Molecular architecture of the Mos1 paired-end complex: the structural basis of DNA transposition in a eukaryote.

A key step in cut-and-paste DNA transposition is the pairing of transposon ends before the element is excised and inserted at a new site in its host genome. Crystallographic analyses of the paired-end complex (PEC) formed from precleaved transposon ends and the transposase of the eukaryotic element Mos1 reveals two parallel ends bound to a dimeric enzyme. The complex has a trans arrangement, with each transposon end recognized by the DNA binding region of one transposase monomer and by the active site of the other monomer. Two additional DNA duplexes in the crystal indicate likely binding sites for flanking DNA. Biochemical data provide support for a model of the target capture complex and identify Arg186 to be critical for target binding. Mixing experiments indicate that a transposase dimer initiates first-strand cleavage and suggest a pathway for PEC formation.

Use of mariner transposases for one-step delivery and integration of DNA in prokaryotes and eukaryotes by transfection.

Delivery of DNA to cells and its subsequent integration into the host genome is a fundamental task in molecular biology, biotechnology and gene therapy. Here we describe an IP-free one-step method that enables stable genome integration into either prokaryotic or eukaryotic cells. A synthetic mariner transposon is generated by flanking a DNA sequence with short inverted repeats. When purified recombinant Mos1 or Mboumar-9 transposase is co-transfected with transposon-containing plasmid DNA, it penetrates prokaryotic or eukaryotic cells and integrates the target DNA into the genome. In vivo integrations by purified transposase can be achieved by electroporation, chemical transfection or Lipofection of the transposase:DNA mixture, in contrast to other published transposon-based protocols which require electroporation or microinjection. As in other transposome systems, no helper plasmids are required since transposases are not expressed inside the host cells, thus leading to generation of stable cell lines. Since it does not require electroporation or microinjection, this tool has the potential to be applied for automated high-throughput creation of libraries of random integrants for purposes including gene knock-out libraries, screening for optimal integration positions or safe genome locations in different organisms, selection of the highest production of valuable compounds for biotechnology, and sequencing.

Structural role of the flanking DNA in mariner transposon excision.

During cut-and-paste mariner/Tc1 transposition, transposon DNA is cut precisely at its junction with flanking DNA, ensuring the transposon is neither shortened nor lengthened with each transposition event. Each transposon end is flanked by a TpA dinucleotide: the signature target site duplication of mariner/Tc1 transposition. To establish the role of this sequence in accurate DNA cleavage, we have determined the crystal structure of a pre-second strand cleavage mariner Mos1 transpososome. The structure reveals the route of an intact DNA strand through the transposase active site before second strand cleavage. The crossed architecture of this pre-second strand cleavage paired-end complex supports our proposal that second strand cleavage occurs in trans. The conserved mariner transposase WVPHEL and YSPDL motifs position the strand for accurate DNA cleavage. Base-specific recognition of the flanking DNA by conserved amino acids is revealed, defining a new role for the WVPHEL motif in mariner transposition and providing a molecular explanation for in vitro mutagenesis data. Comparison of the pre-TS cleavage and post-cleavage Mos1 transpososomes with structures of Prototype Foamy Virus intasomes suggests a binding mode for target DNA prior to Mos1 transposon integration.

Structural Basis for the Inverted Repeat Preferences of mariner Transposases.

The inverted repeat (IR) sequences delimiting the left and right ends of many naturally active mariner DNA transposons are non-identical and have different affinities for their transposase. We have compared the preferences of two active mariner transposases, Mos1 and Mboumar-9, for their imperfect transposon IRs in each step of transposition: DNA binding, DNA cleavage, and DNA strand transfer. A 3.1 Å resolution crystal structure of the Mos1 paired-end complex containing the pre-cleaved left IR sequences reveals the molecular basis for the reduced affinity of the Mos1 transposase DNA-binding domain for the left IR as compared with the right IR. For both Mos1 and Mboumar-9, in vitro DNA transposition is most efficient when the preferred IR sequence is present at both transposon ends. We find that this is due to the higher efficiency of cleavage and strand transfer of the preferred transposon end. We show that the efficiency of Mboumar-9 transposition is improved almost 4-fold by changing the 3' base of the preferred Mboumar-9 IR from guanine to adenine. This preference for adenine at the reactive 3' end for both Mos1 and Mboumar-9 may be a general feature of mariner transposition.

