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J. Biol. Chem., Vol. 276, Issue 13, 9613-9619, March 30, 2001

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Equilibrium Binding of Single-stranded DNA to the Secondary DNA Binding Site of the Bacterial Recombinase RecA^*

Anne-Sophie Gourves, Martine Defais, and Neil P. Johnson

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

The bacterial recombinase RecA forms a nucleoprotein filament in vitro with single-stranded DNA (ssDNA) at its primary DNA binding site, site I. This filament has a second site, site II, which binds ssDNA and double-stranded DNA. We have investigated the binding of ssDNA to the RecA protein in the presence of adenosine 5'-O-(thiotriphosphate) cofactor using fluorescence anisotropy. The RecA protein carried out DNA strand exchange with a 5'-fluorescein-labeled 32-mer oligonucleotide. The anisotropy signal was shown to measure oligonucleotide binding to RecA, and the relationship between signal and binding density was determined. Binding of ssDNA to site I of RecA was stable at high NaCl concentrations. Binding to site II could be described by a simple two-state equilibrium, K = 4.5 ± 1.5 × 10⁵ M¹ (37 °C, 150 mM NaCl, pH 7.4). The reaction was enthalpy-driven and entropy-opposed. It depended on salt concentration and was sensitive to the type of monovalent anion, suggesting that anion-dependent protein conformations contribute to ssDNA binding at site II.

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				M. Defais, E. Phez, and N. P. Johnson Kinetic Mechanism for the Formation of the Presynaptic Complex of the Bacterial Recombinase RecA J. Biol. Chem., January 31, 2003; 278(6): 3545 - 3551. [Abstract] [Full Text] [PDF]