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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

2009; Elsevier BV; Volume: 284; Issue: 38 Linguagem: Inglês

10.1074/jbc.m109.042861

ISSN

1083-351X

Autores

Pawan Singh, Pankaj Tripathi, George H. Silva, Alfred Pingoud, K. Muniyappa,

Tópico(s)

Biochemical and Molecular Research

Resumo

Mycobacterium leprae, which has undergone reductive evolution leaving behind a minimal set of essential genes, has retained intervening sequences in four of its genes implicating a vital role for them in the survival of the leprosy bacillus. A single in-frame intervening sequence has been found embedded within its recA gene. Comparison of the M. leprae recA intervening sequence with the known intervening sequences indicated that it has the consensus amino acid sequence necessary for being a LAGLIDADG-type homing endonuclease. In light of massive gene decay and function loss in the leprosy bacillus, we sought to investigate whether its recA intervening sequence encodes a catalytically active homing endonuclease. Here we show that the purified M. leprae RecA intein (PI-MleI) binds to cognate DNA and displays endonuclease activity in the presence of alternative divalent cations, Mg2+ or Mn2+. A combination of approaches, including four complementary footprinting assays such as DNase I, copper-phenanthroline, methylation protection, and KMnO4, enhancement of 2-aminopurine fluorescence, and mapping of the cleavage site revealed that PI-MleI binds to cognate DNA flanking its insertion site, induces helical distortion at the cleavage site, and generates two staggered double strand breaks. Taken together, these results implicate that PI-MleI possesses a modular structure with separate domains for DNA target recognition and cleavage, each with distinct sequence preferences. From a biological standpoint, it is tempting to speculate that our findings have implications for understanding the evolution of the LAGLIDADG family of homing endonucleases. Mycobacterium leprae, which has undergone reductive evolution leaving behind a minimal set of essential genes, has retained intervening sequences in four of its genes implicating a vital role for them in the survival of the leprosy bacillus. A single in-frame intervening sequence has been found embedded within its recA gene. Comparison of the M. leprae recA intervening sequence with the known intervening sequences indicated that it has the consensus amino acid sequence necessary for being a LAGLIDADG-type homing endonuclease. In light of massive gene decay and function loss in the leprosy bacillus, we sought to investigate whether its recA intervening sequence encodes a catalytically active homing endonuclease. Here we show that the purified M. leprae RecA intein (PI-MleI) binds to cognate DNA and displays endonuclease activity in the presence of alternative divalent cations, Mg2+ or Mn2+. A combination of approaches, including four complementary footprinting assays such as DNase I, copper-phenanthroline, methylation protection, and KMnO4, enhancement of 2-aminopurine fluorescence, and mapping of the cleavage site revealed that PI-MleI binds to cognate DNA flanking its insertion site, induces helical distortion at the cleavage site, and generates two staggered double strand breaks. Taken together, these results implicate that PI-MleI possesses a modular structure with separate domains for DNA target recognition and cleavage, each with distinct sequence preferences. From a biological standpoint, it is tempting to speculate that our findings have implications for understanding the evolution of the LAGLIDADG family of homing endonucleases. Mycobacterium leprae, a Gram-positive rod-shaped bacillus, mostly found in warm