Saxena Alka, Saffery Richard, Wong Lee H, Kalitsis Paul, Choo K H Andy
Murdoch Childrens Research Institute, Royal Children's Hospital, Flemington Rd., Parkville 3052, Australia.
J Biol Chem. 2002 Jul 26;277(30):26921-6. doi: 10.1074/jbc.M200620200. Epub 2002 May 13.
Poly(ADP-ribose) polymerase-1 (PARP-1) is activated by DNA strand breaks during cellular genotoxic stress response and catalyzes poly(ADP-ribosyl)ation of acceptor proteins. These acceptor proteins include those involved in modulation of chromatin structure, DNA synthesis, DNA repair, transcription, and cell cycle control. Thus, PARP-1 is believed to play a pivotal role in maintaining genome integrity through modulation of protein-protein and protein-DNA interactions. We previously described the association of PARP-1 with normal mammalian centromeres and human neocentromeres by affinity purification and immunofluorescence. Here we investigated the interaction of this protein with, and poly(ADP-ribosyl)ation of, three constitutive centromere proteins, Cenpa, Cenpb, and Cenpc, and a spindle checkpoint protein, Bub3. Immunoprecipitation and Western blot analyses demonstrate that Cenpa, Cenpb, and Bub3, but not Cenpc, interacted with PARP-1, and are poly(ADP-ribosyl)ated following induction of DNA damage. The results suggest a role of PARP-1 in centromere assembly/disassembly and checkpoint control. Demonstration of PARP-1-binding and poly(ADP-ribosyl)ation in three of the four proteins tested further suggests that many more centromere proteins may behave similarly and implicates PARP-1 as an important regulator of diverse centromere function.
聚（ADP-核糖）聚合酶-1（PARP-1）在细胞遗传毒性应激反应期间被DNA链断裂激活，并催化受体蛋白的聚（ADP-核糖基）化。这些受体蛋白包括参与染色质结构调节、DNA合成、DNA修复、转录和细胞周期控制的蛋白。因此，PARP-1被认为通过调节蛋白质-蛋白质和蛋白质-DNA相互作用在维持基因组完整性方面发挥关键作用。我们之前通过亲和纯化和免疫荧光描述了PARP-1与正常哺乳动物着丝粒和人类新着丝粒的关联。在这里，我们研究了这种蛋白质与三种组成型着丝粒蛋白Cenpa、Cenpb和Cenpc以及一种纺锤体检查点蛋白Bub3的相互作用及其聚（ADP-核糖基）化情况。免疫沉淀和蛋白质印迹分析表明，Cenpa、Cenpb和Bub3（而非Cenpc）与PARP-1相互作用，并在DNA损伤诱导后发生聚（ADP-核糖基）化。结果表明PARP-1在着丝粒组装/拆卸和检查点控制中发挥作用。在四种测试蛋白中的三种中证明了PARP-1结合和聚（ADP-核糖基）化，这进一步表明更多着丝粒蛋白可能有类似行为，并暗示PARP-1是多种着丝粒功能的重要调节因子。
