Interaction of tyrosine 65 of RecA protein with the first and second DNA strands.

We investigated the structure of the active RecA-DNA complex by analyzing the environment of tyrosine residue 65, which is on the DNA-binding surface of the protein. We prepared a modified RecA protein in which the tyrosine residue was replaced by tryptophan, a natural fluorescent reporter, and measured the change in its fluorescence upon binding of DNA and cofactor. The fluorescence of the inserted tryptophan 65 (Trp65) was centered at 345 nm, indicating a partly exposed residue. Binding cofactor, adenosine 5'-O-3-thiotriphosphate (ATPgammaS), alone at a low salt concentration did not change the fluorescence of Trp65, confirming that the residue is not close to the nucleotide. In contrast, the binding of single-stranded DNA quenched the fluorescence of Trp65 in both the presence and absence of ATPgammaS. Trp65 fluorescence was also quenched upon binding a second DNA strand. The fluorescence change depended upon the presence and absence of ATPgammaS, reflecting the difference in the DNA binding. These results indicate that residue 65 is close to both the first and second DNA strands. The degree of quenching depended upon the base composition of DNA, suggesting that the residue 65 interacts with the DNA bases. Binding of DNA with ATPgammaS as well as binding of ATPgammaS alone at high salt concentration shifted the fluorescence emission peak from 345 to 330 nm, indicating a change from a polar to a non-polar environment. Therefore, the environment change around residue 65 would also be linked to a change in conformation and thus the activation of the protein.