tropical countries, is the bacterium that causes leprosy in humans (1Cole S.T. Eiglmeier K. Parkhill J. James K.D. Thomson N.R. Wheeler P.R. Honoré N. Garnier T. Churcher C. Harris D. Mungall K. Basham D. Brown D. Chillingworth T. Connor R. Davies R.M. Devlin K. Duthoy S. Feltwell T. Fraser A. Hamlin N. Holroyd S. Hornsby T. Jagels K. Lacroix C. Maclean J. Moule S. Murphy L. Oliver K. Quail M.A. Rajandream M.A. Rutherford K.M. Rutter S. Seeger K. Simon S. Simmonds M. Skelton J. Squares R. Squares S. Stevens K. Taylor K. Whitehead S. Woodward J.R. Barrell B.G. Nature. 2001; 409: 1007-1011Crossref PubMed Scopus (1357) Google Scholar). The lack of understanding of the basic biology of M. leprae is believed to be the key factor for the failure of leprosy research to advance. The genome sequence of M. leprae contains 3.27 Mb and has an average G + C content of 57.8%, values much lower than the corresponding values for Mycobacterium tuberculosis, which are ∼4.41 Mb and 65.6% G + C, respectively (2Brosch R. Gordon S.V. Eiglmeier K. Garnier T. Cole S.T. Res. Microbiol. 2000; 151: 135-142Crossref PubMed Scopus (46) Google Scholar). There are some 1500 genes that are common to both M. leprae and M. tuberculosis. The comparative genome analysis suggests that both species of mycobacteria are derived from a common ancestor and, at one stage, had gene pools of similar size. The downsizing of the M. tuberculosis genome from ∼4.41 to 3.27 Mb of M. leprae would account for the loss of some 1200 protein-coding sequences (1Cole S.T. Eiglmeier K. Parkhill J. James K.D. Thomson N.R. Wheeler P.R. Honoré N. Garnier T. Churcher C. Harris D. Mungall K. Basham D. Brown D. Chillingworth T. Connor R. Davies R.M. Devlin K. Duthoy S. Feltwell T. Fraser A. Hamlin N. Holroyd S. Hornsby T. Jagels K. Lacroix C. Maclean J. Moule S. Murphy L. Oliver K. Quail M.A. Rajandream M.A. Rutherford K.M. Rutter S. Seeger K. Simon S. Simmonds M. Skelton J. Squares R. Squares S. Stevens K. Taylor K. Whitehead S. Woodward J.R. Barrell B.G. Nature. 2001; 409: 1007-1011Crossref PubMed Scopus (1357) Google Scholar, 3Cole S.T. Eur. Respir. J. 2002; 36: S78-S86Crossref Google Scholar). There is evidence that many of the genes that were present in the genome of M. leprae have truly been lost (1Cole S.T. Eiglmeier K. Parkhill J. James K.D. Thomson N.R. Wheeler P.R. Honoré N. Garnier T. Churcher C. Harris D. Mungall K. Basham D. Brown D. Chillingworth T. Connor R. Davies R.M. Devlin K. Duthoy S. Feltwell T. Fraser A. Hamlin N. Holroyd S. Hornsby T. Jagels K. Lacroix C. Maclean J. Moule S. Murphy L. Oliver K. Quail M.A. Rajandream M.A. Rutherford K.M. Rutter S. Seeger K. Simon S. Simmonds M. Skelton J. Squares R. Squares S. Stevens K. Taylor K. Whitehead S. Woodward J.R. Barrell B.G. Nature. 2001; 409: 1007-1011Crossref PubMed Scopus (1357) Google Scholar, 3Cole S.T. Eur. Respir. J. 2002; 36: S78-S86Crossref Google Scholar). Comparative genomics of M. leprae with that of M. tuberculosis indicate that the former has undergone substantial downsizing, losing more than 2000 genes, thus suggesting an extreme case of reductive evolution in a microbial pathogen (1Cole S.T. Eiglmeier K. Parkhill J. James K.D. Thomson N.R. Wheeler P.R. Honoré N. Garnier T. Churcher C. Harris D. Mungall K. Basham D. Brown D. Chillingworth T. Connor R. Davies R.M. Devlin K. Duthoy S. Feltwell T. Fraser A. Hamlin N. Holroyd S. Hornsby T. Jagels K. Lacroix C. Maclean J. Moule S. Murphy L. Oliver K. Quail M.A. Rajandream M.A. Rutherford K.M. Rutter S. Seeger K. Simon S. Simmonds M. Skelton J. Squares R. Squares S. Stevens K. Taylor K. Whitehead S. Woodward J.R. Barrell B.G. Nature. 