Ochem A E, Skopac D, Costa M, Rabilloud T, Vuillard L, Simoncsits A, Giacca M, Falaschi A
Molecular Biology Unit, International Centre for Genetic Engineering and Biotechnology, Padriciano 99, 34012 Trieste, Italy.
J Biol Chem. 1997 Nov 21;272(47):29919-26. doi: 10.1074/jbc.272.47.29919.
The Ku antigen consists of two subunits of 70 and 83 kDa and is endowed with both duplex DNA end-binding capacity and helicase activity (human DNA helicase II). HeLa Ku can be isolated from in vitro cultured human cells uniquely as a heterodimer, and the subunits can be separated by electrophoresis only under denaturing conditions. To dissect the molecular functions of the two subunits of the heterodimer, we have cloned and expressed their cDNAs separately in Escherichia coli. The two activities of Ku (DNA binding and unwinding) were reconstituted by mixing and refolding both subunits in equimolar amounts (Tuteja, N., Tuteja, R., Ochem, A., Taneja, P., Huang, N-W., Simoncsits, A., Susic, S., Rahman, K., Marusic, L., Chen, J., Zang, J., Wang, S., Pongor, S., and Falaschi, A. (1994) EMBO J. 13, 4991-5001). Renaturation of the separate subunits can be achieved in the presence of a synthetic solubilizing and stabilizing agent, dimethyl ethylammonium propane sulfonate (NDSB 195). The helicase activity of the Ku protein resides uniquely in the 70-kDa subunit, whereas the DNA end-binding activity can be reconstituted only through renaturation of the two subunits in the heterodimeric form and is practically absent in the separate subunits. The 83-kDa subunit, when refolded in the absence of the 70-kDa subunit, forms homodimers unable to unwind DNA and bind duplex ends. The three separate species (heterodimer, 70-kDa subunit, and 83-kDa subunit homodimer) all have ssDNA-dependent ATPase activity.
Ku抗原由70 kDa和83 kDa的两个亚基组成，具有双链DNA末端结合能力和螺旋酶活性（人DNA螺旋酶II）。HeLa Ku只能从体外培养的人细胞中作为异二聚体分离出来，并且只有在变性条件下亚基才能通过电泳分离。为了剖析异二聚体两个亚基的分子功能，我们分别在大肠杆菌中克隆并表达了它们的cDNA。通过将两个亚基等摩尔混合并复性，Ku的两种活性（DNA结合和解旋）得以重建（图特亚，N.，图特亚，R.，奥切姆，A.，塔内贾，P.，黄，N-W.，西蒙奇西茨，A.，苏西奇，S.，拉赫曼，K.，马鲁西奇，L.，陈，J.，臧，J.，王，S.，庞戈尔，S.，和法拉希，A.（1994年）《欧洲分子生物学组织杂志》13卷，4991 - 5001页）。在合成增溶剂和稳定剂二甲基乙基铵丙烷磺酸盐（NDSB 195）存在的情况下，可以实现单独亚基的复性。Ku蛋白的螺旋酶活性仅存在于70 kDa亚基中，而DNA末端结合活性只有通过异二聚体形式的两个亚基复性才能重建，单独的亚基中几乎不存在这种活性。83 kDa亚基在没有70 kDa亚基的情况下复性时，会形成无法解旋DNA和结合双链末端的同二聚体。三种单独的物种（异二聚体、70 kDa亚基和83 kDa亚基同二聚体）都具有单链DNA依赖性ATP酶活性。
