MUTATIONS INDUCED IN A SHUTTLE VECTOR PLASMID EXPOSED TO MONOFUNCTIONALLY ACTIVATED MITOMYCIN-C

Reductive activation of mitomycin C leads to its covalent binding to D NA, forming monoadducts and cross-links. The cytotoxicity of mitomycin C has been attributed to cross-link formation, whereas monoadducts ar e assumed to cause mutagenicity. We have developed a P-32-postlabeling technique to measure mitomycin C DNA adducts. Using this technique, w e have measured monoadduct formation in the shuttle vector plasmid pSP 189 and have determined mutations induced by monoadduct formation. The shuttle vector plasmid was incubated with mitomycin C under condition s favoring monofunctional activation of mitomycin C. The plasmid was t hen replicated in human Ad293 cells, rescued in bacteria, and analyzed for mutations in the supF tRNA gene sequence of pSP189. One major mit omycin C/DNA adduct was observed by P-32-postlabeling and was characte rized as a monoadduct of guanine. When pSP189 was exposed to monofunct ionally activated mitomycin C, increases in adduct levels and mutation frequency were found to be related to mitomycin C concentration. The majority of the mutations involved single bases, with base substitutio ns making up 59.1% of the total mutations observed. Of the base substi tutions, 67.2% were transversions and 32.8% were transitions, with nea rly 80% of all base substitutions involving G:C base pairs. Deletions, either as single bases or large deletions, also involved G:C base pai rs the majority of the time. The observed bios of mutations at G:C and the formation of a mitomycin C/DNA monoadduct involving guanine sugge sts that monoadduct formation may be responsible For the mutations. (C ) 1997 Wiley-Liss, Inc.