Selective covalent binding of an ethidium analog to mitochondrial DNA with production of petite mutants in yeast by photoaffinity labeling

Abstract

A photosensitive azide derivative of ethidium bromide, 3(8)-amino-8(3)-azido-5-ethyl-6-phenyl phenanthridinium chloride, was prepared which binds covalently to DNA via a light-generated nitrene intermediate. When exposed to light in aqueous solvent the azide loses free nitrogen to yield a reactive nitrene, which reacts with water to form the hydroxylamine derivative. When the drug is photolyzed while tightly bound to DNA, rather than free in solution, a covalent complex is generated in situ due to the attack of the nitrene to form a covalent bond with the nucleic acid. A selectivity for cytoplasmic DNA reported previously for ethidium bromide was also shown for the azido analog by enhanced production of petite mutants in Saccharomyces following the photolysis of the azide in vivo. Isolated mitochondrial DNA following binding of the ethidium analog in vivo also appeared to retain a greater number of drug molecules per nucleotide than nuclear DNA. In vitro binding experiments did not suggest the same extent of preferential binding. This technique of photoaffinity labeling provides a means of studying the precise model of binding nucleic acid active drugs in relation to their biological effects.