BASE ORIENTATION OF 2ND DNA IN RECA-CENTER-DOT-DNA FILAMENTS - ANALYSIS BY COMBINATION OF LINEAR DICHROISM AND SMALL-ANGLE NEUTRON-SCATTERING IN FLOW-ORIENTED SOLUTION

To gain insight into the mechanism of pairing two complementary DNA st rands by the RecA protein, we have determined the nucleobase orientati on of the first and the second bound DNA strands in the RecA DNA filam ent by combined measurements of linear dichroism and small angle neutr on scattering on flow-oriented samples. An etheno-modified DNA, poly(d epsilon A) was adapted as the first DNA and an oligo(dT) as the secon d DNA, making it possible to distinguish between the linear dichroism signals of the two DNA strands, The results indicate that binding of t he second DNA does not alter the nucleobase orientation of the first b ound strand and that the bases of the second DNA are almost coplanar t o the bases of the first strand although somewhat more tilted (60 degr ees relative to the fiber axis compared with 70 degrees for the first DNA strand). Similar results were obtained for the RecA DNA complex fo rmed with unmodified poly(dA) and oligo(dT). An almost coplanar orient ation of nucleobases of two DNA strands in a RecA-DNA filament would f acilitate scanning for, and recognition of, complementary base sequenc es. The slight deviation from co-planarity could increase the free ene rgy of the duplex to facilitate dissociation in case of mismatching ba se sequences.