<p>DNA interstrand cross-links (ICLs) constitute one of the most severe types of DNA damage. ICLs covalently tether both strands of duplex DNA, preventing unwinding and polymerase access during replication and transcription. This obstruction is exploited in cancer chemotherapy since it leads to replication fork collapse, double strand breaks (DSBs), and cell death. Mechanistic understanding of how eukaryotic cells repair these specific lesions, however, is still in its infancy. It is understood that ICL repair consists of a multitude of intersecting and connecting repair pathways that rely on interplay between critical protein factors. Interestingly, Pso2 has been identified as an integral component of the ICL repair pathway in <em>Saccharomyces cerevisiae</em>. Pso2 is a yeast nuclease from the β-CASP family of proteins that function predominantly in the repair of ICLs. It has been recognized as the only protein that does not serve a redundant function in any other DNA repair pathway. It remains unclear how the ICL repair pathway generates DNA intermediates suitable for high fidelity repair dependent on Pso2 nuclease activity. Here we show that Pso2 possesses structure-specific endonuclease activity that may be essential to its role in ICL repair. Direct <em>in vitro</em> activity assessment of the protein on a site-specific ICL proved to be inconclusive due to the heat-labile nature of the cross-linking agent employed. <em>In vitro </em>activity testing was also performed on various substrates resembling intermediates generated during ICL repair. Biochemical analysis demonstrated that Pso2 cleaves hairpins, stem loops, heterologous loops, and symmetrical bubbles. Although the precise cleavage sites vary between substrates, Pso2 demonstrates preference for the single- to double-stranded junction in the DNA backbone, with similar activity to that previously demonstrated for its human homologue, Artemis. This specific endonuclease activity is stimulated by increased concentrations of phosphate. Through two-dimensional gel electrophoresis, the presence of unique DNA intermediates generated in response to ICL damage <em>in </em><em>vivo </em>was also monitored. Results suggest the generation of hairpin-like intermediates that resemble those tested <em>in vitro</em>. These intermediates persist in the absence of Pso2 but are resolved by exogenous addition of control endonucleases. Our findings expand on previous data that established hairpin-opening activity for this protein and suggest that the structure-specific endonuclease activity demonstrated by Pso2 is important for ICL repair. We anticipate that Pso2 acts on a hairpin-containing DNA substrate in the ICL repair pathway and the resolution of this intermediate is uniquely dependent on Pso2 for the effective repair of ICL damage in yeast. Taking into consideration the current models of ICL repair, both in yeast and humans, possible roles for Pso2 have been described. Achieving a complete mechanistic perspective of this pathway is critical for the therapeutic exploitation of the human homologue, SNM1A. Implications include the potential inhibitory target for increased efficacy of chemotherapy with cross-linking agents.</p> / Master of Science (MSc)

No / Degradation of nuclear DNA is a hallmark of programmed cell death. Epidermal keratinocytes die in the course of cornification to function as the dead building blocks of the cornified layer of the epidermis, nails, and hair. Here, we investigated the mechanism and physiological function of DNA degradation during cornification in vivo. Targeted deletion of the keratinocyte-specific endonuclease DNase1-like 2 (DNase1L2) in the mouse resulted in the aberrant retention of DNA in hair and nails, as well as in epithelia of the tongue and the esophagus. In contrast to our previous studies in human keratinocytes, ablation of DNase1L2 did not compromise the cornified layer of the epidermis. Quantitative PCRs showed that the amount of nuclear DNA was dramatically increased in both hair and nails, and that mitochondrial DNA was increased in the nails of DNase1L2-deficient mice. The presence of nuclear DNA disturbed the normal arrangement of structural proteins in hair corneocytes and caused a significant decrease in the resistance of hair to mechanical stress. These data identify DNase1L2 as an essential and specific regulator of programmed cell death in skin appendages, and demonstrate that the breakdown of nuclear DNA is crucial for establishing the full mechanical stability of hair.

