Shieh W M, Amé J C, Wilson M V, Wang Z Q, Koh D W, Jacobson M K, Jacobson E L
Department of Clinical Sciences, University of Kentucky, Lexington, Kentucky 40506-0286, USA.
J Biol Chem. 1998 Nov 13;273(46):30069-72. doi: 10.1074/jbc.273.46.30069.
Poly(ADP-ribose) polymerase (PARP) (EC 2.4.2.30), the only enzyme known to synthesize ADP-ribose polymers from NAD+, is activated in response to DNA strand breaks and functions in the maintenance of genomic integrity. Mice homozygous for a disrupted gene encoding PARP are viable but have severe sensitivity to gamma-radiation and alkylating agents. We demonstrate here that both 3T3 and primary embryo cells derived from PARP-/- mice synthesized ADP-ribose polymers following treatment with the DNA-damaging agent, N-methyl-N'-nitro-N-nitrosoguanidine, despite the fact that no PARP protein was detected in these cells. ADP-ribose polymers isolated from PARP-/- cells were indistinguishable from that of PARP+/+ cells by several criteria. First, they bound to a boronate resin selective for ADP-ribose polymers. Second, treatment of polymers with snake venom phosphodiesterase and alkaline phosphatase yielded ribosyladenosine, a nucleoside diagnostic for the unique ribosyl-ribosyl linkages of ADP-ribose polymers. Third, they were digested by treatment with recombinant poly(ADP-ribose) glycohydrolase, an enzyme highly specific for ADP-ribose polymers. Collectively, these data demonstrate that ADP-ribose polymers are formed in PARP-/- cells in a DNA damage-dependent manner. Because the PARP gene has been disrupted, these results suggest the presence of a previously unreported activity capable of synthesizing ADP-ribose polymers in PARP-/- cells.
聚（ADP-核糖）聚合酶（PARP）（EC 2.4.2.30）是已知唯一能从NAD+合成ADP-核糖聚合物的酶，它在DNA链断裂时被激活，并在维持基因组完整性方面发挥作用。编码PARP的基因被破坏的纯合小鼠是存活的，但对γ射线和烷化剂具有严重的敏感性。我们在此证明，尽管在这些细胞中未检测到PARP蛋白，但用DNA损伤剂N-甲基-N'-硝基-N-亚硝基胍处理后，源自PARP-/-小鼠的3T3细胞和原代胚胎细胞都能合成ADP-核糖聚合物。从PARP-/-细胞中分离出的ADP-核糖聚合物在几个标准上与PARP+/+细胞的聚合物没有区别。首先，它们与对ADP-核糖聚合物具有选择性的硼酸酯树脂结合。其次，用蛇毒磷酸二酯酶和碱性磷酸酶处理聚合物可产生核糖基腺苷，这是一种用于诊断ADP-核糖聚合物独特核糖基-核糖基连接的核苷。第三，用重组聚（ADP-核糖）糖苷水解酶处理可将它们消化，该酶对ADP-核糖聚合物具有高度特异性。总的来说，这些数据表明ADP-核糖聚合物在PARP-/-细胞中以DNA损伤依赖的方式形成。由于PARP基因已被破坏，这些结果表明在PARP-/-细胞中存在一种以前未报道的能够合成ADP-核糖聚合物的活性。