2001; 409: 1007-1011Crossref PubMed Scopus (1357) Google Scholar). With the availability of the M. leprae genome sequence, using functional genomics approaches, it is possible to identify the gene products, elucidate the mechanism of their action, and identify novel drug targets for rational design of new therapeutic regimens and drugs to treat leprosy.Eubacterial RecA proteins catalyze a set of biochemical reactions that are essential for homologous recombination, DNA repair, restoration of stalled replication forks, and SOS response (4Kowalczykowski S.C. Dixon D.A. Eggleston A.K. Lauder S.D. Rehrauer W.M. Microbiol. Rev. 1994; 58: 401-465Crossref PubMed Google Scholar, 5Lusetti S.L. Cox M.M. Annu. Rev. Biochem. 2002; 71: 71-100Crossref PubMed Scopus (349) Google Scholar, 6Schlacher K. Pham P. Cox M.M. Goodman M.F. Chem. Rev. 2006; 106: 406-419Crossref PubMed Scopus (57) Google Scholar, 7Chen Z. Yang H. Pavletich N.P. Nature. 2008; 453: 489-504Crossref PubMed Scopus (493) Google Scholar). RecA protein and the process of homologous recombination, which is the main mechanism of genetic exchange, are evolutionarily conserved among a range of organisms (4Kowalczykowski S.C. Dixon D.A. Eggleston A.K. Lauder S.D. Rehrauer W.M. Microbiol. Rev. 1994; 58: 401-465Crossref PubMed Google Scholar, 7Chen Z. Yang H. Pavletich N.P. Nature. 2008; 453: 489-504Crossref PubMed Scopus (493) Google Scholar). Perhaps the most striking development in the field of RecA protein biology was the discovery of an in-frame insertion of an intein-coding sequence in the recA genes of M. tuberculosis and M. leprae (8Davis E.O. Sedgwick S.G. Colston M.J. J. Bacteriol. 1991; 173: 5653-5662Crossref PubMed Google Scholar, 9Davis E.O. Thangaraj H.S. Brooks P.C. Colston M.J. EMBO J. 1994; 13: 699-703Crossref PubMed Scopus (88) Google Scholar). In these organisms, RecA is synthesized as a large precursor, which undergoes protein splicing to excise the intein, and the two flanking domains called exteins are ligated together to generate a functionally active RecA protein (9Davis E.O. Thangaraj H.S. Brooks P.C. Colston M.J. EMBO J. 1994; 13: 699-703Crossref PubMed Scopus (88) Google Scholar, 10Davis E.O. Jenner P.J. Brooks P.C. Colston M.J. Sedgwick S.G. Cell. 1992; 71: 201-210Abstract Full Text PDF PubMed Scopus (140) Google Scholar). The milieu in which RecA precursor undergoes splicing differs substantially between M. tuberculosis and M. leprae. M. leprae RecA precursor (79 kDa) undergoes splicing only in mycobacterial species, whereas M. tuberculosis RecA precursor (85 kDa) is spliced efficiently in Escherichia coli as well (9Davis E.O. Thangaraj H.S. Brooks P.C. Colston M.J. EMBO J. 1994; 13: 699-703Crossref PubMed Scopus (88) Google Scholar, 10Davis E.O. Jenner P.J. Brooks P.C. Colston M.J. Sedgwick S.G. Cell. 1992; 71: 201-210Abstract Full Text PDF PubMed Scopus (140) Google Scholar, 11Frischkorn K. Springer B. Böttger E.C. Davis E.O. Colston M.J. Sander P. J. Bacteriol. 2000; 182: 3590-3592Crossref PubMed Scopus (8) Google Scholar). Intriguingly, M. tuberculosis and M. leprae RecA inteins differ greatly in their size, primary sequence, and location within the recA gene, thereby suggesting two independent origins during evolution (9Davis E.O. Thangaraj H.S. Brooks P.C. Colston M.J. EMBO J. 1994; 13: 699-703Crossref PubMed Scopus (88) Google Scholar). 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 (9Davis E.O. Thangaraj H.S. Brooks P.C. Colston M.J. EMBO J. 1994; 13: 699-703Crossref PubMed Scopus (88) Google Scholar). Previously, we have shown that M. tuberculosis RecA intein (PI-MtuI), 2The abbreviations used are: PI-MtuIM. tuberculosis RecA inteinPI-MleIM. leprae RecA inteinDTTdithiothreitolDMSdimethyl sulfate2-AP2 aminopurineODNoligonucleotideATPγSadenosine 5′-O-(thiotriphosphate)(OP)2Cu2+phenanthroline-copper. 