Conídios são estruturas especializadas, produzidas assexuadamente pelo micélio de muitas espécies de ascomicetos. A produção dos conídios requer o controle espacial e temporal da expressão gênica e a formação de estruturas específicas durante o desenvolvimento. Os conídios estão envolvidos na reprodução, dispersão e persistência ambiental dos fungos. Em espécies patogênicas como Aspergillus fumigatus e Metarhizium anisopliae, os conídios também são responsáveis pela infecção do hospedeiro. Um dos principais fatores ambientais capazes de matar e / ou danificar os conídios é a radiação solar. Os dímeros de pirimidina ciclobutano (CPDs) são os principais fotoprodutos do DNA induzidos pela radiação UVB. Os principais objetivos deste trabalho foram: (1) estimar as frequências de CPDs em conídios expostos a doses subletais de radiação UVB, (2) correlacionar a frequência de CPDs com a cinética de germinação dos conídios, (3) comparar a frequência de CPDs em conídios selvagens com a frequência em conídios mutantes para a pigmentação, (4) identificar genes diferencialmente expressos durante as fases da conidiogênese de A. fumigatus e (5) identificar genes modulados pela radiação UVB em micélio jovem de A. fumigatus. Conídios de M. anisopliae, A. nidulans e A. fumigatus foram expostos à irradiância de 1000 mW m-2 de UVB por 15, 30, 60 e 90 min. As doses totais ao final das exposições foram 0,9, 1,8, 3,6 e 5,4 kJ m-2. O aumento na frequência de CPDs foi linear e diretamente proporcional à dose, com 0,215, 0,455, 0,803 e 1,628 CPDs 10 kb-1 induzidos pelas doses de 0,9, 1,8, 3,6 e 5,4 kJ m-2 em A. fumigatus, 0,037, 0,077, 0,142 e 0,202 CPDs 10 kb-1 em A. nidulans e 0,041, 0,085, 0,155 e 0,255 CPDs 10 kb-1 em M. anisopliae. A frequência de CPDs no mutante albino de M. anisopliae (0,552 10 kb-1) foi aproximadamente dez vezes maior do que na linhagem selvagem (0.057 10 kb-1) após exposição à dose de 1,8 kJ m-2. Esta é a primeira evidência direta de que a pigmentação dos conídios protege o DNA contra os danos induzidos pela radiação UVB. Microarranjos genômicos de DNA foram utilizados para comparar os transcriptomas de micélios com 20 h (início da conidiogênese), 24 h (fase intermediária) e 25 h (fase final) com o transcriptoma de micélio jovem. Foram identificados 34 genes diferencialmente expressos (7 com aumento e 27 com diminuição) com 20 h de desenvolvimento, 101 genes (12 com aumento e 89 com diminuição) com 24 h e 76 genes (oito com aumento e 68 com diminuição) com 25 h. Alguns genes que apresentaram aumento na expressão (stuA e o gene da scytalone dehydratase) já haviam sido associados com fases específicas da conidiogênese, entretanto a maioria dos genes que apresentou aumento na expressão não tem função conhecida. A análise de transcriptomas com microarranjos de DNA também foi utilizada para identificar genes com expressão modulada por exposições à radiação UVB (1,8 kJ m-2) em micélio jovem de A. fumigatus. Foram identificados 101 genes diferencialmente expressos ao final da exposição à radiação UVB (51 genes com aumento e 50 com redução). O gene radc apresentou o maior aumento na expressão (aproximadamente 16 ×). A maioria dos genes com aumento na expressão não possui função conhecida. Foram identificados 418 genes diferencialmente expressos 30 min após o termino da exposição (51 genes com aumento e 367 com redução na expressão). / Conidia are specialized structures produced asexually during mycelia growth of many ascomycete species. The process of conidiation involves temporal and spatial regulation of gene expression, cell specialization, intercellular communication, and formation of specific structures during fungal growth. Conidia are responsible for the reproduction, dispersal and environmental persistence of many fungal species. In pathogenic