JUNCTION PROBES - SEQUENCE SPECIFIC DETECTION OF NUCLEIC ACIDS VIA TEMPLATE ENHANCED HYBRIDIZATION PROCESSES

As new disease biomarkers such as cancer-linked microRNAs are discovered, the need for new strategies for the detection of these disease biomarkers at isothermal conditions will increase. The junction probe (JP) technology is a restriction endonuclease-based nucleic acids detection platform that achieves isothermal amplified sensing and does not require the presence of an endonuclease recognition sequence in the target analyte. The first generation junction probe platform however suffered from long assay time (several hours). We hypothesized that the slow catalysis in the first-generation JP platform was due to an inhibition cycle. Consequently, a second generation JP platform, which is modified with phosphorothioate moieties in order to suppress the inhibition cycle, was developed. The second-generation JP platform is significantly superior to the first-generation platform and we demonstrated the potential of second-generation JP by detecting microRNA and bacterial ribosomal RNA. Importantly, second-generation JP could detect RNA in crude bacterial cell lysates without extensive sample preparation

Fluorescence Studies of EcoRI Restriction Endonuclease Structure and Dynamics.

Sequence specific protein-DNA interaction involves changes in protein conformation and dynamics. Research work involved in this thesis is the study of structure and dynamics of EcoRI restriction endonuclease N-termini. The N-termini of EcoRI endonuclease has been shown to be essential for DNA cleavage and also stabilize EcoRI-DNA complex. But they are not resolved in the X-ray crystal structure. An Asn3Cys mutation was made at the N-termini by site-directed mutagenesis and the mutant was subjected to cysteine crosslinking, pyrene labeling and fluorescence studies. Chemical crosslinking of Asn3Cys mutant and steady-state fluorescence studies of pyrene-labeled mutant indicated that the N-termini are in close proximity and probably involved in the dimer interface. Time resolved fluorescence measurements revealed dynamics of the N-termini by examining the dissociation and reformation of pyrene excimers as well as the monomer spectral shifts caused by N-terminal segment movements. Fluorescence anisotropy decay analysis indicated the N-termini are on the protein surface and not totally disoriented in solution. Substrate DNA binding, however, causes the N-termini to be more mobile without affecting the proximity relationship. Fluorescence energy transfer experiments were carried out using a double mutant Asn3CysTrp104Tyr. The through space distance between the fluorescent labels, MIANS or 1,5-IAEDANS, and Trp246 is around 30 A. Substrate DNA binding decreased the distance to 26 A but cofactor Mg\sp{2+} ion did not cause any distance change. Single Trp246 fluorescence in the double mutant also revealed possible conformational changes upon substrate DNA or cofactor binding. Experimental evidence indicates that the N-termini are located at the dimer interface but distal to the DNA binding site. The findings provide further insight into the function of the N-termini of EcoRI endonuclease

Etablierung eines Systems zur intrazellulären Generierung Triple-Helix-bildender Oligonukleotide und die sequenzspezifische Inhibition des humanen MCP-1

