The RecA intein of Mycobacterium tuberculosis promotes cleavage of ectopic DNA sites. Implications for the dispersal of inteins in natural populations.
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.
Guhan
N
N
Department of Biochemistry, Indian Institute of Science, Bangalore 560012, India.
Muniyappa
K
K
D000255:Adenosine Triphosphate / Q000378:metabolism; D001483:Base Sequence; D017931:DNA Primers; D004269:DNA, Bacterial / Q000378:metabolism*; D006868:Hydrolysis; D008345:Manganese / Q000378:metabolism; D016297:Mutagenesis, Site-Directed; D009169:Mycobacterium tuberculosis / Q000378:metabolism*; D011938:Rec A Recombinases / Q000235:genetics* / Q000378:metabolism
D017931:DNA Primers; D004269:DNA, Bacterial; D008345:Manganese; D000255:Adenosine Triphosphate; D011938:Rec A Recombinases