species like Aspergillus fumigatus and Metarhizium anisopliae, conidia are also responsible for host infection. One of the main environmental factors that can kill and/or damage conidia is solar UV radiation. Cyclobutane pyrimidine dimers (CPDs) are the major DNA photoproducts induced by UVB. The principal goals of the present study were to: (1) estimate the frequency of CPDs induced by sublethal doses of UVB radiation in conidial DNA of three selected ascomycetes, (2) examine correlation of CPD frequencies with germination speed, and (3) estimate the protective effect of the wild-type green conidial pigmentation on DNA in M. anisopliae var. anisopliae, (4) identify differentially expressed genes during the different phases of A. fumigatus conidiogenesis and (5) identify genes regulated by UVB radiation in A. fumigatus young mycelia. A. fumigatus, A. nidulans, and M. anisopliae conidia were exposed to 1000 mW m-2 UV irradiance for 15, 30, 60 and 90 min. Total Quaite-weighted doses were 0.9, 1.8, 3.6 and 5.4 kJ m-2, respectively. The frequencies of dimers were linear and directly proportional to the doses, with 0.215, 0.455, 0.803 and 1.628 CPDs 10 kb-1 detected at the doses of 0.9, 1.8, 3.6 and 5.4 kJ m-2 in A. fumigatus, 0.037, 0.077, 0.142 and 0.202 CPDs 10 kb-1 in A. nidulans, and 0.041, 0.085, 0.155 and 0.255 CPDs 10 kb-1 in M. anisopliae. The frequency of dimers in the M. anisopliae albino mutant DWR 180 (0.552 10 kb-1) was approximately ten times higher than of the wild-type ARSEF 23 strain (0.057 10 kb-1) after exposure to doses of 1.8 kJ m-2. DNA microarrays carrying sequence of 11,000 genes of A. fumigatus were used to compare transcriptomes of 20 h-old mycelia (initial phase of the conidiogenesis), 24 h (intermediate phase) e 25 h (final phase) with young mycelia transcriptome. Thirty-four genes displayed a statistically significant difference in expression (7 with increase and 27 with decrease mRNA expression) in 20 h-old mycelia, 101 genes (12 with increase and 89 with decrease) in 24 h-old mycelia and 76 genes (8 with increase and 68 with decrease) in 25 h-old mycelia. Some overexpressed genes (stuA ad the scytalone dehydratase gene) were previously related to specific phases of conidiogenesis but the function of most of them is unknown. Transcriptome analysis using microarrays was also used to identify genes modulated by exposures to UVB radiation (1,8 kJ m-2) in A. fumigatus young mycelia. One hundred and one differentially expressed genes were identified at the end of the exposure to UVB radiation (51 genes with increase and 50 with decrease). The radc gene displayed the higher increase in expression (approximately 16 ×). The function of most of the overexpressed genes is unknown. Four hundred and eighteen differentially expressed genes were identified 30 min after the end of exposure (51 genes with increase and 367 with decrease in expression).

Bakterielle Restriktions- und Modifikationssysteme (R/M-Systeme) greifen DNA endonukleolytisch an, die nicht die spezifische Markierung der eigenen Wirtszelle trägt. Zu einem R/M-System gehören eine Restriktionsendonuklease und eine DNA- Methyltransferase gleicher DNA-Spezifität. Die biologische Funktion der Restriktionsendonuklease besteht in der Abwehr von fremder, in die Zelle eindringender DNA, z. B? von Virus-Infektionen. Die korrespondierende DNA-Methyltransferase schützt die zelluläre DNA durch sequenz-spezifische DNA-Methylierung vor der endonukleolytischen Wirkung der Restriktionsendonuklease. Die dimeren TypII- Restriktionsendonukleasen erkennen kurze spezifische, unmethylierte Basensequenzen, die sie in Anwesenheit von Mg2+ Ionen an einer definierten Position endonukleolytisch spalten. Die Restriktionsendonuklease EcoRII braucht die koordinierte Wechselwirkung mit zwei Kopien der