Solution conformations of early intermediates in Mos1 transposition.

DNA transposases facilitate genome rearrangements by moving DNA transposons around and between genomes by a cut-and-paste mechanism. DNA transposition proceeds in an ordered series of nucleoprotein complexes that coordinate pairing and cleavage of the transposon ends and integration of the cleaved ends at a new genomic site. Transposition is initiated by transposase recognition of the inverted repeat sequences marking each transposon end. Using a combination of solution scattering and biochemical techniques, we have determined the solution conformations and stoichiometries of DNA-free Mos1 transposase and of the transposase bound to a single transposon end. We show that Mos1 transposase is an elongated homodimer in the absence of DNA and that the N-terminal 55 residues, containing the first helix-turn-helix motif, are required for dimerization. This arrangement is remarkably different from the compact, crossed architecture of the dimer in the Mos1 paired-end complex (PEC). The transposase remains elongated when bound to a single-transposon end in a pre-cleavage complex, and the DNA is bound predominantly to one transposase monomer. We propose that a conformational change in the single-end complex, involving rotation of one half of the transposase along with binding of a second transposon end, could facilitate PEC assembly.

Biochemical characterization and comparison of two closely related active mariner transposases.

Most DNA transposons move from one genomic location to another by a cut-and-paste mechanism and are useful tools for genomic manipulations. Short inverted repeat (IR) DNA sequences marking each end of the transposon are recognized by a DNA transposase (encoded by the transposon itself). This enzyme cleaves the transposon ends and integrates them at a new genomic location. We report here a comparison of the biophysical and biochemical properties of two closely related and active mariner/Tc1 family DNA transposases: Mboumar-9 and Mos1. We compared the in vitro cleavage activities of the enzymes on their own IR sequences, as well as cross-recognition of their inverted repeat sequences. We found that, like Mos1, untagged recombinant Mboumar-9 transposase is a dimer and forms a stable complex with inverted repeat DNA in the presence of Mg(2+) ions. Mboumar-9 transposase cleaves its inverted repeat DNA in the manner observed for Mos1 transposase. There was minimal cross-recognition of IR sequences between Mos1 and Mboumar-9 transposases, despite these enzymes having 68% identical amino acid sequences. Transposases sharing common biophysical and biochemical properties, but retaining recognition specificity toward their own IR, are a promising platform for the design of chimeric transposases with predicted and improved sequence recognition.

Structure of a complex phosphoglycan epitope from gp72 of Trypanosoma cruzi.

The parasitic protozoan organism Trypanosoma cruzi is the causative agent of Chagas disease. The insect vector-dwelling epimastigote form of the organism expresses a low abundance glycoprotein associated with the flagellum adhesion zone, called gp72. The gp72 glycoprotein was first identified with an anti-carbohydrate IgG3 monoclonal antibody called WIC29.26 and has been shown to have an unusual sugar composition. Here, we describe a new way to isolate the WIC29.26 carbohydrate epitope of gp72. Using (1)H NMR and mass spectrometry before and after derivatization, we provide an almost complete primary chemical structure for the epitope, which is that of a complex phosphosaccharide: Galfß1-4Rhapα1-2Fucpα1-4(Galpß1-3)(Galpα1-2)Xylpß1-4Xylpß1-3(Xylpß1-2Galpα1-4(Galpß1-3)(Rhapα1-2)Fucpα1-4)GlcNAcp, with phosphate attached to one or other of the two Galp terminal residues and in which all residues are of the d-absolute configuration, except for fucose and rhamnose which are l. Combined with previous data (Haynes, P. A., Ferguson, M. A., and Cross, G. A. (1996) Glycobiology 6, 869-878), we postulate that this complex structure and its variants lacking one or more residues are linked to Thr and Ser residues in gp72 via a phosphodiester linkage (GlcNAcpα1-P-Thr/Ser) and that these units may form phosphosaccharide repeats through GlcNAcpα1-P-Galp linkages. The gp72 glycoprotein is associated with the flagellum adhesion zone on the parasite surface, and its ligation has been implicated in inhibiting parasite differentiation from the epimastigote to the metacyclic trypomastigote stage. The detailed structure of the unique phosphosaccharide component of gp72 reported here provides a template for future biosynthetic and functional studies.

