Base Orientation of Second DNA in RecA·DNA Filaments: ANALYSIS BY COMBINATION OF LINEAR DICHROISM AND SMALL ANGLE NEUTRON SCATTERING IN FLOW-ORIENTED SOLUTION

Abstract

To gain insight into the mechanism of pairing two complementary DNA strands by the RecA protein, we have determined the nucleobase orientation of the first and the second bound DNA strands in the RecA.DNA filament by combined measurements of linear dichroism and small angle neutron scattering on flow-oriented samples. An etheno-modified DNA, poly(depsilonA) was adapted as the first DNA and an oligo(dT) as the second DNA, making it possible to distinguish between the linear dichroism signals of the two DNA strands. The results indicate that binding of the second DNA does not alter the nucleobase orientation of the first bound strand and that the bases of the second DNA are almost coplanar to the bases of the first strand although somewhat more tilted (60 degrees relative to the fiber axis compared with 70 degrees for the first DNA strand). Similar results were obtained for the RecA.DNA complex formed with unmodified poly(dA) and oligo(dT). An almost coplanar orientation of nucleobases of two DNA strands in a RecA-DNA filament would facilitate scanning for, and recognition of, complementary base sequences. The slight deviation from co-planarity could increase the free energy of the duplex to facilitate dissociation in case of mismatching base sequences.