Kinetic mechanism for the formation of the presynaptic complex of the bacterial recombinase RecA.
RecA protein from Escherichia coli catalyzes 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-ATPgammaS 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 ATPgammaS (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 ATPgammaS. These results show that the presynaptic complex can assume conformations with different segmental mobilities that could play a role in homologous recombination.
Defais
Martine
M
Institut de Pharmacologie et de Biologie Structurale UMR 5089, CNRS 205, route de Narbonne, 31077 Toulouse Cedex, France.
Phez
Emilie
E
Johnson
Neil P
NP
D000255:Adenosine Triphosphate / Q000031:analogs & derivatives* / Q000378:metabolism; D001483:Base Sequence; D004926:Escherichia coli / Q000201:enzymology*; D007700:Kinetics; D008969:Molecular Sequence Data; D009841:Oligonucleotides; D017729:Presynaptic Terminals / Q000378:metabolism*; D011938:Rec A Recombinases / Q000096:biosynthesis* / Q000378:metabolism
D009841:Oligonucleotides; C022571:adenosine 5'-O-(3-thiotriphosphate); D000255:Adenosine Triphosphate; D011938:Rec A Recombinases