Termination of in vitro DNA synthesis at AAF adducts in the DNA

DNA synthesis catalyzed by E. coli polymerases I or III is inhibited on templates containing N-acetoxy-acetylaminofluorene-reacted adducts. Termination of synthesis occurs just before the site of the adduct. Synthesis on 0X174 templates primed with restriction fragments and treated with AAAF can be visualized on DNA sequencing gels. Comparison of the amounts of the different newly synthesized fragments with those calculated from the probability of termination as determined from the average number of adducts per molecule shows that synthesis terminates, rather than stutters, at each adduct. This method may be useful for detecting the bypass of lesions.