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Nucleic Acids Research, 2003, Vol. 31, No. 14 4184-4191
© 2003 Oxford University Press

Mycobacterium tuberculosis RecA intein, a LAGLIDADG homing endonuclease, displays Mn2+ and DNA-dependent ATPase activity

N. Guhan and K. Muniyappa*

Department of Biochemistry, Indian Institute of Science, Bangalore 560 012, India

*To whom correspondence should be addressed. Tel: +91 80 360 0278; Fax: +91 80 360 0814; Email: kmbc{at}biochem.iisc.ernet.in

Mycobacterium tuberculosis RecA intein (PI-MtuI), a LAGLIDADG homing endonuclease, displays dual target specificity in response to alternative cofactors. While both ATP and Mn2+ were required for optimal cleavage of an inteinless recA allele (hereafter referred to as cognate DNA), Mg2+ alone was sufficient for cleavage of ectopic DNA sites. In this study, we have explored the ability of PI-MtuI to catalyze ATP hydrolysis in the presence of alternative metal ion cofactors and DNA substrates. Our results indicate that PI-MtuI displays maximum ATPase activity in the presence of cognate but not ectopic DNA. Kinetic analysis revealed that Mn2+ was able to stimulate PI-MtuI catalyzed ATP hydrolysis, whereas Mg2+ failed to do so. Using UV crosslinking, limited proteolysis and amino acid sequence analysis, we show that 32P-labeled ATP was bound to a 14 kDa peptide containing the putative Walker A motif. Furthermore, the limited proteolysis approach disclosed that cognate DNA was able to induce structural changes in PI-MtuI. Mutation of the presumptive metal ion-binding ligands (Asp122 and Asp222) in the LAGLIDADG motifs of PI-MtuI impaired its affinity for ATP, thus resulting in a reduction in or loss of its endonuclease activity. Together, these results suggest that PI-MtuI is a (cognate) DNA- and Mn2+-dependent ATPase, unique from the LAGLIDADG family of homing endonucleases, and implies a possible role for ATP hydrolysis in the recognition and/or cleavage of homing site DNA sequence.


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 J. Biol. Chem.Home page
P. Singh, P. Tripathi, G. H. Silva, A. Pingoud, and K. Muniyappa
Characterization of Mycobacterium leprae RecA Intein, a LAGLIDADG Homing Endonuclease, Reveals a Unique Mode of DNA Binding, Helical Distortion, and Cleavage Compared with a Canonical LAGLIDADG Homing Endonuclease
J. Biol. Chem., September 18, 2009; 284(38): 25912 - 25928.
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