JBC Ideal method for primary cell transfection

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Originally published In Press as doi:10.1074/jbc.M204341200 on November 26, 2002

J. Biol. Chem., Vol. 278, Issue 6, 3545-3551, February 7, 2003
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Kinetic Mechanism for the Formation of the Presynaptic Complex of the Bacterial Recombinase RecA*

Martine Defais, Emilie Phez, and Neil P. JohnsonDagger

From the Institut de Pharmacologie et de Biologie Structurale UMR 5089, CNRS 205, route de Narbonne, 31077 Toulouse Cedex, France

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-ATPgamma S 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 ATPgamma S (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 ATPgamma S. These results show that the presynaptic complex can assume conformations with different segmental mobilities that could play a role in homologous recombination.

* This study was supported by Centre National de la Recherche Scientifique.The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

Dagger To whom correspondence should be addressed. Tel.: 033-5-61-17-59-60; Fax: 033-5-61-17-59-97; E-mail: Neil.Johnson@ipbs.fr.

Copyright © 2003 by The American Society for Biochemistry and Molecular Biology, Inc.
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