Biochemical and immunological characterization of Toxoplasma gondii macrophage migration inhibitory factor.

Macrophage migration inhibitory factor (MIF) is a proinflammatory molecule in mammals that, unusually for a cytokine, exhibits tautomerase and oxidoreductase enzymatic activities. Homologues of this well conserved protein are found within diverse phyla including a number of parasitic organisms. Herein, we produced recombinant histidine-tagged Toxoplasma gondii MIF (TgMIF), a 12-kDa protein that lacks oxidoreductase activity but exhibits tautomerase activity with a specific activity of 19.3 µmol/min/mg that cannot be inhibited by the human MIF inhibitor ISO-1. The crystal structure of the TgMIF homotrimer has been determined to 1.82 Å, and although it has close structural homology with mammalian MIFs, it has critical differences in the tautomerase active site that account for the different inhibitor sensitivity. We also demonstrate that TgMIF can elicit IL-8 production from human peripheral blood mononuclear cells while also activating ERK MAPK pathways in murine bone marrow-derived macrophages. TgMIF may therefore play an immunomodulatory role during T. gondii infection in mammals.

Fluorescence-resonance-energy-transfer-based assay to estimate modulation of TDP1 activity through arginine methylation.

Tyrosyl DNA phosphodiesterase (TDP1) is a DNA repair enzyme that hydrolyzes the phosphotyrosyl linkage between 3'-DNA-protein crosslinks such as stalled topoisomerase 1 cleavage complexes (Top1cc). Here, we present a fluorescence-resonance-energy-transfer-(FRET) based assay to estimate modulation of TDP1 activity through arginine methylation. We describe steps for TDP1 expression and purification and estimating TDP1 activity using fluorescence-quenched probes mimicking Top1cc. We then detail data analysis of real-time TDP1 activity and screening of TDP1-selective inhibitors. For complete details on the use and execution of this protocol, please refer to Bhattacharjee et al. (2022).1.

Chemical structure of Trichomonas vaginalis surface lipoglycan: a role for short galactose (ß1-4/3) N-acetylglucosamine repeats in host cell interaction.

The extracellular parasite Trichomonas vaginalis contains a surface glycoconjugate that appears to mediate parasite-host cell interaction via binding to human galectin-1. This glycoconjugate also elicits cytokine production from human vaginal epithelial cells, implicating its role in modulation of host immune responses. We have analyzed the structure of this glycoconjugate, previously described to contain the sugars rhamnose (Rha), N-acetylglucosamine (GlcNAc), galactose (Gal), xylose (Xyl), N-acetylgalactosamine (GalNAc), and glucose (Glc), using gas chromatograph mass spectrometry (GC-MS), matrix-assisted laser desorption/ionization time of flight mass spectrometry (MALDI-TOF), electrospray MS/MS, and nuclear magnetic resonance (NMR), combined with chemical and enzymatic digestions. Our data reveal a complex structure, named T. vaginalis lipoglycan (TvLG), that differs markedly from Leishmania lipophosphoglycan and Entamoeba lipopeptidophosphoglycan and is devoid of phosphosaccharide repeats. TvLG is composed of an α1-3 linked polyrhamnose core, where Rha residues are substituted at the 2-position with either ß-Xyl or chains of, on average, five N-acetyllactosamine (-3Galß1-4GlcNAcß1-) (LacNAc) units and occasionally lacto-N-biose (-3Galß1-3GlcNAcß1-) (LNB). These chains are themselves periodically substituted at the Gal residues with Xyl-Rha. These structural analyses led us to test the role of the poly-LacNAc/LNB chains in parasite binding to host cells. We found that reduction of poly-LacNAc/LNB chains decreased the ability of TvLG to compete parasite binding to host cells. In summary, our data provide a new model for the structure of TvLG, composed of a polyrhamnose backbone with branches of Xyl and poly-LacNAc/LNB. Furthermore, the poly-LacNAc side chains are shown to be involved in parasite-host cell interaction.