Chemokines play a key role in the cellular infiltration of inflamed tissue. They are released by a wide variety of cell types during the initial phase of host response to injury, allergens, antigens, or invading microorganisms, and selectively attract leukocytes to inflammatory foci, inducing both migration and activation. Monocyte chemoattractant protein-1 (MCP-1), a member of the CC chemokine superfamily, functions in attracting monocytes, T lymphocytes, and basophils to sites of inflammation. MCP-1 is produced by monocytes, fibroblasts, vascular endothelial cells and smooth muscle cells in response to various stimuli such as tumour necrosis factor-a (TNF-a), interferon-g (IFN-g), and interleukin-1b (IL-1b). It also plays an important role in the pathogenesis of chronic inflammation, and overexpression of MCP-1 has been implicated in diseases including glomerulonephritis and rheumatoid arthritis. Oligonucleotide-directed triple helix formation offers a means to target specific sequences in DNA and interfere with gene expression at the transcriptional level. Triple helix-forming oligonucleotides (TFOs) bind to homopurine/homopyrimidine sequences, forming a stable, sequence-specific complex with the duplex DNA. Purine-rich sequences are frequent in gene regulatory regions and TFOs directed to promoter sequences have been shown to prevent binding of transcription factors and inhibit transcription initiation and elongation. Exogenous TFOs that bind homopurine/ homopyrimidine DNA sequences and form triple-helices can be rationally designed, while the intracellular delivery of single-stranded RNA TFOs has not been studied in detail before. In this study, expression vectors were constructed which directed transcription of either a 19 nt triplex-forming pyrimidine CU-TFO sequence targeting the human MCP-1 or two different 19 nt GU- or CA-control sequences, respectively, together with the vector encoded hygromycin resistance mRNA as one fusion transcript. HEK 293 cells were stable transfected with these vectors and several TFO and control cell lines were generated. Functional relevant triplex formation of a TFO with a corresponding 19 bp GC-rich AP-1/SP-1 site of the human MCP-1 promoter was shown. Binding of synthetic 19 nt CUTFO to the MCP-1 promoter duplex was verified by triplex blotting at pH 6.7. Underlining binding specificity, control sequences, including the GU- and CA-sequence, a TFO containing one single mismatch and a MCP-1 promoter duplex containing two mismatches, did not participate in triplex formation. Establishing a magnetic capture technique with streptavidin microbeads it was verified that at pH 7.0 the 19 nt TFO embedded in a 1.1 kb fusion transcript binds to a plasmid encoded MCP-1 promoter target duplex three times stronger than the controls. Finally, cell culture experiments revealed 76 ± 10.2% inhibition of MCP-1 protein secretion in TNF-a stimulated CU-TFO harboring cell lines and up to 88% after TNF-a and IFN-g costimulation in comparison to controls. Expression of interleukin-8 (IL-8) as one TNF-a inducible control gene was not affected by CU-TFO, demonstrating both highly specific and effective chemokine gene repression. Furthermore, another chemokine target, regulated upon activation normal T cell expressed and secreted (RANTES), which plays an essential role in inflammation by recruiting T lymphocytes, macrophages and eosinophils to inflammatory sites, was analysed using the triplex approach. A 28 nt TFO was designed targeting the murine RANTES gene promoter, and gel mobility shift assays demonstrated that the phosphodiester TFO formed a sequencespecific triplex with the