Control of directionality in the DNA strand-exchange reaction catalysed by the tyrosine recombinase TnpI

In DNA site-specific recombination catalysed by tyrosine recombinases, two pairs of DNA strands are sequentially exchanged between separate duplexes and the mechanisms that confer directionality to this theoretically reversible reaction remain unclear. The tyrosine recombinase TnpI acts at the internal resolution site (IRS) of the transposon Tn4430 to resolve intermolecular transposition products. Recombination is catalysed at the IRS core sites (IR1–IR2) and is regulated by adjacent TnpI-binding motifs (DR1 and DR2). These are dispensable accessory sequences that confer resolution selectivity to the reaction by stimulating synapsis between directly repeated IRSs. Here, we show that formation of the DR1–DR2-containing synapse imposes a specific order of activation of the TnpI catalytic subunits in the complex so that the IR1-bound subunits catalyse the first strand exchange and the IR2-bound subunits the second strand exchange. This ordered pathway was demonstrated for a complete recombination reaction using a TnpI catalytic mutant (TnpI-H234L) partially defective in DNA rejoining. The presence of the DR1- and DR2-bound TnpI subunits was also found to stabilize transient recombination intermediates, further displacing the reaction equilibrium towards product formation. Implication of TnpI/IRS accessory elements in the initial architecture of the synapse and subsequent conformational changes taking place during strand exchange is discussed

Organisation moléculaire et assemblage du complexe de recombinaison TnpI/IRS du transposon Tn4430

The transposon Tn4430 from Bacillus thuringiensis encodes a DNA site-specific recombination system (TnpI/IRS system) that functions to resolve cointegrate intermediates arising from replicative transposition. The IRS site-specific recombination site (116 bp) contains four TnpI recombinase binding motifs. The first two motifs (IR1 and IR2) constitute the recombination core site at which TnpI acts to cleave and rejoin the DNA strands. The DR1 and DR2 motifs repeated in direct orientation downstream the core region are accessory sequences. The function of these two accessory TnpI binding sites is to prevent intermolecular recombination by restricting recombination to sites that are present on the same DNA molecule. The deletion products exhibits a specific topology (two-noded catenane), demonstrating that the accessory motifs DR1-DR2 contribute to the formation of a complex with a specific architecture. Without DR1 and DR2, TnpI displays relaxed recombination activity, leading to inter- and intramolecular recombination events in vivo and to topologically variable products in vitro. The aim of the first part of this work was to determine the architecture of the TnpI/IRS synaptic complex. Hybrid recombination sites between the TnpI/IRS system and the Cre/lox system of bacteriophage P1 were constructed. Topological study of the products arising form recombination at these hybrid sites allowed us to propose a model for the synaptic complex in which the DNA is wrapped around the TnpI subunits, trapping 3 negative supercoils form the initial substrate. It was also demonstrated that the recombinase tetramer bound to the core sites orients their C-terminal domain towards the accessory sequences DR1-DR2. Bandshift experiments performed with deleted version of the IRS showed that TnpI cooperatively binds to the IR1-IR2 core site and to the DR1-DR2 accessory motifs with the overall same affinity. This suggests that TnpI binding to the core and accessory sequences may occur independently during complex assembly. Communication between the core and regulatory sequences of the IRS was investigated by changing the spacing between the two regions. This analysis showed that the spacing, but not the phasing, between IR1-IR2 and DR1-DR2 can be altered without affecting the topological selectivity of the reaction. In addition, out-of-phase recombination sites were shown to inhibit recombination. This supports a dynamic pathway for recombination complex assembly, in which the TnpI subunits bound to the accessory motifs interact first to form the synapse, and then position the catalytic subunits bound to the core sites. Formation of the synapse requires a sharp bend in the DNA between the core and accessory regions of the complex.Le transposon Tn4430 de Bacillus thuringiensis encode un système de recombinaison site-spécifique (système TnpI/IRS) dont la fonction est de résoudre les cointégrats générés par son mode de transposition réplicatif. Le site de recombinaison IRS (116 pb) contient quatre motifs de fixation pour la recombinase TnpI. Les deux premiers motifs (IR1 et IR2) constituent le site coeur de recombinaison au niveau duquel la recombinase catalyse les réactions de coupure et d'échange de brins. Les motifs DR1 et DR2, répétés en orientation directe en aval du site coeur, sont quant à eux des éléments accessoires dont la fonction est de contrôler la réaction en favorisant le rapprochement de deux sites en orientation directe sur une même molécule d'ADN. Ce contrôle s'opère par la formation d'un complexe nucléoprotéique (complexe synaptique) de géométrie fixe. La formation d'un tel complexe produit, après échange des brins d'ADN, un caténat à deux croisements. En absence de DR1 et DR2, la TnpI présente une activité relâchée produisant différents types de réarrangements inter- et intramoléculaires. La première partie de ce travail de thèse a consisté à déterminer l'architecture du complexe synaptique TnpI/IRS. Pour cela, des sites de recombinaison hybrides entre le système Cre/lox du bactériophage P1 et le système TnpI/IRS ont été construits. L'étude de la topologie des produits issus d'une réaction entre deux sites hybrides a permis de présenter un modèle de complexe dans lequel trois croisements de super-enroulements sont emprisonnés au niveau des sites accessoires DR1 et DR2. Il a également été démontré que les quatre sous-unités de TnpI fixées sur les coeurs positionnaient leur domaine C-terminal vers les séquences accessoires DR1-DR2. Des expériences de retard sur gel effectuées avec des versions délétées du site IRS ont démontré que la recombinase TnpI se fixait avec une affinité similaire au niveau des séquences coeurs et accessoires, suggérant que la liaison de la protéine au niveau de ces deux régions pourrait se faire de manière indépendante lors de la formation du complexe de recombinaison. La communication entre les séquences coeurs et accessoires du site IRS a été investiguée en changeant la distance entre ces deux régions. Des analyses in vivo et in vitro ont démontré que l'espacement entre IR1-IR2 et DR1-DR2 pouvait être altéré sans affecter la sélectivité topologique de la réaction, à condition que ces deux régions restent en phase l'une par rapport à l'autre. Il a également été montré que les sites de recombinaison possédant un déphasage complet entre les régions coeur et accessoires inhibaient la recombinaison. Cette dernière donnée supporte un mode d'assemblage du complexe synaptique en deux temps, dans lequel les sous-unités de recombinases fixées sur les motifs accessoires interagissent en premier lieu et positionnent, ensuite, les sous-unités catalytiques fixées sur les sites coeurs. D'autre part, des expériences de recombinaison asymétrique entre un site IRS sauvage et un site IRS modifié, montrent que l'assemblage de la synapse implique la formation d'une boucle dans l'ADN entre les régions coeurs et accessoires du complexe.Doctorat en sciences (sciences biologiques) (BIOL 3)--UCL, 200

