Mobile inteins and introns are genetic elements capable of self-propagation by “homing” into host genes and occur in entire taxonomy: eubacteria, eukarya, archaea and viruses. The process of “homing” is promoted by an endonuclease encoded by the open reading frame (ORF) embedded within the genetic element. Homing endonucleases are encoded by group I and group II introns, archaeal introns, inteins, and free standing ORFs. They are believed to play a central role in rearrangement of organelle as well as nuclear genomes. Inteins are genetic elements present within protein-coding genes with dual function: protein-splicing and homing endonuclease activities. One hallmark of homing endonucleases is their ability to recognize and cleave extended degenerate asymmetric sequences (14 - 40 bp) in intein- or intron-less alleles. Homing endonucleases are classified into four families based on the presence of LAGLIDADG, GIY-YIG, His-Cys box, or H-N-H conserved motifs. Among these, LAGLIDADG family is the largest, widespread and well-studied class. 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. In vitro, these enzymes are extremely specific for their recognition sites and they prefer Mg2+ as the metal-ion cofactor. Unlike Escherichia coli, recA of Mycobacterium tuberculosis and Mycobacterium leprae contain in-frame insertion of an intein-coding sequence. In addition to recA,scrutiny of M. tuberculosis genome revealed that intein-coding sequences are present in the ORFs of dnaA and Rv1461 (pps1). M. tuberculosis recA encodes a 85 kDa precursor protein. Amino acid sequence comparison between M. tuberculosis RecA precursor and the prototype E. coli RecA displayed high degree of homology at the amino-terminal (1 -254 amino acid residues) and carboxyl-terminal (694 - 790 amino acid residues)domains of the RecA precursor. The central domain comprising 440 amino acid residues showed significant homology to the members of the LAGLIDADG super-family of intein homing endonucleases. Following the synthesis of precursor protein, RecA intein and active RecA are generated by protein splicing reaction. The protein splicing reaction of RecA intein has been studied extensively; however, its endonuclease activity remained obscure. To identify the biochemical function of M. tuberculosis RecA intein (PI-MtuI), the intervening sequence from recA was cloned, overexpressed in E. coli and purified to homogeneity. The identity of PI-MtuI was ascertained by sequencing 10 amino acid residues at the amino-terminal end and by Western blot analysis using polyclonal antibodies raised against precursor RecA.

Evolutionary relationships amongst strains of the fungal genus Leptographium and related taxa were inferred using the internal transcribed spacer (ITS) region of the nuclear ribosomal DNA repeat. To generate robust sequence alignments for phylogenetic analysis the relationship between DNA sequence variability and RNA structural conservation of ITS segments was examined. The results demonstrate that structural conservation of helical regions is facilitated by compensatory base changes, compensating insertions/deletions, and, possibly, RNA strand slippage. A high mol % G+C bias for ITS1 and ITS2 and structural constraints at the RNA level appear to limit the types of changes observed. Fifty strains of Leptographium were screened for the presence of introns within mitochondrial genes. Superimposing intron survey data onto the ITS-derived phylogenetic tree reveals that introns are absent from the small ribosomal RNA (rns) gene of all strains of L. procerum yet are found in all strains of L. lundbergii. Amongst members of L. wingfieldii, L. terebrantis, and L. truncatum intron distribution is stochastic and is not correlated to the evolutionary relationships amongst strains. A group II intron/LAGLIDADG homing endonuclease gene (HEG) composite element from the mt rns gene of L. truncatum strain CBS929.85 was characterized. Intron-catalyzed splicing was tested using ORF-less and ORF-containing precursor transcripts, and both versions of the intron readily self-splice under moderate temperature and ionic conditions (37 °C and 6 mM MgCl2). Cleavage activity of the intron-encoded protein (I-LtrII) was tested using an N-terminal His6-tagged and near native protein. The homing endonuclease cleaves double-stranded DNA 2 nucleotides upstream of the intron insertion site within the exon, generating 4 nucleotide 3’ OH overhangs. Intron splicing is not enhanced by the addition of I-LtrII and RNA-binding assays indicate that the His6-tagged protein does not bind to the intron. Phylogenetic relationships amongst the rns gene, intron, and amino acid sequences were inferred. An evolutionary model of the composite element is proposed in which the HEG invaded a group II intron and mobilized it. The mobile genetic element may be transmitted vertically amongst L. lundbergii strains and horizontally through lateral gene transfer amongst strains of L. wingfieldii, L. terebrantis, and L. truncatum.

