Roshan B. Christensen

Specificity and frequency of ultraviolet-induced reversion of an iso-1-cytochrome c ochre mutant in radiation-sensitive strains of yeast☆

The basis for the specific pattern of ultraviolet-induced reversion of cyc1-9, an ochre allele of the structural gene for iso-1-cytochrome c, has been examined in radiation-sensitive strains of yeast. Previous analysis, using RAD+ strains, showed that 21 out of 23 cyc1-9 revertants induced by ultraviolet light arose by A · T to G · C transition at the first position in the UAA codon, the remaining two occurring by A · T to T · A transversion at the second position (Stewart et al., 1972; Sherman & Stewart, 1974). All possible base-pair substitutions could be obtained with the aid of other mutagens. It has now been shown that this specificity depends largely on the action of the RAD6 locus, since ultraviolet-induced revertants of cyc1-9 arose by a variety of base-pair substitutions in a strain carrying the rad6-1 allele. Induced reversion frequencies in strains carrying this allele are much lower than normal, though significantly higher than the spontaneous frequency, and the strains are more sensitive to the lethal effects of both ultraviolet and X-irradiation. The phenotypically similar rad18-2 mutation, which appears to block the same repair pathway as rad6-1, also has some effect on the reversion specificity, but its action depends on the presence of other, unidentified, mutations. Specificity was, however, completely unaltered in an excision-defective strain carrying the rad1-2 allele. Induced reversion frequency of cyc1-9 was much higher than normal in this strain. Photoreactivation studies indicated that pyrimidine dimers were responsible for most of the revertants in RAD+, rad1 and rad6 strains. These experiments show that the RAD6+ locus is intimately concerned with error-prone repair, and suggest that excision repair is substantially error-free.

Ultraviolet-induced reversion of cyc1 alleles in radiation-sensitive strains of yeast: I. rev1 mutant strains☆

Radiation-sensitive strains of the yeast, Saccharomyces cerevisiae, that carry the rev1-1 mutation exhibit greatly reduced frequencies of reversion of a number of auxotrophic mutations (Lemontt, 1971a), much lower frequencies of forward mutation to auxotrophy (Lemontt, 1972) and substantially reduced frequencies of reversion of cyc1-9 (Lawrence and Christensen, 1976), an ochre allele of the structural gene for iso-1-cytochrome c (Stewart et al., 1972), when exposed to ultraviolet light. In contrast, the u. v. -induced reversion of cyc1-131, whose message contains the valine codon GUG in place of the AUG initiation codon (Stewart et al., 1971), is unaffected by the presence of the rev1-1 mutations (Lawrence and Christensen, 1976). We have confirmed this observation using the rev1-1 as well as the rev1-1 mutation and have also identified four other cyc1 alleles out of 15 examined that behave in the same way. It is clear, therefore, that while the REV1 gene function is required for the production of many mutational alterations at this locus, it is not for the production of certain specific events. The reason for this appears to depend on the genetic nature of these events rather than the kind of premutational lesion but, despite almost complete information on the base-pair changes and surrounding nucleotide sequences at these sites, it has not yet been possible to define their special nature precisely, indicating that the mutational process is surprisingly complex. The REV1 gene function is not required for the production of most base-pair additions or deletions and although necessary for the formation of most base-pair substitutions it is also not required for the substitutions that lead to the reversion of cyc1-131 and the proline missense mutant, cyc1-115. The REV1 gene function is necessary to produce identical substitutions at other sites, however, and also to produce different substitutions at the cyc1-131 site, suggesting that the special character of cyc1-115 and cyc1-131 is the requirement for a specific base-pair alteration at a specific site. Whatever the explanation, the evidence of this kind of allele-specific control of u.v. -induced reversion must be accounted for by any proposed model of the mutagenic process and must also be accommodated by any scheme to test environmental mutagens.

REV7, a new gene concerned with UV mutagenesis in yeast

SummaryThree allelic mutations of a new yeast gene, which we have named REV7, have been isolated by testing 313 methyl methane sulfonate sensitive mutants for UV-induced reversion of a lys2 allele. Rev7 mutants are markedly deficient with respect to UV-induced reversion of lys2, are slightly sensitive to UV and appear to be in the RAD6 epistasis group for UV survival. Rev7-1, which is probably an amber mutation, does not appear to affect sporulation in homozygous diploids. The REV7 gene is located about 12 cM distal to HIS5 on chromosome IX.

The mechanism of untargeted mutagenesis in UV-irradiated yeast.

