DNA sequence analysis of spontaneous mutations in the SUP4-o gene of Saccharomyces cerevisiae.

A collection of 196 spontaneous mutations in the SUP4-o gene of the yeast Saccharomyces cerevisiae was analyzed by DNA sequencing. The classes of mutation identified included all possible types of base-pair substitution, deletions of various lengths, complex alterations involving multiple changes, and insertions of transposable elements. Our findings demonstrate that at least several different mechanisms are responsible for spontaneous mutagenesis in S. cerevisiae.

Mutational specificities of 1,3-bis(2-chloroethyl)-1-nitrosourea and nitrogen mustard in the SUP4-o gene of Saccharomyces cerevisiae.

A collection of 346 mutations arising in the SUP4-o gene of the yeast Saccharomyces cerevisiae following treatment with 1,3-bis(2-chloro-ethyl)-1-nitrosourea (BCNU) or nitrogen mustard was analyzed by DNA sequencing. Both agents induced all possible types of base-pair substitution as well as deletions and double mutations. The base-pair changes consisted primarily of events at G.C pairs and were distributed throughout the gene. However, the distributions differed for the two drugs, and a prominent substitution hotspot was detected for nitrogen mustard. BCNU induced a substantial fraction of deletions the majority of which were recovered at a hotspot encompassing a tract of five G.C pairs. In contrast, nitrogen mustard generated relatively few deletions, but substantially more double mutations were recovered than with treatment with BCNU. Neither agent exhibited a preference for contiguous G.C sites, and more than one quarter of the mutations occurred at G.C sites, flanked by A.T pairs or at A.T pairs indicating that mutagenesis was not restricted to G.C runs. The data indicate that for BCNU and nitrogen mustard, monoadducts may play a role in mutagenesis, and site-specific mutability is influenced by factors in addition to the G.C richness of the sequence involved.

Influence of DNA repair defects (rad1, rad52) on nitrogen mustard mutagenesis in yeast.

Nitrogen mustard (HN2) mutagenesis of a plasmid-borne copy of the Saccharomyces cerevisiae SUP4-o gene was examined in a repair-proficient yeast strain and isogenic derivatives defective for excision (rad1) or DNA double-strand break (rad52) repair. The excision repair deficiency sensitized the cells to killing by HN2 and abolished mutation induction. Inactivation of RAD52 had no influence on the lethality of HN2 treatment but diminished the induced mutation frequency by 50% at all doses tested. DNA sequence analysis of HN2-induced SUP4-o mutations suggested that RAD52 contributed to the production of basepair substitutions at G.C sites. The rad52 defect appeared to alter the distribution of G.C-->A.T transitions in SUP4-o relative to the distribution for the wild-type strain. This difference did not seem to be due to an effect of RAD52 on the relative fractions of HN2-induced transitions at localized (flanked by A.T pairs) or contiguous (flanked by at least one G.C pair) G.C sites but instead to an influence on the strand specificity of HN2 mutagenesis. In the repair-proficient strain, the transitions showed a small bias for sites having the guanine on the transcribed strand and this preference was eliminated by inactivation of RAD52.

Analysis of mutations induced in the SUP4-o gene of Saccharomyces cerevisiae by cis-diammine dichloroplatinum(II).

A collection of 196 mutations induced in the SUP4-o gene of yeast by treatment with cis-diammine dichloroplatinum(II) (cis-DDP) was characterized by DNA sequencing. All possible types of base pair substitution were identified as well as deletions, insertions and double mutations. Base pair changes at G.C sites predominated and were distributed throughout the gene. The majority of substitutions occurred at 5'-GG-3' and 5'-GA-3' sequences, potential sites of cis-DDP adducts. However, mutations were also detected at a number of other DNA sequences where cis-DDP has been found to bind in vitro or form adducts in vivo including 5'-AA-3', 5'-AG-3', 5'-GNG-3' and 5'-AAA-3'. The site specificity of cis-DDP mutagenesis argues that some of these sequences are significant targets for the induction of mutation in vivo despite the fact that they were considered to be weak binding sites for cis-DDP in vitro. In addition, the distribution of the substitutions within the SUP4-o gene indicates that DNA sequence context influences cis-DDP mutagenesis in vivo. Finally, our results suggest that intrastrand cross-links formed by cis-DDP might facilitate the gain or loss of single base pairs by stabilizing strand misalignments that template these events.

DNA sequence analysis of the mutational specificity of u.v. light in the SUP4-o gene of yeast.

We have characterized mutations induced in the SUP4-o gene of Saccharomyces cerevisiae by u.v. irradiation. Mutants were selected following treatment with 60 J/m2 u.v. light which reduced cell survival to 10% and increased the SUP4-o mutation frequency 100-fold above background. DNA sequence analysis of 120 mutants revealed that u.v. induced all types of base substitutions, although transitions, in particular G:C----A:T events predominated. In addition, a small number of single base pair deletions and double mutations, occurring in tandem or separated by a few base pairs, were recovered. The base pair substitutions were not distributed randomly in the SUP4-o gene and, with one exception, were all located at sites of adjacent pyrimidines, suggesting that they were targeted by u.v. photolesions. A substantial fraction of the mutations were detected at hotspots for u.v. mutagenesis. The majority of changes occurred at the 3' base of dipyrimidine sequences where both cyclobutane dimers and [6-4]-photoproducts could form. Approximately one-third of the induced base substitutions were found at potential pyrimidine dimer sites where [6-4]-photoproducts would be expected to occur rarely. The possible origins of the induced mutations and the role of cyclobutane dimers as premutational u.v. lesions in yeast are considered.