Structural Adaptation of DNA to Small Ligands and Recombination Proteins
In order to contribute to the understanding of mechanisms of recognition in nucleic acid systems, the interaction of DNA with a variety of ligands, representing different cases of adaptation of the double-helix structure, was studied using flow linear dichroism (LD), circular dichroism (CD), fluorescence and absorption spectroscopic techniques. A chiral hydrophobic metal complex, [Ru(dpphen)3]2+ was examined, since this had been reported to be the most efficient molecule to discriminate DNA handedness. However, opposite enantiomers of the complex were found to have practically identical binding affinities and binding modes for B-DNA in a random non-intercalative binding.
Various other small ligands, such as intercalators (9-aminoacridine (9AA), bis-9AA) and groove binders (DAPI, Hoechst 33258), were also examined with respect to their binding mode and ability to stabilise double and triple helical DNA. Intercalators and groove binders were found to interact with triplex DNA in the same binding modes as those observed with duplex DNA. However, while intercalators stabilise both triplex and duplex DNA, the minor groove binders studied seem to destabilise triplex but stabilise duplex DNA.
Structural adaptation is believed to be important for the recognition between homologous DNAs and DNA strand exchange reactions mediated by recombination enzymes. Both eukaryotic and prokaryotic recombination proteins (RecA, XRad51, HsRad51) were studied in the presence of various nucleotide cofactors and double or single stranded DNAs with respect to structural changes and stabilities. A secondly bound, complementary ssDNA entering into the RecA fiber is found to have its bases slightly more tilted than those of the first bound template ssDNA according to LD and smallangle neutron scattering (SANS) data. Active cofactors were confirmed to elongate the filament of RecA and XRad51 more effectively than inactive cofactors and DNA adapts its structure by elongating its length by approximately 50% compared to B-DNA according to the SANS results. Fluorescence measurements indicate that active cofactors generally promote more stable protein-ssDNA complexes than inactive cofactors, with the exception of the inactive cofactor ADP which stabilises the HsRad51-ssDNA complex as much as the active cofactor ATP. This suggests that the roles of nucleotide cofactors in the strand exchange reaction may vary among the RecA, XRad51 and HsRad51systems.