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.     

Lack of Chemically Induced Mutation in Repair-Deficient Mutants of Yeast

Two genes, rad6 and rad9, that confer radiation sensitivity in the yeast Saccharomyces cerevisiae also greatly reduce the frequency of chemically-induced reversions of a tester mutant cyc1-131, which is a chain initiation mutant in the structural gene determining iso-1-cytochrome c. Mutations induced by ethyl methanesulfonate (EMS), diethyl sulfate (DES), methyl methanesulfonate (MMS), dimethyl sulfate (DMS), nitroquinoline oxide (NQO), nitrosoguanidine (NTG), nitrogen mustard (HN2), beta-propiolactone, and tritiated uridine, as well as mutations induced by ultraviolet light (UV) and ionizing radiation were greatly diminished in strains homozygous for either the rad6 or rad9 gene. Nitrous acid and nitrosoimidazolidone (NIL), on the other hand, were highly mutagenic in these repair-deficient mutants, and at low doses, these mutagens acted with about the same efficiency as in the normal RAD strain. At high doses of either nitrous acid or NIL, however, reversion frequencies were significantly reduced in the two rad mutants compared to normal strains. Although both rad mutants are immutable to about the same extent, the rad9 strains tend to be less sensitive to the lethal effect of chemical mutagens than rad6 strains. It is concluded that yeast requires a functional repair system for mutation induction by chemical agents.     

Plate assay for chemical- and radiation-induced mutagenesis of CAN1 in yeast as a function of post-treatment DNA replication: the effect of rad6-1

An agar post-treatment method was used to monitor levels of ultraviolte light-and hydrazine-induced mutagenesis at CAN1 in Saccharomyces cerevisiae as a function of post-treatment cell division prior to selection for canavanine-resistant mutants with a top-agar overlay containing canavanine. The advantage of this method is that its permits reliable measurements of mutation induction during the early period before, during, and after the first round of post-treatment DNA replication. In strains that are wild-type for DNA repair, ultraviolet light mutagenesis appears to be a pre-replicative phenomenon, while mutation by hydrazine involves a replicative or post-replicative mechanism. Most chemical mutagenesis in yeast requires a functional RAD6 gene. Hydrazine mutability is also reduced by rad6-1, suggesting a possible misrepair mechanism.     

DNA repair and the genetic effects of thymidylate stress in yeast

Folate antagonists, such as aminopterin, methotrexate and various sulfonamides, block de novo thymidylate biosynthesis in Saccharomyces cerevisiae. The resulting starvation for thymine nucleotides is lethal and recombinagenic in RAD wild-type strains. In this paper we report our studies of these effects in repair-deficient yeast. Antifolate treatment of various rad mutants revealed that repair defects influence the killing and recombination caused by thymidylate deprivation. Compared to a RAD wild-type strain, diploids homozygous for rad3, rad6 or rad18 were more resistant to cell killing. Thus, contrary to findings with conventional DNA-damaging agents, the lethal effects of thymidylate starvation appear to be ameliorated by certain DNA repair deficiencies. On the other hand, a rad50 strain was extremely sensitive to the antifolates. Within this series of diploids, increasing sensitivity to thymidylate starvation was accompanied by an increase in recombination frequencies. The degrees of lethality and recombination, induced by thymidylate depletion, were correlated with the severity of DNA-strand breakage in the RAD and rad50 strains. Experiments with diploids homozygous for rad52, rad54 or rad57 suggested that aborted recombination events, provoked by thymidylate deprivation, caused chromosome loss. Furthermore, the repair defects in these mutants indicated that double-strand breaks are among the lethal lesions induced by thymine nucleotide starvation. Finally, we discuss the possibility that the recombinagenicity of thymidylate stress may account for one type of acquired resistance to methotrexate in mammalian cells.     

A yeast protein analogous to Escherichia coli RecA protein whose cellular level is enhanced after UV irradiation

Summary In Saccharomyces cerevisiae, a protein was recognized by polyclonal antibodies raised against homogeneous Escherichia coli K12 RecA protein. The cellular level of the yeast protein called RecAsc (molecular weight 44 kDa, pI 6.3), was transiently enhanced after UV irradiation. Protease inhibitors were required to minimize degradation of the RecAsc protein during cell lysis. The RecAsc protein exhibited similar basal levels and similar kinetics of increase after UV irradiation in DNA-repair proficient (RAD ⁺) strains carrying mitochondrial DNA or not (rho ⁰). This was also true for the following DNA-repair deficient (rad ^-) strains: rad2-6 rad6-1 rad52-1, a triple mutant blocked in three major repair pathways; rad6-, a mutant containing an integrative deletion in a gene playing a central role in mutagenesis; pso2-1, a mutant that exhibits a reduced rate of mutagenesis and recombination after exposure to DNA cross-linking agents.     

