Elimination of the Yeast Rad6 Ubiquitin Conjugase Enhances Base-Pair Transitions and G.c -> T.a Transversions as Well as Transposition of the Ty Element: Implications for the Control of Spontaneous Mutation

The RAD6 gene of the yeast Saccharomyces cerevisiae encodes an enzyme that conjugates ubiquitin to other proteins. Defects in RAD6 confer a mutator phenotype due, in part, to an increased rate of transposition of the yeast Ty element. To further delineate the role of protein ubiquitination in the control of spontaneous mutagenesis in yeast, we have characterized 202 mutations that arose spontaneously in the SUP4-o gene carried on a centromere vector in a RAD6 deletion strain. The resulting mutational spectrum was compared to that for 354 spontaneous SUP4-o mutations isolated in the isogenic wild-type parent. This comparison revealed that the rad6 mutator enhanced the rate of single base-pair substitution, as well as Ty insertion, but did not affect the rates of the other mutational classes detected. Relative to the wild-type parent, Ty inserted at considerably more SUP4-o positions in the rad6 strain with a significantly smaller fraction detected at a transposition hotspot. These findings suggest that, in addition to the rate of transposition, protein ubiquitination might influence the target site specificity of Ty insertion. The increase in the substitution rate accounted for approximately 90% of the rad6 mutator effect but only the two transitions and the G. C----T.A transversion were enhanced. Analysis of the distribution of these events within SUP4-o suggested that the site specificity of the substitutions was influenced by DNA sequence context. Transformation of heteroduplex plasmid DNAs into the two strains demonstrated that the rad6 mutator did not reduce the efficiency of correcting mismatches that could give rise to the transitions or transversion nor did it bias restoration of the mismatches to the incorrect base-pairs. These results are discussed in relation to possible mechanisms that might link ubiquitination of proteins to spontaneous mutation rates.     

Specificity of the mutator effect caused by disruption of the RAD1 excision repair gene of Saccharomyces cerevisiae.

Disruption of RAD1, a gene controlling excision repair in the yeast Saccharomyces cerevisiae, increased the frequency of spontaneous forward mutation in a plasmid-borne copy of the SUP4-o gene. To characterize this effect in detail, a collection of 249 SUP4-o mutations arising spontaneously in the rad1 strain was analyzed by DNA sequencing. The resulting mutational spectrum was compared with that derived from an examination of 322 spontaneous SUP4-o mutations selected in an isogenic wild-type (RAD1) strain. This comparison revealed that the rad1 mutator phenotype was associated with increases in the frequencies of single-base-pair substitution, single-base-pair deletion, and insertion of the yeast retrotransposon Ty. In the rad1 strain, the relative fractions of these events and their distributions within SUP4-o exhibited features similar to those for spontaneous mutagenesis in the isogenic RAD1 background. The increase in the frequency of Ty insertion argues that Ty transposition can be activated by unrepaired spontaneous DNA damage, which normally would be removed by excision repair. We discuss the possibilities that either translesion synthesis, a reduced fidelity of DNA replication, or a deficiency in mismatch correction might be responsible for the majority of single-base-pair events in the rad1 strain.     

Disruption of the Rad52 Gene Alters the Spectrum of Spontaneous Sup4-O Mutations in Saccharomyces Cerevisiae

Defects in the RAD52 gene of the yeast Saccharomyces cerevisiae confer a mutator phenotype. To characterize this effect in detail, a collection of 238 spontaneous SUP4-o mutations arising in a strain having a disrupted RAD52 gene was analyzed by DNA sequencing. The resulting mutational spectrum was compared to that derived from an examination of 222 spontaneous mutations selected in a nearisogenic wild-type (RAD52) strain. This comparison revealed that the mutator phenotype was associated with an increase in the frequency of base-pair substitutions. All possible types of substitution were detected but there was a reduction in the relative fraction of A.T----G.C transitions and an increase in the proportion of G.C----C.G transversions. These changes were sufficient to cause a twofold greater preference for substitutions at G.C sites in the rad52 strain despite a decrease in the fraction of G.C----T.A transversions. There were also considerable differences between the distributions of substitutions within the SUP4-o gene. Base-pair changes occurred at fewer sites in the rad52 strain but the mutated sites included several that were not detected in the RAD52 background. Only two of the four sites that were mutated most frequently in the rad52 strain were also prominent in the wild-type strain and mutation frequencies at almost all sites common to both strains were greater for the rad52 derivative. Although single base-pair deletions occurred in the two strains with similar frequencies, several classes of mutation that were recovered in the wild-type background including multiple base-pair deletions, insertions of the yeast transposable element Ty, and more complex changes, were not detected in the rad52 strain.(ABSTRACT TRUNCATED AT 250 WORDS)     

Excision repair influences the site and strand specificity of sunlight mutagenesis in yeast.

