Multiple mutations in a shuttle vector modified by ultraviolet irradiation, (+/-)-7 beta,8 alpha-dihydroxy-9 alpha,10 alpha-epoxy-7,8,9,10-tetrahydrobenzo[a]pyrene, and aflatoxin B(1) have different properties than single mutations and may be generated during translesion synthesis.

Abstract

Shuttle vector-based systems are extensively employed to study the mutational properties of various mutagens in mammalian cells. Such vectors are designed for the detection of point mutations, that is small deletions and single base and tandem substitutions. However, mutant target genes carrying two or more point mutations, referred to as multiple mutations, can also be found in various proportions depending on the mutagen and the cells used. To evaluate the frequency and characteristics of multiple mutations, we used a system where the plasmid, pYZ289, was treated by ultraviolet irradiation, aflatoxin B(1) or (+/-)-7 beta,8 alpha-dihydroxy-9 alpha, 10 alpha-epoxy-7,8,9,10-tetrahydrobenzo[a]pyrene before transfection into mouse fibroblast cells. The kinds of mutations and the mutational spectra were different for single and multiple mutations. In addition, in at least 75% of the cases, mutations of multiples appeared to arise in the same strand. Furthermore, mutational spectra for multiple mutations were different for 5' and 3' members of multiple sets. These observations suggest that multiple mutations arise via a different mechanism than single mutations. Moreover, these findings suggest that multiples arise during translesion DNA synthesis and involve an error-prone polymerase able to introduce a base opposite misinstructive or noninstructional DNA lesions and subject to subsequent misincorporation errors.