Interplay between symmetric arginine dimethylation and ubiquitylation regulates TDP1 proteostasis for the repair of topoisomerase I-DNA adducts.

Tyrosyl-DNA phosphodiesterase (TDP1) hydrolyzes the phosphodiester bond between a DNA 3' end and a tyrosyl moiety and is implicated in the repair of trapped topoisomerase I (Top1)-DNA covalent complexes (Top1cc). Protein arginine methyltransferase 5 (PRMT5) catalyzes arginine methylation of TDP1 at the residues R361 and R586. Here, we establish mechanistic crosstalk between TDP1 arginine methylation and ubiquitylation, which is critical for TDP1 homeostasis and cellular responses to Top1 poisons. We show that R586 methylation promotes TDP1 ubiquitylation, which facilitates ubiquitin/proteasome-dependent TDP1 turnover by impeding the binding of UCHL3 (deubiquitylase enzyme) with TDP1. TDP1-R586 also promotes TDP1-XRCC1 binding and XRCC1 foci formation at Top1cc-damage sites. Intriguingly, R361 methylation enhances the 3'-phosphodiesterase activity of TDP1 in real-time fluorescence-based cleavage assays, and this was rationalized using structural modeling. Together, our findings establish arginine methylation as a co-regulator of TDP1 proteostasis and activity, which modulates the repair of trapped Top1cc.

Phosphorylation of NANOG by casein kinase I regulates embryonic stem cell self-renewal.

The self-renewal efficiency of mouse embryonic stem cells (ESCs) is determined by the concentration of the transcription factor NANOG. While NANOG binds thousands of sites in chromatin, the regulatory systems that control DNA binding are poorly characterised. Here, we show that NANOG is phosphorylated by casein kinase I, and identify target residues. Phosphomimetic substitutions at phosphorylation sites within the homeodomain (S130 and S131) have site-specific functional effects. Phosphomimetic substitution of S130 abolishes DNA binding by NANOG and eliminates LIF-independent self-renewal. In contrast, phosphomimetic substitution of S131 enhances LIF-independent self-renewal, without influencing DNA binding. Modelling the DNA-homeodomain complex explains the disparate effects of these phosphomimetic substitutions. These results indicate how phosphorylation may influence NANOG homeodomain interactions that underpin ESC self-renewal.

Structures of Leishmania major orthologues of macrophage migration inhibitory factor.

Leishmania major, an intracellular parasitic protozoon that infects, differentiates and replicates within macrophages, expresses two closely related MIF-like proteins. To ascertain the roles and potential differences of these two Leishmania proteins, recombinant L. major MIF1 and MIF2 have been produced and the structures resolved by X-ray crystallography. Each has a trimeric ring architecture similar to mammalian MIF, but with some structurally distinct features. LmjMIF1, but not LmjMIF2, has tautomerase activity. LmjMIF2 is found in all life cycle stages whereas LmjMIF1 is found exclusively in amastigotes, the intracellular stage responsible for mammalian disease. The findings are consistent with parasite MIFs modulating or circumventing the host macrophage response, thereby promoting parasite survival, but suggest the LmjMIFs have potentially different biological roles. Analysis of the Leishmania braziliensis genome showed that this species lacks both MIF genes. Thus MIF is not a virulence factor in all species of Leishmania.

