PURIFICATION AND PROPERTIES OF HUMAN DNA HELICASE-VI

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 wa s recovered, free of DNA polymerase, ligase, topoisomerase, nicking an d 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-P-32]ATP labelled the 128 kDa pro tein, Only ATP or dATP hydrolysis supports the unwinding activity for which a divalent cation (Mg2+ > Mn2+) is required, HDH VI unwinds excl usively DNA duplexes with an annealed portion <32 bp and prefers a rep lication fork-like structure of the substrate, It cannot unwind blunt- end duplexes and is inactive also on DNA-RNA or RNA-RNA hybrids. HDH V I unwinds DNA unidirectionally by moving in the 3' to 5' direction alo ng the bound strand.