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DNA-dependent ATPase from HeLa cells is related to human Ku autoantigen
1Worcester Foundation for Experimental Biology, Shrewsbury, Massachusetts 01545.
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.
Insights
This study identifies an 83/68-kDa DNA-dependent ATPase in HeLa cells as human Ku protein. Phosphorylation by a cofractionating kinase upregulates its DNA-dependent ATPase activity.
Area of Science:
- Molecular Biology
- Biochemistry
- Cell Biology
Background:
- A 150-kDa DNA-dependent ATPase associated with DNA synthesis enzymes was previously identified in HeLa cells.
- This ATPase was reported to be a 21S complex of enzymes.
Purpose of the Study:
- To purify and characterize the 150-kDa DNA-dependent ATPase from HeLa cells.
- To determine the subunit composition and identity of the purified ATPase.
- To investigate the regulatory mechanisms of the ATPase activity.
Main Methods:
- Subcellular fractionation and poly(ethylene-glycol) precipitation.
- Chromatography including Q-Sepharose, DNA-celluloses, and Mono-S FPLC.
- Polyacrylamide gel electrophoresis, peptide sequencing, immunoblot analysis, and immunoprecipitation.
Main Results:
- The purified 150-kDa ATPase consists of equimolar 83- and 68-kDa subunits.
- Peptide sequence analysis revealed high homology to human Ku protein subunits (80/70 kDa).
- Immunoblot and immunoprecipitation confirmed the ATPase subunits are part of human Ku protein.
- Both subunits are phosphorylated by a cofractionating DNA-dependent protein kinase, upregulating ATPase activity.
Conclusions:
- The 150-kDa DNA-dependent ATPase purified from HeLa cells is identified as human Ku protein.
- Phosphorylation by a DNA-dependent protein kinase regulates and enhances the ATPase activity of human Ku protein.