double-stranded target DNA with a Kd of 2.5 x 10-7 M. It was analysed whether RANTES expression could be inhibited at the transcriptional level testing the TFO in two different cell lines, T helper-1 lymphocytes and brain microvascular endothelial cells (bend3 cells). Although there was a sequence-specific binding of the TFO detectable in the gel shift assays, there was no inhibitory effect of the exogenously added and phosphorothioate stabilised TFO on endogenous RANTES gene expression visible. Additionally, the small interfering RNA (siRNA) approach was tested as another strategy to inhibit expression of the pro-inflammatory chemokines MCP-1 and RANTES. Two different methods were pursuit, describing transient transfection with vector derived and synthetic siRNA. The vector pSUPER containing the siRNA coding sequence was used to suppress endogenous MCP-1 in HEK 293 cells. An empty vector without RNA sequence served as a control. Inhibition due to the siRNA was measured in stimulated and unstimulated cells. In TNF-a stimulated cells MCP-1 protein synthesis was decreased by 35 ± 11% after siRNA transfection. Using a synthetic double-stranded siRNA, the TNF-a induced MCP-1 protein secretion could be successfully inhibited about 62.3 ± 10.3% in HEK 293 cells, indicating that the siRNA is functional in these cells to suppress chemokine expression. The siRNA approach targeting murine RANTES in Th1 cells and b-end3 cells revealed no inhibition of endogenous gene expression. Gene therapy approaches rely on efficient transfer of genes to the desired target cells. A wide variety of viral and nonviral vectors have been developed and evaluated for their efficiency of transduction, sustained expression of the transgene, and safety. Among them, lentiviruses have been widely used for gene therapy applications. In order to improve the delivery of TFOs or siRNAs into the target cells, cloning of the lentiviral transfer vector SEW, the production of lentiviral particles by transient transfection were performed with the aim to generate lentiviral vector-derived TFOs in further experiments. Here, Th1 cells were transduced with infectious lentiviral particles and transduction efficacy was measured. Transduction efficacy higher than 82% could be achieved using the lentiviral vector SEW, opening optimal possibilities for the TFO or siRNA approach.Die Chemokine sind eine Unterfamilie der Zytokine mit starker chemotaktischer Aktivität und übernehmen bei der Positionierung von Zellen im Organismus wichtige Aufgaben. Im Falle einer Infektion mobilisieren sie Effektorzellen und leiten sie zu den Infektionsherden und ins lymphatische Gewebe. Darüber hinaus spielen Chemokine in der Organentwicklung, Wundheilung, Angiogenese, Angiostase, bei der homöostatischen Leukozytenrezirkulation und Immunregulation eine Rolle. MCP-1 (monocyte chemoattractant protein-1) ist ein Mitglied der CC-Chemokin Familie und lockt eine Vielzahl von Zelltypen wie Monozyten, T-Lymphozyten und Basophile zum Entzündungsherd. MCP-1 wird von verschiedenen Zellen wie Monozyten, Fibroblasten, vaskulären Endothelzellen und glatten Muskelzellen nach Stimulation mit Tumornekrosefaktor-alpha (TNF-alpha) Interferon-gamma (IFN-gamma) oder Interleukin-1 beta (IL-1 beta) produziert. Eine Überexpression von MCP-1 wird in vielen entzündlichen Erkrankungen wie rheumatoider Arthritis, Multipler Sklerose und Glomerulonephritis gefunden. Ziel dieser Arbeit war es, molekulare Strategien auf dem Weg zu einer therapeutischen Anwendung zu entwickeln, um die Aktivität z.B. pro-inflammatorischer Chemokine wie MCP-1 und RANTES (regulated upon activation normal T cell expressed