Evolutionary relationships amongst strains of the fungal genus Leptographium and related taxa were inferred using the internal transcribed spacer (ITS) region of the nuclear ribosomal DNA repeat. To generate robust sequence alignments for phylogenetic analysis the relationship between DNA sequence variability and RNA structural conservation of ITS segments was examined. The results demonstrate that structural conservation of helical regions is facilitated by compensatory base changes, compensating insertions/deletions, and, possibly, RNA strand slippage. A high mol % G+C bias for ITS1 and ITS2 and structural constraints at the RNA level appear to limit the types of changes observed. Fifty strains of Leptographium were screened for the presence of introns within mitochondrial genes. Superimposing intron survey data onto the ITS-derived phylogenetic tree reveals that introns are absent from the small ribosomal RNA (rns) gene of all strains of L. procerum yet are found in all strains of L. lundbergii. Amongst members of L. wingfieldii, L. terebrantis, and L. truncatum intron distribution is stochastic and is not correlated to the evolutionary relationships amongst strains. A group II intron/LAGLIDADG homing endonuclease gene (HEG) composite element from the mt rns gene of L. truncatum strain CBS929.85 was characterized. Intron-catalyzed splicing was tested using ORF-less and ORF-containing precursor transcripts, and both versions of the intron readily self-splice under moderate temperature and ionic conditions (37 °C and 6 mM MgCl2). Cleavage activity of the intron-encoded protein (I-LtrII) was tested using an N-terminal His6-tagged and near native protein. The homing endonuclease cleaves double-stranded DNA 2 nucleotides upstream of the intron insertion site within the exon, generating 4 nucleotide 3’ OH overhangs. Intron splicing is not enhanced by the addition of I-LtrII and RNA-binding assays indicate that the His6-tagged protein does not bind to the intron. Phylogenetic relationships amongst the rns gene, intron, and amino acid sequences were inferred. An evolutionary model of the composite element is proposed in which the HEG invaded a group II intron and mobilized it. The mobile genetic element may be transmitted vertically amongst L. lundbergii strains and horizontally through lateral gene transfer amongst strains of L. wingfieldii, L. terebrantis, and L. truncatum.

This work presents a survey of blue-stain fungi found in Northwestern Ontario, characterization of a homing endonuclease gene within Grosmannia piceiperda and finally an examination of the introns and homing endonuclease genes found in the large ribosomal subunit gene in species of Ceratocystis; using molecular techniques and phylogenetic analysis, we studied the molecular evolution of these mobile genetic elements. The blue-stain fungi of Northwestern Ontario were identified based on phylogenic analysis of rDNA internal transcribed spacer region sequences. This data was supplemented with morphological characteristics of the fungal cultures. The second project was an examination of a LAGLIDADG homing endonuclease and its IC2 group I intron. This intron is uniquely positioned within the group I intron-encoded rps3 gene of the large subunit ribosomal RNA gene. The final chapter is an investigation of the large subunit ribosomal RNA gene in species of Ceratocystis. The 3’ segment of this gene contains several novel introns and homing endonuclease genes. There is also much diversity between strains despite their close relation on the rDNA internal transcribed spacer region phylogenetic tree. Further, our data also suggest that the single motif LAGLIDADG homing endonuclease of the rDNA mL1923 intron is likely to be an ancestor to other homing endonucleases in the area. The results of these studies demonstrate the role that these elements play in the genetic diversity observed in the blue-stain fungi.