Induction of forward mutations in mutationally defective yeast

Summary The 3 rev loci that reduce ultraviolet light (UV)-induced reversion in S. cerevisiae had a similar effect on forward mutation to auxotrophy induced by a single 400 erg/mm² UV dose: rev1-1, rev2-1 and rev3-1 reduced average frequencies of auxotrophs to 4%, 64% and 4% that in wild type and reduced frequencies of mutants at ade1 or ade2 to 19%, 88% and 2% wild type, respectively. The rev2-1 strain exhibited high frequencies of spontaneous mutation. It is suggested that rev1-1 and rev3-1 block steps in a general UV mutation mechanism controlling forward and reverse mutation throughout the genome. The small effect of rev2-1, compared to the effect of rev1-1 or rev3-1, is consistent with previously obtained data on UV reversion and could be due to a specificity for induced mutation involving only certain types of UV damage or, on the other hand, it may be related to mutator activity. Although rev caused varying degrees of sensitivity to ethylmethanesulfonate (EMS), there was little or no significant effect on mutation induced by a single 30 min. dose of 3% EMS. Auxotroph frequencies were 79%, 109% and 94% wild type, whild frequencies at ade1 or ade2 were 82%, 56% and 51% wild type in the respective strains. It is suggested that steps blocked by rev, although they may participate in repair of lethal EMS damage, do not themselves generate EMS-induced mutations.     

The dependence of HNO2 mutagenesis in phage T4 on ligase and the lack of dependence of 2AP mutagenesis on repair functions

Summary A temperature sensitive ligase allele of phage T4 reduced or eliminated HNO₂ induced reversion of am mutants. Since at the temperatures used, the ligase mutant is defective in the repair of some types of lethal lesions (i.e., UV, MMS and EMS induced lesions) these results indicate that HNO₂ mutagenesis may occur through a ligase dependent repair pathway. In contrast, 2AP induced mutation was not inhibited by mutants defective in the gene 30 ligase or in genes 32, 39, 41, 44, 45, 46, 47, 49, 52, 56, 58–61 and v. This indicates that 2AP mutagenesis probably does not depend on a repair pathway in phage T4.     

Differentiation between Amber and Ochre Mutants of Yeast by Reversion with 4-Nitroquinoline-1-Oxide

4-nitroquinoline-1-oxide (NQO) induces high frequencies of intragenic revertants of amber (UAG) but not ochre (UAA) mutants of yeast. Distinction of the amber and ochre codons was made with well-characterized nonsense mutants of the iso-1-cytochrome c gene (cyc1 mutants) as well as with nonsense mutants having nutritional requirements. Thus the NQO-induced reversion frequencies corroborated the assignments that were based on the pattern of amino acid replacements in intragenic revertants and on the speficity of suppression. It was concluded from these results and from the results of a previous investigation with other cyc1 mutants (Prakash, Stewart and Sherman 1974) that NQO induces transversions of G:C base pairs at many sites and that the specificity is not strongly influenced by neighboring base pairs in at least the strains examined in these studies. NQO was previously shown to induce G:C → A:T transitions at least at one site and this and the previous study established that it does not significantly mutate A:T base pairs at numerous sites. Thus NQO can be used to selectively mutate G:C base pairs and to determine if the pathways of reverse mutations involve G:C base pairs. Suppressors that act on either amber or ochre mutants were induced with NQO, indicating that they can arise by mutations of G:C base pairs.     

UV and chemical mutagenesis in rev7 mutants of yeast

Summary We have examined induced mutagenesis in rev7-1 mutants of Baker's yeast' Saccharomyces cerevisiae, using a variety of contrasting test systems and several different mutagens. UV-induced reversion frequencies of the ochre allele arg4-17, the putative missense allele ilv1-92 and the frameshift allele his4-38 were 10 to 200 fold lower in haploid and diploid rev7-1 mutants compared with wild type strains, but UV-induced reversion frequencies of the frameshift allele leu2-3 and the proline missense allele cyc1-115 were reduced only a few fold. Ilv1-92 reversion frequencies induced by methyl methane sulfonate or by N-methyl-N-nitro-N-nitrosoguanidine were 10 to 20 times lower in rev7-1 mutants, but normal frequencies of these revertants were induced with ethyl methane sulfonate, even though rev7-1 strains are slightly sensitive to this mutagen as well as to the others tested. We conclude that the rev7 mutants, like the rev3 mutants they closely resemble, have a substantial but not total deficiency concerning induced mutagenesis.     

