1. Functional Properties of the Separate Subunits of Human DNA Helicase II/Ku Autoantigen
- Author
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Thierry Rabilloud, Doris Skopac, Mario Costa, András Simoncsits, Alexander Ochem, Mauro Giacca, Arturo Falaschi, Laurent Vuillard, A. E., Ochem, D., Skopac, M., Costa, T., Rabilloud, L., Vuillard, A., Simoncsit, Giacca, Mauro, and A., Falaschi
- Subjects
Adenosine Triphosphatase ,Protein Denaturation ,Protein Folding ,DNA Repair ,metabolism, Antigen ,Protein Conformation ,Protein subunit ,Autoantigens ,Biochemistry ,metabolism, Adenosine Triphosphate ,HeLa ,Adenosine Triphosphatases ,metabolism, Antigens ,Nuclear, Autoantigens ,metabolism, DNA Helicases, DNA Repair, DNA-Binding Proteins ,metabolism, HeLa Cells, Humans, Kinetics, Nuclear Proteins ,metabolism, Protein Conformation, Protein Denaturation, Protein Folding, Recombinant Proteins ,metabolism, Solubility, Transcription Factors ,metabolism ,chemistry.chemical_compound ,Adenosine Triphosphate ,metabolism, HeLa Cells, Humans, Kinetics, Nuclear Protein ,metabolism, Solubility, Transcription Factor ,Humans ,Nuclear, Autoantigen ,Nuclear ,Antigens ,Ku Autoantigen ,Molecular Biology ,metabolism, Protein Conformation, Protein Denaturation, Protein Folding, Recombinant Protein ,biology ,DNA Helicases ,Nuclear Proteins ,Helicase ,Antigens, Nuclear ,Cell Biology ,Metabolism ,biology.organism_classification ,Molecular biology ,Recombinant Proteins ,In vitro ,Ku Protein ,DNA-Binding Proteins ,Kinetics ,Solubility ,chemistry ,metabolism, DNA Helicases, DNA Repair, DNA-Binding Protein ,Duplex (building) ,biology.protein ,DNA ,HeLa Cells ,Transcription Factors - Abstract
The Ku antigen consists of two subunits of 70 and 83 kDa and is endowed with both duplex DNA end-binding capacity and helicase activity (human DNA helicase II). HeLa Ku can be isolated from in vitro cultured human cells uniquely as a heterodimer, and the subunits can be separated by electrophoresis only under denaturing conditions. To dissect the molecular functions of the two subunits of the heterodimer, we have cloned and expressed their cDNAs separately in Escherichia coli. The two activities of Ku (DNA binding and unwinding) were reconstituted by mixing and refolding both subunits in equimolar amounts (Tuteja, N., Tuteja, R., Ochem, A., Taneja, P., Huang, N-W., Simoncsits, A., Susic, S., Rahman, K., Marusic, L., Chen, J., Zang, J., Wang, S., Pongor, S., and Falaschi, A. (1994) EMBO J. 13, 4991-5001). Renaturation of the separate subunits can be achieved in the presence of a synthetic solubilizing and stabilizing agent, dimethyl ethylammonium propane sulfonate (NDSB 195). The helicase activity of the Ku protein resides uniquely in the 70-kDa subunit, whereas the DNA end-binding activity can be reconstituted only through renaturation of the two subunits in the heterodimeric form and is practically absent in the separate subunits. The 83-kDa subunit, when refolded in the absence of the 70-kDa subunit, forms homodimers unable to unwind DNA and bind duplex ends. The three separate species (heterodimer, 70-kDa subunit, and 83-kDa subunit homodimer) all have ssDNA-dependent ATPase activity.
- Published
- 1997
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