Specificity of spontaneous mutations induced in mutA mutator cells

Escherichia coli cells expressing the mutA allele of a glyV (glycine tRNA) gene express a strong mutator phenotype. The mutA allele differs from the wild type glyV gene by a base substitution in the anticodon such that the resulting tRNA misreads certain aspartate codons as glycine, resulting in random, low-level Asp-->Gly substitutions in proteins. Subsequent work showed that many types of mistranslation can lead to a very similar phenotype, named TSM for translational stress-induced mutagenesis. Here, we have determined the specificity of forward mutations occurring in the lacI gene in mutA cells as well as in wild type cells. Our results show that in comparison to wild type cells, base substitutions are elevated 23-fold in mutA cells, as against a eight-fold increase in insertions and a five-fold increase in deletions. Among base substitutions, transitions are elevated 13-fold, with both G:C-->A:T and A:T-->G:C mutations showing roughly similar increases. Transversions are elevated 35-fold, with G:C-->T:A, G:C-->C:G and A:T-->C:G elevated 28-, 13- and 27-fold, respectively. A:T-->T:A mutations increase a striking 348-fold over parental cells, with most occurring at two hotspot sequences that share the G:C-rich sequence 5'-CCGCGTGG. The increase in transversion mutations is similar to that observed in cells defective for dnaQ, the gene encoding the proofreading function of DNA polymerase III. In particular, the relative proportions and sites of occurrence of A:T-->T:A transversions are similar in mutA and mutD5 (an allele of dnaQ) cells. Interestingly, transversions are also the predominant base substitutions induced in dnaE173 cells in which a missense mutation in the alpha subunit of polymerase III abolishes proofreading without affecting the 3'-->5' exonuclease activity of the epsilon subunit.     

Stabilization of yeast artificial chromosome clones in a rad54-3 recombination-deficient host strain.

The cloning and propagation of large fragments of DNA on yeast artificial chromosomes (YACs) has become a routine and valuable technique in genome analysis. Unfortunately, many YAC clones have been found to undergo rearrangements or deletions during the cloning process. The frequency of transformation-associated alterations and mitotic instability can be reduced in a homologous recombination-deficient yeast host strain such as a rad52 mutant. RAD52 is one member of an epistatic group of genes required for the recombinational repair of double-strand breaks in DNA. rad52 mutants grow more slowly and transform less efficiently than RAD + strains and are therefore not ideal hosts for YAC library construction. We have investigated the ability of both null and temperature-sensitive alleles of RAD54 , another member of the RAD52 epistasis group, to prevent rearrangements of human YAC clones containing tandemly repeated DNA sequences. Our results show that the temperature-sensitive rad54-3 allele blocks mitotic recombination between tandemly repeated DYZ3 satellite sequences and significantly stabilizes a human DYZ5 satellite-containing YAC clone. Yeast carrying the rad54-3 mutation can undergo meiosis, have growth and transformation rates comparable with RAD + strains, and therefore represent improved YAC cloning hosts.     

Dose-dependent changes in the spectrum of mutations induced by ionizing radiation

We examined the influence of dose on the spectrum of mutations induced at the hypoxanthine guanine phosphoribosyltransferase (Hprt) locus in Chinese hamster ovary (CHO) cells. Independent CHO-K1 cell mutants at the Hprt locus were isolated from cells exposed to 0, 0.5, 1.5, 3.0 and 6.0 Gy (137)Cs gamma rays, and the genetic changes responsible for the mutations were determined by multiplex polymerase chain reaction (PCR)-based exon deletion analysis. We observed dose-dependent changes in mutation spectra. At low doses, the principal radiation-induced mutations were point mutations. With increasing dose, multibase deletion mutations became the predominant mutation type such that by 6.0 Gy, there were almost three times more deletion mutations than point mutations. The dose response for induction of point mutations was linear while that for multibase deletions fit a linear-quadratic response. There was a biphasic distribution of deletion sizes, and different dose responses for small compared to large deletions. The frequency of large (>36 kb) total gene deletions increased exponentially, implying that they develop from the interaction between two independent events. In contrast, the dose response for deletion mutations of less than 10 kb was nearly linear, suggesting that these types of mutations develop mostly from single events and not the interactions between two independently produced lesions. The observation of dose-dependent changes in radiation-induced mutation spectra suggests that the types of alterations and therefore the risks from low-dose radiation exposure cannot be easily extrapolated from high-dose effects.     

