IS911 transpososome assembly as analysed by tethered particle motion.
Initiation of transposition requires formation of a synaptic complex between both transposon ends and the transposase (Tpase), the enzyme which catalyses DNA cleavage and strand transfer and which ensures transposon mobility. We have used a single-molecule approach, tethered particle motion (TPM), to observe binding of a Tpase derivative, OrfAB[149], amputated for its C-terminal catalytic domain, to DNA molecules carrying one or two IS911 ends. Binding of OrfAB[149] to a single IS911 end provoked a small shortening of the DNA. This is consistent with a DNA bend introduced by protein binding to a single end. This was confirmed using a classic gel retardation assay with circularly permuted DNA substrates. When two ends were present on the tethered DNA in their natural, inverted, configuration, Tpase not only provoked the short reduction in length but also generated species with greatly reduce effective length consistent with DNA looping between the ends. Once formed, this 'looped' species was very stable. Kinetic analysis in real-time suggested that passage from the bound unlooped to the looped state could involve another species of intermediate length in which both transposon ends are bound. DNA carrying directly repeated ends also gave rise to the looped species but the level of the intermediate species was significantly enhanced. Its accumulation could reflect a less favourable synapse formation from this configuration than for the inverted ends. This is compatible with a model in which Tpase binds separately to and bends each end (the intermediate species) and protein-protein interactions then lead to synapsis (the looped species).
Pouget
N
N
Laboratoire de Microbiologie et Génétique Moléculaire, UMR CNRS 5100, 118 route de Narbonne, 31062 Toulouse cedex, France.
Turlan
C
C
Destainville
N
N
Salomé
L
L
Chandler
M
M
D001665:Binding Sites; D004251:DNA Transposable Elements; D004269:DNA, Bacterial / Q000737:chemistry / Q000378:metabolism; D007700:Kinetics; D008856:Microscopy, Fluorescence; D008857:Microscopy, Interference; D018715:Microscopy, Video; D008863:Microspheres; D009038:Motion; D009690:Nucleic Acid Conformation; D009698:Nucleoproteins / Q000737:chemistry* / Q000378:metabolism; D012091:Repetitive Sequences, Nucleic Acid; D019895:Transposases / Q000378:metabolism*
D004251:DNA Transposable Elements; D004269:DNA, Bacterial; D009698:Nucleoproteins; D019895:Transposases