Kinetic mechanism for the formation of the presynaptic complex of the bacterial recombinase RecA

被引:7
|
作者
Defais, M [1 ]
Phez, E [1 ]
Johnson, NP [1 ]
机构
[1] CNRS, UMR 5089, Inst Pharmacol & Biol Struct, F-31077 Toulouse, France
关键词
D O I
10.1074/jbc.M204341200
中图分类号
Q5 [生物化学]; Q7 [分子生物学];
学科分类号
071010 ; 081704 ;
摘要
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.
引用
收藏
页码:3545 / 3551
页数:7
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