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) 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.
Tuteja
N
N
International Centre for Genetic Engineering and Biotechnology, Area Science Park, Trieste, Italy.
Tuteja
R
R
Ochem
A
A
Taneja
P
P
Huang
N W
NW
Simoncsits
A
A
Susic
S
S
Rahman
K
K
Marusic
L
L
Chen
J
J
D000251:Adenosine Triphosphatases / Q000737:chemistry* / Q000276:immunology / Q000302:isolation & purification; D000595:Amino Acid Sequence; D034961:Antigens, Nuclear; D001483:Base Sequence; D015153:Blotting, Western; D003001:Cloning, Molecular; D004265:DNA Helicases; D004268:DNA-Binding Proteins / Q000737:chemistry* / Q000276:immunology / Q000302:isolation & purification; D006367:HeLa Cells; D006801:Humans; D000072200:Ku Autoantigen; D008969:Molecular Sequence Data; D009500:Neutralization Tests; D009687:Nuclear Proteins / Q000737:chemistry* / Q000276:immunology / Q000302:isolation & purification; D017386:Sequence Homology, Amino Acid; D013379:Substrate Specificity
D034961:Antigens, Nuclear; D004268:DNA-Binding Proteins; D009687:Nuclear Proteins; D000251:Adenosine Triphosphatases; D004265:DNA Helicases; C106893:XRCC5 protein, human; C000606509:Xrcc6 protein, human; D000072200:Ku Autoantigen