Kinetic analysis of pairing and strand exchange catalyzed by RecA. Detection by fluorescence energy transfer

Abstract

RecA is a 38-kDa protein from Escherichia coli that polymerizes on single-stranded DNA, forming a nucleoprotein filament that pairs with homologous duplex DNA and carries out strand exchange in vitro. In this study, we measured RecA-catalyzed pairing and strand exchange in solution by energy transfer between fluorescent dyes on the ends of deoxyribo-oligonucleotides. By varying the position of the dyes in separate assays, we were able to detect the pairing of single-stranded RecA filament with duplex DNA as an increase in energy transfer, and strand displacement as a decrease in energy transfer. With these assays, the kinetics of pairing and strand displacement were studied by stopped-flow spectrofluorometry. The data revealed a rapid, second order, reversible pairing step that was followed by a slower, reversible, first order strand exchange step. These data indicate that an initial unstable intermediate exists which can readily return to reactants, and that a further, rate-limiting step (or steps) is required to effect or complete strand exchange.