MUTAGENIC REPLICATION IN HUMAN CELL-EXTRACTS OF DNA CONTAINING SITE-SPECIFIC N-2-ACETYLAMINOFLUORENE ADDUCTS

We have analyzed the effects of site-specific N-2-acetylaminofluorene (AAF) adducts on the efficiency and frameshift fidelity of bidirection al replication of double-stranded DNA in a human cell extract. Plasmid vectors were constructed containing the simian virus 40 origin of rep lication and single AAF adducts at one of three guanines in the Nar I sequence GGCGCC in a lacZ reporter gene. The presence of an AAF adduct diminishes replication efficiency in HeLa cell extracts by 70-80%. Re plication product analyses reveal unique termination sites with each d amaged vector, suggesting that when the replication fork encounters an AAF adduct, it often stops before incorporation opposite the adduct. We also observed a higher proportion of products representing replicat ion of the undamaged strand compared to the damaged strand. This sugge sts that the undamaged strand is replicated more readily, either by un coupling the first fork to encounter the lesion or by replication usin g the fork arriving from the other direction. Also included among repl ication products are covalently closed monomer-length molecules resist ant to cleavage at the AAF-modified Nor I site. This resistance is cha racteristic of substrates containing the AAF adduct, suggesting that t ranslesion bypass had occurred. Transformation of Escherichia coli cel ls with the replicated damaged DNA yielded lacZ alpha revertant freque ncies significantly above values obtained with undamaged DNA or with d amaged DNA not replicated in vitro. This increase was only seen with t he substrate modified at the third guanine position. Analysis of mutan t DNA demonstrated the loss of a GC dinucleotide at the Nar I sequence . Generation of this position-dependent AAF-induced frameshift error i n a human replication system is consistent with previous observations in E. coil suggesting that, after incorporation of dCMP opposite modif ied guanine in the third position, realignment of the template-primer occurs to form an intermediate with two unpaired nucleotides in the te mplate strand.