Self-control in DNA site-specific recombination mediated by the tyrosine recombinase TnpI.

Tn4430 is a distinctive transposon of the Tn3 family that encodes a tyrosine recombinase (TnpI) to resolve replicative transposition intermediates. The internal resolution site of Tn4430 (IRS, 116 bp) contains two inverted repeats (IR1 and IR2) at the crossover core site, and two additional TnpI binding motifs (DR1 and DR2) adjacent to the core. Deletion analysis demonstrated that DR1 and DR2 are not required for recombination in vivo and in vitro. Their function is to provide resolution selectivity to the reaction by stimulating recombination between directly oriented sites on a same DNA molecule. In the absence of DR1 and/or DR2, TnpI-mediated recombination of supercoiled DNA substrates gives a mixture of topologically variable products, while deletion between two wild-type IRSs exclusively produces two-noded catenanes. This demonstrates that TnpI binding to the accessory motifs DR1 and DR2 contributes to the formation of a specific synaptic complex in which catalytically inert recombinase subunits act as architectural elements to control recombination sites pairing and strand exchange. A model for the organization of TnpI/IRS recombination complex is presented

the tyrosine recombinase TnpI

strand-exchange reaction catalysed b

The Tn3-family of replicative transposons

Transposons of the Tn3 family form a widespread and remarkably homogeneous group of bacterial transposable elements in terms of transposition functions and an extremely versatile system for mediating gene reassortment and genomic plasticity owing to their modular organization. They have made major contributions to antimicrobial drug resistance dissemination or to endowing environmental bacteria with novel catabolic capacities. Here, we discuss the dynamic aspects inherent to the diversity and mosaic structure of Tn3-family transposons and their derivatives. We also provide an overview of current knowledge of the replicative transposition mechanism of the family, emphasizing most recent work aimed at understanding this mechanism at the biochemical level. Previous and recent data are put in perspective with those obtained for other transposable elements to build up a tentative model linking the activities of the Tn3-family transposase protein with the cellular process of DNA replication, suggesting new lines for further investigation. Finally, we summarize our current view of the DNA site-specific recombination mechanisms responsible for converting replicative transposition intermediates into final products, comparing paradigm systems using a serine recombinase with more recently characterized systems that use a tyrosine recombinase

The Tn3-family of Replicative Transposons

Transposons of the Tn3 family form a widespread and remarkably homogeneous group of bacterial transposable elements in terms of transposition functions and an extremely versatile system for mediating gene reassortment and genomic plasticity owing to their modular organization. They have made major contributions to antimicrobial drug resistance dissemination or to endowing environmental bacteria with novel catabolic capacities. Here, we discuss the dynamic aspects inherent to the diversity and mosaic structure of Tn3-family transposons and their derivatives. We also provide an overview of current knowledge of the replicative transposition mechanism of the family, emphasizing most recent work aimed at understanding this mechanism at the biochemical level. Previous and recent data are put in perspective with those obtained for other transposable elements to build up a tentative model linking the activities of the Tn3-family transposase protein with the cellular process of DNA replication, suggesting new lines for further investigation. Finally, we summarize our current view of the DNA site-specific recombination mechanisms responsible for converting replicative transposition intermediates into final products, comparing paradigm systems using a serine recombinase with more recently characterized systems that use a tyrosine recombinase