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

The spectrum of mutations induced by the carcinogen N-2-acetylaminoflu orene (AAF) was analysed in Saccharomyces cerevisiae using a forward m utation assay, namely the inactivation of the URA3 gene. The URA3 gene , carried on a yeast/bacterial shuttle vector, was randomly modified i n vitro using N-acetoxy-N-2-acetylaminofluorene (N-AcO-AAF) as a model reactive metabolite of the carcinogen AAF. The binding spectrum of AA F to the URA3 gene was determined and found to be essentially random, as all guanine residues reacted about equally well with N-AcO-AAF. Ind ependent 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 adduc ts per plasmid molecule. At this level of modification the mutation fr equency was equal to approximate to 70 x 10(-4), i.e. approximate to 5 0-fold above the background mutation frequency. UV irradiation of the yeast cells did not further stimulate the mutagenic response, indicati ng the lack of an SOS-like mutagenic response in yeast. Sequence analy sis of the URA3 mutants revealed approximate to 48% frameshifts, appro ximate to 44% base substitutions and approximate to 8% complex events. While most base substitutions (74%) were found to be targeted at G re sidues where AAF is known to form covalent C8 adducts, frameshift muta tions were observed at GC base pairs in only approximate to 24% of cas es. Indeed, more than 60% of frameshift events occurred at sequences s uch 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 the se mutations as semi-targeted events and present a potential mechanism that explains their occurrence.