Structure topologique et moléculaire du complexe de recombinaison site-spécifique 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 contains four DNA binding motifs for the recombinase TnpI. The IR1 and IR2 motifs form the recombination core site at which TnpI acts to cleave and rejoin the DNA strands. The DR1 and DR2 motifs are accessory sequences whose function is to control and stimulate the resolution of cointegrates by restricting recombination to sites that are present on a same DNA molecule. This control takes place by the formation of a synaptic complex with a specific architecture, in which the DNA is intertwined around the recombinases with a specific topology. In this study, complementary biochemical approaches were developed to determine the role of the TnpI/IRS accessory sequences and proteins in the control of the recombination reaction and the molecular organisation of the recombination complex. In the first part of the work, the topological architecture of the TnpI/IRS synaptic complex was investigated using two independent approaches. The results obtained with both approaches converge to a model for the topological architecture of the synaptic complex in which the DNA is wrapped around the TnpI protein, trapping 3 negative supercoils form the initial substrate. In the second part of the thesis, the molecular steps leading to the assembly of the recombination complex and its structure organisation were investigated in further detail Together, the data support a dynamic model for the assembly of TnpI/IRS recombination complex, in which formation of a synapse between the accessory sequences of the partners acts as a checkpoint for controlling the outcome of the recombination reaction. The molecular organisation of the complex was also modelled based on our results and structural data available in the literature. This model was validated by examining the recombination activity of specific IRS variants.