This work presents a survey of blue-stain fungi found in Northwestern Ontario, characterization of a homing endonuclease gene within Grosmannia piceiperda and finally an examination of the introns and homing endonuclease genes found in the large ribosomal subunit gene in species of Ceratocystis; using molecular techniques and phylogenetic analysis, we studied the molecular evolution of these mobile genetic elements. The blue-stain fungi of Northwestern Ontario were identified based on phylogenic analysis of rDNA internal transcribed spacer region sequences. This data was supplemented with morphological characteristics of the fungal cultures. The second project was an examination of a LAGLIDADG homing endonuclease and its IC2 group I intron. This intron is uniquely positioned within the group I intron-encoded rps3 gene of the large subunit ribosomal RNA gene. The final chapter is an investigation of the large subunit ribosomal RNA gene in species of Ceratocystis. The 3’ segment of this gene contains several novel introns and homing endonuclease genes. There is also much diversity between strains despite their close relation on the rDNA internal transcribed spacer region phylogenetic tree. Further, our data also suggest that the single motif LAGLIDADG homing endonuclease of the rDNA mL1923 intron is likely to be an ancestor to other homing endonucleases in the area. The results of these studies demonstrate the role that these elements play in the genetic diversity observed in the blue-stain fungi.

Mobile genetic elements are DNA sequences that move around to different positions within one genome or between different genomes. Mobile DNA elements were initially considered as selfish DNA sequences parasitizing the organism’s genome. However, this view has changed with the discovery of several mobile genetic elements which play important evolutionary and functional roles. Such understanding has led to a new connotation for these genetic elements such as drivers or natural molecular tools of genome evolution. Extensive research over the past several years has also led to the identification of several new mobile genetic elements including transposons, segregation distorters, heritable organisms, introns and inteins. Homing endonucleases (HEnases) are a group of rare cutting site-specific doublestranded DNA endonucleases encoded by open reading frames within introns, inteins or free standing genes in all the three forms of life including viruses. These enzymes confer mobility to themselves and their encoding sequences by a gene conversion event termed “homing”. During the homing process, the endonuclease inflicts a double-strand break at or near the homing site of the intein-/intron-less allele, which is subsequently repaired by the host DNA repair machinery resulting in the inheritance of intein/intron. The first homing endonuclease identified was the Saccharomyces cerevisiae mitochondrial genetic marker ‘ω’, which affects the polarity of recombination. This genetic marker, which was later shown to be a mobile group I intron, was present in the mitochondrial 21S rRNA gene and encodes a homing endonuclease. HEnases are distinguished for being able to recognise long DNA sequences (14-40 bp), and display disparate cleavage mechanisms. Unlike restriction endonucleases, these enzymes tolerate sequence polymorphism in their recognition region which provides a mechanism for increasing their genetic diversity. Substantial efforts are underway to explore the possibility of utilizing HEnases as tools for genome mapping, cloning of megabase DNA fragments and gene targeting. HEnases are divided into five sub-families on the basis of their conserved sequence and structural motifs: LAGLIDADG, GIY-YIG, H-N-H, His-Cys box and PD-(D/E)-XK families. Among these, LAGLIDADG family is the largest, most prevalent and well-studied class of HEnases. Homing enzymes that contain a single copy of LAGLIDADG motif per polypeptide chain, such as ICreI, I-MsoI and I-CeuI function as homodimers and recognize and cleave palindromic and pseudo-palindromic DNA sequences. On the other hand, HEnases that harbour two copies of LAGLIDADG motifs including I-AniI, PI-SceI and I-SceI act as monomers and recognize and cleave their DNA target sites with considerable asymmetry. Eubacterial RecA proteins are important for a number of cellular processes such as homologous recombination, DNA repair, restoration of stalled replication forks and SOS response. RecA protein and the process of homologous recombination, which is the main mechanism of genetic exchange, are evolutionarily conserved among a range of organisms. However, few mycobacterial species such as Mycobacterium tuberculosis and Mycobacterium leprae were found to be an exception as they harboured in-frame insertion of an intein-coding sequence in their recA genes. In these organisms, RecA is synthesized as a large precursor, which undergoes protein splicing resulting in the formation of an intein and functionally active RecA protein. 