HUMAN DNA HELICASE-II - A NOVEL DNA UNWINDING ENZYME IDENTIFIED AS THE KU AUTOANTIGEN

Human DNA helicase II (HDH II) is a novel ATP-dependent DNA unwinding enzyme, purified to apparent homogeneity from HeLa cells, which (i) un winds exclusively DNA duplexes, (ii) prefers partially unwound substra tes 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 DN A binding capacity. All these activities comigrate in gel filtration a nd glycerol gradients, giving a sedimentation coefficient of 7.4S and a Stokes radius of similar to 46 Angstrom, corresponding to a native m olecular weight of 158 kDa. The antibodies raised in rabbit against ei ther polypeptide can remove from the solution all the activities of HD H II. Photoaffinity labelling with [alpha-P-32]ATP labelled both polyp eptides. Microsequencing of the separate polypeptides of HDH II and cr ossreaction with specific antibodies showed that this enzyme is identi cal to Ku, an autoantigen recognized by the sera of scleroderma and lu pus erythematosus patients, which binds specifically to duplex DNA end s and is a regulator of a DNA-dependent protein kinase. Recombinant HD H II/Ku protein expressed in and purified from Escherichia coli cells showed DNA binding and helicase activities indistinguishable from thos e 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 mol ecule in DNA metabolism.