The Role of Radiation (rad) Genes in Meiotic Recombination in Yeast

In yeast, the functions controlled by radiation-repair genes RAD6, RAD50, RAD52 and RAD57 are essential for normal meiosis; diploids with lesions in these genes either fail to sporulate (rad6) or sporulate but produce inviable spores (rad50, 52, 57). Since RAD genes may control aspects of DNA metabolism, we attempted to define more precisely the role of each gene in meiosis, especially with regard to possible roles in premeiotic DNA replication and recombination. We constructed diploids singly homozygous for each of the four rad mutations, heteroallelic at his1 and heterozygous for a recessive canavanine-resistance marker. Each strain was exposed to sporulation-inducing conditions and monitored for (1) completion of mitotic cell cycles, (2) cell viability, (3) utilization of acetate for mass increases, (4) premeiotic DNA synthesis, (5) intragenic recombination at his1, and (6) formation of viable haploid spores. Control strains heterozygous for the rad mutations completed mitosis, metabolized acetate, replicated their DNA, and showed typically high levels of gene conversion and viable-spore formation. The mutant diploids also completed mitosis, utilized acetate, and carried out premeiotic DNA replication. The mutants, however, showed little or no meiotic gene conversion. The rad50, 52 and 57 strains sporulated, but the spores were inviable. The rad6 strain did not sporulate. The rad50, 52 and 57 strains exhibited viability losses that coincided with the period of DNA synthesis, but not with later meiotic events; the rad6 strain did not lose viability. We propose that the normal functions specified by RAD50, 52 and 57 are not essential for either the initial or terminal steps in meiosis, but are required for successful recombination. The rad6 strain may be recombination-defective, or it may fail to progress past DNA replication in the overall sequence leading to formation and recovery of meiotic recombinants.     

Rat hepatic vinyl chloride metabolites induce gene conversion in the yeast strain D7RAD in vitro and in vivo

We have tested the genetic activity of gaseous vinyl chloride in vitro and in vivo using the gene-conversion system trp5-12/trp5-27 → TRP⁺) in the yeast strain D7RAD. To induce, in vitro, TRP⁺ convertants with 2.5% gaseous vinyl chloride, a rat-liver microsomal system for metabolic activation of the vinyl chloride and dividing yeast cells are required. Neither a deficiency in excision repair (rad3) nor in the error-prone repair pathway (rad6) increased the vinyl-chloride-induced conversion frequencies compared with the repair-competent D7RAD strain.When logarithmically growing cells of the D7RAD strain were injected intravenously into male Wistar rats which inhaled 1% vinyl chloride in air for 24 h, a significant enhancement of the TRP⁺ conversion frequencies was found compared with that in cells re-isolated from untreated rats. These results indicate that vinyl chloride metabolites from the metabolizing hepatocytes diffuse into yeast cells, which accumulate in the liver capillaries. This supports the hypothesis that the endothelial cells of the liver sinuses, which have hardly any metabolic activity, but give rise to vinyl-chloride-induced hemangiotheliomas (rate type of liver tumor), are transformed by diffusible metabolites of the procarcinogen vinyl chloride.     

Repair of 8-methoxypsoralen photoinduced cross-links in yeast

Summary The repair of interstrand cross-links induced by 8-methoxypsoralen plus UVA (365 nm) radiation DNA was analyzed in diploid strains of the yeast Saccharomyces cerevisiae. The strains employed were the wild-type D₇ and derivatives homozygous for the rad18-1 or the rad3-12 mutation. Alkaline step-elution and electron microscopy were performed to follow the process of induction and removal of photoinduced crosslinks. In accordance with previous reports, the D₇ rad3-12 strain failed to remove the induced lesions and could not incise cross-links. The strain D₇ rad18-1 was nearly as efficient in the removal of 8-MOP photoadducts after 2 h of post-treatment incubation as the D₇ RAD⁺ wild-type strain. However, as demonstrated by alkaline step-elution and electron microscopic analysis, the first incision step at DNA cross-links was three times more effective in D₇ rad18-1 than in D₇ RAD⁺. This is consistent with the hypothesis that the RAD18 gene product is involved in the filling of gaps resulting from persistent non-informational DNA lesions generated by the endonucleolytic processing of DNA cross-links. Absence of this gene product may lead to extensive strand breakage and decreased recognition of such lesions by structural repair systems.     

