Mycobacterium tuberculosis RecA intein possesses a novel ATP-dependent site-specific double-stranded DNA endonuclease activity

Abstract

Mycobacterium tuberculosis recAharbors 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 Mn²⁺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 recAallele. 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 Mn²⁺-ATP-dependent double-strand specific endonuclease, which is likely to be important for homing processin vivo.