Secondary structural modifications as a consequence of in vitro acetylation and phenanthrylation of DNA by the ultimate carcinogen N-acetoxy-N-2-acetylaminophenanthrene

The acetic acid ester of the proximate carcinogen N-hydroxy-N-2-acetylaminophenanthrene was reacted in vitro with native and heat-denatured calf thymus DNA under various conditions. We showed that besides the phenanthrylation of the DNA bases there is an acetylation reaction of the DNA during its reaction with the ultimate carcinogen. Heat-denatured DNA is 5 to 10 times more acetylated than native DNA. This result suggests that most of the acetylation sites are nonreactive in the double-helical structure of DNA. On the other hand, the phenanthrylation of the bases is shown not to depend on the DNA secondary structure, suggesting that the phenanthrylation sites of the bases are accessible in the grooves of the DNA double helix. The influence of the DNA dynamic structure on the reactions of acetylation and phenanthrylation has been investigated by increasing the ionic strength of the incubation buffer. The melting temperature of different DNA samples, which have been reacted with different concentrations of N-acetoxy-N-2-acetylaminophenanthrene, decreases as the extent of the DNA modifications increases. This thermal destabilization of the double helix is tentatively attributed to the phenanthrylation rather than to the acetylation reaction.