Mycobacterium tuberculosis RecA Intein Possesses a Novel ATP-dependent Site-specific Double-stranded DNA Endonuclease Activity

Guhan, N and Muniyappa, K (2002) Mycobacterium tuberculosis RecA Intein Possesses a Novel ATP-dependent Site-specific Double-stranded DNA Endonuclease Activity. In: The Journal of Biological Chemistry, 277 (18). pp. 16257-16264.

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Abstract

Mycobacterium tuberculosis recA harbors an intervening sequence in its open reading frame, presumed to encode an endonuclease (PI-MtuI) required for intein homing in inteinless recA allele. Although the protein-splicing ability of PI-MtuI has been characterized, the identification of its putative endonuclease activity has remained elusive. To investigate whether PI-MtuI possesses endonuclease activity, recA intervening sequence was cloned, overexpressed, and purified to homogeneity. Here we show that PI-MtuI bound both single- and double-stranded DNA with similar affinity but failed to cleave DNA in the absence of cofactors. Significantly, PI-MtuI nicked supercoiled DNA in the presence of alternative cofactors but required both $Mn^{2+}$ and ATP to generate linear double-stranded DNA. We observed that PI-MtuI was able to inflict a staggered double-strand break 24 bp upstream of the insertion site in the inteinless recA allele. Similar to a few homing endonucleases, DNA cleavage by PI-MtuI was specific with an exceptionally long cleavage site spanning 22 bp. The kinetic mechanism of PI-MtuI promoted cleavage supports a sequential rather than concerted pathway of strand cleavage with the formation of nicked double-stranded DNA as an intermediate. Together, these results reveal that RecA intein is a novel $Mn^{2+}$-ATP-dependent double-strand specific endonuclease, which is likely to be important for homing process in vivo.

Item Type:	Journal Article
Publication:	The Journal of Biological Chemistry
Publisher:	American Society for Biochemistry and Molecular Biology
Additional Information:	Copyright of this article belongs to American Society for Biochemistry and Molecular Biology.
Department/Centre:	Division of Biological Sciences > Biochemistry
Date Deposited:	05 Oct 2007
Last Modified:	19 Sep 2010 04:40
URI:	http://eprints.iisc.ac.in/id/eprint/12097

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