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. 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. The occurrence of inteins in the obligate mycobacterial pathogens M. tuberculosis, M. leprae and M. microti, initially suggested that RecA inteins might play a role in pathogenesis or virulence, however this was found to be not the case due to the subsequent identification of these intervening sequences in several non pathogenic mycobacterial strains. Sequence comparison of RecA inteins suggested that they belong to the LAGLIDADG class of homing endonucleases. Accordingly, we have shown earlier that M. tuberculosis RecA intein (PI-MtuI), is a novel LAGLIDADG homing endonuclease, which displays dual target specificity in the presence of alternative cofactors in an ATP-dependent manner. The genome of M. leprae, a gram positive bacillus reveals that in contrast to the genomes of other mycobacterial species, it has undergone extensive deletions and decay and thereby represents an extreme case of reductive evolution. In such a scenario of massive gene decay and function loss in the leprosy bacillus, and dissimilarities in size and primary structures among mycobacterial RecA inteins, it was of interest to examine whether M. leprae recA intervening sequence can encode a catalytically active homing endonuclease. To this end, the intervening sequence corresponding to M. leprae recA intein was PCR amplified, cloned, overexpressed and purified to homogeneity using IMPACT protocol. The identity of the purified RecA intein was ascertained by sequencing 9 amino acid residues at the N-terminal end and Western blot analysis using anti-PI-MleI antibodies. Purified enzyme was found to be devoid of any contaminating exonuclease. Protein crosslinking experiments using glutaraldehyde suggested that PI-MleI exists in solution as a monomer, consistent with double-motif LAGLIDADG enzymes. To test whether the purified PI-MleI can bind to the DNA and display any DNA-binding specificity, we carried out electrophoretic mobility shift assays with both single-stranded and double-stranded cognate DNA. The enzyme displayed robust binding to cognate doublestranded DNA, compared to the cognate single-stranded DNA. DNA binding was further found to be sequence independent though the presence of the cognate sequence was required for maximal binding. The stability and specificity of PI-MleI-cognate DNA complexes were further examined by salt titration and competition experiments, which indicated high stability and specificity. After establishing the stable binding of recombinant PI-MleI to the cognate duplex DNA, we next investigated its endonuclease activity on the cognate plasmid pMLR containing the intein-less recA allele, in the absence or presence of divalent cations. The cleavage was monitored by the conversion of supercoiled pMLR to nicked circular as well as linear duplex DNA. PI-MleI exhibited both single-stranded nicking and double-stranded DNA cleavage activity. PI- MleI exhibits endonuclease activity both in the presence of Mg2+ or Mn2+ through a two step reaction. PI-MleI mediated cleavage though was found to be divalent cation dependent however was nucleotide cofactor independent, unlike PI-MtuI, which cleaves the cognate DNA substrate in the presence of ATP. PI-MleI endonuclease activity was assayed under different conditions and found to display a broad divalent cation, pH and temperature dependence. The kinetic experiments revealed slow turnover rate of PI-MleI suggesting its weak endonuclease activity in contrast to robust cleavage activity displayed by several other known LAGLIDADG homing endonucleases. An intriguing observation emerged from the cleavage site mapping of PI-MleI at singlenucleotide resolution. PI-MleI displayed a staggered double- strand break in the homing site by nicking in the left flanking sequence 44 to 47 bp and in the right flanking sequence 16 to 25 bp, away from the intein insertion site. Similar cleavage patterns have been earlier observed for few GIY-YIG homing endonucleases. To gain further mechanistic insights into the PI-MleI mediated catalysis, we examined the binding of PI-MleI to the cognate DNA by DNase I and (OP)2 Cu footprinting experiments. Both the footprinting approaches revealed interaction of PI-MleI with a region upstream and downstream of its own insertion site, conferring protection to 16 nucleotide residues on the upper and 12 nucleotide residues on the lower strand, respectively. The asymmetric footprints have been earlier observed for some members of LAGLIDADG-type homing endonucleases wherein protection on the complementary strands was found to be out of register by 2 to 3 nucleotides, respectively. In case of PI-MleI, however the footprint formed on the complementary strands of the homing site is non-overlapping, indicating the asymmetric mode of interaction of the enzyme. Surprisingly, PI-MleI footprint was not evident at the cleavage sites and this could be due to the unstable binding of the intein at these regions. To