Transposon-mediated site-specific recombination: a defined in vitro system

Abstract

Transposition of the insertion element gamma delta is thought to involve formation of intermediates in which the element is present at each junction between donor and target replicons. In vivo these cointegrate structures are rapidly converted to the end products of transposition by site-specific recombination at a defined sequence, res, that is present in each directly repeated gamma delta element. Resolvase, an element encoded protein of molecular weight 21,000 is required for cointegrate resolution. I have demonstrated site-specific recombination in vitro using purified resolvase and a cointegrate analog substrate. The required components of the system described here are resolvase, negatively supercoiled substrate DNA, buffer and Mg2+. Neither host-encoded products nor high energy cofactors appear to be required for resolution in vitro. Catenated, resolved molecules are the major products of the reaction. Elimination of Mg2+ from the reaction yields different product molecules. The in vitro system described here provides an opportunity for detailed study of the resolution reaction.