The central aromatic residue in loop L2 of RecA interacts with DNA. Quenching of the fluorescence of a tryptophan reporter inserted in L2 upon binding to DNA.
To determine the role of the central aromatic residue in one of the DNA binding domains in Escherichia coli RecA protein, we have constructed a protein in which a tryptophan fluorescence reporter is inserted in the place of phenylalanine residue 203 in loop L2, a putative DNA binding site, and measured its fluorescence. The modified protein is active both in vivo and in vitro. The binding of nucleotide cofactor (ATP or its analog adenosine 5'-O-3-thiotriphosphate) does not modify the fluorescence. By contrast, the binding of DNA, both in the absence and presence of cofactor, strongly decreases the fluorescence in intensity (40-65%) and shifts the emission peak from 344 to 337 nm. The change occurs both with single- and double-stranded DNA and also upon the binding of a second single-stranded DNA. The results indicate that the residue 203 is in fact close to the first and second DNA binding sites. However, the quenching is not total and depends only slightly on the nature of DNA bases, thus suggesting an indirect interaction with DNA bases.
Maraboeuf
F
F
Groupe d'Etude Mutagénèse et Cancérogénèse URA 1342 CNRS, Orsay, France.
Voloshin
O
O
Camerini-Otero
R D
RD
Takahashi
M
M
D001665:Binding Sites; D004269:DNA, Bacterial / Q000378:metabolism*; D004268:DNA-Binding Proteins / Q000737:chemistry / Q000378:metabolism*; D005454:Fluorescence Polarization; D011938:Rec A Recombinases / Q000737:chemistry / Q000378:metabolism*; D014364:Tryptophan / Q000737:chemistry*
D004269:DNA, Bacterial; D004268:DNA-Binding Proteins; D014364:Tryptophan; D011938:Rec A Recombinases