Analysis of lethal mutations induced in a mutator strain that activates transposable elements in Caenorhabditis elegans

A screen was conducted for lethal mutations in the nematode Caenorhabditis elegans in a strain containing the mutator mut-4 (st700)I to examine the nature of mutator-induced lethal mutations within two large chromosomal regions comprising a total of 49 map units (linkage group IV (right) and linkage group V (left)). The genetic analysis of 28 lethal mutations has revealed that the mutator locus mut-4(st700)I causes both putative single-gene mutations and deficiencies. We have identified lethal mutations in three different genes, in addition to seven deficiencies. There is a mutational hot spot on linkage group V (left) around the lin-40 locus. Six mutations appear to be alleles of lin-40. In addition, 5 of 7 deficiencies have breakpoints at or very near lin-40. All seven deficiencies delete the left-most known gene on linkage group V (left) and thus appear to delete the tip of the chromosome. This is in contrast to gamma ray and formaldehyde induced deficiencies, which infrequently delete the closest known gene to the tip of a chromosome.     

The yeast rad18 mutator specifically increases G.C----T.A transversions without reducing correction of G-A or C-T mismatches to G.C pairs.

Inactivation of the Saccharomyces cerevisiae RAD18 gene confers a mutator phenotype. To determine the specificity of this effect, a collection of 212 spontaneous SUP4-o mutants arising in a rad18 strain was characterized by DNA sequencing. Comparison of the resulting mutational spectrum with that for an isogenic wild-type (RAD18) strain revealed that the rad18 mutator specifically enhanced the frequency of single base pair substitutions. Further analysis indicated that an increase in the frequency of G.C----T.A transversions accounted for the elevated SUP4-o mutation frequency. Thus, rad18 is the first eucaryotic mutator found to generate only a particular base pair substitution. The majority of G.C pairs that were not mutated in the rad18 background were at sites where G.C----T.A events can be detected in SUP4-o, suggesting that DNA sequence context influences the rad18 mutator effect. Transformation of heteroduplex plasmid DNAs into the two strains demonstrated that the rad18 mutator did not reduce the efficiency of correcting G-A or C-T mismatches to G.C pairs or preferentially correct the mismatches to A.T pairs. We propose that the RAD18 gene product might contribute to the fidelity of DNA replication in S. cerevisiae by involvement in a process that serves to limit the formation of G-A and C-T mismatches at template guanine and cytosine sites during DNA synthesis.     

Frameshift mutations induced by an Escherichia coli strain carrying a mutator gene, mutD5

We have studied eight frameshift mutations induced by the Escherichia coli mutator allele mutD5 in a derivative of the bacteriophage M13mp8, carrying an insertion of 91 base pairs derived from the tetR gene of pBR 322. All mutations were analyzed by the dideoxy sequencing method and were found to be deletions of a GC base pair which occurred in regions characterized by the presence of at least two GC base pairs. We have attempted to explain these results by the looping-out model, which was previously proposed to unify the results obtained with mutD5.     

Activity of the Escherichia coli mutT mutator allele in an anaerobic environment.

Mutation frequencies for an Escherichia coli mutT strain were measured in both aerobic and anaerobic environments. When cells were grown in a rich medium (L broth), mutation frequencies were similar in both aerobic and anaerobic conditions. In contrast, when grown in a minimal medium, mutT anaerobic mutation frequencies were reduced dramatically compared with aerobic values, which were similar to L broth frequencies. L broth mutT cultures treated with a commercial enzyme complex that reduces free oxygen in the medium also showed strongly reduced anaerobic mutation frequencies. These results indicate that the biological role of the MutT protein is to prevent oxidative damage from becoming mutagenic.     

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.     

