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.
Vanhooff
Virginie
V
Unité de Génétique, Institut des Sciences de la Vie, UCLouvain, 5/6 Place Croix du Sud, B-1348 Louvain-la-Neuve, Belgium.
Normand
Christophe
C
Galloy
Christine
C
Segall
Anca M
AM
Hallet
Bernard
B
D020816:Amino Acid Motifs; D055162:Biocatalysis; D053837:DNA Cleavage; D004254:DNA Nucleotidyltransferases / Q000737:chemistry* / Q000378:metabolism; D008958:Models, Molecular; D045522:Recombinases / Q000737:chemistry* / Q000378:metabolism; D011995:Recombination, Genetic; D020449:Repetitive Sequences, Amino Acid
D045522:Recombinases; D004254:DNA Nucleotidyltransferases; C069598:tnpI recombinase