ROLE OF DNA INTERCALATORS IN THE BINDING OF RECA TO DOUBLE-STRANDED DNA

RecA protein can bind to double-stranded DNA even without the cofactor ATP if a DNA intercalator such as ethidium bromide is present (Thresh er R. J., and Griffith, J. D. (1990) Proc. Natl. Acad. Sci. U. S. A. 8 7, 5056-5060). We have studied the structure and association kinetics of the ethidium-promoted DNA-RecA complex in order to understand the r ole of this intercalator in the DNA-RecA association process, informat ion that could provide insight about the binding mechanism of RecA to DNA. Both linear dichroism and fluorescence measurements show that eth idium remains intercalated between the DNA bases in the RecA-DNA compl ex in the absence of ATP. Even in the presence of the ATP analog, aden osine 5'-O-(3-thiotriphosphate) (ATPgammaS), ethidium bromide shows so me stimulating effect on the binding of RecA to DNA. The results indic ate that the destacking of DNA bases is an important limiting step in the association of RecA to DNA (DNA is stretched in the ATPgammaS-RecA -DNA complex). In the presence of ATPgammaS, however, ethidium was ext ruded from DNA upon the binding of RecA. This result suggests that the binding mechanism of RecA to DNA may involve intercalation of one or more amino acid residues of RecA between the DNA bases. Such an interc alation would also be consistent with the stretching of DNA and the ob servation that the DNA bases remain in a (virtually stacked) perpendic ular geometry (Takahashi, M., Kubista, M., and Norden, B. (1991) Bioch emie (Paris) 73, 219-226; Norden, B., Elvingson, C., Kubista, M., Sjob erg, B., Ryberg, H., Ryberg, M., Mortensen, K., and Takahashi, M. (199 2b) J. Mol. Biol. 226, 1175-1191).