MUTATIONS IN A SHUTTLE VECTOR EXPOSED TO ACTIVATED MITOMYCIN-C

The cytotoxicity of the potent antibiotic and antitumor agent mitomyci n C (MMC) is due to its irreversible binding to DNA. Alkylating specie s generated by bioreductive activation of MMC are known to cause monoa dducts and cross-links in DNA by specifically binding to guanine resid ues. To gain insight into how these lesions lead to base- and sequence -specific mutations, shuttle vector pSP189 was treated with MMC chemic ally reduced by treatment with sodium borohydride, replicated in human Ad293 cells, rescued in bacteria, and analyzed for mutations in the s upF tRNA gene sequence. The MMC-induced mutations were predominantly b ase substitutions. Eighty-four percent of the base substitutions were transversions, with G:C-->T:A the major transversion. Single base dele tions were the other major mutational event, and 77% of these were G:C deletions. Base positions 115, 123, and 163 were mutational hot spots based on the frequency of independent mutations. Identification of a single MMC adduct (presumed to be a modified G on the basis of its R(f ) value) and clustering of MMC-induced mutations at three GC-rich area s (nt 100-123, 152-163, and 168-176) suggested that the mutational spe ctrum we found was due to binding of MMC to guanine on either strand o f the plasmid DNA. (C) 1994 Wiley-Liss, Inc.