Structural basis for DNA 3'-end processing by human tyrosyl-DNA phosphodiesterase 1.

Tyrosyl-DNA phosphodiesterase (Tdp1) is a DNA 3'-end processing enzyme that repairs topoisomerase 1B-induced DNA damage. We use a new tool combining site-specific DNA-protein cross-linking with mass spectrometry to identify Tdp1 interactions with DNA. A conserved phenylalanine (F259) of Tdp1, required for efficient DNA processing in biochemical assays, cross-links to defined positions in DNA substrates. Crystal structures of Tdp1-DNA complexes capture the DNA repair machinery after 3'-end cleavage; these reveal how Tdp1 coordinates the 3'-phosphorylated product of nucleosidase activity and accommodates duplex DNA. A hydrophobic wedge splits the DNA ends, directing the scissile strand through a channel towards the active site. The F259 side-chain stacks against the -3 base pair, delimiting the junction of duplexed and melted DNA, and fixes the scissile strand in the channel. Our results explain why Tdp1 cleavage is non-processive and provide a molecular basis for DNA 3'-end processing by Tdp1.

Crystallization of a Mos1 transposase-inverted-repeat DNA complex: biochemical and preliminary crystallographic analyses.

A complex formed between Mos1 transposase and its inverted-repeat DNA has been crystallized. The crystals diffract to 3.25 A resolution and exhibit monoclinic (P2(1)) symmetry, with unit-cell parameters a = 120.8, b = 85.1, c = 131.6 A, beta = 99.3 degrees . The X-ray diffraction data display noncrystallographic twofold symmetry and characteristic dsDNA diffraction at approximately 3.3 A. Biochemical analyses confirmed the presence of DNA and full-length protein in the crystals. The relationship between the axis of noncrystallographic symmetry, the unit-cell axes and the DNA diffraction pattern are discussed. The data are consistent with the previously proposed model of the paired-ends complex containing a dimer of the transposase.

A bend, flip and trap mechanism for transposon integration.

Cut-and-paste DNA transposons of the mariner/Tc1 family are useful tools for genome engineering and are inserted specifically at TA target sites. A crystal structure of the mariner transposase Mos1 (derived from Drosophila mauritiana), in complex with transposon ends covalently joined to target DNA, portrays the transposition machinery after DNA integration. It reveals severe distortion of target DNA and flipping of the target adenines into extra-helical positions. Fluorescence experiments confirm dynamic base flipping in solution. Transposase residues W159, R186, F187 and K190 stabilise the target DNA distortions and are required for efficient transposon integration and transposition in vitro. Transposase recognises the flipped target adenines via base-specific interactions with backbone atoms, offering a molecular basis for TA target sequence selection. Our results will provide a template for re-designing mariner/Tc1 transposases with modified target specificities.

Structural basis of Mos1 transposase inhibition by the anti-retroviral drug Raltegravir.

DNA transposases catalyze the movement of transposons around genomes by a cut-and-paste mechanism related to retroviral integration. Transposases and retroviral integrases share a common RNaseH-like domain with a catalytic DDE/D triad that coordinates the divalent cations required for DNA cleavage and integration. The anti-retroviral drugs Raltegravir and Elvitegravir inhibit integrases by displacing viral DNA ends from the catalytic metal ions. We demonstrate that Raltegravir, but not Elvitegravir, binds to Mos1 transposase in the presence of Mg(2+) or Mn(2+), without the requirement for transposon DNA, and inhibits transposon cleavage and DNA integration in biochemical assays. Crystal structures at 1.7 Å resolution show Raltegravir, in common with integrases, coordinating two Mg(2+) or Mn(2+) ions in the Mos1 active site. However, in the absence of transposon ends, the drug adopts an unusual, compact binding mode distinct from that observed in the active site of the prototype foamy virus integrase.

Cloning, expression and characterisation of FKB-6, the sole large TPR-containing immunophilin from C. elegans.