and secreted) zu modulieren. In der Literatur sind verschiedene erfolgreiche Prinzipien beschrieben, die auf dem Einsatz von kurzen Oligonukleotiden basieren, um die Expression spezifischer Gene zu hemmen. Hierzu gehören die Antisense-Oligonukleotide, triple helix-forming oligonucleotides (TFOs) und auch die small interfering RNA (siRNA). Im Unterschied zur Wirkung von siRNA und Antisense-Oligonukleotiden auf die mRNA Translation geht es in dieser Arbeit um das Design von therapeutischen Agenzien, die auf der DNA-Ebene durch sequenzspezifische Interaktionen die Transkription verändern können. In der Promoterregion des humanen MCP-1 Gens befindet sich eine geeignete TFO Zielsequenz in einer Länge von 19 bp. Diese Zielsequenz umfasst die Bindungsstellen für die Transkriptionsfaktoren SP-1 und AP-1, welche sowohl bei der basalen als auch der induzierten Genexpression von MCP-1 eine entscheidende Rolle spielen. Generelle Schwierigkeiten der Oligonukleotid-Technologien wie der Einsatz hoher Konzentrationen, Notwendigkeit chemischer Modifikationen und die Transfektionseffizienz konnten durch die Etablierung von Vektoren zur intrazellulären Generierung von TFOs auf RNA-Basis maßgeblich verbessert werden. Verschiedene Kontrollen wurden in vitro durchgeführt, so auch der Nachweis der spezifischen Triplex-Formation mit synthetischen 19 nt Oligoribonukleotiden und dem 39 bp MCP-1 Promoterfragment. In Zelllinien, die konstitutiv TFOs generieren, konnte die endogene TNF-alpha induzierte MCP-1 Protein-Sekretion um 76 ± 10,2% im Vergleich zu Kontrollzelllinien inhibiert werden, die TNF-alpha induzierte IL-8 Expression blieb durch das TFO dagegen vollkommen unbeeinflusst. Auch in IFN-gamma stimulierten Zellen konnte eine Inhibition bis zu 83% erreicht werden, in TNF-alpha und IFN-gamma kostimulierten Zellen bis zu 88%. Die Präsenz der TFOs und der Kontrollen in den verschiedenen Zelllinien konnte über RT-PCR und Northern-Blots bestätigt werden. Ein weiteres Target war das murine RANTES, welches eine wichtige Rolle in der Chemoattraktion von Leukozyten während des Entzündungsprozesses spielt. Ein 28 nt TFO mit Purin-Bindungsmotiv (GT) wurde synthetisiert, und Triplex-Bildung mit der RANTES Promoterzielsequenz konnte im EMSA gezeigt werden. Für eine sequenzspezifische Triplex-Formation konnte ein Kd-Wert von 2.5 x 10-7 M bestimmt werden. Neben der TFO-Strategie wurde in dieser Arbeit auch die Möglichkeit von siRNAs untersucht, um die Expression des Chemokins MCP-1 zu inhibieren. Eine synthetische siRNA gegen MCP-1 erzielte in HEK 293 Zellen eine Inhibition der MCP-1 Protein-Sekretion von 62.3 ± 10.3%. Ein weiteres Ziel dieser Arbeit war es, den potentiellen Einsatz von TFO oder siRNA als gentherapeutisches Instrument durch geeignete Transfervektoren zu verbessern. In dieser Arbeit wurden lentivirale Partikel durch eine transiente Transfektion von 239T Zellen mit dem Transfervektor SEW und den notwendigen Verpackungsvektoren hergestellt. Der SEW Vektor enthält als Marker das grüne Fluoreszenzprotein (GFP). Im Anschluss wurden Th1 Zellen mit den infektiösen lentiviralen Vektoren transduziert. Die relative Frequenz an GFP-produzierenden Zellen konnte mittels FACS-Analyse bestimmt werden. Die Transduktionseffizienz lag bei 82%. Durch diese hohe Transduktionseffizienz ergeben sich auch optimale Bedingungen für den Einsatz von TFO und siRNA. Die erfolgreichen TFO- und siRNA-Sequenzen gegen das humane MCP-1 könnten weiterhin auch lentiviral exprimiert werden, um den Gentransfer zu verbessern. Die Effizienz der beiden Strategien könnte in verschiedenen Zelllinien und letztendlich auch im Tiermodell überprüft werden

