EFFECTS OF MINOR AND MAJOR GROOVE-BINDING DRUGS AND INTERCALATORS ON THE DNA ASSOCIATION OF MINOR GROOVE-BINDING PROTEINS RECA AND DEOXYRIBONUCLEASE-I DETECTED BY FLOW LINEAR DICHROISM

Linear and circular dichroic spectroscopies have been employed to inve stigate the effects of small DNA ligands on the interactions of two pr oteins which bind to the minor groove of DNA, viz. RecA protein from E scherichia coli and deoxyribonuclease I (bovine pancreas). Ligands rep resenting three specific non-covalent binding modes were investigated: 4',6-diamidino-2-phenylindole and distamycin A (minor groove binders) , methyl green (major groove binder), and methylene blue, ethidium bro mide and ethidium dimer (intercalators). Linear dichroism was demonstr ated to be an excellent detector, in real time, of DNA double-strand c leavage by deoxyribonuclease I. Ligands bound in all three modes inter fered with the deoxyribonuclease I digestion of dsDNA, although the le vel of interference varied in a manner which could be related to the l igand binding site, the ligand charge appearing to be less important. In particular, the retardation of deoxyribonuclease I cleavage by the major groove binder methyl green demonstrates that accessibility to th e minor groove can be affected by occupancy of the opposite groove. Bi nding of all three types of ligand also had marked effects on the inte raction of RecA with dsDNA in the presence of non-hydrolyzable cofacto r adenosine 5'-O-3-thiotriphosphate, decreasing the association rate t o varying extents but with the strongest effects from ligands having s ome minor groove occupancy. Finally, each ligand was displaced from it s DNA binding site upon completion of RecA association, again demonstr ating that modification of either groove can affect the properties and behaviour of the other. The conclusions are discussed against the bac kground of previous work on the use of small DNA ligands to probe DNA- protein interactions.