We have cloned, expressed, purified and characterised ceFKB-6, the only large tetratricopeptide repeat motif-containing immunophilin in Caenorhabditis elegans which is similar to the human orthologues FKBP51 and FKBP52. It shows increased peptidyl prolyl isomerase activity, the measured k(cat)/K(m) of 1.3 x 10(6) M(-1) s(-1)is twofold greater than that of hFKBP12 and hFKBP51. NMR studies of the interaction between FKB-6 and the C-terminal DAF-21 pentapeptide MEEVD show interactions consistent with those found between the large human immunophilin TPR domains and human Hsp90. In vivo localisation studies show that the fkb-6 gene is expressed in all stages from embryo to adult with predominant expression being noted in the adult dorsal and ventral nerve cords.

Mechanism of Mos1 transposition: insights from structural analysis.

We present the crystal structure of the catalytic domain of Mos1 transposase, a member of the Tc1/mariner family of transposases. The structure comprises an RNase H-like core, bringing together an aspartic acid triad to form the active site, capped by N- and C-terminal alpha-helices. We have solved structures with either one Mg2+ or two Mn2+ ions in the active site, consistent with a two-metal mechanism for catalysis. The lack of hairpin-stabilizing structural motifs is consistent with the absence of a hairpin intermediate in Mos1 excision. We have built a model for the DNA-binding domain of Mos1 transposase, based on the structure of the bipartite DNA-binding domain of Tc3 transposase. Combining this with the crystal structure of the catalytic domain provides a model for the paired-end complex formed between a dimer of Mos1 transposase and inverted repeat DNA. The implications for the mechanisms of first and second strand cleavage are discussed.

Trypanosoma brucei glycoproteins contain novel giant poly-N-acetyllactosamine carbohydrate chains.

The flagellar pocket of the bloodstream form of the African sleeping sickness parasite Trypanosoma brucei contains material that binds the beta-d-galactose-specific lectin ricin (Brickman, M. J., and Balber, A. E. (1990) J. Protozool. 37, 219-224). Glycoproteins were solubilized from bloodstream form T. brucei cells in 8 M urea and 3% SDS and purified by ricin affinity chromatography. Essentially all binding of ricin to these glycoproteins was abrogated by treatment with peptide N-glycosidase, showing that the ricin ligands are attached to glycoproteins via N-glycosidic linkages to asparagine residues. Glycans released by peptide N-glycosidase were resolved by Bio-Gel P-4 gel filtration into two fractions: a low molecular mass mannose-rich fraction and a high molecular mass galactose and N-acetylglucosamine-rich fraction. The latter fraction was further separated by high pH anion exchange chromatography and analyzed by gas chromatography mass spectrometry, one- and two-dimensional NMR, electrospray mass spectrometry, and methylation linkage analysis. The high molecular mass ricin-binding N-glycans are based on a conventional Manalpha1-3(Manalpha1-6)Manbeta1-4-GlcNAcbeta1-4GlcNAc core structure and contain poly-N-acetyllactosamine chains. A significant proportion of these structures are extremely large and of unusual structure. They contain an average of 54 N-acetyllactosamine (Galbeta1-4GlcNAc) repeats per glycan, linked mostly by -4GlcNAcbeta1-6Galbeta1-interrepeat linkages, with an average of one -4GlcNAcbeta1-3(-4GlcNAcbeta1-6)Galbeta1- branch point in every six repeats. These structures, which also bind tomato lectin, are twice the size reported for the largest mammalian poly-N-acetyllactosamine N-linked glycans and also differ in their preponderance of -4GlcNAcbeta1-6Galbeta1- over -4GlcNacbeta1-3Galbeta1- interrepeat linkages. Molecular modeling suggests that -4GlcNAcbeta1-6Galbeta1- interrepeat linkages produce relatively compact structures that may give these giant N-linked glycans unique physicochemical properties. Fluorescence microscopy using fluorescein isothiocyanatericin indicates that ricin ligands are located mainly in the flagellar pocket and in the endosomal/lysosomal system of the trypanosome.