Nucleoside diphosphate kinase from Mycobacterium tuberculosis cleaves single strand DNA within the human c-myc promoter in an enzyme-catalyzed reaction.
The reason for secretion of nucleoside diphosphate kinase (NdK), an enzyme involved in maintaining the cellular pool of nucleoside triphosphates in both prokaryotes and eukaryotes, by Mycobacterium tuberculosis is intriguing. We recently observed that NdK from M.tuberculosis (mNdK) localizes within nuclei of HeLa and COS-1 cells and also nicks chromosomal DNA in situ (A. K. Saini, K. Maithal, P. Chand, S. Chowdhury, R. Vohra, A. Goyal, G. P. Dubey, P. Chopra, R. Chandra, A. K. Tyagi, Y. Singh and V. Tandon (2004) J. Biol. Chem., 279, 50142-50149). In the current study, using a molecular beacon approach, we demonstrate that the mNdK catalyzes the cleavage of single strand DNA. It displays Michaelis-Menten kinetics with a kcat/K(M) of 9.65 (+/-0.88) x 10(6) M(-1) s(-1). High affinity (K(d) approximately K(M) of approximately 66 nM) and sequence-specific binding to the sense strand of the nuclease hypersensitive region in the c-myc promoter was observed. This is the first study demonstrating that the cleavage reaction is also enzyme-catalyzed in addition to the enzymatic kinase activity of multifunctional NdK. Using our approach, we demonstrate that GDP competitively inhibits the nuclease activity with a K(I) of approximately 1.9 mM. Recent evidence implicates mNdK as a potent virulence factor in tuberculosis owing to its DNase-like activity. In this context, our results demonstrate a molecular mechanism that could be the basis for assessing in situ DNA damage by secretory mNdK.
Kumar
Praveen
P
Institute of Genomics and Integrative Biology, CSIR Mall Road, Delhi 110007, India.
Verma
Anjali
A
Saini
Adesh Kumar
AK
Chopra
Puneet
P
Chakraborti
Pradip K
PK
Singh
Yogendra
Y
Chowdhury
Shantanu
S
D001665:Binding Sites; D002384:Catalysis; D003596:Cytosine / Q000378:metabolism; D004277:DNA, Single-Stranded / Q000378:metabolism*; D003851:Deoxyribonucleases / Q000378:metabolism*; D005470:Fluorometry; D016259:Genes, myc; D006801:Humans; D007700:Kinetics; D009169:Mycobacterium tuberculosis / Q000201:enzymology*; D009701:Nucleoside-Diphosphate Kinase / Q000378:metabolism*; D011401:Promoter Regions, Genetic; D011743:Pyrimidines / Q000378:metabolism
D004277:DNA, Single-Stranded; D011743:Pyrimidines; D003596:Cytosine; D009701:Nucleoside-Diphosphate Kinase; D003851:Deoxyribonucleases