Abstract
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).
Highlights
From the $Departmentof Physical Chemistry, Chelmers Universityof Technology, s-412 96 Gothenburg, Sweden and the IURA 1342 Centre National de la Recherche Scientifique, Znstitut Curie, Batiment 110, Universite ParisSud,F-91405 Orsay, France
With dsDNA nocomplex is formed in the absenceof stretching of DNA and the observation that theDNA cofactor, complex formation can be promoted by bases remain in aperpendicular ethidium bromide
The formationof complexes of RecA and DNA is observed as a large increase of linear dichroism (LD) intensity at 260 nm, from almost zero LD of the free DNA, which is too flexible to become significantlyaligned by the small shear gradient, to a subwithout ethidium bromide, are very similar but with a somewhat faster initiatiown ith ethidium, whereas the RecA binding to DNA induced by ethidium in the absence of ATP-yS is an order of magnitude faster
Summary
RecA-DNA complex in the absence of ATP. Even in kinetics. With dsDNA nocomplex is formed in the absenceof stretching of DNA and the observation that theDNA cofactor, complex formation can be promoted by bases remain in a (virtuallystacked)perpendicular ethidium bromide. RecA is an essential protein of the DNA repair system in RecA to dsDNA in the absence of ATP/ATPyS. Similar proteins are found in various prokaert-hidium bromide cannot bind to already formed ATP+ yotic organisms (forreviews, see Walker (1984), Smit(h1989), RecA-dsDNA complex (Dombroski etal., 1983). That, in the absence of ATPyS, ethidium bromide remains intercalated between the DNA bases in theRecA-DNA complex and, should notdirectly interact with RecA. 0.05 extrusion of the intercalated ethidium from DNA, despite the fact that the complex formation is found to be more quickly initiated by the presenceof the intercalator
Sign-in/Register to view highlights & summary Sign-in/Register to access full text options 
Talk to us
Join us for a 30 min session where you can share your feedback and ask us any queries you have
Schedule a callDisclaimer: All third-party content on this website/platform is and will remain the property of their respective owners and is provided on "as is" basis without any warranties, express or implied. Use of third-party content does not indicate any affiliation, sponsorship with or endorsement by them. Any references to third-party content is to identify the corresponding services and shall be considered fair use under The CopyrightLaw.