Heteroduplex Repair as an Intermediate Step of Uv Mutagenesis in Yeast

We have measured UV-induced mutation frequencies in yeast in a forward, nonselective assay system by scoring white adex ade2 double auxotrophs among parental red-pigmented ade2 clones. The frequencies of sectored and pure mutant clones were determined separately. In excision-defective strains carrying the genes rad1–1, rad3–2 and rad4–4, as well as in the double mutants, rad 1–1 rad 3–2 and rad 1–1 rad 4–4, considerably more sectored than pure clones are induced in the low-dose range; in repair-competent strains, pure mutant clones substantially outnumber the sectored clones. These results can be explained on the basis of known differences in the timing of error-prone repair during the cell division cycle; that is, we assume that error-prone repair occurs primarily before replication in RAD wild-type strains but after replication in excision-deficient mutants. It has been suggested that excision deficiency has a pleiotropic effect on heteroduplex repair and nucleotide excision repair; however, the high percentage (36.6%) of half-sectored clones found in the rad1–1 strain is hard to reconcile with this hypothesis. We propose that heteroduplex repair occurs subsequent to error-prone repair in both excision-proficient and excision-deficient strains.     

The isolation and characterization of ngm2, a mutation that affects nitrosoguanidine mutagenesis in yeast

Summary We have isolated and characterized a new mutant of Saccharomyces cerevisiae, carrying a single mutant allele that we designate ngm2-1, which is defective with respect to induced mutagenesis. This mutant was isolated by screening mutagenized clones for reduced frequencies of reversion of the his1-7 allele, induced by N-methyl-N-nitro-N-nitrosoguanidine. As judged by the reversion of his1-7 and ilv1-92, ngm2-1 mutant strains are also deficient with respect to mutability induced by methyl methane sulfonate, ethyl methane sulfonate and, at least partially, by UV. UV-induced reversion of the ochre mutation arg4-17 and the frameshift mutation his4-38 was not much affected by ngm2-1, however. Like rev3 and rev7 mutations, ngm2-1 also has little influence on the reversion of the proline missense allele, cyc1-115. Ngm2-1 mutants are only at best very slightly more sensitive to the toxicity of the four mutagens used, and homozygous diploids sporulate normally.     

Role of DNA polymerase eta in the bypass of abasic sites in yeast cells

Abasic (AP) sites are major DNA lesions and are highly mutagenic. AP site-induced mutagenesis largely depends on translesion synthesis. We have examined the role of DNA polymerase η (Polη) in translesion synthesis of AP sites by replicating a plasmid containing a site-specific AP site in yeast cells. In wild-type cells, AP site bypass resulted in preferred C insertion (62%) over A insertion (21%), as well as −1 deletion (3%), and complex event (14%) containing multiple mutations. In cells lacking Polη (rad30), Rev1, Polζ (rev3), and both Polη and Polζ, translesion synthesis was reduced to 30%, 30%, 15% and 3% of the wild-type level, respectively. C insertion opposite the AP site was reduced in rad30 mutant cells and was abolished in cells lacking Rev1 or Polζ, but significant A insertion was still detected in these mutant cells. While purified yeast Polα effectively inserted an A opposite the AP site in vitro, purified yeast Polδ was much less effective in A insertion opposite the lesion due to its 3′→5′ proofreading exonuclease activity. Purified yeast Polη performed extension synthesis from the primer 3′ A opposite the lesion. These results show that Polη is involved in translesion synthesis of AP sites in yeast cells, and suggest that an important role of Polη is to catalyze extension following A insertion opposite the lesion. Consistent with these conclusions, rad30 mutant cells were sensitive to methyl methanesulfonate (MMS), and rev1 rad30 or rev3 rad30 double mutant cells were synergistically more sensitive to MMS than the respective single mutant strains.     

Poleta, Polzeta and Rev1 together are required for G to T transversion mutations induced by the (+)- and (-)-trans-anti-BPDE-N2-dG DNA adducts in yeast cells

