein complexes. Tools to sensitively quantify PAR in live cells have been lacking. We recently described the LivePAR probe (EGFP fused to the RNF146-encoded WWE PAR binding domain) to measure PAR formation at sites of laser micro-irradiation in live cells. Here, we present two methods that expand on the use of LivePAR and its WWE domain. First, LivePAR enriches in the nucleus of cells following genotoxic challenge. Image quantitation can identify single-cell PAR formation following genotoxic stress at concentrations lower than PAR ELISA or PAR immunoblot, with greater sensitivity to genotoxic stress than CometChip. In a second approach, we used the RNF146-encoded WWE domain to develop a split luciferase probe for analysis in a 96-well plate assay. We then applied these PAR analysis tools to demonstrate their broad applicability. First, we show that both approaches can identify genetic modifications that alter PARylation levels, such as hyper-PARylation in BRCA2-deficient cancer cells. Second, we demonstrate the utility of the WWE split luciferase assay to characterize the cellular response of genotoxins, PARP inhibitors, and PARG inhibitors, thereby providing a screening method to identify PAR modulating compounds.
聚（ADP - 核糖）（PAR）是一种分子支架，有助于DNA修复蛋白复合物的形成。一直缺乏在活细胞中灵敏定量PAR的工具。我们最近描述了LivePAR探针（与RNF146编码的WWE PAR结合域融合的EGFP），用于测量活细胞中激光微照射部位的PAR形成。在此，我们介绍两种扩展LivePAR及其WWE结构域应用的方法。首先，在遗传毒性刺激后，LivePAR在细胞核中富集。图像定量可以在低于PAR ELISA或PAR免疫印迹的浓度下识别遗传毒性应激后的单细胞PAR形成，对遗传毒性应激的敏感性高于彗星芯片。在第二种方法中，我们使用RNF146编码的WWE结构域开发了一种用于96孔板分析的分裂荧光素酶探针。然后我们应用这些PAR分析工具来证明它们的广泛适用性。首先，我们表明这两种方法都可以识别改变PAR化水平的基因修饰，例如BRCA2缺陷癌细胞中的高PAR化。其次，我们证明了WWE分裂荧光素酶测定在表征遗传毒素、PARP抑制剂和PARG抑制剂的细胞反应方面的实用性，从而提供了一种筛选方法来鉴定PAR调节化合物。
