Kinetic studies of recA protein binding to a fluorescent single-stranded polynucleotide.
Fluorescence spectroscopy was used to investigate the binding of Escherichia coli recA protein to a single-stranded polynucleotide. Poly(deoxy-1,N6-ethenoadenylic acid) was prepared by reaction of chloroacetaldehyde with poly(deoxyadenylic acid). The fluorescence of poly(deoxy-1,N6-ethenoadenylic acid) was enhanced upon recA protein binding. The kinetics of the binding process were studied as a function of several parameters: ionic concentration (KCl and MgCl2), pH, nature of the nucleoside triphosphate [adenosine 5'-triphosphate or adenosine 5'-O-(gamma-thiotriphosphate)], protein and polynucleotide concentrations, polynucleotide chain length, and order of sequential additions. The observed kinetic curves exhibited a lag phase followed by a slow binding process characteristic of a nucleation-elongation mechanism with an additional slow step governing the rate of the association process. The lag phase reflecting the nucleation step was not observed when the protein was first bound to the polynucleotide before addition of adenosine 5'-triphosphate. Adenosine 5'-triphosphate induced a dissociation of the recA protein, which was immediately followed by binding of the recA-adenosine 5'-triphosphate-Mg2+ ternary complex. The origin of this "mnemonic effect" and of the different kinetic steps is discussed with respect to protein conformational changes and aggregation phenomena.
Chabbert
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M
Cazenave
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Hélène
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C
D000255:Adenosine Triphosphate / Q000031:analogs & derivatives; D004926:Escherichia coli / Q000378:metabolism; D005456:Fluorescent Dyes; D007700:Kinetics; D008274:Magnesium; D009994:Osmolar Concentration; D011061:Poly A; D011938:Rec A Recombinases / Q000378:metabolism*; D013050:Spectrometry, Fluorescence / Q000379:methods
D005456:Fluorescent Dyes; C040459:poly(d-epsilon A); D011061:Poly A; C022571:adenosine 5'-O-(3-thiotriphosphate); D000255:Adenosine Triphosphate; D011938:Rec A Recombinases; D008274:Magnesium