2The abbreviations used are: PI-MtuIM. tuberculosis RecA inteinPI-MleIM. leprae RecA inteinDTTdithiothreitolDMSdimethyl sulfate2-AP2 aminopurineODNoligonucleotideATPγSadenosine 5′-O-(thiotriphosphate)(OP)2Cu2+phenanthroline-copper. which contains Walker A motif, displays dual target specificity in the presence of alternative cofactors in an ATP-dependent manner (12Guhan N. Muniyappa K. J. Biol. Chem. 2002; 277: 16257-16264Abstract Full Text Full Text PDF PubMed Scopus (14) Google Scholar, 13Guhan N. Muniyappa K. J. Biol. Chem. 2002; 277: 40352-40361Abstract Full Text Full Text PDF PubMed Scopus (6) Google Scholar).Since their discovery in Saccharomyces cerevisiae (14Kane P.M. Yamashiro C.T. Wolczyk D.F. Neff N. Goebl M. Stevens T.H. Science. 1990; 250: 651-657Crossref PubMed Scopus (384) Google Scholar, 15Hirata R. Ohsumk Y. Nakano A. Kawasaki H. Suzuki K. Anraku Y. J. Biol. Chem. 1990; 265: 6726-6733Abstract Full Text PDF PubMed Google Scholar), a large number of putative homing endonucleases have been found in a diverse range of proteins in all the three domains of life (16Pietrokovski S. Trends Genet. 2001; 17: 465-472Abstract Full Text Full Text PDF PubMed Scopus (99) Google Scholar, 17Perler F.B. Nucleic Acids Res. 2002; 30: 383-384Crossref PubMed Scopus (317) Google Scholar, 18Belfort M. Derbyshire V. Stoddard B.L. Wood D.W. Homing Endonucleases and Inteins (Nucleic Acids and Molecular Biology). Vol. 16. Springer-Verlag, Berlin Heidelberg, Germany2005: 1-377Google Scholar, 19Guhan N. Muniyappa K. Crit. Rev. Biochem. Mol. Biol. 2003; 38: 199-248Crossref PubMed Scopus (23) Google Scholar). The majority of inteins possess the protein splicing and homing endonuclease activities (18Belfort M. Derbyshire V. Stoddard B.L. Wood D.W. Homing Endonucleases and Inteins (Nucleic Acids and Molecular Biology). Vol. 16. Springer-Verlag, Berlin Heidelberg, Germany2005: 1-377Google Scholar, 19Guhan N. Muniyappa K. Crit. Rev. Biochem. Mol. Biol. 2003; 38: 199-248Crossref PubMed Scopus (23) Google Scholar). Homing endonucleases are a class of diverse rare-cutting enzymes that promote site-specific transposition of their encoding genetic elements by inflicting double-stranded DNA breaks via different cleavage mechanisms in alleles lacking these elements (18Belfort M. Derbyshire V. Stoddard B.L. Wood D.W. Homing Endonucleases and Inteins (Nucleic Acids and Molecular Biology). Vol. 16. Springer-Verlag, Berlin Heidelberg, Germany2005: 1-377Google Scholar, 19Guhan N. Muniyappa K. Crit. Rev. Biochem. Mol. Biol. 2003; 38: 199-248Crossref PubMed Scopus (23) Google Scholar, 20Belfort M. Roberts R.J. Nucleic Acids Res. 1997; 25: 3379-3388Crossref PubMed Scopus (393) Google Scholar, 21Jurica M.S. Stoddard B.L. Cell. Mol. Life Sci. 1999; 55: 1304-1326Crossref PubMed Scopus (137) Google Scholar, 22Chevalier B.S. Stoddard B.L. Nucleic Acids Res. 2001; 29: 3757-3774Crossref PubMed Scopus (376) Google Scholar, 23Stoddard B.L. Q. Rev. Biophys. 2005; 38: 49-95Crossref PubMed Scopus (339) Google Scholar). In addition, these are characterized by their ability to bind long DNA target sites (14–40 bp), and their tolerance of minor sequence changes in their binding region. These have been divided into highly divergent subfamilies on the basis of conserved sequence and structural motifs as follows: LAGLIDADG, GIY-YIG, HNH, His-Cys box, and the more recently identified PD(D/E)XK families (18Belfort M. Derbyshire V. Stoddard B.L. Wood D.W. Homing Endonucleases and Inteins (Nucleic Acids and Molecular Biology). Vol. 16. Springer-Verlag, Berlin Heidelberg, Germany2005: 1-377Google Scholar, 19Guhan N. Muniyappa K. Crit. Rev. Biochem. Mol. Biol. 2003; 38: 199-248Crossref PubMed Scopus (23) Google Scholar, 20Belfort M. Roberts R.J. Nucleic Acids Res. 1997; 25: 3379-3388Crossref PubMed Scopus (393) Google Scholar, 21Jurica M.S. Stoddard B.L. Cell. Mol. Life Sci. 1999; 55: 1304-1326Crossref PubMed Scopus (137) Google Scholar, 22Chevalier B.S. Stoddard B.L. Nucleic Acids Res. 2001; 29: 3757-3774Crossref PubMed Scopus (376) Google Scholar, 23Stoddard B.L. Q. Rev. Biophys. 