Mechanisms of Metal Ion Action in Tn10 Transposition

Tn10/IS10 transposition involves assembly of a synaptic complex (or transpososome) in which two transposon ends are paired, followed by four distinct chemical steps at each transposon end. The chemical steps are dependent on the presence of a suitable divalent metal cation (Me2+). Transpososome assembly and structure are also affected by Me2+. To gain further insight into the mechanisms of Me2+ action in Tn10/IS10 transposition we have investigated the effects of substituting Mn2+ for Mg2+, the physiologic Me2+, in transposition. We have also investigated the significance of an Me2+-assisted conformational change in transpososome structure. We show that Mn2+ has two previously unrecognized effects on the Tn10 donor cleavage reaction. It accelerates the rates of hairpin formation and hairpin resolution without significantly affecting the rate of the first chemical step, first strand nicking. Mn2+ also relaxes the specificity of first strand nicking. We also show that Me2+-assisted transpososome unfolding coincides with a structural transition in the transposon–donor junction that may be necessary for hairpin formation. Possible mechanisms for these observations are considered.