Sequenz 5 CCWGG, um katalytisch aktiv sein zu können, wobei eine der beiden Sequenzen als allosterischer Effektor wirkt und nicht gespalten werden muß. Die zwei Kopien der 5 CCWGG Sequenz können sowohl auf demselben als auch auf verschiedenen Molekülen lokalisiert sein. Die Interaktion von EcoRII mit verschiedenen DNA-Molekülen ist durch deren Länge und Konzentration, die Interaktion innerhalb eines DNA-Moleküls durch den Abstand zwischen beiden Sequenzen limitiert. Die durch Proteolyse nachgewiesene Zwei-Domänen-Struktur von EcoRII scheint diese besondere Form der Protein-DNA-Wechselwirkung zu ermöglichen. Die C-terminale Domäne von EcoRII stellt eine neue Restriktionsendonuklease (EcoRII-C) dar. Im Gegensatz zum Wildtyp-Enzym spaltet EcoRII-C an singulären 5 CCWGG Sequenzen. Die trunkierte Endonuklease spaltet DNA spezifisch und unabhängig von einem zweiten EcoRII-Erkennungsort. Die Reaktion verläuft deutlich schneller als die des kompletten EcoRII-Proteins. Die N-terminale Domäne bindet spezifisch DNA, attenuiert die endonukleolytische Aktivität von EcoRII und macht das Enzym abhängig von einer zweiten Kopie der Sequenz 5 CCWGG. EcoRII Wildtyp könnte demzufolge ein evolutionäres Intermediat zwischen einer sequenz-spezifischen Endonuklease und einem Protein sein, das spezifisch mit zwei Orten auf der DNA interagiert, wie z. B. Rekombinasen oder Transposasen. Durch die Kombination beider Funktionen könnte EcoRII selbst die Verbreitung der EcoRII-codierenden DNA-Sequenz in neue Populationen, ähnlich einem transponiblen Element, realisieren. / Bacterial restriction and modification systems (R/M-systems) endonucleolytically attack DNA that is not host cell-specifically modified. R/M-systems comprise a restriction endonuclease and a DNA methyltransferase exhibiting the same DNA sequence specificity. The biological function of the restriction endonuclease is the protection of the cell against invading foreign DNA, e. g. virus infection. The corresponding DNA methyltransferase renders cellular DNA resistent against the endonucleolytic action of the restriction endonuclease by sequence-specific DNA methylation. Dimeric type II- restriction endonucleases recognize short, specific, and unmethylated base sequences that they cut at a defined position in the presence of Mg2+ ions. Restriction endonuclease EcoRII requires the co- ordinated interaction with two copies of the sequence 5 CCWGG for catalytic activity. One of these sequences serves as an allosteric activator site and has not to be cleaved. The two copies of the sequence 5 CCWGG can be located as well on the same as on different DNA molecule(s). EcoRII interaction with two sites on different DNA molecules is limited by their length and concentration, EcoRII interaction within one DNA molecule is limited by the distance between the two sites. The two- domain structure of EcoRII figured out by limited proteolysis studies probably allows this particular form of protein-DNA interaction. The C-terminal domain of EcoRII represents a new restriction endonuclease (EcoRII-C). In contrast to EcoRII wild type, EcoRII-C cleaves DNA at single 5 CCWGG sites. The truncated endonuclease cleaves DNA specifically and independent of a second site. The enzymatic reaction passes well more rapid than that of the complete enzyme. The N-terminal domain binds DNA specifically, attenuates the endonucleolytic activity of EcoRII and makes it dependent on a second copy of the sequence 5 CCWGG. Therefore, the current EcoRII could be an evolutionary intermediate between a site-specific endonuclease and a protein that functions specifically with two DNA sites on the DNA such as recombinases and transposases. The combination of both functions may enable EcoRII to accomplish its own propagation similarly to transposable elements.

