Kinetic Mechanism for the Formation of the Presynaptic Complex of the Bacterial Recombinase RecA

Abstract RecA protein from Escherichia colicatalyzes DNA strand exchange during homologous recombination in a reaction that requires nucleoside triphosphate cofactor. In the first step of this reaction RecA protein polymerizes on single-stranded DNA to form a filament with a stoichiometry of three nucleotides/RecA monomer called the presynaptic complex. We have used fluorescence anisotropy of a fluorescein-labeled oligonucleotide to investigate presynaptic complex formation. RecA-ATPγS bound to oligonucleotide by a two-step process. Kinetic studies revealed an intermediate in the polymerization reaction that had greater mobility than the final product filament. The intermediate was transformed into the final product by a process that was independent of filament concentration and temperature, k = 0.3 ± 0.1 min−1. This process had the same rate as that reported for a step in the isomerization of presynaptic complex by ATPγS (Paulus, B. F., and Bryant, F. R. (1997) Biochemistry 36, 7832–7838). Judging from anisotropy measurements, the intermediate had hydrodynamic properties similar to a mixed filament containing RecA monomers with and without ATPγS. These results show that the presynaptic complex can assume conformations with different segmental mobilities that could play a role in homologous recombination.