Mycobacterium tuberculosis RecA intein possesses a novel ATP-dependent site-specific double-stranded DNA endonuclease activity.
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.
Guhan
N
N
Department of Biochemistry, Indian Institute of Science, Bangalore 560 012, India.
Muniyappa
K
K
D000255:Adenosine Triphosphate / Q000378:metabolism*; D001483:Base Sequence; D001665:Binding Sites; D004269:DNA, Bacterial / Q000737:chemistry / Q000378:metabolism*; D003850:Deoxyribonuclease I / Q000378:metabolism*; D007438:Introns; D007700:Kinetics; D008345:Manganese / Q000378:metabolism; D008969:Molecular Sequence Data; D009169:Mycobacterium tuberculosis / Q000201:enzymology / Q000235:genetics / Q000378:metabolism*; D016366:Open Reading Frames; D011938:Rec A Recombinases / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D011993:Recombinant Fusion Proteins / Q000378:metabolism
D004269:DNA, Bacterial; D011993:Recombinant Fusion Proteins; D008345:Manganese; D000255:Adenosine Triphosphate; D011938:Rec A Recombinases; D003850:Deoxyribonuclease I