Conídios são estruturas especializadas, produzidas assexuadamente pelo micélio de muitas espécies de ascomicetos. A produção dos conídios requer o controle espacial e temporal da expressão gênica e a formação de estruturas específicas durante o desenvolvimento. Os conídios estão envolvidos na reprodução, dispersão e persistência ambiental dos fungos. Em espécies patogênicas como Aspergillus fumigatus e Metarhizium anisopliae, os conídios também são responsáveis pela infecção do hospedeiro. Um dos principais fatores ambientais capazes de matar e / ou danificar os conídios é a radiação solar. Os dímeros de pirimidina ciclobutano (CPDs) são os principais fotoprodutos do DNA induzidos pela radiação UVB. Os principais objetivos deste trabalho foram: (1) estimar as frequências de CPDs em conídios expostos a doses subletais de radiação UVB, (2) correlacionar a frequência de CPDs com a cinética de germinação dos conídios, (3) comparar a frequência de CPDs em conídios selvagens com a frequência em conídios mutantes para a pigmentação, (4) identificar genes diferencialmente expressos durante as fases da conidiogênese de A. fumigatus e (5) identificar genes modulados pela radiação UVB em micélio jovem de A. fumigatus. Conídios de M. anisopliae, A. nidulans e A. fumigatus foram expostos à irradiância de 1000 mW m-2 de UVB por 15, 30, 60 e 90 min. As doses totais ao final das exposições foram 0,9, 1,8, 3,6 e 5,4 kJ m-2. O aumento na frequência de CPDs foi linear e diretamente proporcional à dose, com 0,215, 0,455, 0,803 e 1,628 CPDs 10 kb-1 induzidos pelas doses de 0,9, 1,8, 3,6 e 5,4 kJ m-2 em A. fumigatus, 0,037, 0,077, 0,142 e 0,202 CPDs 10 kb-1 em A. nidulans e 0,041, 0,085, 0,155 e 0,255 CPDs 10 kb-1 em M. anisopliae. A frequência de CPDs no mutante albino de M. anisopliae (0,552 10 kb-1) foi aproximadamente dez vezes maior do que na linhagem selvagem (0.057 10 kb-1) após exposição à dose de 1,8 kJ m-2. Esta é a primeira evidência direta de que a pigmentação dos conídios protege o DNA contra os danos induzidos pela radiação UVB. Microarranjos genômicos de DNA foram utilizados para comparar os transcriptomas de micélios com 20 h (início da conidiogênese), 24 h (fase intermediária) e 25 h (fase final) com o transcriptoma de micélio jovem. Foram identificados 34 genes diferencialmente expressos (7 com aumento e 27 com diminuição) com 20 h de desenvolvimento, 101 genes (12 com aumento e 89 com diminuição) com 24 h e 76 genes (oito com aumento e 68 com diminuição) com 25 h. Alguns genes que apresentaram aumento na expressão (stuA e o gene da scytalone dehydratase) já haviam sido associados com fases específicas da conidiogênese, entretanto a maioria dos genes que apresentou aumento na expressão não tem função conhecida. A análise de transcriptomas com microarranjos de DNA também foi utilizada para identificar genes com expressão modulada por exposições à radiação UVB (1,8 kJ m-2) em micélio jovem de A. fumigatus. Foram identificados 101 genes diferencialmente expressos ao final da exposição à radiação UVB (51 genes com aumento e 50 com redução). O gene radc apresentou o maior aumento na expressão (aproximadamente 16 ×). A maioria dos genes com aumento na expressão não possui função conhecida. Foram identificados 418 genes diferencialmente expressos 30 min após o termino da exposição (51 genes com aumento e 367 com redução na expressão). / Conidia are specialized structures produced asexually during mycelia growth of many ascomycete species. The process of conidiation involves temporal and spatial regulation of gene expression, cell specialization, intercellular communication, and formation of specific structures during fungal growth. Conidia are responsible for the reproduction, dispersal and environmental persistence of many fungal species. In pathogenic species like Aspergillus fumigatus and Metarhizium anisopliae, conidia are also responsible for host infection. One of the main environmental factors that can kill and/or damage conidia is solar UV radiation. Cyclobutane pyrimidine dimers (CPDs) are the major DNA photoproducts induced by UVB. The principal goals of the present study were to: (1) estimate the frequency of CPDs induced by sublethal doses of UVB radiation in conidial DNA of three selected ascomycetes, (2) examine correlation of CPD frequencies with germination speed, and (3) estimate the protective effect of the wild-type green conidial pigmentation on DNA in M. anisopliae var. anisopliae, (4) identify differentially expressed genes during the different phases of A. fumigatus conidiogenesis and (5) identify genes regulated by UVB radiation in A. fumigatus young mycelia. A. fumigatus, A. nidulans, and M. anisopliae conidia were exposed to 1000 mW m-2 UV irradiance for 15, 30, 60 and 90 min. Total Quaite-weighted doses were 0.9, 1.8, 3.6 and 5.4 kJ m-2, respectively. The frequencies of dimers were linear and directly proportional to the doses, with 0.215, 0.455, 0.803 and 1.628 CPDs 10 kb-1 detected at the doses of 0.9, 1.8, 3.6 and 5.4 kJ m-2 in A. fumigatus, 0.037, 0.077, 0.142 and 0.202 CPDs 10 kb-1 in A. nidulans, and 0.041, 0.085, 0.155 and 0.255 CPDs 10 kb-1 in M. anisopliae. The