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 (Thresher R. J., and Griffith, J. D. (1990) Proc. Natl. Acad. Sci. U.S.A. 87, 5056-5060). We have studied the structure and association kinetics of the ethidium-promoted DNA-RecA complex in order to understand the role of this intercalator in the DNA-RecA association process, information that could provide insight about the binding mechanism of RecA to DNA. Both linear dichroism and fluorescence measurements show that ethidium remains intercalated between the DNA bases in the RecA-DNA complex in the absence of ATP. Even in the presence of the ATP analog, adenosine 5'-O-(3-thiotriphosphate) (ATP gamma S), ethidium bromide shows some stimulating effect on the binding of RecA to DNA. The results indicate that the destacking of DNA bases is an important limiting step in the association of RecA to DNA (DNA is stretched in the ATP gamma S-RecA-DNA complex). In the presence of ATP gamma S, however, ethidium was extruded 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 intercalation would also be consistent with the stretching of DNA and the observation that the DNA bases remain in a (virtually stacked) perpendicular geometry (Takahashi, M., Kubista, M., and Nordén, B. (1991) Biochemie (Paris) 73, 219-226; Nordén, B., Elvingson, C., Kubista, M., Sjöberg, B., Ryberg, H., Ryberg, M., Mortensen, K., and Takahashi, M. (1992b) J. Mol. Biol. 226, 1175-1191).
Kim
S K
SK
Department of Physical Chemistry, Chalmers University of Technology, Gothenburg, Sweden.
Nordén
B
B
Takahashi
M
M
D000255:Adenosine Triphosphate / Q000031:analogs & derivatives / Q000737:chemistry; D004247:DNA / Q000187:drug effects / Q000378:metabolism*; D004268:DNA-Binding Proteins / Q000378:metabolism*; D004996:Ethidium / Q000494:pharmacology; D007364:Intercalating Agents / Q000494:pharmacology*; D007700:Kinetics; D011938:Rec A Recombinases / Q000378:metabolism*; D013050:Spectrometry, Fluorescence
D004268:DNA-Binding Proteins; D007364:Intercalating Agents; C022571:adenosine 5'-O-(3-thiotriphosphate); D000255:Adenosine Triphosphate; D004247:DNA; D011938:Rec A Recombinases; D004996:Ethidium