Functional Properties of the Separate Subunits of Human DNA Helicase II/Ku Autoantigen*
- Alexander E. Ochem‡,
- Doris Skopac‡,
- Mario Costa‡,
- Thierry Rabilloud§,
- Laurent Vuillard¶,
- András Simoncsits‖,
- Mauro Giacca**‡ and
- Arturo Falaschiत
- From the ‡Molecular Biology Unit, ‖Protein Structure and Function Group, and **Molecular Medicine Unit, International Centre for Genetic Engineering and Biotechnology, Padriciano 99, 34012 Trieste, Italy, §Departement de Biologie Moléculaire et Structurale, Centre d’Etudes Nucleaires-Grenoble, 17 Rue des Martyrs, 38042 Grenoble Cedex 09, France, ¶Institut Laue-Langevin, BP 156, 38042 Grenoble Cedex 09, France, and ‡Istituto di Genetica Biochimica ed Evoluzionistica del Consiglio Nazionale delle Ricerche, Via Abbiategrasso 207, 27100 Pavia, Italy
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 fromin 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 inEscherichia 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.
Footnotes
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↵* The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked “advertisement” in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
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↵§§ To whom correspondence should be addressed. Tel.: 39-40-3757303; Fax: 39-40-3757353; E-mail:falaschi{at}icgeb.trieste.it.
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↵1 The abbreviations used are: HDH II, human DNA helicase II; PAGE, polyacrylamide gel electrophoresis; ssDNA, single-stranded DNA; NDSB, dimethyl ethylammonium propane sulfonate.
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- Received April 29, 1997.
- Revision received August 13, 1997.
