DNA-dependent ATPase from HeLa cells is related to human Ku autoantigen.
A 150-kDa DNA-dependent ATPase composed of 83/68-kDa subunits was previously reported to cofractionate with a 21S complex of enzymes for DNA synthesis from HeLa cells (Vishwanatha, J. K., & Baril, E. F. (1990) Biochemistry 29, 8753-8759). The DNA-dependent ATPase was purified to electrophoretic homogeneity from a HeLa cell homogenate by a modified procedure that involves subcellular fractionation, poly(ethylene-glycol) precipitation of the combined nuclear extract/cytosol, and chromatography on Q-Sepharose and native and denatured DNA/celluloses followed by Mono-S fast protein liquid chromatography. The purified enzyme showed equimolar amounts of 83- and 68-kDa polypeptides following polyacrylamide gel electrophoresis under denaturing conditions. Sequence analysis of peptide fragments derived from the separated 83- and 68-kDa polypeptides showed 90-100% homology with the corresponding 80- and 70-kDa subunits of human Ku protein. Immunoblot analysis of the ATPase during the course of its purification and immunoprecipitation with antibodies to the 80- and 70-kDa subunits of human Ku protein confirmed the relationship of the 83- and 68-kDa polypeptides of the human DNA-dependent ATPase to the subunits of human Ku protein. Both the 83- and 68-kDa polypeptides are phosphorylated by a DNA-dependent protein kinase that cofractionates with the ATPase. The DNA-dependent ATPase activity is up regulated by phosphorylation.
Worcester Foundation for Experimental Biology, Shrewsbury, Massachusetts 01545.
Q P
QP
Cao
S
S
Pitt
J
J
Leszyk
E F
EF
Baril
D000251:Adenosine Triphosphatases / Q000737:chemistry* / Q000302:isolation & purification; D000595:Amino Acid Sequence; D034961:Antigens, Nuclear; D011232:Chemical Precipitation; D002845:Chromatography; D002851:Chromatography, High Pressure Liquid; D004265:DNA Helicases; D004268:DNA-Binding Proteins / Q000737:chemistry*; D004591:Electrophoresis, Polyacrylamide Gel; D006367:HeLa Cells / Q000737:chemistry*; D006801:Humans; D006863:Hydrogen-Ion Concentration; D015151:Immunoblotting; D007163:Immunosorbent Techniques; D000072200:Ku Autoantigen; D008969:Molecular Sequence Data; D009687:Nuclear Proteins / Q000737:chemistry*; D010766:Phosphorylation; D017421:Sequence Analysis
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