decipher the interaction of PI-MleI at the cleavage sites and to ascertain if these interactions have any functional implications in terms of alterations in base-pairing positioning or strand separation to mediate DNA catalysis, we probed the structure of PI-MleI-DNA complexes with KMnO4. KMnO4 treatment of PI-MleI-cognate DNA complexes revealed the presence of hypersensitive T residues on both the strands at the cleavage sites, but showed no such reactive T residues within the PI-MleI-binding regions. Also, hyper-sensitive T residues were not seen at or near the intein-insertion site or in the region between binding and cleavage sites suggesting that PI-MleI upon binding its cognate DNA induces distortions selectively at the cleavage region. To validate these findings and to test whether such alterations occurred on all substrate DNA molecules or on a small sub-population of target molecules, we used a more sensitive 2-aminopurine fluorescence approach. To this end, six cognate duplex DNA molecules each containing 2-aminopurine (2-AP) at different positions such as at the insertion site, in the DNAbinding region, at or near to the cleavage sites were synthesized to monitor helical distortions in the target DNA. The 2-AP containing cognate DNA duplexes were incubated with increasing concentrations of PI-MleI in the assay buffer and monitored the changes in 2-AP fluorescence intensity in the spectral region from 330 to 450 nm. Out of the 2-AP placed at several positions within the cognate substrate, only the 2-aminopurines at the cleavage site showed enhanced fluorescence with PI-MleI addition, consistent with the hyper-sensitivity of T residues during KMnO4 probing. The findings suggest that DNA distortion might assist PI-MleI in widening the minor groove at the cleavage site and make the scissile phosphates accessible to the enzyme active site similar to what has been seen with other LAGLIDADG homing enzymes. These observations suggest that PI-MleI binds to cognate DNA flanking its insertion site, induces helical distortion at the cleavage sites and generates two staggered double-strand breaks. Together, these finding indicate the modular structure of PI-MleI having separate domains for DNA target recognition and cleavage and a bipartite structure of its homing site. After demonstrating the endonuclease activity of PI-MleI, we next examined the active site residues of PI-MleI involved in double-stranded DNA cleavage, which would further provide insights into its catalytic mechanism. Previously, sequence alignment analyses of LAGLIDADG enzymes carried out using different alignment programs identified the presence of 115VLGSLMGDGP123 sequence as DOD motif I (Block C) and 185LQRAVYLGDG194 or 210VLAIWYMDDG219C sequences as catalytic DOD motif II (Block E) in M. leprae RecA intein (PI-MleI). The bioinformatics analyses though on one hand identified the catalytic motifs in PI-MleI, on the other hand led to conflicting data in regard to the identity and the specific position of the catalytic DOD motif II within the PI-MleI polypeptide. We therefore, performed site-directed mutagenesis of key residues in these catalytic motifs and examined their effect on PI-MleI mediated catalysis. A wealth of mutagenesis and structural data, which exists concerning HEnases, suggests that catalytic centers carry essential aspartate residues, one in each of the LAGLIDADG motifs Accordingly, we chose to mutate conserved aspartates that have been previously implicated in catalysis. By site-directed mutagenesis, we constructed five mutant proteins, in which Asp122 was mutated to alanine, cysteine and threonine; whereas Asp193 and Asp218 were mutated to alanine. The identity of each mutant was ascertained by determining the complete nucleotide sequence of the mutant gene. Mutant proteins were further purified to >95% homogeneity using the purification strategy developed for wild type PI-MleI and were found to be devoid of any contaminating exonuclease. To study the effect of mutations in PI-MleI active site residues on its DNA-binding affinity, we examined the binding characteristics of the wild type PI-MleI and its aspartate variants with the intein-less recA substrate and the stability of protein-DNA complexes. All the mutants displayed similar binding affinity to the cognate DNA as that of the wild type PI-MleI, as judged by the comparison of their binding constants (Kd) which were found to be of the same order. Comparison of salt titration isotherms of wild type PI-MleI and its aspartate variants further revealed the similar salt titration midpoint for most of the mutants as that of wild type enzyme suggesting similar protein-DNA complexes stability. Although these results indicate the occurrence of stable complexes between PI-MleI variants and target DNA, to further define the DNA-binding properties of each mutant protein, wild-type PI-MleI and its variants were assayed by DNase I footprinting. All the mutants (D122A, D122C, D122T, D193A and D218A) showed an asymmetric