A RecB-family nuclease motif in the Type I restriction endonuclease EcoR124I

The Type I restriction-modification enzyme EcoR124I is an ATP-dependent endonuclease that uses dsDNA translocation to locate and cleave distant non-specific DNA sites. Bioinformatic analysis of the HsdR subunits of EcoR124I and related Type I enzymes showed that in addition to the principal PD-(E/D)xK Motifs, I, II and III, a QxxxY motif is also present that is characteristic of RecB-family nucleases. The QxxxY motif resides immediately C-terminal to Motif III within a region of predicted α-helix. Using mutagenesis, we examined the role of the Q and Y residues in DNA binding, translocation and cleavage. Roles for the QxxxY motif in coordinating the catalytic residues or in stabilizing the nuclease domain on the DNA are discussed

Transcription factor Sp3 as target for SUMOylation in vivo

A group of sequence-specific DNA-binding proteins related to the transcription factor Sp1 (specificity protein 1) has been implicated in the regulation of many different genes, since binding sites for these transcription factors (GC/GT boxes) are a recurrent motif in regulatory sequences of these genes. In contrast to the transcriptional activators Sp1 and Sp4, the ubiquitously expressed Sp3 protein can both activate and repress transcription. The complex activity of Sp3 depends on two glutamine-rich activation domains, similar to those found in Sp1 and Sp4, and, adjacent to these, on an inhibitory domain unique to Sp3. The critical lysine residue in the Sp3 inhibitory domain lies within a consensus motif (IK551EE) that targets proteins for SUMO modification. SUMO (small ubiquitin-related modifier) is covalently attached to lysine residues in target proteins via an isopeptide linkage in a multi-step process that is analogous to ubiquitination. The present work analyses various aspects of SUMO conjugation to Sp3 in vivo. Studying modification of Sp3 by SUMO is complicated by the existence of a number of Sp3 isoforms. Immunoblot analyses revealed four distinct Sp3 proteins, two slow migrating of more than 100 kDa and two fast migrating species. Seven to eight Sp3 bands appeared, when cells were lysed in denaturing conditions. The additional protein species represent SUMO modified Sp3 isoforms. Currently, it is not known whether the relative distribution of the different Sp3 isoforms is regulated. However, a significant shift towards the long isoforms of Sp3, however, is observed in Sp1-/- ES cells demonstrating that Sp3 isoform expression principally can change in vivo. In addition, this observation suggests that the long isoforms of Sp3 may take over Sp1 functions under Sp1 knockout conditions. When Sp3 is overexpressed along with SUMO1 and SUMO2 in cells in culture, attachment of both SUMO paralogues to Sp3 occurred with almost equal efficiency. Beside lysine 551 within the inhibitory domain, there are two other potential SUMOylation sites in Sp3 (VKQE at position 9 and IKDE at position 120). This study revealed that SUMOylation takes place exclusively at K551, present in all four isoforms. Visualization of endogenous Sp3 by immumofluorescence showed a sponge-like, diffuse appearance, located predominantly in the nucleus. Evolutionally closely related Sp family members Sp1 and Sp2 are also located in the nucleus and the subcellular localization patterns are similar to Sp3. Ectopic expression of SUMO1 fused to GFP (green fluorescent protein) led to the accumulation of this fusion protein within subnuclear “dots” or PODs (promyelocytic leukemia oncogenic domains), whereas endogenous Sp3 remained diffusely distributed throughout nuclei. In addition, the wild-type Sp3 isoforms and the SUMOylation-deficient mutants of Sp3were located in the nucleus exhibiting also a sponge-like, diffuse appearance. Analyzing the Sp3 expression in different cell lines and mouse organs revealed that the relative level of Sp3 modification by SUMO is not cell line or organ dependent. In addition, no variation in Sp3 expression pattern after serum starvation, serum induction and heat shock was observed. Ultraviolet radiation or Tumor Necrosis Factor alpha and Cycloheximide treatment of mammalian cells did not alter the SUMOylation level of Sp3 protein in our experimental conditions. A significant reduction in Sp3 SUMO modification was observed upon treatment with MG-132, a cell-permeable inhibitor of the proteasome. Possibly this proteasome inhibitor prevents proteasome degradation of SUMO specific isopeptidase, which subsequently remove the Sp3-SUMO moiety. PIAS1 (protein inhibitor of activated STAT) was previously cloned in a two-hybrid screen by using the inhibitory domain of Sp3. Moreover, it was shown that PIAS1 strongly enhances SUMO-modification of Sp3 in vitro and thus acts as an SUMO E3 ligase towards Sp3. PIAS1-associated proteins might confer substrate specificity towards Sp3 and other transcription factors and/or regulate PIAS1 activity in vivo. For the purification and identification of PIAS1-associated proteins, a number of C-terminal tagged expression plasmids were constructed for constitutive and inducible expression. The dual-tag affinity purification system established in this thesis work contains a small 15 amino acid artificial tag (BiotinTAG) that becomes biotinylated by the BirA ligase upon co-transfection of an appropriate expression construct. To enhance specificity, a second tag was included in the expression vectors (Calmodulin Binding Peptide or alternatively FLAG or Triple-FLAG). In addition, dual tags expression plasmids for Sp3 were constructed. The establishment of stable cell lines expressing these fusion proteins in an inducible manner was initiated. Such cell lines might be ideal for further analyzes of PIAS1 activities and to purify PIAS1 (Sp3) associated factors

