聚(ADP-核糖)聚合酶1(PARP1)在自身聚(ADP-核糖)化后或在脱嘌呤嘧啶核酸内切酶1(APE1)存在的情况下,从三维DNA损伤搜索转变为一维扩散。
PARP1 changes from three-dimensional DNA damage searching to one-dimensional diffusion after auto-PARylation or in the presence of APE1.
作者信息
Liu Lili, Kong Muwen, Gassman Natalie R, Freudenthal Bret D, Prasad Rajendra, Zhen Stephanie, Watkins Simon C, Wilson Samuel H, Van Houten Bennett
机构信息
Department of Pharmacology and Chemical Biology, University of Pittsburgh, Pittsburgh, PA 15213, USA.
The University of Pittsburgh Cancer Institute, Hillman Cancer Center, Pittsburgh, PA 15213, USA.
出版信息
Nucleic Acids Res. 2017 Dec 15;45(22):12834-12847. doi: 10.1093/nar/gkx1047.
PARP1-dependent poly-ADP-ribosylation (PARylation) participates in the repair of many forms of DNA damage. Here, we used atomic force microscopy (AFM) and single molecule fluorescence microscopy to examine the interactions of PARP1 with common DNA repair intermediates. AFM volume analysis indicates that PARP1 binds to DNA at nicks, abasic (AP) sites, and ends as a monomer. Single molecule DNA tightrope assays were used to follow the real-time dynamic behavior of PARP1 in the absence and presence of AP endonuclease (APE1) on AP DNA damage arrays. These experiments revealed that PARP1 conducted damage search mostly through 3D diffusion. Co-localization of APE1 with PARP1 on DNA was found capable of inducing 1D diffusion of otherwise nonmotile PARP1, while excess APE1 also facilitated the dissociation of DNA-bound PARP1. Moreover, auto-PARylation of PARP1 allowed the protein to switch its damage search strategy by causing a 3-fold increase in linear diffusion. Finally, we demonstrated that PARP inhibitor olaparib did not significantly alter the rate of PARP1 dissociation from DNA, but instead resulted in more motility of DNA-bound PARP1 molecules.
聚(ADP-核糖)聚合酶1(PARP1)依赖性多聚ADP核糖基化(PARylation)参与多种形式的DNA损伤修复。在此,我们使用原子力显微镜(AFM)和单分子荧光显微镜来检测PARP1与常见DNA修复中间体的相互作用。AFM体积分析表明,PARP1以单体形式结合于缺口、无碱基(AP)位点及DNA末端。利用单分子DNA走钢丝检测法,追踪在有无AP核酸内切酶(APE1)存在时,PARP1在AP DNA损伤阵列上的实时动态行为。这些实验表明,PARP1主要通过三维扩散进行损伤搜索。研究发现,APE1与PARP1在DNA上的共定位能够诱导原本不移动的PARP1进行一维扩散,而过量的APE1也促进了与DNA结合的PARP1的解离。此外,PARP1的自身PARylation使该蛋白通过使线性扩散增加3倍来切换其损伤搜索策略。最后,我们证明PARP抑制剂奥拉帕利不会显著改变PARP1从DNA上解离的速率,反而导致与DNA结合的PARP1分子的移动性增强。