A collection of 384 mutations recovered in a tRNA gene (SUP4-o) following exposure of isogenic excision-repair-proficient (RAD1) or deficient (rad1) strains of the yeast Saccharomyces cerevisiae to sunlight was characterized by DNA sequencing. In each case, greater than 90% of the mutations were single base-pair substitutions with events at G.C pairs constituting most of the changes. However, more than half of these substitutions were transversions in the RAD1 strain whereas transitions predominated in the rad1 strain. Tandem double substitutions were recovered in both strains and the individual changes were exclusively G.C----A.T transitions. The majority of single substitutions, and all tandem double changes, were at base-pairs where the pyrimidine(s) was part of a dipyrimidine sequence and the site specificities were consistent with cyclobutane dimers and/or pyrimidine (6-4) pyrimidone photoproducts contributing to sunlight mutagenesis. Yet, the data also pointed to an important role for lesions that form at G.C pairs and give rise to transversions. Analysis of the strand specificity of sunlight mutagenesis indicated that transitions or transversions at G.C pairs occurred preferentially in SUP4-o at sites where a dipyrimidine or a guanine, respectively, was on the transcribed strand. These biases required a functional excision-repair system.     

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

Summary 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 (radl) 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.     

The spectrum of spontaneous mutations in a Saccharomyces cerevisiae uracil-DNA-glycosylase mutant limits the function of this enzyme to cytosine deamination repair.

Uracil-DNA-glycosylase has been proposed to function as the first enzyme in strand-directed mismatch repair in eukaryotic organisms, through removal of uracil from dUMP residues periodically inserted into the DNA during DNA replication (Aprelikova, O. N., V. M. Golubovskaya, T. A. Kusmin, and N. V. Tomilin, Mutat. Res. 213:135-140, 1989). This hypothesis was investigated with Saccharomyces cerevisiae. Mutation frequencies and spectra were determined for an ung1 deletion strain in the target SUP4-o tRNA gene by using a forward selection scheme. Mutation frequencies in the SUP4-o gene increased about 20-fold relative to an isogenic wild-type S. cerevisiae strain, and the mutator effect was completely suppressed in the ung1 deletion strain carrying the wild-type UNG1 gene on a multicopy plasmid. Sixty-nine independently derived mutations in the SUP4-o gene were sequenced. All but five of these were due to GC----AT transitions. From this analysis, we conclude that the mutator phenotype of the ung1 deletion strain is the result of a failure to repair spontaneous cytosine deamination events occurring frequently in S. cerevisiae and that the UNG1 gene is not required for strand-specific mismatch repair in S. cerevisiae.     

Highly mutable sites for ICR-170-induced frameshift mutations are associated with potential DNA hairpin structures: studies with SUP4 and other Saccharomyces cerevisiae genes.

The majority of the mutations induced by ICR-170 in both the CYC1 gene (J. F. Ernst et al. Genetics 111:233-241, 1985) and the HIS4 gene (L. Mathison and M. R. Culbertson, Mol. Cell. Biol. 5:2247-2256, 1985) of the yeast Saccharomyces cerevisiae were recently shown to be single G . C base-pair insertions at monotonous runs of two or more G . C base pairs. However, not all sites were equally mutable; in both the CYC1 and HIS4 genes there is a single highly mutable site where a G . C base pair is preferentially inserted at a [sequence in text]. Here we report the ICR-170 mutagen specificity at the SUP4-o tyrosine tRNA gene of yeast. Genetic fine structure analysis and representative DNA sequence determination of ICR-170-induced mutations revealed that there is also a single highly mutable site in SUP4-o and that the mutation is a G . C base-pair insertion at a monotonous run of G . C base pairs. Analysis of DNA sequences encompassing the regions of highly mutable sites for all three genes indicated that the mutable sites are at the bases of potential hairpin structures; this type of structure could not be found at any of the other, less mutable G . C runs in SUP4, CYC1, and HIS4. Based on these results and recent information regarding novel DNA structural conformations, we present a mechanism for ICR-170-induced mutagenesis. (i) ICR-170 preferentially binds to DNA in the beta conformation; factors that increase the temporal stability of this structure, such as adjacent stem-and-loop formation, increase the frequency of ICR-170 binding; (ii) the observed mutagen specificity reflects formation of a preferred ICR-170 intercalative geometry at [sequence in text] sites; (iii) during replication or repair, ICR-170 remains associated with the single-stranded template; (iv) stuttering or strand slippage by the polymerization complex as it encounters the mutagen results in nucleotide duplication; (v) subsequent replication or mismatch repair fixes the insertion into the genome. This mechanism accounts for both the IRC-170 mutagenic specificity and the molecular basis of the highly mutable sites in S. cerevisiae.     

