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J. Biol. Chem., Vol. 279, Issue 48, 50142-50149, November 26, 2004

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Nuclear Localization and in Situ DNA Damage by Mycobacterium tuberculosis Nucleoside-diphosphate Kinase^*

Adesh Kumar Saini, Kapil Maithal, Prem Chand¶, Shantanu Chowdhury, Reena Vohra, Anita Goyal, Gyanendra P. Dubey, Puneet Chopra, Ramesh Chandra, Anil K. Tyagi||, Yogendra Singh**, and Vibha Tandon

From the Dr. B. R. Ambedkar Center for Biomedical Research, University of Delhi, Delhi 110 007, India, Institute of Genomics and Integrative Biology, Mall Road, Delhi 110 007, India, ^¶Department of Physics, Indian Institute of Technology, Kanpur 208076, Uttar Pradesh, India, and ^||Department of Biochemistry, University of Delhi, Benito Juarez Road, New Delhi 110021, India

Nucleoside-diphosphate kinase of Mycobacterium tuberculosis (mNdK) is a secretory protein, but the rationale behind secreting an enzyme involved in the maintenance of cellular pool of nucleoside triphosphates is not clearly understood. To elucidate the biological significance of mNdK secretion, we expressed mNdK fused to green fluorescent protein in HeLa and COS-1 cells. Interestingly, mNdK was detected in the nuclei of HeLa and COS-1 cells. Incubation of mNdK with nuclei isolated from HeLa and COS-1 cells led to in situ damage of chromosomal DNA. Surface plasmon resonance studies demonstrated that mNdK binds supercoiled plasmid DNA lacking apurinic/apyrimidinic sites with a dissociation constant of 30 ± 3.2 µM. Plasmid cleavage by mNdK was found to be dependent on the specific divalent metal ion and inhibited by a metal ion chelator. Moreover, the metal ion-dependent DNA cleavage by mNdK was mediated by superoxide radicals as detected by electron paramagnetic resonance. The cleavage reaction was inhibited under nitrogen atmosphere confirming the necessity of molecular oxygen for DNA cleavage. In view of the findings that mNdK is secreted by intracellular mycobacteria and damages the nuclear DNA, it can be postulated that mNdK may cause cell death that could help in the dissemination of the pathogen.

Received for publication, August 30, 2004 , and in revised form, September 15, 2004.

^* This work was supported by Grant NMITLI/SMM0003 from the Council of Scientific and Industrial Research, India. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

^** To whom correspondence may be addressed. Tel.: 91-11-2766-6156; Fax: 91-11-2766-7471; E-mail: ysingh{at}igib.res.in. To whom correspondence may be addressed. Tel.: 91-11-2766-6272; Fax: 91-11-2766-6248; E-mail: vtandon{at}acbrdu.edu.

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