frequency of dimers in the M. anisopliae albino mutant DWR 180 (0.552 10 kb-1) was approximately ten times higher than of the wild-type ARSEF 23 strain (0.057 10 kb-1) after exposure to doses of 1.8 kJ m-2. DNA microarrays carrying sequence of 11,000 genes of A. fumigatus were used to compare transcriptomes of 20 h-old mycelia (initial phase of the conidiogenesis), 24 h (intermediate phase) e 25 h (final phase) with young mycelia transcriptome. Thirty-four genes displayed a statistically significant difference in expression (7 with increase and 27 with decrease mRNA expression) in 20 h-old mycelia, 101 genes (12 with increase and 89 with decrease) in 24 h-old mycelia and 76 genes (8 with increase and 68 with decrease) in 25 h-old mycelia. Some overexpressed genes (stuA ad the scytalone dehydratase gene) were previously related to specific phases of conidiogenesis but the function of most of them is unknown. Transcriptome analysis using microarrays was also used to identify genes modulated by exposures to UVB radiation (1,8 kJ m-2) in A. fumigatus young mycelia. One hundred and one differentially expressed genes were identified at the end of the exposure to UVB radiation (51 genes with increase and 50 with decrease). The radc gene displayed the higher increase in expression (approximately 16 ×). The function of most of the overexpressed genes is unknown. Four hundred and eighteen differentially expressed genes were identified 30 min after the end of exposure (51 genes with increase and 367 with decrease in expression).

Restriction endonucleases occur ubiquitously among bacteria, archaea and in viruses of certain unicellular algae, and they are usually accompanied by a modification enzyme of identical specificity; together, the two activities form a restriction-modification (R-M) system- the prokaryotic equivalent of an immune system. More than 3,800 R-M enzymes have been characterized so far and they manifest 262 unique recognition specificities. These enzymes represent the largest family of functionally related enzymes. Based on the number and organization of subunits, cofactor requirements, catalytic mechanism, and sequence specificity, restriction enzymes have been classified into different types, Types I, II, III, and IV. R-M systems are important model systems for studying highly specific DNA-Protein interactions and serve as excellent systems for investigating structure-function relationship and for understanding the evolution of functionally similar enzymes with highly dissimilar sequence. In bacteria, DNA methyltransferases (MTases) associated with R-M systems protects the host DNA from cleavage by the cognate restriction endonuclease recognizing the same sequence and provides the integrity of host cell genome against foreign DNA invasion. The modification MTases catalyses the addition of a methyl group to one nucleotide in each strand of the recognition sequence using S-adenosyl-L-methionine (AdoMet) as the methyl group donor. Based on the chemistry of the methylation reaction catalyzed, DNA MTases are classified as C5 enzymes (endocyclic MTases), which transfer the methyl group to C5 position of cytosine, and N6 and N4 enzymes (exocyclic amino MTases), which transfer the methyl group to the exocyclic amino group of adenine or cytosine, respectively. DNA MTases of all three types contain conserved regions, which are responsible for catalysis and AdoMet binding, and variable regions known as target recognition domains (TRD), which determine the substrate specificity of a particular enzyme. Ten conserved amino acid motifs (I–X) are found in C5 MTases. Exocyclic DNA MTases are subdivided further into six groups (namely α, β, γ, ζ, δ and ε), according to the linear arrangements of three conserved motifs, the AdoMet-binding domain (FXGXG), the TRD (target recognition domain) and the catalytic domain (D/N/S)PP(Y/F). Base flipping has been proposed as a general mechanism used by all MTases in which the target base to be methylated is rotated 180º out of the DNA into a catalytic domain (motif IV). EcoP15I restriction enzyme (R.EcoP15I) belongs to the Type III restriction-modification (R-M) family. These enzymes are composed of two subunits, Res (Restriction) and Mod (Modification). The Mod subunit alone functions as a DNA methyltransferase in presence of AdoMet and magnesium and determines the specificity for restriction and methylation, whereas restriction activity requires the cooperation of both the Res and Mod subunits. EcoP15I methyltransferase (M.EcoP15I), a homodimeric enzyme catalyzes the transfer of a methyl group from AdoMet to the second adenine residue in the recognition sequence, 5’-CAGCAG-3’, in presence of magnesium ions. M.EcoP15I belongs to the β-subfamily of N6-adenine methyltransferases. In addition to the two highly conserved sequence motifs, FXGXG (motif 1) involved in AdoMet binding and DPPY (motif IV) involved in catalysis, the amino acid residues of the region 355-377 contains a PD(X)n(D/E)XK-like motif involved in metal binding. A Mutation in the Mod Subunit of EcoP15I Restriction Enzyme Converts the DNA Methyltransferase to a Site-Specific Endonuclease An