Direct observation of complexes formed between recA protein and a fluorescent single-stranded deoxyribonucleic acid derivative.
The reaction of chloroacetaldehyde with single-stranded DNA (ssDNA) yields epsilon DNA, a highly fluorescent substance. The binding of recA protein to epsilon DNA nearly doubles its fluorescence yield. The enhanced fluorescence signals the formation of a recA-epsilon DNA complex. This complex exhibits an ATPase activity as great as that of the corresponding recA-ssDNA complex. Addition of a saturating concentration of adenosine 5'-O-(3-thiotriphosphate) (ATP gamma S) to a solution of the recA-epsilon DNA complex yields a further rise in fluorescence. Saturation with ATP produces the same rise. The nucleotide triphosphates have converted the recA-epsilon DNA complex into the respective ATP gamma S-recA-epsilon DNA and ATP-recA-epsilon DNA complexes. The fluorescence changes that accompany the formation of the three complexes have enabled us to (1) establish by titration that recA protein binds to 6.0 +/- 0.3 nucleotides of epsilon DNA, (2) show that the binding of ATP to the recA-epsilon DNA complex is highly cooperative under various conditions, with a Hill coefficient of 2.4-4.9 and Kapp = 25 +/- 2 micro M, (3) show that the binding of ATP gamma S is also highly cooperative, with a Hill coefficient of 3.3-4.2 and Kapp congruent to 0.5 micro M, and (4) perform initial measurements on the rate at which recA protein transfers between polynucleotides. The experiments provide the first direct observation of an ATP-recA-ssDNA-like complex, and they illuminate some of the properties of such complexes.
D000255:Adenosine Triphosphate / Q000378:metabolism; D001426:Bacterial Proteins / Q000378:metabolism*; D004277:DNA, Single-Stranded / Q000378:metabolism*; D004926:Escherichia coli / Q000235:genetics*; D007700:Kinetics; D011485:Protein Binding; D011938:Rec A Recombinases; D013050:Spectrometry, Fluorescence; D013816:Thermodynamics
D001426:Bacterial Proteins; D004277:DNA, Single-Stranded; D000255:Adenosine Triphosphate; D011938:Rec A Recombinases