Functional coupling between the two active sites during Tn10 transposition buffers the mutation of sequences critical for DNA hairpin processing

Abstract

DNA processing reactions often involve multiple components acting in concert to achieve the desired outcome. However, it is usually difficult to know how the components communicate and cooperate to orchestrate an ordered series of events. We address this question in the context of the Tn 10 transposition reaction, in which the DNA cleavage and joining events occur within a higher-order complex containing a transposase dimer, two transposon ends and the DNA-bending host-factor IHF (Integration Host Factor). Previously it was shown that the complex is asymmetric. The a side consists of an IHF protomer initially immobilized by a DNA-loop, but subsequently used to promote conformational changes required for the cleavage steps. The beta side of the complex was considered to fulfil a more passive role. Here we show that the a side of the complex promotes coupled conformational changes at both transposon ends, while the a and beta sides communicate and cooperate to dominate different phases of the transposition reaction. Together, these effects provide for a robust response to critical changes in the transposon end. These findings also explain the intriguing genetic phenotypes of a series of previously reported Tn10 mutants and have consequences for the evolution of new elements.