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Mycobacterium tuberculosis RecA intein, a LAGLIDADG homing endonuclease, displays $Mn^{2+}$ and DNA-dependent ATPase activity

Guhan, N and Muniyappa, K (2003) Mycobacterium tuberculosis RecA intein, a LAGLIDADG homing endonuclease, displays $Mn^{2+}$ and DNA-dependent ATPase activity. In: Nucleic Acids Research, 31 (14). pp. 4184-4191.

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Abstract

Mycobacterium tuberculosis RecA intein (PI-MtuI), a LAGLIDADG homing endonuclease, displays dual target specificity in response to alternative cofactors. While both ATP and $Mn^{2+}$ were required for optimal cleavage of an inteinless recA allele (hereafter referred to as cognate DNA), $Mg^{2+}$ 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 $Mn^{2+}$ was able to stimulate PI-MtuI catalyzed ATP hydrolysis, whereas $Mg^{2+}$ failed to do so. Using UV crosslinking, limited proteolysis and amino acid sequence analysis, we show that $^{32}P$-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 $Mn^{2+}$-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.

Item Type: Journal Article
Additional Information: Copyright of this article belongs to Oxford University Press.
Department/Centre: Division of Biological Sciences > Biochemistry
Date Deposited: 15 Oct 2007
Last Modified: 19 Sep 2010 04:39
URI: http://eprints.iisc.ernet.in/id/eprint/11993

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