MUTATIONAL SPECIFICITY OF 1-(2-CHLOROETHYL)-3-CYCLOHEXYL-1-NITROSOUREA IN ESCHERICHIA-COLI COMPARISON OF IN-VIVO WITH IN-VITRO EXPOSURE OF THE SUPF GENE

Forward mutations induced by 1-(2-chloroethyl)-3-cyclohexyl-1-nitrosou rea (CCNU) in the supF gene of Escherichia coil were recovered from ba cteria deficient in nucleotide excision repair one in DNA-alkyltransfe rase activity. Bacteria were exposed to 0.4 mM CCNU (in vivo supF muta genesis), increasing the overall mutation Frequency 15.7-fold above th e spontaneous value. A total of 73 independent supF(-) mutants were se quenced. The resulting mutation spectrum was compared with those obtai ned in bacteria and mammalian cells Following the classical shuttle-ve ctor approach (in vitro supF mutagenesis). In vivo CCNU mutagenesis in E. coil yielded a large number of deletions (20/73), in agreement wit h mammalian data but distinct from in vitro bacterial spectra, which a re almost exclusively composed of G:C --> A:T transitions. A substanti al proportion (6/18) of CCNU-induced deletions (>3 bp) involved repeat ed DNA sequences, suggesting a contribution of a slippage-misalignment process in the generation of this mutation class. Substitutions occur red primarily at G:C bose pairs (44/53) and were predominantly G:C --> A:T transitions (39/53). This mutational change was attributed to the mispair potential of the O-6-chloroethylguanine lesion with thymine. Most G:C --> A:T transitions (34/39) were located at three 5'-GG-3' ho tspot sites (positions 123, 160, and 168). The distribution of hotspot sites for G:C --> A:T substitutions differed as a function of the in vivo or in vitro chemical modification of the supF-bearing plasmids an d revealed significant differences in the DNA strand distribution of t his mutational event. Our data suggest that the transcriptional status of the target gene has strong influence on the probability of O-6-chl oroethylguanine formation, reducing its incidence in the transcribed D NA strand. (C) 1997 Wiley-Liss, Inc.