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J. Biol. Chem., Vol. 277, Issue 43, 40352-40361, October 25, 2002

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The RecA Intein of Mycobacterium tuberculosis Promotes Cleavage of Ectopic DNA Sites
IMPLICATIONS FOR THE DISPERSAL OF INTEINS IN NATURAL POPULATIONS^*

N. Guhan and K. Muniyappa^§

From the Department of Biochemistry, Indian Institute of Science, Bangalore 560012, India

The RecA intein of Mycobacterium tuberculosis, a novel double-stranded DNA endonuclease, requires both Mn²⁺ and ATP for efficient cleavage of the inteinless recA allele. In this study, we show that Mg²⁺ alone was sufficient to stimulate PI-MtuI to cleave double-stranded DNA at ectopic sites. In the absence of Mg²⁺, PI-MtuI formed complexes with topologically different forms of DNA containing ectopic recognition sequences with equal affinity but failed to cleave DNA. We observed that PI-MtuI was able to inflict double-strand breaks robustly within the ectopic recognition sequence to generate either a blunt end or 1-2-nucleotide 3'-hydroxyl overhangs. Mutational analyses of the presumptive metal ion-binding ligands (Asp¹²², Asp²²², and Glu²²⁰) together with immunoprecipitation assays provided compelling evidence to link both the Mg²⁺- and Mn²⁺ and ATP-dependent endonuclease activities to PI-MtuI. The kinetic mechanism of PI-MtuI promoted cleavage of ectopic DNA sites proceeded through a sequential mechanism with transient accumulation of nicked circular duplex DNA as an intermediate. Together, these data suggest that PI-MtuI, like group II introns, might mediate ectopic DNA transposition and hence its lateral transfer in natural populations.

This article has been cited by other articles:

				N. Guhan and K. Muniyappa Mycobacterium tuberculosis RecA intein, a LAGLIDADG homing endonuclease, displays Mn2+ and DNA-dependent ATPase activity Nucleic Acids Res., July 15, 2003; 31(14): 4184 - 4191. [Abstract] [Full Text] [PDF]