Guhan, N and Muniyappa, K (2002) The RecA intein of Mycobacterium tuberculosis promotes cleavage of ectopic DNA sites. Implications for the dispersal of inteins in natural populations. In: Journal of Biological Chemistry, 277 (43). pp. 40352-61.
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The RecA intein of Mycobacterium tuberculosis, a novel double-stranded DNA endonuclease, requires both Mn(2+) and ATP for efficient cleavage of the inteinless recA allele. In this study, we show that Mg(2+) alone was sufficient to stimulate PI-MtuI to cleave double-stranded DNA at ectopic sites. In the absence of Mg(2+), 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 (Asp(122), Asp(222), and Glu(220)) together with immunoprecipitation assays provided compelling evidence to link both the Mg(2+)- and Mn(2+) 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.
|Item Type:||Journal Article|
|Additional Information:||Copyright of this article belogs to The American Society for Biochemistry and Molecular Biology.|
|Department/Centre:||Division of Biological Sciences > Biochemistry|
|Date Deposited:||24 Feb 2010 05:21|
|Last Modified:||19 Sep 2010 05:55|
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