2005; 38: 49-95Crossref PubMed Scopus (339) Google Scholar, 24Zhao L. Bonocora R.P. Shub D.A. Stoddard B.L. EMBO J. 2007; 26: 2432-2442Crossref PubMed Scopus (48) Google Scholar). LAGLIDADG homing enzymes, which include the largest family, contain one or two copies of the conserved dodecapeptide motif and utilize an extended protein-DNA interface covering up to 40 bp to acquire their necessary specificity (18Belfort M. Derbyshire V. Stoddard B.L. Wood D.W. Homing Endonucleases and Inteins (Nucleic Acids and Molecular Biology). Vol. 16. Springer-Verlag, Berlin Heidelberg, Germany2005: 1-377Google Scholar, 19Guhan N. Muniyappa K. Crit. Rev. Biochem. Mol. Biol. 2003; 38: 199-248Crossref PubMed Scopus (23) Google Scholar, 20Belfort M. Roberts R.J. Nucleic Acids Res. 1997; 25: 3379-3388Crossref PubMed Scopus (393) Google Scholar, 21Jurica M.S. Stoddard B.L. Cell. Mol. Life Sci. 1999; 55: 1304-1326Crossref PubMed Scopus (137) Google Scholar, 22Chevalier B.S. Stoddard B.L. Nucleic Acids Res. 2001; 29: 3757-3774Crossref PubMed Scopus (376) Google Scholar). The LAGLIDADG sequence is a part of the conserved 10- or 12-residue sequence motif defining the family of LAGLIDADG-type homing endonucleases; therefore, it is designated as deca- or dodecapeptide motif (19Guhan N. Muniyappa K. Crit. Rev. Biochem. Mol. Biol. 2003; 38: 199-248Crossref PubMed Scopus (23) Google Scholar).Comparison of the M. leprae recA intervening sequence with known intervening sequences indicated that it has the consensus amino acid sequence necessary for being a LAGLIDADG-type homing endonuclease (25Perler F.B. Olsen G.J. Adam E. Nucleic Acids Res. 1997; 25: 1087-1093Crossref PubMed Scopus (178) Google Scholar, 26Dalgaard J.Z. Klar A.J. Moser M.J. Holley W.R. Chatterjee A. Mian I.S. Nucleic Acids Res. 1997; 25: 4626-4638Crossref PubMed Scopus (174) Google Scholar). In light of massive gene decay and function loss in the leprosy bacillus, and dissimilarities in size and primary structures among mycobacterial inteins, we sought to investigate whether M. leprae recA intervening sequence encodes a catalytically active homing endonuclease. In this study, we show that the purified M. leprae RecA intein (PI-MleI) binds to cognate DNA and displays endonuclease activity in the presence of alternative divalent cations Mg2+ or Mn2+. Furthermore, using a variety of approaches, we have mapped the positions of PI-MleI binding as well as cleavage in the cognate DNA, thus providing the most comprehensive analysis of PI-MleI. Taken together, these results suggest that PI-MleI possesses a modular structure with functionally separable domains for DNA target recognition and cleavage, each with distinct sequence preferences. These results provide insights into understanding the function and evolution of the family of LAGLIDADG homing endonucleases.DISCUSSIONIn this study, we show that PI-MleI binds to cognate DNA containing the homing site and displays endonuclease activity in the presence of alternative divalent cations, Mg2+ or Mn2+. A combination of approaches, including four complementary footprinting assays (DNase I, copper-phenanthroline, KMnO4, and methylation protection), enhancement of 2-AP fluorescence, and mapping of the cleavage site revealed that PI-MleI binds to cognate DNA flanking its insertion site, induces helical distortion at the cleavage site(s) but not at the binding site(s), and generates two staggered double strand breaks (Fig. 13). These results suggest that PI-MleI cleavage sites are located at 20 and 4 bp away (on the upper and lower DNA strands, respectively) from its binding site, and implicate that PI-MleI possesses a modular structure with separate domains for DNA target recognition and cleavage (Fig. 13). In summary, these findings disclose that the structural and mechanistic aspects of PI-MleI are distinct from other well characterized LAGLIDADG-type homing endonucleases.Inteins in MycobacteriaSince the first discovery of an intein in S. cerevisiae TFP1 gene (also designated VMA1), which encodes the 69-kDa catalytic subunit of the vacuolar H+-ATPase (14Kane P.M. Yamashiro C.T. Wolczyk D.F. Neff N. Goebl M. Stevens T.H. Science. 1990; 250: 651-657Crossref PubMed Scopus (384) Google Scholar, 15Hirata R. Ohsumk Y. Nakano A. Kawasaki H. Suzuki K. Anraku Y. J. Biol. Chem. 1990; 265: 6726-6733Abstract Full Text PDF PubMed Google Scholar), intein-encoding sequences have been reported from all three phylogenetic domains as follows: bacteria, eukarya, and archaea (16Pietrokovski S. Trends Genet. 2001; 17: 465-472Abstract Full Text Full Text PDF PubMed Scopus (99) Google Scholar, 17Perler F.B. Nucleic Acids Res. 2002; 30: 383-384Crossref PubMed Scopus (317) Google Scholar, 18Belfort M. Derbyshire V. Stoddard B.L. Wood D.W. Homing Endonucleases and Inteins (Nucleic Acids and Molecular Biology). Vol. 16. Springer-Verlag, Berlin Heidelberg, Germany2005: 1-377Google Scholar, 19Guhan N. Muniyappa K. Crit. Rev. Biochem. Mol. Biol. 2003; 38: 199-248Crossref PubMed Scopus (23) Google Scholar). Sequence analysis of the genomes of 39 mycobacterial strains revealed that intein-encoding sequences are found embedded in their recA gene. However, they are inserted at two distinct sites, RecA-a and RecA-b, respectively, at the RecA-a site in M. tuberculosis and at the RecA-b site in Mycobacterium chitae, Mycobacterium fallax, Mycobacterium gastri, Mycobacterium shimodei, and Mycobacterium thermoresistibile. The latter corresponds with the M. leprae RecA allelic family (62Saves I. Lanéelle M.A. Daffé M. Masson J.M. FEBS Lett. 2000; 480: 221-225Crossref PubMed Scopus (20) Google Scholar). The occurrence of inteins in the two obligate mycobacterial pathogens, M. tuberculosis and M. leprae, was initially thought to be associated with virulence (9Davis E.O. Thangaraj H.S. Brooks P.C. Colston M.J. EMBO J. 1994; 13: 699-703Crossref PubMed Scopus (88) Google Scholar). However, subsequent studies showed that the presence of inteins does not correlate with specific characteristics of the species such as pathogenicity or growth rate (62Saves I. Lanéelle M.A. Daffé M. Masson J.M. FEBS Lett. 2000; 480: 221-225Crossref PubMed Scopus (20) Google Scholar). Comparative analysis revealed that the degree of primary sequence conservation between PI-MtuI and PI-MleI is 22%, although both belong to the large family of LAGLIDADG homing endonucleases. M. tuberculosis inactive RecA precursor undergoes splicing in E. coli, whereas splicing of M. leprae precursor RecA does not occur in E. coli, but mature RecA protein is generated in Mycobacterium smegmatis (9Davis E.O. Thangaraj H.S. Brooks P.C. Colston M.J. EMBO J. 1994; 13: 699-703Crossref PubMed Scopus (88) Google Scholar, 10Davis E.O. Jenner P.J. Brooks P.C. Colston M.J. Sedgwick S.G. Cell. 1992; 71: 201-210Abstract Full Text PDF PubMed Scopus (140) Google Scholar, 11Frischkorn K. Springer B. Böttger E.C. Davis E.O. Colston M.J. Sander P. J. Bacteriol. 