Mécanismes de transposition et de régulation de la transposase de l'élément mariner Mos1

L élément mariner Mos1 est un élément transposable de classe II qui code une transposase, l enzyme permettant aux transposons de se déplacer dans les génomes. Cette transposase possède un coeur catalytique DDE similaire à celui des intégrases rétrovirales. Mon travail de thèse a consisté en l étude de la transposase de Mos1, sous différents aspects. Mes résultats complètent le modèle de transposition précédemment établi au laboratoire et apportent une nouvelle vision de la formation du complexe synaptique qui permet l excision de transposon du site donneur. Mes travaux ont également permis d identifier le domaine de liaison à l ADN de la transposase comme un domaine de type CRO. L étude de la régulation de l activité de la transposase, qui est phosphorylée, m a permis d élargir le modèle de transposition de Mos1 au contexte cellulaire eucaryote, et des études d ingénierie de la protéine posent la question de l impact des facteurs d hôtes lors de la transposition de Mos1. Enfin, des inhibiteurs de la transposase de Mos1 ont été identifiés et leur mode d action caractérisé. Ces composés présentent également une activité sur l intégrase du VIH-1 et une autre transposase à cœur catalytique DDE.The Mos1 mariner element is a Class II transposable element, encoding a transposase, which is the enzyme allowing them to move in the genomes. This transposase has a DDE catalytic core like the retroviral integrases. My work consisted in studying the Mos1 transposase, under different aspects. My results complete the model of transposition previously established in the laboratory and bring a new vision of the formation of synaptic complex, which allows excision of the transposon donor site. The DNA-binding of Mos1 transposase has also been identified as a CRO-like domain. Work on regulation of the activity of Mos1 transposase, which is phosphorylated, allowed me to expand the model of Mos1 transposition in a eukaryotic cell context. The engineering of the protein were also conducted and questions about the impact of host factors on Mos1 transposition. Inhibitors of Mos1 transposase have been identified and characterized. These compounds also inhibit HIV-1 integrase and an other DDE transposase.TOURS-Bibl.électronique (372610011) / SudocSudocFranceF

Molecular Evidence for the Evolution of Ichnovirus from Ascoviruses by Symbiogenesis

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In Vitro Recombination and Inverted Terminal Repeat Binding Activities of the Mcmar1 Transposase.

International audienceThe Mcmar1 mariner element (MLE) presents some intriguing features with two large, perfectly conserved, 355 bp inverted terminal repeats (ITRs) containing two 28 bp direct repeats (DRs). The presence of a complete ORF in Mcmar1 makes it possible to explore the transposition of this unusual M LE. Mcmar1 transposase (MCMAR1) was purified, and in vitro transposition assays showed that it is able to promote ITR-dependent DNA cleavages and recombination events, which correspond to plasmid fusions and transpositions with imprecise ends. Further analyses indicated that MCMAR1 is able to interact with the 355 bp ITR through two DRs: the EDR (external DR) is a high-affinity binding site for MCMAR1, whereas the IDR (internal DR) is a low-affinity binding site. The main complex detected within the EDR contained a transposase dimer and only one DNA molecule. We hypothesize that the inability of MCMAR1 to promote precise in vitro transposition events could be due to mutations in its ORF sequence or to the specific features of transposase binding to the ITR. Indeed, the ITR region spanning from EDR to IDR resembles a MITE and could be bent by specific host factors. This suggests that the assembly of the transposition complex is more complex than that of those involved in the mobility of the Mos1 and Hintar1 mariner elements

Site-directed integration of transgenes: transposons revisited using DNA-binding-domain technologies

International audienceIn the last 20 years, tools derived from DNA transposons have made major contributions to genetic studies from gene delivery to gene discovery. Various complementary and fairly ubiquitous DNA vehicles have been developed. Although many transposons are efficient DNA vehicles, they appear to have limited ability to target specific sequences, since all that is required at the integration locus is the presence of a short 2- to 4-bp sequence. Consequently, insertions mediated by transposon-based vectors occur somewhat randomly. In the past 5 years, strategies have emerged to enhance the site-specificity of transposon-based vectors, and to avoid random integrations. The first proposes that new target site specificity could be grafted onto a transposase by adding a new DNA-binding domain. Alternative strategies consist of indirectly targeting either the transposase or the transposon to a chosen genomic locus. The most important information available about each strategy are presented, and limitations and future prospects are discussed

Transposase-Transposase Interactions in MOS1 Complexes: A Biochemical Approach.