SummaryThe SOS error-prone repair hypothesis proposes that untargeted and targeted mutations in E. coli both result from the inhibition of polymerase functions that normally maintain fidelity, and that this is a necessary precondition for translesion synthesis. Using mating experiments with excision deficient strains of Bakers yeast, Saccharomyces cerevisiae, we find that up to 40% of cyc1–91 revertants induced by UV are untargeted, showing that a reduction in fidelity is also found in irradiated cells of this organism. We are, however, unable to detect the induction or activation of any diffusible factor capable of inhibiting fidelity, and therefore suggest that untargeted and targeted mutations are the consequence of largely different processes. We propose that these observations are best explained in terms of a limited fidelity model. Untargeted mutations are thought to result from the limited capacity of processes which normally maintain fidelity, which are active during replication on both irradiated and unirradiated templates. Even moderate UV fluences saturate this capacity, leading to competition for the limited resource. Targeted mutations are believed to result from the limited, though far from negligible, capacity of lesions like pyrimidine dimers to form Watson-Crick base pairs.

New mutations affecting induced mutagenesis in yeast

Previously isolated mutations in bakers yeast, Saccharomyces cerevisiae, that impair induced mutagenesis were all identified with the aid of tests that either exclusively or predominantly detect base-pair substitutions. To avoid this bias, we have screened 11 366 potentially mutant clones for UV-induced reversion of the frameshift allele, his4-38, and have identified 10 mutants that give much reduced yields of revertants. Complementation and recombination tests show that 6 of these carry mutations at the previously known REV1, REV1 and REV3 loci, while the remaining 4 define 3 new genes, REV4 (2 mutations), REV5 and REV6. The rev4 mutations are readily suppressed in many genetic backgrounds and, like the rev5 mutation, impart only a limited deficiency for induced mutagenesis: it is likely, therefore that the REV4+ and REV5+ gene functions are only remotely concerned with this process. The rev6 mutants have a more general deficiency, however, as well as marked sensitivity to UV and an increased spontaneous mutation rate, properties that suggest the REV6 gene is directly involved in mutation induction. The REV5 gene is located about 1 cM proximal to CYC1 on chromosome X.

UmuC function is not essential for the production of all targeted lacI mutations induced by ultraviolet light.

Up to a quarter or more of the normal yield of lacI- mutations could be induced by ultraviolet light in a uvrA6 umuC122:: Tn5 strain if they were detected by plating on 5-bromo-4-chloro-3-indolyl-beta-D-galactoside medium, where all surviving cells can form colonies. Using phenyl beta-D-galactoside selection, which curtails post-irradiation growth, only low yields of mutations were induced. Nucleotide sequence analysis of 134 spontaneous and 145 ultraviolet light-induced mutations shows that broadly similar kinds of mutations were induced in the umuC mutant and its uvrA6 umuC+ counterpart. In particular, these data offer no reason for believing that most of the mutations induced in the umuC mutant were other than normal, targeted events. We conclude that UmuC function, rather than being essential, facilitates recovery and specifically, following the model of Bridges & Woodgate, that it facilitates the prompt resumption of chain elongation.

Absence of relationship between UV-induced reversion frequency and nucleotide sequence at the CYC1 locus of yeast

SummaryThe UV induced mutation frequency of a given base pair located at different sites within the CYC1 gene of Saccharomyces cerevisiae was found to vary by more than fifty fold, indicating the existence of hotspots and coldspots typical of those found in other organisms. We were unable, however, to find any feature of the nucleotide sequence at or near the sites of mutation that explains this variability. These and other data suggest that hotspots are not located within regions particularly susceptible to the formation of premutational lesions. More probably the variation in mutability depends on differences in the activity of enzymes responsible for producing mutations, though the reasons for these differences are not understood and may depend on factors not directly related to nucleotide sequence.

Conjugation-dependent enhancement of induced and spontaneous mutation in the lacI gene of E. coli

SummaryThe frequency of lac- mutations induced in an F′ lacIS plasmid, transferred by conjugation from UV-irradiated, excision-deficient donors to excision-deficient, Δ pro lac recipients, is 2-3 fold higher than that typical of non-mating cells which contain the plasmid. These additional induced mutations can probably be ascribed to errors made during the first, or repliconation, synthesis that takes place in the recipient during the course of plasmid transfer. We also find that spontaneous mutation rates are enhanced in conjugating cells, indicating that fewer errors are corrected, or more made, during transfer replication.

Untargeted mutagenesis induced by UV in the lacI gene of Escherichia coli

SummaryUsing a nonselective method, we have estimated the proportion of untargeted mutations in the lacI gene of E. coli by transferring either irradiated or unirradiated F′ pro lac plasmids from an excision deficient donor to an excision deficient pro lac deleted recipient that had been irradiated and allowed to induce recA dependent functions for 30 min. We find that about 10 percent of the mutations induced by either 3.5 Jm-2 or 7 Jm-2 UV are untargeted.

Mechanisms of UV Mutagenesis in Yeast

UV mutagenesis in yeast depends on the function of the RAD6 locus, a gene that is also responsible for a substantial fraction of wild-type resistance, suggesting that this eukaryote may possess a misrepair mechanism analogous to that proposed for Escherichia coli. The molecular mechanism responsible for RAD6 repair or recovery is not yet known, but it is different from either excision or recombination-dependent repair, processes carried out by the other two main repair pathways in yeast. RAD6-dependent mutagenesis has been found to have the following characteristics.