<p>Homing endonuclease genes (HEGs) are selfish genetic elements that mediate their own super-Mendelian inheritance. This is mediated by the homing endonuclease cleavage of a HEG^-allele followed by recombination-repair with a HEG⁺ allele.</p><p>The majority of the HEGs are encoded in intervening sequences (IVSs). The insertion of the IVS interrupts the endonuclease recognition site, making the genome with the IVS resistant to further cleavage by homing endonucleases with specificity for that particular sequence, but susceptible for homing endonucleases with a target not affected by the IVS insert. In 39 studied strains of the <i>Bacillus cereus</i> group, 28 IVSs were found in the <i>nrdIEF</i> operon. Phylogenetic studies of these sequences showed a scattered distribution of the IVSs, indicating a frequent horizontal gene transfer and that there might be competition between the different IVSs in the <i>nrdIEF</i> operon in the <i>Bacillaceae</i> family. One novel group I intron was shown to encode a functional homing endonuclease with a GIY-(X)₈-YIG motif, expanding the family motif to GIY-(X)₈-₁₁-YIG. Interestingly, by studying the known insertion sites for IVSs in the ribonuclotide reductase genes, we show that the majority of the insertions are at conserved motifs, indicating that conservation is important for IVS survival.</p><p>Most freestanding HEGs in bacteriophage T4 cleave both HEG⁺ and HEG^- alleles, possibly providing a competitive advantage for the host organism when two phages infect the same bacterium. Two novel freestanding HEGs replace two putative HEGs in T4 in some T-even-like phages. The characterisation of these HEGs showed that both cleave double stranded DNA. SegH was shown to promote homing of its gene. Hef showed no homing, possibly due to general exclusion of other phages. The <i>mobE</i> putative HEG was shown to be homing proficient and showed strong general DNA degradation when expressed in <i>Escherichia coli.</i></p>

Homing endonuclease genes (HEGs) are selfish genetic elements that mediate their own super-Mendelian inheritance. This is mediated by the homing endonuclease cleavage of a HEG- allele followed by recombination-repair with a HEG+ allele. The majority of the HEGs are encoded in intervening sequences (IVSs). The insertion of the IVS interrupts the endonuclease recognition site, making the genome with the IVS resistant to further cleavage by homing endonucleases with specificity for that particular sequence, but susceptible for homing endonucleases with a target not affected by the IVS insert. In 39 studied strains of the Bacillus cereus group, 28 IVSs were found in the nrdIEF operon. Phylogenetic studies of these sequences showed a scattered distribution of the IVSs, indicating a frequent horizontal gene transfer and that there might be competition between the different IVSs in the nrdIEF operon in the Bacillaceae family. One novel group I intron was shown to encode a functional homing endonuclease with a GIY-(X)8-YIG motif, expanding the family motif to GIY-(X)8-11-YIG. Interestingly, by studying the known insertion sites for IVSs in the ribonuclotide reductase genes, we show that the majority of the insertions are at conserved motifs, indicating that conservation is important for IVS survival. Most freestanding HEGs in bacteriophage T4 cleave both HEG+ and HEG- alleles, possibly providing a competitive advantage for the host organism when two phages infect the same bacterium. Two novel freestanding HEGs replace two putative HEGs in T4 in some T-even-like phages. The characterisation of these HEGs showed that both cleave double stranded DNA. SegH was shown to promote homing of its gene. Hef showed no homing, possibly due to general exclusion of other phages. The mobE putative HEG was shown to be homing proficient and showed strong general DNA degradation when expressed in Escherichia coli.