2000; 182: 3590-3592Crossref PubMed Scopus (8) Google Scholar). Intriguingly, in contrast to the genomes of other obligate parasites, the degenerate M. leprae genome retains the recA intervening sequence; it would thus be interesting to check if it encodes a catalytically active LAGLIDADG-type homing endonuclease. The dodecapeptide sequence is positioned not only in homing endonucleases encoded by group I and archaeal introns but also in homothallic switching endonuclease (63Bakhrat A. Jurica M.S. Stoddard B.L. Raveh D. Genetics. 2004; 166: 721-728Crossref PubMed Scopus (21) Google Scholar). Enzymes possessing the LAGLIDADG motif cleave DNA within their recognition sequences to leave 4-base 3′-hydroxyl overhangs (42Komori K. Fujita N. Ichiyanagi K. Shinagawa H. Morikawa K. Ishino Y. Nucleic Acids Res. 1999; 27: 4167-4174Crossref PubMed Scopus (29) Google Scholar, 48Saves I. Westrelin F. Daffé M. Masson J.M. Nucleic Acids Res. 2001; 29: 4310-4318Crossref PubMed Scopus (13) Google Scholar, 49Saves I. Morlot C. Thion L. Rolland J.L. Diétrich J. Masson J.M. Nucleic Acids Res. 2002; 30: 4158-4165Crossref PubMed Scopus (11) Google Scholar). The recognition sequences are generally asymmetrical and long, with sizes of 12–40 bp (18Belfort M. Derbyshire V. Stoddard B.L. Wood D.W. Homing Endonucleases and Inteins (Nucleic Acids and Molecular Biology). Vol. 16. Springer-Verlag, Berlin Heidelberg, Germany2005: 1-377Google Scholar, 19Guhan N. Muniyappa K. Crit. Rev. Biochem. Mol. Biol. 2003; 38: 199-248Crossref PubMed Scopus (23) Google Scholar, 20Belfort M. Roberts R.J. Nucleic Acids Res. 1997; 25: 3379-3388Crossref PubMed Scopus (393) Google Scholar, 21Jurica M.S. Stoddard B.L. Cell. Mol. Life Sci. 1999; 55: 1304-1326Crossref PubMed Scopus (137) Google Scholar, 22Chevalier B.S. Stoddard B.L. Nucleic Acids Res. 2001; 29: 3757-3774Crossref PubMed Scopus (376) Google Scholar).DNA Binding Properties of PI-MleISeveral lines of evidence suggest that homing endonucleases bind DNA in a site-specific but sequence-tolerant fashion (18Belfort M. Derbyshire V. Stoddard B.L. Wood D.W. Homing Endonucleases and Inteins (Nucleic Acids and Molecular Biology). Vol. 16. Springer-Verlag, Berlin Heidelberg, Germany2005: 1-377Google Scholar, 19Guhan N. Muniyappa K. Crit. Rev. Biochem. Mol. Biol. 2003; 38: 199-248Crossref PubMed Scopus (23) Google Scholar, 20Belfort M. Roberts R.J. Nucleic Acids Res. 1997; 25: 3379-3388Crossref PubMed Scopus (393) Google Scholar, 21Jurica M.S. Stoddard B.L. Cell. Mol. Life Sci. 1999; 55: 1304-1326Crossref PubMed Scopus (137) Google Scholar, 22Chevalier B.S. Stoddard B.L. Nucleic Acids Res. 2001; 29: 3757-3774Crossref PubMed Scopus (376) Google Scholar). Sequence homology comparisons revealed that PI-MleI belongs to the family of LAGLIDADG homing endonucleases (25Perler F.B. Olsen G.J. Adam E. Nucleic Acids Res. 1997; 25: 1087-1093Crossref PubMed Scopus (178) Google Scholar, 26Dalgaard J.Z. Klar A.J. Moser M.J. Holley W.R. Chatterjee A. Mian I.S. Nucleic Acids Res. 1997; 25: 4626-4638Crossref PubMed Scopus (174) Google Scholar). Our electrophoretic mobility shift assay results showed that PI-MleI binds specifically to cognate duplex DNA. A distinct complex was seen in these gel shift experiments, and the complex was disrupted at relatively low concentrations of NaCl. The quantitative assessment of its substrate specificity indicates that the presence of the homing site in the cognate DNA is required to achieve maximal binding affinity. Using a similar strategy, we observed that PI-MleI binds to single-stranded DNA containing the homing site, albeit less efficiently, which is in good agreement with the substrate specificity of PI-MtuI (12Guhan N. Muniyappa K. J. Biol. Chem. 2002; 277: 16257-16264Abstract Full Text Full Text PDF PubMed Scopus (14) Google Scholar) and other LAGLIDADG-type homing endonucleases. This reduction in binding specificity to single-stranded DNA correlates with its inability to exhibit single-strand nicking activity. These