Development of a yeast system to assay mutational specificity

We have developed a system wherein DNA alterations occurring in a target gene in the yeast Saccharomyces cerevisiae can be determined by DNA sequencing. The target gene, SUP4-o, an ochre suppressor allele of a yeast tyrosine tRNA gene, has been inserted into a shuttle vector (YCpMP2) which is maintained in yeast at a copy number of one per cell Mutations in SUP4-o are selected by virtue of their inactivation of suppressor activity. Rapid DNA preparations from these mutants are used to transform an appropriate bacterial strain. Since YCpMP2 also carries the M13 phage replication origin, superinfection of bacterial cells containing the plasmid with wild-type M13 phage yields single stranded YCpMP2 DNA suitable for dideoxynucleotide chain termination sequencing. We have used this system to examine mutations arising spontaneously in the SUP4-o gene. The spontaneous mutants occurred at a frequency of 3.2 X 10(-6)/viable cell, corresponding to a rate of 2.7 X 10(-7) events/cell division. Following bacterial transformation, 16% of the recovered plasmids tested displayed altered gel mobility consistent with loss of significant portions of the plasmid. Hybridization analysis of total yeast DNA and use of purified YCpMP2 revealed that these very large deletions were not generated in yeast but were associated with bacterial transformation. Among the SUP4-o mutants analyzed by DNA sequencing, we identified each type of single base pair substitution (transitions and transversions), small deletions of varying length (1-32 base pairs) and more extensive deletions of undetermined size. These results demonstrate that the SUP4-o system can be used to detect various types of mutation at numerous sites in a single eukaryotic gene and to characterize the DNA sequence changes responsible for the mutations selected.     

Specificity of the Yeast Rev3Δ Antimutator and Rev3 Dependency of the Mutator Resulting from a Defect (Rad1Δ) in Nucleotide Excision Repair

The yeast REV3 gene has been predicted to encode a DNA polymerase specializing in translesion synthesis. This polymerase likely participates in spontaneous mutagenesis, as rev3 mutants have an antimutator phenotype. Translesion synthesis also may be necessary for the mutator caused by a RAD1 (nucleotide excision repair) deletion (rad1Δ). To further examine the role of REV3 in spontaneous mutagenesis, we characterized SUP4-o mutations that arose spontaneously in strains having combinations of normal or mutant REV3 and RAD1 alleles. The largest fraction of the rev3Δ-dependent mutation rate decrease was observed for single base-pair substitutions and deletions, although the rates of all mutational classes detected in the RAD1 background were reduced by at least 30%. Interestingly, inactivation of REV3 was associated with a doubling of the number of sites at which the retrotransposon Ty inserted. rev3Δ also greatly diminished the magnitude of the rad1Δ mutator, but not to the rev3Δ antimutator level, implicating REV3-dependent and independent processes in the rad1Δ mutator effect. However, the specificity of the rev3Δ antimutator suggested that the same REV3-dependent processes gave rise to the majority of spontaneous mutations in the RAD1 and rad1Δ strains.     

Dissociation kinetics of 19 base paired oligonucleotide-DNA duplexes containing different single mismatched base pairs.

The dissociation kinetics of 19 base paired oligonucleotide-DNA duplex containing a various single mismatched base pair are studied on dried agarose gels. The kinetics of the dissociation are first order under our experimental conditions. The incorporation of a single mismatched base pair destabilizes the DNA duplexes to some extent, the amount depending on the nature of the mismatched base pair. G-T and G-A mismatches slightly destabilize a duplex, while A-A, T-T, C-T and C-A mismatches significantly destabilize it. The activation energy for the overall dissociation processes for these oligonucleotide-DNA duplexes containing 19 base pairs is 52 +/- 2 Kcal mol-1 as determined from the slope of Arrhenius plot.