Tuteja N, Ochem A, Taneja P, Tuteja R, Skopác D, Falaschi A
International Centre for Genetic Engineering and Biotechnology, Trieste, Italy.
Nucleic Acids Res. 1995 Jul 11;23(13):2457-63. doi: 10.1093/nar/23.13.2457.
A novel ATP-dependent DNA unwinding enzyme, called human DNA helicase VI (HDH VI), was purified to apparent homogeneity from HeLa cells and characterized. From 327 g of cultured cells, 0.44 mg of pure enzyme was recovered, free of DNA polymerase, ligase, topoisomerase, nicking and nuclease activities. The enzyme behaves as a monomer having an M(r) of 128 kDa, whether determined with SDS-PAGE, or in native conditions. Photoaffinity labelling with [alpha-32P]ATP labelled the 128 kDa protein. Only ATP or dATP hydrolysis supports the unwinding activity for which a divalent cation (Mg2+ > Mn2+) is required. HDH VI unwinds exclusively DNA duplexes with an annealed portion < 32 bp and prefers a replication fork-like structure of the substrate. It cannot unwind blunt-end duplexes and is inactive also on DNA-RNA or RNA-RNA hybrids. HDH VI unwinds DNA unidirectionally by moving in the 3' to 5' direction along the bound strand.
一种名为人类DNA解旋酶VI（HDH VI）的新型ATP依赖性DNA解旋酶从HeLa细胞中纯化至表观均一，并进行了特性分析。从327克培养细胞中回收了0.44毫克纯酶，该酶不含DNA聚合酶、连接酶、拓扑异构酶、切口酶和核酸酶活性。无论是通过SDS-PAGE还是在天然条件下测定，该酶均表现为单体，分子量为128 kDa。用[α-32P]ATP进行光亲和标记标记了128 kDa的蛋白质。只有ATP或dATP水解支持解旋活性，该活性需要二价阳离子（Mg2+>Mn2+）。HDH VI仅解开退火部分<32 bp的DNA双链体，并且更喜欢底物的复制叉状结构。它不能解开平端双链体，对DNA-RNA或RNA-RNA杂交体也无活性。HDH VI通过沿着结合链从3'向5'方向移动来单向解开DNA。