interesting aspect of M.EcoP15I is that the methylation requires magnesium and magnesium binding to the PD(X)n(D/E)XK-like motif participates in base flipping. The PD-(D/E)XK superfamily of Mg2+-dependent nucleases were initially identified in structurally characterized Type II REases and later found in many enzymes involved in DNA replication, recombination and repair. The charged residues from the catalytic triads are implicated in metal ion mediated DNA cleavage. In EcoP15I DNA methyltransferase, a PD(X)n(D/E)XK like motif is present in which the partially conserved proline is replaced by methionine (MD(X)18(D/E)XK). Using site-directed mutagenesis methionine at 357 was changed to proline (M357P), which resulted in the formation of a Mg2+ binding/catalytic motif similar to several Mg2+-dependent endonucleases. Substitution of methionine at position 357 by proline converts EcoP15I DNA methyltransferase to a site-specific endonuclease. The mutant protein specifically binds to the recognition sequence 5’-CAGCAG-3’ and cleaves DNA in presence of Mg2+. The engineered EcoP15I-M357P is an active, sequence-dependent restriction endonuclease that cleaves DNA 10/1 nucleotide away from its recognition sequence in the presence of Mg2+. Unlike the holoenzyme, R.EcoP15I, the engineered endonuclease neither requires AdoMet or ATP nor requires two sites in the inverted orientation for DNA cleavage. It is of potential interest to use such an engineered enzyme as a genetic manipulation tool. Dimerisation of EcoP15I DNA Methyltransferase is Required for Sequence Recognition and Catalysis In the cell, after each round of replication, substrate for any DNA MTase is hemimethylated DNA and therefore, only a single methylation event restores the fully methylated state. This is in agreement with the fact that most of the DNA MTases studied exist as monomers in solution. The peculiar feature of M.EcoP15I is that it methylates only one strand of the DNA, at the N6-position of the adenine residue. Earlier studies using gel filtration and glutaraldehyde cross-linking demonstrated that M.EcoP15I exists as dimer in solution. However, the significance of dimerisation in the reaction mechanism of EcoP15I MTase is not clear. Therefore, experiments have been performed to determine whether M.EcoP15I could function as a monomer and the significance of dimerisation, if any, in catalysis. Towards this a homology model of the M.EcoP15I was generated by “FRankenstein monster” approach. Residues D223, V225, and V392, the side chains of which are present in the putative dimerisation interface in the model were targeted for site-directed mutagenesis. These residues were mutated to lysine and their importance was studied. Methylation and in vitro restriction assays showed that the triple mutant was catalytically inactive. Interestingly, the mutations resulted in weakening of the interaction between the monomers leading to both monomeric and dimeric species. M.EcoP15I was inactive in the monomeric form and therefore, dimerisation might be the initial step in its function. This must be required for positioning of the target base of the DNA in the active-site pocket of the M.EcoP15I. A part of this interface may be involved in site-specific DNA binding. Dimerisation of M.EcoP15I is, therefore, a prerequisite for the high-affinity substrate binding needed for efficient catalysis. Understanding the role(s) of Amino and Carboxyl-terminal Domains of EcoP15I DNA Methyltransferase in DNA Recognition and Catalysis N-terminal and C- terminal domains (NTD and CTD) of proteins are known to play many important roles such as folding, stability, dimerisation, regulation of gene expression, enzyme activity and substrate binding. From the modeled dimeric structure of M.EcoP15I, it was hypothesized that N- and C-termini are in close proximity with each other. In addition, it was predicted that each monomer can bind to AdoMet and DNA. Towards understanding the role(s) of the N- and C-terminal domains of M.EcoP15I in its structure and function, N-, and C-terminal deletions were created. Interestingly, deletion of N-terminal 53 amino acids and C-terminal 127 amino acids from of EcoP15I MTase converted the dimeric enzyme to a stable, monomeric protein that was structurally stable but enzymatically inactive. Each monomer could bind single-stranded DNA but dimerisation was required for double-stranded DNA binding and methylation. This indicated that amino acids at the N- and C-termini are important for maintaining a proper dimeric structure for M.EcoP15I functions. Therefore, it can be proposed that in a complex three-dimensional structure, the NTD and CTD should be properly maintained in order to execute its function, including dimerisation and DNA binding. However, since the 3D structure of M.EcoP15I has not yet been determined, the biochemical, biophysical and bioinformatics approaches may serve to provide useful information on the relative contributions of the electrostatic forces and hydrophobic contacts to the structural stability. Understanding the structural organization and folding of M.EcoP15I is crucial to elucidation of the mechanism of action.

