PMID:12456687
| Citation |
Defais, M, Phez, E and Johnson, NP (2003) Kinetic mechanism for the formation of the presynaptic complex of the bacterial recombinase RecA. J. Biol. Chem. 278:3545-51 |
|---|---|
| Abstract |
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. |
| Links |
PubMed Online version:10.1074/jbc.M204341200 |
| Keywords |
Adenosine Triphosphate/analogs & derivatives; Adenosine Triphosphate/metabolism; Base Sequence; Escherichia coli/enzymology; Kinetics; Molecular Sequence Data; Oligonucleotides; Presynaptic Terminals/metabolism; Rec A Recombinases/biosynthesis; Rec A Recombinases/metabolism |
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