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 DN A binding domains in Escherichia coil RecA protein, we have constructe d a protein in which a tryptophan fluorescence reporter is inserted in the place of phenylalanine residue 203 in loop L2, a putative DNA bin ding site, and measured its fluorescence, The modified protein is acti ve both in vivo and in vitro, The binding of nucleotide cofactor (ATP or its analog adenosine 5'-O-3-thiotriphosphate) does not modify the f luorescence, 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 bin ding of a second single-stranded DNA. The results indicate that the re sidue 203 is in fact close to the first and second DNA binding sites. However, the quenching is not total and depends only slightly can the nature of DNA bases, thus suggesting an indirect interaction with DNA bases.