Human mitochondrial DNA (mtDNA) is located in discrete DNA-protein complexes, so called nucleoids. These structures can be easily visualized in living cells by utilizing the fluorescent stain PicoGreen®. In contrary, cells devoid of endogenous mitochondrial genomes (ρ0 cells) display no mitochondrial staining in the cytoplasm. A modified restriction enzyme can be targeted to mitochondria to cleave the mtDNA molecules in more than two fragments, thereby activating endogenous nucleases. By applying this novel enzymatic approach to generate mtDNA-depleted cells the destruction of mitochondrial nucleoids in cultured cells could be detected in a time course. It is clear from these experiments that mtDNA-depleted cells can be seen as early as 48 h post-transfection using the depletion system. To prove that mtDNA is degraded during this process, mtDNA of transfected cells was quantified by real-time PCR. A significant decline could be observed 24 h post-transfection. Combination of both results showed that mtDNA of transfected cells is completely degraded and, therefore, ρ0 cells were generated within 48 h. Thus, the application of a mitochondrially-targeted restriction endonuclease proves to be a first and fast, but essential step towards a therapy for mtDNA disorders.

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.

Au cours de ces 10 dernières années une nouvelle classe de petits ARN non-codants nommés "tRNA-derived fragments" (tRFs) a été caractérisée. Tandis que le rôle canonique des tRNA est bien connu, les raisons pour lesquels des fragments de tRNA s'accumulent dans la cellule restent inconnues. Actuellement, peu d'informations sont disponibles sur leurs biogenèses et leurs rôles biologiques, mais les preuves montrant leur importance dans la régulation de l'expression des gènes augmente régulièrement. Cependant, peu de données sont disponibles chez les plantes. A l'aide d’expérience de "deep-sequencing" et de northern blot nous avons confirmé l'existence d'une grande population en tRFs d'origine variée. A la suite de ces observations, trois questions sont établies. Tout d'abord, quelles sont les enzymes responsables de la biogenèse des tRFs. Ensuite, où les tRFs sont générés. Enfin, est-ce que les tRFs sont des sous-produits de la dégradation des tRNA ou ont-ils une fonction biologique? / During the last decade, a new class of small non-coding RNAs called tRNA-derived fragments (tRFs) has emerged. Whilst the canonic role of tRNA is well-known, the reason(s) why stable tRFs remains in the cell is unknown. Indeed, the number of tRFs has rapidly increased in various evolutionary divergent organisms. To date, only few data on their biogenesis and on their biological roles is known but their importance in the regulation of gene expression and in cell life is expanding. In plants, the existence of tRFs has also been reported but only few data are available. Using deep-sequencing on various small RNA libraries from Arabidopsis thaliana and Northern blots experiments, we confirmed the existence of a large but specific population of tRFs. Following these observations, three questions are addressed. First, what are the enzymes responsible for tRFs biogenesis, second where are tRFs generated and third, are tRFs merely degredation by-products or do they have biological functions?