Transcription factor Sp3 as target for SUMOylation in vivo

A group of sequence-specific DNA-binding proteins related to the transcription factor Sp1 (specificity protein 1) has been implicated in the regulation of many different genes, since binding sites for these transcription factors (GC/GT boxes) are a recurrent motif in regulatory sequences of these genes. In contrast to the transcriptional activators Sp1 and Sp4, the ubiquitously expressed Sp3 protein can both activate and repress transcription. The complex activity of Sp3 depends on two glutamine-rich activation domains, similar to those found in Sp1 and Sp4, and, adjacent to these, on an inhibitory domain unique to Sp3. The critical lysine residue in the Sp3 inhibitory domain lies within a consensus motif (IK551EE) that targets proteins for SUMO modification. SUMO (small ubiquitin-related modifier) is covalently attached to lysine residues in target proteins via an isopeptide linkage in a multi-step process that is analogous to ubiquitination. The present work analyses various aspects of SUMO conjugation to Sp3 in vivo. Studying modification of Sp3 by SUMO is complicated by the existence of a number of Sp3 isoforms. Immunoblot analyses revealed four distinct Sp3 proteins, two slow migrating of more than 100 kDa and two fast migrating species. Seven to eight Sp3 bands appeared, when cells were lysed in denaturing conditions. The additional protein species represent SUMO modified Sp3 isoforms. Currently, it is not known whether the relative distribution of the different Sp3 isoforms is regulated. However, a significant shift towards the long isoforms of Sp3, however, is observed in Sp1-/- ES cells demonstrating that Sp3 isoform expression principally can change in vivo. In addition, this observation suggests that the long isoforms of Sp3 may take over Sp1 functions under Sp1 knockout conditions. When Sp3 is overexpressed along with SUMO1 and SUMO2 in cells in culture, attachment of both SUMO paralogues to Sp3 occurred with almost equal efficiency. Beside lysine 551 within the inhibitory domain, there are two other potential SUMOylation sites in Sp3 (VKQE at position 9 and IKDE at position 120). This study revealed that SUMOylation takes place exclusively at K551, present in all four isoforms. Visualization of endogenous Sp3 by immumofluorescence showed a sponge-like, diffuse appearance, located predominantly in the nucleus. Evolutionally closely related Sp family members Sp1 and Sp2 are also located in the nucleus and the subcellular localization patterns are similar to Sp3. Ectopic expression of SUMO1 fused to GFP (green fluorescent protein) led to the accumulation of this fusion protein within subnuclear “dots” or PODs (promyelocytic leukemia oncogenic domains), whereas endogenous Sp3 remained diffusely distributed throughout nuclei. In addition, the wild-type Sp3 isoforms and the SUMOylation-deficient mutants of Sp3were located in the nucleus exhibiting also a sponge-like, diffuse appearance. Analyzing the Sp3 expression in different cell lines and mouse organs revealed that the relative level of Sp3 modification by SUMO is not cell line or organ dependent. In addition, no variation in Sp3 expression pattern after serum starvation, serum induction and heat shock was observed. Ultraviolet radiation or Tumor Necrosis Factor alpha and Cycloheximide treatment of mammalian cells did not alter the SUMOylation level of Sp3 protein in our experimental conditions. A significant reduction in Sp3 SUMO modification was observed upon treatment with MG-132, a cell-permeable inhibitor of the proteasome. Possibly this proteasome inhibitor prevents proteasome degradation of SUMO specific isopeptidase, which subsequently remove the Sp3-SUMO moiety. PIAS1 (protein inhibitor of activated STAT) was previously cloned in a two-hybrid screen by using the inhibitory domain of Sp3. Moreover, it was shown that PIAS1 strongly enhances SUMO-modification of Sp3 in vitro and thus acts as an SUMO E3 ligase towards Sp3. PIAS1-associated proteins might confer substrate specificity towards Sp3 and other transcription factors and/or regulate PIAS1 activity in vivo. For the purification and identification of PIAS1-associated proteins, a number of C-terminal tagged expression plasmids were constructed for constitutive and inducible expression. The dual-tag affinity purification system established in this thesis work contains a small 15 amino acid artificial tag (BiotinTAG) that becomes biotinylated by the BirA ligase upon co-transfection of an appropriate expression construct. To enhance specificity, a second tag was included in the expression vectors (Calmodulin Binding Peptide or alternatively FLAG or Triple-FLAG). In addition, dual tags expression plasmids for Sp3 were constructed. The establishment of stable cell lines expressing these fusion proteins in an inducible manner was initiated. Such cell lines might be ideal for further analyzes of PIAS1 activities and to purify PIAS1 (Sp3) associated factors

Exploring the structure and function of bacterial cytosine specific DNA methyltransferases using site-directed mutagenesis.