results suggest that binding of PI-MleI to double-stranded cognate DNA is of potential functional significance, whereas its binding to single-stranded cognate DNA could be fortuitous.DNA Target Recognition and Cleavage by PI-MleIMany homing endonucleases are known to have a single biologically relevant DNA target sequence, the homing site, centered on the intron/intein insertion site of intron/intein-less alleles (18Belfort M. Derbyshire V. Stoddard B.L. Wood D.W. Homing Endonucleases and Inteins (Nucleic Acids and Molecular Biology). Vol. 16. Springer-Verlag, Berlin Heidelberg, Germany2005: 1-377Google Scholar, 19Guhan N. Muniyappa K. Crit. Rev. Biochem. Mol. Biol. 2003; 38: 199-248Crossref PubMed Scopus (23) Google Scholar). Our results suggest that PI-MleI, like other LAGLIDADG-type homing enzymes, is a site-specific endonuclease. Like other members of the family of homing endonucleases, the activity was seen in the presence of both magnesium and manganese; however, slightly higher activity was observed in the presence of manganese. Homing endonucleases recognize and cleave widely divergent intron/intein insertion sites ranging from 15 to 40 bp (18Belfort M. Derbyshire V. Stoddard B.L. Wood D.W. Homing Endonucleases and Inteins (Nucleic Acids and Molecular Biology). Vol. 16. Springer-Verlag, Berlin Heidelberg, Germany2005: 1-377Google Scholar, 19Guhan N. Muniyappa K. Crit. Rev. Biochem. Mol. Biol. 2003; 38: 199-248Crossref PubMed Scopus (23) Google Scholar, 20Belfort M. Roberts R.J. Nucleic Acids Res. 1997; 25: 3379-3388Crossref PubMed Scopus (393) Google Scholar, 21Jurica M.S. Stoddard B.L. Cell. Mol. Life Sci. 1999; 55: 1304-1326Crossref PubMed Scopus (137) Google Scholar, 22Chevalier B.S. Stoddard B.L. Nucleic Acids Res. 2001; 29: 3757-3774Crossref PubMed Scopus (376) Google Scholar, 23Stoddard B.L. Q. Rev. Biophys. 2005; 38: 49-95Crossref PubMed Scopus (339) Google Scholar). The longer recognition sequence of these enzymes is believed to ensure that the cleavage of the host genome is minimized, because the target sequences are likely to be present in a single or few copies (18Belfort M. Derbyshire V. Stoddard B.L. Wood D.W. Homing Endonucleases and Inteins (Nucleic Acids and Molecular Biology). Vol. 16. Springer-Verlag, Berlin Heidelberg, Germany2005: 1-377Google Scholar, 19Guhan N. Muniyappa K. Crit. Rev. Biochem. Mol. Biol. 2003; 38: 199-248Crossref PubMed Scopus (23) Google Scholar, 20Belfort M. Roberts R.J. Nucleic Acids Res. 1997; 25: 3379-3388Crossref PubMed Scopus (393) Google Scholar, 21Jurica M.S. Stoddard B.L. Cell. Mol. Life Sci. 1999; 55: 1304-1326Crossref PubMed Scopus (137) Google Scholar, 22Chevalier B.S. Stoddard B.L. Nucleic Acids Res. 2001; 29: 3757-3774Crossref PubMed Scopus (376) Google Scholar, 23Stoddard B.L. Q. Rev. Biophys. 2005; 38: 49-95Crossref PubMed Scopus (339) Google Scholar). PI-MleI introduced staggered double strand breaks in the homing site by nicking in the left flanking sequence 44–47 bp and in the right flanking sequence 16–25 bp, away from the intein insertion site. Interestingly, PI-MleI shows differences in the extent of cleavage between the left and right sites flanking the intein insertion site. One explanation for the reactivity differences is that PI-MleI initiates the process of insertion of the intein into the intein-less allele on the left cleavage site.Although it seems unusual, we note that similar cleavage patterns have been observed for several other homing endonucleases. For example, I-TevI cleaves its homing substrate at 23 bp in the left flanking sequence (64Bell-Pedersen D. Quirk S. Clyman J. Belfort M. Nucleic Acids Res. 1990; 18: 3763-3770Crossref PubMed Scopus (86) Google Scholar, 6

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