Nuclear and extranuclear mutations in yeast induced by ethyl methanesulfonate

When zero-point mutations were induced in the yeastSaccharomyces cerevisiae using ethyl methanesulfonate (EMS) no differences were found in the frequency of auxotrophic mutants formed by a short and a prolonged treatment of the agent at equal survival level. The expression of a part of the mutations induced by a prolonged EMS treatment was delayed by one or two division cycles. The total frequency of auxotrophs due to both the zero point and delayed mutations, however, is still considerably lower than the frequency of auxotrophs induced by a prolonged treatment of EMS in some bacterial species. Both the prolonged and short EMS treatment induces in yeast also extranuclear respiration-deficient (RD) mutants at a relatively high frequency; in wild strains at equal survival level the prolonged treatment produces a higher number of RD mutants than the short one. In strain which is more susceptible to the lethal EMS effect than wild strain the number of RD mutants produced by the agent is much higher than in the wild strain. The results support the assumption of the different DNA arrangement in yeast nuclei and mitochondria and indicate the possible effect of repair mechanisms during the induction of mutations causing the respiration deficiency.     

A similar defect in UV-induced mutagenesis conferred by the rad6 and rad18 mutations of Saccharomyces cerevisiae

The single rad6 and rad18 yeast mutants share a number of physiological and biochemical properties related to DNA repair, suggesting that they affect closely related steps. However, it has been reported that UV-induced mutagenesis is considerably more depressed in rad6 than it is in rad18 cells. In an attempt to better understand the role of these genes, a genetic system believed to differentiate between targeted and untargeted events was used. The data are interpreted to mean that both mutations prevent the occurrence of targeted events, as if they prevent error-prone replication in front of pyrimidine dimers. The number of non-targeted mutants per survivor in each mutant was increased by UV irradiation. This may correspond to a stimulation of the error-prone replication.     

A novel allele of fission yeast rad11 that causes defects in DNA repair and telomere length regulation

Replication protein A (RPA) is a heterotrimeric single-stranded DNA-binding protein involved in DNA replication, recombination and repair. In Saccharomyces cerevisiae, several mutants in the RFA1 gene encoding the large subunit of RPA have been isolated and one of the mutants with a missense allele, rfa1-D228Y, shows a synergistic reduction in telomere length when combined with a yku70 mutation. So far, only one mutant allele of the rad11⁺ gene encoding the large subunit of RPA has been reported in Schizosaccharomyces pombe. To study the role of S.pombe RPA in DNA repair and possibly in telomere maintenance, we constructed a rad11-D223Y mutant, which corresponds to the S.cerevisiae rfa1-D228Y mutant. rad11-D223Y cells were methylmethane sulfonate, hydroxyurea, UV and γ-ray sensitive, suggesting that rad11-D223Y cells have a defect in DNA repair activity. Unlike the S.cerevisiae rfa1-D228Y mutation, the rad11-D223Y mutation itself caused telomere shortening. Moreover, Rad11-Myc bound to telomere in a ChIP assay. These results strongly suggest that RPA is directly involved in telomere maintenance.     

The yeast prion [URE3] can be greatly induced by a functional mutated URE2 allele

The non-Mendelian element [URE3] of yeast is considered to be a prion form of the Ure2 protein. The [URE3] phenotype occurs at a frequency of 10(-5) in haploid yeast strains, is reversible, and its frequency is increased by overexpressing the URE2 gene. We created a new mutant of the Ure2 protein, called H2p, which results in a 1000-fold increase in the rate of [URE3] occurrence. To date, only the overexpression of various C-terminal truncated mutants of Ure2p gives rise to a comparable level. The h2 allele is, thus, the first characterized URE2 allele that induces prion formation when expressed at a low level. By shuffling mutated and wild-type domains of URE2, we also created the first mutant Ure2 protein that is functional and induces prion formation. We demonstrate that the domains of URE2 function synergistically in cis to induce [URE3] formation, which highlights the importance of intramolecular interactions in Ure2p folding. Additionally, we show using a green fluorescent protein (GFP) fusion protein that the h2 allele exhibits numerous filiform structures that are not generated by the wild-type protein.     

Dominant negative mutator mutations in the mutL gene of Escherichia coli.