Benzo[a]pyrene is an important environmental mutagen and carcinogen. Its metabolism in cells yields the mutagenic, key ultimate carcinogen 7R,8S,9S,10R-anti-benzo[a]pyrene-7,8-dihydrodiol-9,10-epoxide, (+)-anti-BPDE, which reacts via its 10-position with N²-dG in DNA to form the adduct (+)-trans-anti-BPDE-N²-dG. To gain molecular insights into BPDE-induced mutagenesis, we examined in vivo translesion synthesis and mutagenesis in yeast cells of a site-specific 10S (+)-trans-anti-BPDE-N²-dG adduct and the stereoisomeric 10R (−)-trans-anti-BPDE-N²-dG adduct. In wild-type cells, bypass products consisted of 76% C, 14% A and 7% G insertions opposite (+)-trans-anti-BPDE-N²-dG; and 89% C, 4% A and 4% G insertions opposite (−)-trans-anti-BPDE-N²-dG. Translesion synthesis was reduced by ~26–37% in rad30 mutant cells lacking Polη, but more deficient in rev1 and almost totally deficient in rev3 (lacking Polζ) mutants. C insertion opposite the lesion was reduced by ~24–33% in rad30 mutant cells, further reduced in rev1 mutant, and mostly disappeared in the rev3 mutant strain. The insertion of A was largely abolished in cells lacking either Polη, Polζ or Rev1. The insertion of G was not detected in either rev1 or rev3 mutant cells. The rad30 rev3 double mutant exhibited a similar phenotype as the single rev3 mutant with respect to translesion synthesis and mutagenesis. These results show that while the Polζ pathway is generally required for translesion synthesis and mutagenesis of the (+)- and (−)-trans-anti-BPDE-N²-dG DNA adducts, Polη, Polζ and Rev1 together are required for G→T transversion mutations, a major type of mutagenesis induced by these lesions. Based on biochemical and genetic results, we present mechanistic models of translesion synthesis of these two DNA adducts, involving both the one-polymerase one-step and two-polymerase two-step models.     

Expression of a human cytochrome p450 in yeast permits analysis of pathways for response to and repair of aflatoxin-induced DNA damage

Aflatoxin B1 (AFB1) is a human hepatotoxin and hepatocarcinogen produced by the mold Aspergillus flavus. In humans, AFB1 is primarily bioactivated by cytochrome P450 1A2 (CYP1A2) and 3A4 to a genotoxic epoxide that forms N7-guanine DNA adducts. A series of yeast haploid mutants defective in DNA repair and cell cycle checkpoints were transformed with human CYP1A2 to investigate how these DNA adducts are repaired. Cell survival and mutagenesis following aflatoxin B1 treatment was assayed in strains defective in nucleotide excision repair (NER) (rad14), postreplication repair (PRR) (rad6, rad18, mms2, and rad5), homologous recombinational repair (HRR) (rad51 and rad54), base excision repair (BER) (apn1 apn2), nonhomologous end-joining (NHEJ) (yku70), mismatch repair (MMR) (pms1), translesion synthesis (TLS) (rev3), and checkpoints (mec1-1, mec1-1 rad53, rad9, and rad17). Together our data suggest the involvement of homologous recombination and nucleotide excision repair, postreplication repair, and checkpoints in the repair and/or tolerance of AFB1-induced DNA damage in the yeast model. Rev3 appears to mediate AFB1-induced mutagenesis when error-free pathways are compromised. The results further suggest unique roles for Rad5 and abasic endonuclease-dependent DNA intermediates in regulating AFB1-induced mutagenicity.     

Decreased UV mutagenesis in cdc8, a DNA replication mutant of Saccharomyces cerevisiae.

Summary A DNA replication mutant of yeast, cdc8, was found to decrease UV-induced reversion of lys2-1, arg4-17, tyr1 and ura1. This effect was observed with all three alleles of cdc8 tested. Survival curves obtained following UV irradiation in cdc8 rad double mutants show that cdc8 is epistatic to rad6, as well as to rad1; cdc8 rad51 double mutants seem to be more sensitive than the single mutants. Since UV-induced reversion in cdc8 rad1 and cdc8 rad51 double mutants is like that of the cdc8 single mutants, we conclude that CDC8 plays a direct role in error-prone repair. To test whether CDC8 codes for a DNA polymerase, we have purified both DNA polymerase I and DNA polymerase II from cdc8 and CDC+ cells. The purified DNA polymerases from cdc8 were no more heat labile than those from CDC+, suggesting that CDC8 is not a structural gene for either enzyme.     

UV-induced reversion of his4 frameshift mutations in rad6, rev1, and rev3 mutants of yeast

Summary The UV-induced reversion of two his4 frameshift alleles was much reduced in rad6 mutants of Saccharomyces cerevisiae, an observation that is consistent with the hypothesis that RAD6 function is required for the induction of all types of genetic alteration in misrepair mutagenesis. The reversion of these his4 alleles, together with two others of the same type, was also reduced in rev1 and rev3 mutant strains; in these, however, the extent of the reduction varied considerably with test allele used, in a manner analogous to the results in these strains for base repair substitution test alleles. The general features of UV-induced frameshift and substitution mutagenesis therefore appear quite similar, indicating that they may depend on related processes. If this conclusion is correct, greater attention must be given to integrating models which account for the production of nucleotide additions and deletions into those concerning misrepair mutagenesis.