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J Bacteriol. 1971 October; 108(1): 202-212
Copyright © 1971 American Society for Microbiology. All Rights Reserved.

Mutagenic Action, Loss of Transforming Activity, and Inhibition of Deoxyribonucleic Acid Template Activity In Vitro Caused by Chemical Linkage of Carcinogenic Polycyclic Hydrocarbons to Deoxyribonucleic Acid

¹ Marygrove College and Michigan Cancer Foundation, Detroit, Michigan 48201, and Department of Radiological Sciences, The Johns Hopkins University, Baltimore, Maryland 21205

ABSTRACT

In a solvent system of 10^–2M phosphate buffer (pH 6.8)-ethanol (2:1, v/v) and in an iodine-induced reaction, the polycyclic hydrocarbons [³H]3,4-benzpyrene and [³H]3,4-BP/[³H]9, 10-dimethyl-1,2-benzanthracene (DMBA) can be covalently linked to deoxyribonucleic acid (DNA) at room temperature. By stepwise addition of the hydrocarbon and repeating the reaction two to three times after isolating the hydrocarbon DNA adduct, it was possible to introduce as many as one covalently bound hydrocarbon molecule per 100 nucleotide bases. When 3,4-BP and DMBA were linked in this way to biologically active transforming DNA of Bacillus subtilis, they caused (i) reduction of the transforming activity of the DNA accompanied by (ii) significant increases in the frequency of forward mutations. The majority of these hydrocarbon-induced mutations were not able to revert spontaneously. These samples of DNA covalently linked with hydrocarbons showed much lower levels of survival of biological activity when assayed in recipient strains (hcr^–) which are known to be deficient in the enzymes required for repair of ultraviolet light-induced damage to DNA. 3,4-BP covalently linked to calf thymus DNA at a level of approximately one hydrocarbon molecule per 330 bases was shown to cause up to 80% inhibition of the in vitro transcription of the DNA by highly purified ribonucleic acid polymerase prepared from Micrococcus luteus under the experimental condition of template saturation. The presence of 3,4-BP and DMBA molecules covalently bound to B. subtilis DNA samples was also found to prevent complete denaturation of the bihelical structure of certain DNA molecules and thus appears to effect a cross-link in these DNA molecules.