Multiple roles for divalent metal ions in DNA transposition: distinct stages of Tn10 transposition have different Mg2+ requirements

Abstract

Tn10 transposition takes place by a non-replicative mechanism in which the transposon is excised from donor DNA and integrated into a target site. Mg2+ is an essential cofactor in this reaction. We have examined the Mg2+ requirements at various steps in Tn10 transposition. Results presented here demonstrate that Tn10 excision can occur efficiently at a 16-fold lower Mg2+ concentration than strand transfer and that, at Mg2+ concentrations in the range of 60-fold below the wildt-ype optimum, double strand cleavage events at the two transposon ends are completely uncoupled. These experiments identify specific breakpoints in Tn10 transposition which are sensitive to Mg2+ concentration. Whereas the uncoupling of double strand cleavage events at the two transposon ends most likely reflects the inability of two separate IS10 transposase monomers in the synaptic complex to bind Mg2+, the uncoupling of transposon excision from strand transfer is expected to reflect either a conformational change in the active site or the existence of an Mg2+ binding site which functions specifically in target interactions. We also show that Mn2+ relaxes target specificity in Tn10 transposition and suppresses a class of mutants which are blocked specifically for integration. These observations can be explained by a model in which sequence-specific target site binding is tightly coupled to a conformational change in the synaptic complex which is required for catalysis of strand transfer.