footprint and protection of ~16 nucleotide residues on the upper and 12 nucleotide residues on the lower strand, respectively, near the intein-insertion site similar to the wild type PI-MleI. Together, these observations suggest that the aspartate substitutions in the catalytic motifs do not alter DNA recognition specificity of PI-MleI or its variants, and may not play a direct role in protein-DNA interactions, again implicating the existence of a modular structure of PI-MleI with distinct DNA-binding and catalytic domains. Wild-type PI-MleI although binds near the intein insertion site, but however was found to induce helical distortions only at the cleavage sites. To explore, if aspartate substitutions have any effect on the structural modifications in target DNA sequence, we carried out 2-aminopurine fluorescence with wild type PI-MleI and its variants. In agreement with the wild type enzyme, all the mutants showed increase in fluorescence with target DNA containing 2-AP only at the cleavage sites, but not at the binding sites. However, quantitative measurements of fluorescence change suggested that D122A and D193A mutants show nearly two-fold decrease in the magnitudes of spectral change at the cleavage site compared to wild type and other variants suggesting their involvement in the helical distortion process. To study the effect of Asp substitutions on the catalytic activity of PI-MleI, we performed cleavage assays using cognate plasmid pMLR DNA, with increasing concentrations of wild-type PI-MleI, or its variants and measured the double-stranded cleavage activity. Whereas, D122A and D193A mutants were completely inactive in double-stranded DNA cleavage under the conditions of the cleavage assay, D218A showed DNA cleavage activity comparable to that of the wild type PI-MleI. Similarly, D122T showed decrease in doublestranded DNA cleavage activity. Interestingly, D122C variant showed ~2-fold enhanced DNA cleavage, compared to the wild-type enzyme.Together, these findings provide compelling evidence to conclude that 115VLGSLMGDGP123 and 185LQRAVYLGDG194 motifs (Blocks C and E, respectively), but not 210VLAIWYMDDG219 motif (Block E), and that residues Asp122 and Asp193 play a direct role with respect to the catalytic mechanism of PI-MleI. In summary, these results suggest that the structural and mechanistic aspects of PI-MleI catalysis are distinct from other well-characterized LAGLIDADG-type homing endonucleases and thus provide further insights into understanding the function and evolution of LAGLIDADG homing enzymes.

The small ribosomal subunit gene of the Chaetomium thermophilum DSM 1495 is invaded by a nested intron at position mS1247, which is composed of a group I intron encoding a LAGLIDADG open reading frame interrupted by an internal group II intron. The first objective was to examine if splicing of the internal intron could reconstitute the coding regions and facilitate the expression of an active homing endonuclease. Using in vitro transcription assays, the group II intron was shown to self-splice only under high salt concentration. Both in vitro endonuclease and cleavage mapping assays suggested that the nested intron encodes an active homing endonuclease which cleaves near the intron insertion site. This composite arrangement hinted that the group II intron could be regulatory with regards to the expression of the homing endonuclease. Constructs were generated where the codon-optimized open reading frame was interrupted with group IIA1 or IIB introns. The concentration of the magnesium in the media sufficient for splicing was determined by the Reverse Transcriptase-Polymerase Chain Reaction analyses from the bacterial cells grown under various magnesium concentrations. Further, the in vivo endonuclease assay showed that magnesium chloride stimulated the expression of a functional protein but the addition of cobalt chloride to the growth media antagonized the expression. This study showed that the homing endonuclease expression in Escherichia coli can be regulated by manipulating the splicing efficiency of the group II introns which may have implications in genome engineering as potential ‘on/off switch’ for temporal regulation of homing endonuclease expression . Another objective was to characterize native homing endonucleases, cytb.i3ORF and I-OmiI encoded within fungal mitochondrial DNAs, which were difficult to express and purify. For these, an alternative approach was used where two compatible plasmids, HEase.pET28b (+)-kanamycin and substrate.pUC57-chloramphenicol, based on the antibiotic markers were maintained in Escherichia coli BL21 (DE3). The in vivo endonuclease assays demonstrated that these homing endonucleases were able to cleave the substrate plasmids when expressed, leading to the loss of the antibiotic markers and thereby providing an indirect approach to screen for potential active homing endonucleases before one invests effort into optimizing protein overexpression and purification strategies. / October 2016