International audienceTransposases are proteins that have assumed the mobility of class II transposable elements. In order to map the interfaces involved in transposase transposase interactions, we have taken advantage of 12 transposase mutants that impair mariner transposase-transposase interactions taking place during transposition. Our data indicate that transposase transposase interactions regulating Mos1 transposition are sophisticated and result from (i) active MOS1 dimerization through the first HTH of the N-terminal domain, which leads to inverted terminal repeat (ITR) binding; (ii) inactive dimerization carried by part of the C-terminal domain, which prevents ITR binding; and (iii) oligomerization. Inactive dimers are nonpermissive in organizing complexes that produce ITR binding, but the interfaces (or interactions) supplied in this state could play a role in the various rearrangements needed during transposition. Oligomerization is probably not due to a specific MOS1 domain, but rather the result of nonspecific interactions resulting from incorrect folding of the protein. Our data also suggest that the MOS1 catalytic domain is a main actor in the overall organization of MOS1, thus playing a role in MOS1 oligomerization. Finally, we propose that MOS1 behaves as predicted by the pre-equilibrium existing model, whereby proteins are found to exist simultaneously in populations with diverse conformations, monomers and active and inactive dimers for MOS1. We were able to identify several MOS1 mutants that modify this pre-existing equilibrium. According to their properties, some of these mutants will be useful tools to break down the remaining gaps in our understanding of mariner transposition. (C) 2010 Elsevier Ltd. All rights reserved

Physical properties of DNA components affecting the transposition efficiency of the mariner Mos1 element

International audiencePrevious studies have shown that the transposase and the inverted terminal repeat (ITR) of the Mos1 mariner elements are suboptimal for transposition; and that hyperactive transposases and transposon with more efficient ITR configurations can be obtained by rational molecular engineering. In an attempt to determine the extent to which this element is suboptimal for transposition, we investigate here the impact of the three main DNA components on its transposition efficiency in bacteria and in vitro. We found that combinations of natural and synthetic ITRs obtained by systematic evolution of ligands by exponential enrichment did increase the transposition rate. We observed that when untranslated terminal regions were associated with their respective natural ITRs, they acted as transposition enhancers, probably via the early transposition steps. Finally, we demonstrated that the integrity of the Mos1 inner region was essential for transposition. These findings allowed us to propose prototypes of optimized Mos1 vectors, and to define the best sequence features of their associated marker cassettes. These vector prototypes were assayed in HeLa cells, in which Mos1 vectors had so far been found to be inactive. The results obtained revealed that using these prototypes does not circumvent this problem. However, such vectors can be expected to provide new tools for the use in genome engineering in systems such as Caenorhabditis elegans in which Mos1 is very active

Mariner Mos1 transposase optimization by rational mutagenesis

International audienceMariner transposons are probably the most widespread transposable element family in animal genomes. To date, they are believed not to require species-specific host factors for transposition. Despite this, Mos1, one of the most-studied mariner elements (with Himar1), has been shown to be active in insects, but inactive in mammalian genomes. To circumvent this problem, one strategy consists of both enhancing the activity of the Mos1 transposase (MOS1), and making it insensitive to activity-altering post-translational modifications. Here, we report rational mutagenesis studies performed to obtain hyperactive and non-phosphorylable MOS1 variants. Transposition assays in bacteria have made it possible to isolate numerous hyperactive MOS1 variants. The best mutant combinations, named FETY and FET, are 60- and 800-fold more active than the wild-type MOS1 version, respectively. However, there are serious difficulties in using them, notably because they display severe cytotoxicity. On the other hand, three positions lying within the HTH motif, T88, S99, and S104 were found to be sensitive to phosphorylation. Our efforts to obtain active non-phosphorylable mutants at S99 and S104 positions were unsuccessful, as these residues, like the co-linear amino acids in their close vicinity, are critical for MOS1 activity. Even if host factors are not essential for transposition, our data demonstrate that the host machinery is essential in regulating MOS1 activity

cAMP protein kinase phosphorylates the Mos1 transposase and regulates its activity: evidences from mass spectrometry and biochemical analyses

International audienceGenomic plasticity mediated by transposable elements can have a dramatic impact on genome integrity. To minimize its genotoxic effects, it is tightly regulated either by intrinsic mechanisms (linked to the element itself) or by host-mediated mechanisms. Using mass spectrometry, we show here for the first time that MOS1, the transposase driving the mobility of the mariner Mos1 element, is phosphorylated. We also show that the transposition activity of MOS1 is downregulated by protein kinase AMP cyclic-dependent phosphorylation at S170, which renders the transposase unable to promote Mos1 transposition. One step in the transposition cycle, the assembly of the paired-end complex, is specifically inhibited. At the cellular level, we provide evidence that phosphorylation at S170 prevents the active transport of the transposase into the nucleus. Our data suggest that protein kinase AMP cyclic-dependent phosphorylation may play a double role in the early stages of genome invasion by mariner elements

The epigenetic regulation of HsMar1, a human DNA transposon

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First identification and characterization of inhibitors of the eukaryotic Mos1 transposase.

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