Point mutations were engineered into the sequence of the multispecific DNA methyltransferase (Mtase) M. SPRI in motif IX, in order to mimic the corresponding motif IX of mono-specific Mtase. A similar approach was adopted to modify the sequence of the monospecific enzyme M. HhaI in motifs IX and X based on the available structure and as a consequence the enzyme regained methylation potential. It was thought that these changes might be sufficient to enable functional exchange of the target recognition domains (TRDs) between a mono- and a multispecific enzyme. However, insertion of various segments of TRD region from M. SPRI into the M. HhaI was not successful (Chapter 4). To establish whether mono- and multispecific Mtases are incompatible in terms of sequence exchanges, a systematic "swapping" of motifs was carried out (Chapter 5). These experiments suggested that there are some enzyme-specific structural interactions between different subunits within each class of Mtases. In second half of this thesis a bacterial two-hybrid system based on the reversible assembly of an engineered form of M. SPRI was developed (Chapter 6). However the Mtase protein does not assemble into an active species until a DNA segment encoding a leucine zipper motif is fused to each of the two halves. Co-transformation of E. coli with the plasmids expressing the C-terminal and N-terminal domains respectively resulted in the abolition of colonies on double antibiotic plates, when an mcr strain was used as host. High performance liquid chromatography was used to estimate the extent of modification of plasmids indirectly. The extent of methylation at specific sequences within a plasmid molecule was readily detected by the corresponding differential susceptibility to digestion by specific restriction enzymes. Using this approach it proved possible to detect different levels of activity produced by wild type and mutant recombinant DNA Methyltransferases with sensitivity and in a semi quantitative manner. In order to analyse the biochemical properties of Mtase, I have developed an in vitro translation-modification assay. Binary studies with the mutants (from Chapter 3 and 5) showed that there were no detectable sequence-specific recognition differences between these enzymes. Taken together, these results suggest that motif IX plays a role in general stabilisation of the enzyme core structure and has a less significant role in DNA recognition

Aminoacyl-tRNA-Charged Eukaryotic Elongation Factor 1A Is the Bona Fide Substrate for Legionella pneumophila Effector Glucosyltransferases

Legionella pneumophila, which is the causative organism of Legionnaireś disease, translocates numerous effector proteins into the host cell cytosol by a type IV secretion system during infection. Among the most potent effector proteins of Legionella are glucosyltransferases (lgt's), which selectively modify eukaryotic elongation factor (eEF) 1A at Ser-53 in the GTP binding domain. Glucosylation results in inhibition of protein synthesis. Here we show that in vitro glucosylation of yeast and mouse eEF1A by Lgt3 in the presence of the factors Phe-tRNAPhe and GTP was enhanced 150 and 590-fold, respectively. The glucosylation of eEF1A catalyzed by Lgt1 and 2 was increased about 70-fold. By comparison of uncharged tRNA with two distinct aminoacyl-tRNAs (His-tRNAHis and Phe-tRNAPhe) we could show that aminoacylation is crucial for Lgt-catalyzed glucosylation. Aminoacyl-tRNA had no effect on the enzymatic properties of lgt's and did not enhance the glucosylation rate of eEF1A truncation mutants, consisting of the GTPase domain only or of a 5 kDa peptide covering Ser-53 of eEF1A. Furthermore, binding of aminoacyl-tRNA to eEF1A was not altered by glucosylation. Taken together, our data suggest that the ternary complex, consisting of eEF1A, aminoacyl-tRNA and GTP, is the bona fide substrate for lgt's