UV-induced cross-linking of tet repressor to DNA containing tet operator sequences and 8-azidoadenines

Abstract

The synthesis of 8-azldo-2′-deoxyadenosine-5′-tri-phosphate is described. The photoreactive dATP analog was characterized by thin layer chromatography, proton resonance spectroscopy, infrared spectroscopy and UV spectroscopy. Its photolysis upon UV irradiation was studied. After incorporation of this dATP analog into DNA containing the tet operator sequence the investigation of the interactions between tet operator DNA and Tet repressor protein by UV photocross-linking becomes possible. Photocross-linking of protein to DNA was demonstrated by the reduced migration of the DNA in SDS polyacrylamlde gel electrophoresis. Addition of the inducer tetracycllne prior to UV irradiation significantly reduces the DNA-protein cross-linking rate. The long wave UV light applied here does not significantly alter the DNA or the protein under the photocross-linking conditions. © 1990 Oxford University Press.