Binding of DNA quenches tyrosine fluorescence of RecA without energy transfer to DNA bases.
The binding of single- as well as double-stranded DNA to RecA, in the presence of the cofactor analog ATP gamma S (adenosine 5'-O-(3-thiotriphosphate)), leads to about 20% quenching of the tyrosine fluorescence of the protein but to no essential change of the tryptophan fluorescence. The excitation spectrum of the fluorescent DNA analog poly(d epsilon A), complexed with RecA, shows no sign of energy transfer from the tyrosine residues of RecA to the etheno-modified adenine bases of the polynucleotide. From this observation we reject stacking interaction between tyrosine residues and DNA bases. The RecA filament may bind up to three molecules of single-stranded DNA; however, the observed fluorescence change occurs only upon the binding of the first DNA strand, indicating that the binding mode of this first strand is different from those of the others. The fluorescence change is interpreted in terms of a conformational change of the RecA protein promoted by cooperative binding to DNA. A larger quenching (40%) upon the binding of single-stranded DNA is observed in the absence of cofactor. At high salt condition, which induces ATPase activity in RecA just as DNA binding does, the tyrosine fluorescence is more pronounced than at low salt conditions, indicating that the effect induced by high salt is different from the conformational change induced by DNA binding.
Eriksson
S
S
Department of Physical Chemistry, Chalmers University of Technology, Göteborg, Sweden.
Nordén
B
B
Takahashi
M
M
D000255:Adenosine Triphosphate / Q000031:analogs & derivatives / Q000494:pharmacology; D004247:DNA / Q000378:metabolism*; D004277:DNA, Single-Stranded / Q000378:metabolism; D004735:Energy Transfer; D004926:Escherichia coli; D005453:Fluorescence; D011938:Rec A Recombinases / Q000378:metabolism*; D012965:Sodium Chloride / Q000494:pharmacology; D013050:Spectrometry, Fluorescence; D014364:Tryptophan / Q000737:chemistry; D014443:Tyrosine / Q000737:chemistry*
D004277:DNA, Single-Stranded; C022571:adenosine 5'-O-(3-thiotriphosphate); D014443:Tyrosine; D012965:Sodium Chloride; D014364:Tryptophan; D000255:Adenosine Triphosphate; D004247:DNA; D011938:Rec A Recombinases