Simbulan C M, Suzuki M, Izuta S, Sakurai T, Savoysky E, Kojima K, Miyahara K, Shizuta Y, Yoshida S
Laboratory of Cancer Cell Biology, Nagoya University School of Medicine, Aichi, Japan.
J Biol Chem. 1993 Jan 5;268(1):93-9.
The direct effect of the eukaryotic nuclear DNA-binding protein poly(ADP-ribose) polymerase on the activity of DNA polymerase alpha was investigated. Homogenously purified poly(ADP-ribose) polymerase (5 to 10 micrograms/ml) stimulated the activity of immunoaffinity-purified calf or human DNA polymerase alpha by about 6 to 60-fold in a dose-dependent manner. It had no effect on the activities of DNA polymerase beta, DNA polymerase gamma, and primase, indicating that its effect is specific for DNA polymerase alpha. Apparently, poly(ADP-ribosyl)ation of DNA polymerase alpha was not necessary for the stimulation. The stimulatory activity is due to poly(ADP-ribose) polymerase itself since it was immunoprecipitated with a monoclonal antibody directed against poly(ADP-ribose) polymerase. Kinetic analysis showed that, in the presence of poly(ADP-ribose) polymerase, the saturation curve for DNA template primer became sigmoidal; at very low concentrations of DNA, it rather inhibited the reaction in competition with template DNA, while, at higher DNA doses, it greatly stimulated the reaction by increasing the Vmax of the reaction. By the automodification of poly(ADP-ribose) polymerase, however, both the inhibition at low DNA concentration and the stimulation at high DNA doses were largely lost. Furthermore, stimulation by poly(ADP-ribose) polymerase could not be attributed to its DNA-binding function alone since its fragment, containing only the DNA-binding domain, could not exert full stimulatory effect on DNA polymerase, as of the intact enzyme. Poly(ADP-ribose) polymerase is co-immunoprecipitated with DNA polymerase alpha, using anti-DNA polymerase alpha antibody, clearly showing that poly(ADP-ribose) polymerase may be physically associated with DNA polymerase alpha. In a crude extract of calf thymus, a part of poly(ADP-ribose) polymerase activity existed in a 400-kDa, as well as, a larger 700-kDa complex containing DNA polymerase alpha, suggesting the existence in vivo of a complex of these two enzymes.
研究了真核细胞核DNA结合蛋白聚（ADP - 核糖）聚合酶对DNA聚合酶α活性的直接影响。均匀纯化的聚（ADP - 核糖）聚合酶（5至10微克/毫升）以剂量依赖的方式将免疫亲和纯化的小牛或人DNA聚合酶α的活性刺激约6至60倍。它对DNA聚合酶β、DNA聚合酶γ和引发酶的活性没有影响，表明其作用对DNA聚合酶α具有特异性。显然，DNA聚合酶α的聚（ADP - 核糖基）化对于这种刺激不是必需的。刺激活性归因于聚（ADP - 核糖）聚合酶本身，因为它能用针对聚（ADP - 核糖）聚合酶的单克隆抗体进行免疫沉淀。动力学分析表明，在聚（ADP - 核糖）聚合酶存在下，DNA模板引物的饱和曲线变为S形；在非常低的DNA浓度下，它在与模板DNA竞争中反而抑制反应，而在较高的DNA剂量下，它通过增加反应的Vmax极大地刺激反应。然而，通过聚（ADP - 核糖）聚合酶的自身修饰，低DNA浓度下的抑制和高DNA剂量下的刺激在很大程度上都丧失了。此外，聚（ADP - 核糖）聚合酶的刺激不能仅归因于其DNA结合功能，因为其仅包含DNA结合结构域的片段不能像完整酶那样对DNA聚合酶发挥完全的刺激作用。使用抗DNA聚合酶α抗体，聚（ADP - 核糖）聚合酶与DNA聚合酶α共免疫沉淀，清楚地表明聚（ADP - 核糖）聚合酶可能与DNA聚合酶α在物理上相关联。在小牛胸腺粗提物中，一部分聚（ADP - 核糖）聚合酶活性存在于一个400 kDa以及一个更大的包含DNA聚合酶α的700 kDa复合物中，表明这两种酶在体内存在复合物。
