The RecA Intein of Mycobacterium tuberculosisPromotes Cleavage of Ectopic DNA Sites

Abstract

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 doublestranded 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. Mobile inteins and introns are genetic elements capable of self-propagation by "homing" into host genes in a wide variety of organisms: eubacteria, eukarya, archaea, and viruses (reviewed in Ref. 1). The process is promoted by a homing endonuclease, which is encoded by an open reading frame embedded within the genetic element. The genes for homing endonucleases are found among group I and group II introns, archaeal introns, intein-coding sequences, and free standing open reading frames (reviewed in Refs. 1-6). Inteins are genetic elements present within protein-coding sequences with dual function: protein-splicing and homing endonuclease activities. They are believed to play a central role in rearrangement of organelle as well as nuclear genomes (1-6). The hallmark of homing endonucleases is their ability to recognize and cleave extended asymmetric sequences (14 -40 bp) that are generally centered on the intein insertion site in inteinless alleles (1, 7). Homing endonucleases can be classified into four families based on the presence of conserved motifs: LAGLIDADG, GIY-YIG, His-Cys box, and H-N-H (1,5,6,8). Among these, the LAGLIDADG family is the largest, widespread and much studied class of homing endonucleases. Structural and biochemical studies have demonstrated that homing endonucleases with one LAGLIDADG motif act as homodimers, whereas enzymes with two such motifs function as monomers during catalysis (9 -12). Under standard assay conditions in vitro, these enzymes are extremely specific for their recognition sites. However, recent evidence suggests that self-splicing group II introns transpose into ectopic DNA sites that resemble their natural homing sites (13,14). The transposition of group II introns involves reverse splicing of the intron into the intronless allele, which are then reverse transcribed to give complementary DNA. Following the synthesis of the second strand, the intron is incorporated into the genomic DNA by homologous recombination (1-6). But equivalent information is not available for any intein endonuclease. The exact sequence of events that lead to recognition and cleavage of DNA by homing endonucleases is poorly understood. In addition, in no case the molecular mechanism underlying cleavage of ectopic DNA sites by an intein endonuclease has been elucidated.Mycobacterium tuberculosis RecA intein (PI-MtuI) 1 is a member of the LAGLIDADG...

Characterization of Mycobacterium leprae RecA Intein, a LAGLIDADG Homing Endonuclease, Reveals a Unique Mode of DNA Binding, Helical Distortion, and Cleavage Compared with a Canonical LAGLIDADG Homing Endonuclease

“…Each constant represents the average of three experiments. These values are similar to those reported previously for PIMtuI cleavage of form I DNA containing its noncognate substrate (13). Furthermore, the extremely low k cat value corroborates with that of a number of intein-encoded and intronencoded homing endonucleases (13,41,47).…”

“…Effect of Ionic Strength on DNA Cleavage by PI-MleI-The assay buffer contained 25 mM Tris-HCl and 3 mM MnCl 2 or MgCl 2 , which is comparable with standard conditions used in studies with other homing endonucleases (12,13,41). We therefore wished to test the effect of increasing ionic strength on PI-MleI endonuclease activity.…”

“…To this end, we monitored the con- version of negatively supercoiled DNA (form I) to form II and/or form III DNA, a widely used sensitive method for quantification of endonuclease activity (12,13). Cleavage assays were carried out by mixing PI-MleI with form I pMLR DNA containing the intein-less recA allele, the natural recognition sequence of PI-MleI, as the substrate in the presence of 5 mM Mg 2ϩ or Mn 2ϩ .…”

“…The occurrence of inteins in the obligate mycobacterial pathogens, M. tuberculosis, M. leprae, and Mycobacterium microti, suggested that RecA inteins might play a role in mycobacterial functions related to pathogenesis or virulence (9). Previously, we have shown that M. tuberculosis RecA intein (PI-MtuI), 2 which contains Walker A motif, displays dual target specificity in the presence of alternative cofactors in an ATP-dependent manner (12,13).…”

Mutational analysis of active‐site residues in the Mycobacterium leprae RecA intein, a LAGLIDADG homing endonuclease: Asp122 and Asp193 are crucial to the double‐stranded DNA cleavage activity whereas Asp218 is not

“…37 These results are also in agreement with the studies from other enzymes in this family. 47 Alignment of amino acid sequence of PIMleI with other HEases in the LAGLIDADG family revealed the presence of single copy of DOD motif (sequence) in Block C, two copies of the same motif in Block E, and absence of Block D (Fig. 1).…”