Purification and properties of human DNA helicase VI

Abstract

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, llgase, 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. Photoaffinlty labelling wfth [α⁻³²PJATP 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 RNARNA hybrids. HDH VI unwinds DNA unidirectionally by moving In the 3' to 5' direction along the bound strand.