Novel mutagenic properties of abasic sites in Saccharomyces cerevisiae

Abstract

Abasic sites are particularly important in mutation research because they are frequently the ultimate lesion in chemical mutagenesis, and because they are believed to be a paradigm for non-pairing lesions. Although preferential insertion of dAMP ("A-rule") opposite the lesion has been observed in almost all previous studies with other organisms, we find that in budding yeast, Saccharomyces cerevisiae, the preferred nucleotide is dCMP, suggesting that yeast has a "C-rule", at least with respect to the vector constructs used. These constructs contained a single abasic site specifically located within a 28 nucleotide single-stranded region in an otherwise duplex vector. Nucleotide insertions were determined by sequence analysis of replicated vectors taken from a random set of yeast transformants. In three different sequence contexts, the frequencies of dCMP and dAMP insertion were 83% and 13%, 62% and 31%, and 85% and 8%, respectively. A similar bias in favor of cytosine insertion was found using vectors that were entirely single-stranded. However, a preference for dAMP insertion was found when Escherichia coli, rather than yeast, was transfected with samples of the same gapped duplex vector DNA. Preferential insertion of dCMP is not likely to have arisen by previously proposed mechanisms, but is also unlikely to have occurred by a primer/template misalignment mechanism, in which a nearby template guanine directs the insertion of cytosine. Predominant dCMP insertion was observed even when template guanine bases were excluded from a region extending 19 nucleotides 5', and 13 nucleotides 3', to the abasic site.