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 prev iously 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(ethyleneglycol ) precipitation of the combined nuclear extract/cytosol, and chromatog raphy on Q-Sepharose and native and denatured DNA/celluloses followed by Mono-S fast protein liquid chromatography. The purified enzyme show ed equimolar amounts of 83- and 68-kDa polypeptides following polyacry lamide gel electrophoresis under denaturing conditions. Sequence analy sis of peptide fragments derived from the separated 83- and 68-kDa pol ypeptides showed 90-100% homology with the corresponding 80- and 70-kD a subunits of human Ku protein. Immunoblot analysis of the ATPase duri ng the course of its purification and immunoprecipitation with antibod ies to the 80- and 70-kDa subunits of human Ku protein confirmed the r elationship of the 83- and 68-kDa polypeptides of the human DNA-depend ent ATPase to the subunits of human Ku protein. Both; the 83- and 68-k Da polypeptides are phosphorylated by a DNA-dependent protein kinase t hat cofractionates with the ATPase. The DNA-dependent ATPase activity is up regulated by phosphorylation.