The mutL gene product is part of the dam-directed mismatch repair system of Escherichia coli but has no known enzymatic function. It forms a complex on heteroduplex DNA with the mismatch recognition MutS protein and with MutH, which has latent endonuclease activity. An N-terminal hexahistidine-tagged MutL was constructed which was active in vivo. As a first stop to determine the functional domains of MutL, we have isolated 72 hydroxylamine-induced plasmid-borne mutations which impart a dominant-negative phenotype to the wild-type strain for increased spontaneous mutagenesis. None of the mutations complement a mutL deletion mutant, indicating that the mutant proteins by themselves are inactive. All the dominant mutations but one could be complemented by the wild-type mutL at about the same gene dosage. DNA sequencing indicated that the mutations affected 22 amino acid residues located between positions 16 and 549 of the 615 amino acid protein. In the N-terminal half of the protein, 12 out of 15 amino acid replacements occur at positions conserved in various eukaryotic MutL homologs. All but one of the sequence changes affecting the C-terminal end of the protein are nonsense mutations.     

Dominant negative mutator mutations in the mutS gene of Escherichia coli.

The MutS protein of Escherichia coli is part of the dam-directed MutHLS mismatch repair pathway which rectifies replication errors and which prevents recombination between related sequences. In order to more fully understand the role of MutS in these processes, dominant negative mutS mutations on a multicopy plasmid were isolated by screening transformed wild-type cells for a mutator phenotype, using a Lac+ papillation assay. Thirty-eight hydroxylamine- and 22 N-methyl-N'-nitro-N-nitrosoguanidine-induced dominant mutations were isolated. Nine of these mutations altered the P-loop motif of the ATP-binding site, resulting in four amino acid substitutions. With one exception, the remaining sequenced mutations all caused substitution of amino acids conserved during evolution. The dominant mutations in the P-loop consensus caused severely reduced repair of heteroduplex DNA in vivo in a mutS mutant host strain. In a wild-type strain, the level of repair was decreased by the dominant mutations to between 12 to 90% of the control value, which is consistent with interference of wild-type MutS function by the mutant proteins. Increasing the wild-type mutS gene dosage resulted in a reversal of the mutator phenotype in about 60% of the mutant strains, indicating that the mutant and wild-type proteins compete. In addition, 20 mutant isolates showed phenotypic reversal by increasing the gene copies of either mutL or mutH. There was a direct correlation between the levels of recombination and mutagenesis in the mutant strains, suggesting that these phenotypes are due to the same function of MutS.     

The ultrastructural meiotic phenotype of the radiation sensitive mutant rad 6-1 in yeast

An ultrastructural analysis of three yeast rad 6-1/rad 6-1 diploids on sporulation medium for 0, 6, 10, and 24 h shows that arrest occurs at meiotic prophase. Two strains, CL 139 and PU 6, fail to complete chromosome synapsis based on the continued presence of single chromosomal cores in arrested nuclei. A clone derived from CL 139, however, showed complete pairing as evident from the presence of 17 synaptonemal complexes. All three strains underwent spindle pole body duplication but the poles failed to form a proper metaphase I spindle. A revertant Rad 6⁺ isolated from CL 139 showed normal chromosome behaviour and normal kinetic functions. It is concluded that the absence of meiotic recombination in some Rad 6^– strains may result from asynapsis, but that in other strains (e.g., CL 139s) recombination fails in spite of complete synapsis. In all cases the lack of sporulation is adequately explained by failure of the kinetic apparatus to form a metaphase I spindle.     

Specificity of mutations induced by carbon ions in budding yeast Saccharomyces cerevisiae

To investigate the nature of mutations induced by accelerated ions in eukaryotic cells, the effects of carbon-ion irradiation were compared with those of γ-ray irradiation in the budding yeast Saccharomyces cerevisiae.

The mutational effect and specificity of carbon-ion beams were studied in the URA3 gene of the yeast. Our experiments showed that the carbon ions generated more than 10 times the number of mutations induced by γ-rays, and that the types of base changes induced by carbon ions include transversions (68.7%), transitions (13.7%) and deletions/insertions (17.6%). The transversions were mainly G:C → T:A, and all the transitions were G:C → A:T. In comparison with the surrounding sequence context of mutational base sites, the C residues in the 5′-AC(A/T)-3′ sequence were found to be easily changed. Large deletions and duplications were not observed, whereas ion-induced mutations in Arabidopsis thaliana were mainly short deletions and rearrangements. The remarkable feature of yeast mutations induced by carbon ions was that the mutation sites were localized near the linker regions of nucleosomes, whereas mutations induced by γ-ray irradiation were located uniformly throughout the gene.