Structure of RecA-DNA complexes studies by combination of linear dichroism and small-angle neutron scattering measurements on flow-oriented samples

By combining anisotropy of small-angle neutron scattering (SANS) and optical anisotropy (linear dichroism, 1.d.) on flow-oriented RecA-DNA complexes, the average DNA-base orientation has been determined in RecA complexes with double-stranded (ds) as well as single-stranded (ss) DNA. From the anisotrophy of the two-dimensional SANS intensity representation, the secnd moment orientation function S is obtained. Knowledge of S is crucial for the interpretation of 1.d. spectra in terms of orientation of the DNA bases and the aromatic amino acid residues. The DNA-base planes are essentially perpendicular to the fibre axis of the complex between RecA and dsDNA in the presence of cofactor ATPγS. A somewhat tilted base geometry is found for the RecA-ATPγS complexes with single-stranded poly(dT) and poly(dεA). This behaviour contrasts the RecA-ssDNA complex formed without cofactor which displasy a poor orientation of the bases. Well-ordered bases in the ssDNA-RecA complex is possibly reflecting the role of RecA in preparing a nucleotide strand for base-printing in the search-for-homology process. While the central SANS intensity is essentially independent of the pitch of the helical complex, a secondary intensity maximu, which becomes focused upo flow orientation, is found to be a sensitive measure of the pitch. The pitch values for the complexes compare wellwith cryo-electron microscopy results but are slightly larger than those seen for uranyl-stained samples.