Equilibrium binding of single-stranded DNA to the secondary DNA binding site of the bacterial recombinase RecA.
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 x 10(5) m(-1) (37 degrees 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.
Gourves
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AS
Institut de Pharmacologie et de Biologie Structurale, UMR 5089, CNRS, 205 Route de Narbonne, 31077 Toulouse Cedex, France.
Defais
M
M
Johnson
N P
NP
D000255:Adenosine Triphosphate / Q000031:analogs & derivatives* / Q000378:metabolism*; D016880:Anisotropy; D001665:Binding Sites; D004247:DNA / Q000378:metabolism*; D004305:Dose-Response Relationship, Drug; D004926:Escherichia coli / Q000378:metabolism; D007700:Kinetics; D015636:Magnesium Chloride / Q000494:pharmacology; D011485:Protein Binding; D011938:Rec A Recombinases / Q000378:metabolism*; D012965:Sodium Chloride / Q000494:pharmacology; D013050:Spectrometry, Fluorescence; D013696:Temperature; D013816:Thermodynamics; D013997:Time Factors; D014466:Ultraviolet Rays
D015636:Magnesium Chloride; C022571:adenosine 5'-O-(3-thiotriphosphate); D012965:Sodium Chloride; D000255:Adenosine Triphosphate; D004247:DNA; D011938:Rec A Recombinases