DNA-Dependent ATPase from HeLa Cells Is Related to Human Ku Autoantigen

Abstract

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.

High salt- and SDS-stable DNA binding protein complexes with ATPase and protein kinase activity retained in chromatin-depleted nuclei

“…The tight association of these enzymatic activities with DNA are reminiscent of a DNA-dependent protein kinase/ATPase complex detected in HeLa cells (15)(16)(17)(18). The latter complex has been suggested to be involved in the regulation of transcription, DNA replication and cell growth (17).…”

“…The latter complex has been suggested to be involved in the regulation of transcription, DNA replication and cell growth (17). Although the physical constants ofthe previously described complex are clearly different (16)(17)(18), it is of interest that a combined protein kinase/ATPase activity has been correlated with DNA transcription and replication (17), functions that are also expected to be associated with the bases of DNA loops (2,3).…”

Human normal peripheral blood B-lymphocytes are deficient in DNA-dependent protein kinase activity due to the expression of a variant form of the Ku86 protein

“…Biochemical analyses of Ku demonstrated that it bound in vitro to DNA termini or other discontinuities in the DNA structure without apparent sequence speci®city (Minori and Hardin, 1986;Blier et al, 1993;Falzon et al, 1993). Ku has also been shown to possess DNA-dependent ATPase (Cao et al, 1994) and helicase (Tuteja et al, 1994) activities. Lastly, Ku has also been reported to be capable of sequence-speci®c binding within the promoter elements of genes (Gri n et al, 1996).…”

Transfer of Ku86 RNA antisense decreases the radioresistance of human fibroblasts

“…22 Biochemical analyses of Ku demonstrated that it bound in vitro to DNA termini in the DNA structure without apparent sequence specificity. [23][24][25] Ku has also been shown to possess DNA-dependent adenosine triphosphatase 26 and helicase 27 activities. In vitro, heterodimerization of active Ku requires the cotranslation of both subunits.…”