Tuteja N, Tuteja R, Ochem A, Taneja P, Huang N W, Simoncsits A, Susic S, Rahman K, Marusic L, Chen J
International Centre for Genetic Engineering and Biotechnology, Area Science Park, Trieste, Italy.
EMBO J. 1994 Oct 17;13(20):4991-5001. doi: 10.1002/j.1460-2075.1994.tb06826.x.
Human DNA helicase II (HDH II) is a novel ATP-dependent DNA unwinding enzyme, purified to apparent homogeneity from HeLa cells, which (i) unwinds exclusively DNA duplexes, (ii) prefers partially unwound substrates and (iii) proceeds in the 3' to 5' direction on the bound strand. HDH II is a heterodimer of 72 and 87 kDa polypeptides. It shows single-stranded DNA-dependent ATPase activity, as well as double-stranded DNA binding capacity. All these activities comigrate in gel filtration and glycerol gradients, giving a sedimentation coefficient of 7.4S and a Stokes radius of approximately 46 A, corresponding to a native molecular weight of 158 kDa. The antibodies raised in rabbit against either polypeptide can remove from the solution all the activities of HDH II. Photoaffinity labelling with [alpha-32P]ATP labelled both polypeptides. Microsequencing of the separate polypeptides of HDH II and cross-reaction with specific antibodies showed that this enzyme is identical to Ku, an autoantigen recognized by the sera of scleroderma and lupus erythematosus patients, which binds specifically to duplex DNA ends and is regulator of a DNA-dependent protein kinase. Recombinant HDH II/Ku protein expressed in and purified from Escherichia coli cells showed DNA binding and helicase activities indistinguishable from those of the isolated protein. The exclusively nuclear location of HDH II/Ku antigen, its highly specific affinity for double-stranded DNA, its abundance and its newly demonstrated ability to unwind exclusively DNA duplexes, point to an additional, if still unclear, role for this molecule in DNA metabolism.
人DNA解旋酶II（HDH II）是一种新型的依赖ATP的DNA解旋酶，从HeLa细胞中纯化至表观均一，它（i）仅解旋DNA双链体，（ii）偏好部分解旋的底物，并且（iii）在结合链上沿3'至5'方向进行。HDH II是由72 kDa和87 kDa多肽组成的异二聚体。它表现出单链DNA依赖性ATP酶活性以及双链DNA结合能力。所有这些活性在凝胶过滤和甘油梯度中共同迁移，沉降系数为7.4S，斯托克斯半径约为46 Å，对应天然分子量为158 kDa。用针对任一多肽的兔抗体制备的抗体可从溶液中去除HDH II的所有活性。用[α-32P]ATP进行光亲和标记可标记这两种多肽。对HDH II单独多肽的微量测序以及与特异性抗体的交叉反应表明，该酶与Ku相同，Ku是硬皮病和红斑狼疮患者血清识别的自身抗原，它特异性结合双链DNA末端，是一种DNA依赖性蛋白激酶的调节剂。在大肠杆菌细胞中表达并纯化的重组HDH II/Ku蛋白表现出与分离蛋白无法区分的DNA结合和解旋酶活性。HDH II/Ku抗原仅位于细胞核内，对双链DNA具有高度特异性亲和力，含量丰富，并且新证明仅能解旋DNA双链体，这表明该分子在DNA代谢中具有另外一个（尽管仍不清楚）作用。