Endonuclease II (EndoII) of bacteriophage T4 is a GIY-YIG enzyme involved in host DNA breakdown during phage infection of E. coli. EndoII combines features of restriction endonucleases with those of homing endonucleases in that it breaks down DNA foreign to itself but recognizes a 16 bp long asymmetric and ambiguous sequence. This investigation addresses the biological function of EndoII, its mode of interaction with its substrate and roles of individual residues in catalysis, sequence recognition and binding. It is shown here that EndoII increases the frequency of non-homologous recombination in phage-infected cells, showing that EndoII indeed can induce recombinational events. Although single-stranded nicks are frequent in in vitro reactions with purified protein, the enzyme is found to produce mostly double-stranded breaks in vivo, since nicks are repaired. Mutations of residues positioned on the putative catalytic surface result in severely reduced catalytic activity, while residues in the N-terminal region and a middle region (MR) appear to mainly contribute to substrate binding. Mutation of the putatively magnesium-binding residue E118 renders the enzyme catalytically inactive. Residues K76 (in the MR and positioned on the catalytic surface) and G49 and R57 (on the catalytic surface) also contribute to substrate recognition. All mutants bind as tetramers to two DNA molecules, indicating that the wildtype would also bind as a tetramer. EndoII E118A alone can bind also in monomeric and dimeric form to one DNA molecule, possibly because the glutamate charge normally repels the DNA. The solved crystal structure of tetrameric EndoII E118A shows a striking X-shape with two putative catalytic surfaces to each side positioned so that double-stranded cleavage would require severe DNA distortion. Combination of all data suggests that upon binding in vivo EndoII scans the DNA for a second binding site, binding to both sites but nicking or cleaving only one of them.

Homing endonucleases (HEs) typically have one of four types of catalytic domains (LAGLIDADG, GIY-YIG, H-N-H, His-Cys), and a DNA-binding region(s) that provides specificity. I-CreII, which is encoded by the psbA4 intron from Chlamydomonas reinhardtii, is unusual in containing two catalytic motifs: H-N-H and GIY-YIG. A previous study showed that I-CreII cleavage leaves 2-nt 3' OH overhangs similar to GIYYIG endonucleases, but that it also has a flexible metal requirement like H-N-H enzymes. Also, alanine substitution of several conserved residues in the GIY-YIG motif and two in the H-N-H motif did not produce a clear catalytic mutant, although some variants had strongly reduced DNA binding. Thus, in order to identify the catalytic motif, I substituted additional amino acids in both domains with alanine, and identified three histidines in the H-N-H motif that are likely to be involved in catalysis. To gain insight into how I-CreII interacts with its ~30-bp homing-site DNA, three types of DNA protection analysis were performed. Hydroxyl-radical footprinting, which reveals regions of tight DNA binding, indicated that I-CreII binds strongly to a region downstream of the cleavage and intron-insertion sites, corresponding to bp 2-10 of exon 5. There was also partial protection around the cleavage site, but only on the top strand, which is consistent with the enzyme's tendency to cleave this strand first. DNase I protection, which can reveal less closely-bound regions of target DNA, gave a larger footprint than hydroxyl-radical protection, with the additional region lying upstream of the cleavage site. These results also suggest that DNA backbone-binding downstream of the cleavage site involves sugars and phosphates, whereas upstream it is mainly with phosphates. DMS protection, which probes guanines on the N-7 position in the major groove, did not provide any evidence of major groove binding (at least not through guanines). DNase I protection could also be performed on the I-CreII variants that had reduced DNA affinity. The N161A variant was instructive in that it showed reduced protection of a T-A bp very close to the cleavage site, providing support for a catalytic role for the H-N-H motif and a possible constraint for modeling. Of the GIY-YIG motif variants, the footprint of the G231E/K245A variant was distinctly useful in that it was preferentially effected downstream of the cleavage site. This result suggested the H-N-H and GIY-YIG motifs are co-linear with their targets in the homing site. Structural modeling of the GIY-YIG domain of I-CreII using the solved I-TevI domain as template provided evidence for a unique insertion in the I-CreII structure that disrupted a catalytic α-helix; the insertion is predicted to be a positively charged, hairpinlike loop anchored by two antiparallel β-strands. I propose that this insertion can explain the evolutionary conversion of this catalytic endonuclease domain into a DNA-binding domain. These findings should also help to understand other dual-motif H-N-H/GIY-YIG endonucleases, such as I-CmoeI.

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.