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Absence of relaxase-associated ssDNA binding relieves sequence-specific inhibition of oriT unwinding. Duplex unwinding catalysed by increasing concentrations of TraI (•) and TraI M2L/E153D/Q193R/R201Q (○) on heteroduplex oriT substrates that present nic and the IR for TraI binding as ssDNA (A), or constrained in dsDNA (B). The indicated per cent of substrate unwound represents the mean of three independent assays. Standard deviations are shown.
