MUTATIONAL SPECTRUM INDUCED IN SACCHAROMYCES-CEREVISIAE BY THE CARCINOGEN N-2-ACETYLAMINOFLUORENE

The spectrum of mutations induced by the carcinogen N-2-acetylaminofluorene (AAF) was analysed in Saccharomyces cerevisiae using a forward mutation assay, namely the inactivation of the URA3 gene. The URA3 gene, carried on a yeast/bacterial shuttle vector, was randomly modified in vitro using N-acetoxy-N-2-acetylaminofluorene (N-AcO-AAF) as a model reactive metabolite of the carcinogen AAF. The binding spectrum of AAF to the URA3 gene was determined and found to be essentially random, as all guanine residues reacted about equally well with N-AcO-AAF. Independent Ura(-) mutants were selected in vivo after transformation of the modified plasmid into a ura3 Delta yeast strain. Plasmid survival decreased as a function of AAF modification, leading to one lethal hit (37% relative survival) for an average of approximate to 50 AAF adducts per plasmid molecule. At this level of modification the mutation frequency was equal to approximate to 70 x 10(-4), i.e. approximate to 50-fold above the background mutation frequency. UV irradiation of the yeast cells did not further stimulate the mutagenic response, indicating the lack of an SOS-like mutagenic response in yeast. Sequence analysis of the URA3 mutants revealed approximate to 48% frameshifts, approximate to 44% base substitutions and approximate to 8% complex events. While most base substitutions (74%) were found to be targeted at G residues where AAF is known to form covalent C8 adducts, frameshift mutations were observed at GC base pairs in only approximate to 24% of cases. Indeed, more than 60% of frameshift events occurred at sequences such as 5'-(A/T)(n)G-3' where a short (n = 2 or 3) monotonous run of As or Ts is located on the 5' side of a guanine residue. We refer to these mutations as semi-targeted events and present a potential mechanism that explains their occurrence.