Hanna Baranowska

Induction by manganese of mitochondrial antibiotic resistance mutations in yeast.

SummaryManganese added to the growth medium of Saccharomyces cerevisiae to a final concentration of 4–8 mM induces not only mitochondrial respiratory-deficient mutations (Fig. 1), but also mitochondrial mutations to chloramphenicol- and erythromycin-resistance (Fig. 1, Tables 1–3). This is the first mutagen shown to be capable of inducing mitochondrial antibiotic resistance mutations in yeast. It is assumed that manganese induces mutations through its interaction with mitochondrial DNA polymerase.

Manganese mutagenesis in yeast. VI. Mn2+ uptake, mitDNA replication and ER induction: comparison with other divalent cations.

A medium was found in which manganese efficiently induces erythromycin-resistant mitochondrial mutations, and which is suitable for measuring Mn2+ uptake and the labelling of DNA (fig. 1). Mn2+ uptake is stimulated by glucose and slowed down by cycloheximide (Fig 2). Mg2+ competes with Mn2+ uptake much stronger than does Zn2+ (Fig. 3). All of the conditions which favour Mn2+ uptake also favour induction of erythromycin-resistant mutations (Tables 3, 4). Mn2+ strongly inhibits protein synthesis (Table 1). Nuclear DNA replication is also strongly inhibited by this cation, while mitochondrial DNA replication is only weakly inhibited during the first 3 h of labelling, but there is small if any increase of the label incorporation between the 3rd 6th h of labelling (Table 2). The relation between label incorporation into mitDNA and mutation induction by manganese is not straightforward (Table 5). From among 11 divalent cations tested, only Mn2+ was capable of inducing mitochondrial erythromycin-resistant mutations (Table 6).

The influence of the mismatch-repair system on stationary-phase mutagenesis in the yeast Saccharomyces cerevisiae

Abstract. Stationary-phase (also called adaptive) mutation occurs in non-dividing cells during prolonged non-lethal selective pressure, e.g. starvation for an essential amino acid. Because in such conditions no DNA replication is observed, mutations probably arise as a result of inefficient DNA repair. In order to understand the role of the yeast mismatch-repair (MMR) system in the mutagenesis of stationary-phase cells, we studied the effects of deletions in genes encoding MutS- and MutL-related proteins on the reversion frequency of the lys2ΔBgl frameshift mutation. We found that the level of Lys+ reversion was increased in all MMR mutants, with the strongest effect observed in a MSH2 (MUTS homologue)-deprived strain. Disruption of the MSH3 or MSH6 genes (also MUTS homologues) resulted in elevation of the mutation frequency and rate, but to a lesser degree than that caused by the inactivation of MSH2. MutL-related proteins were also required for mutation avoidance in stationary-phase cells, but to a lesser extent than MutS homologues. Among MutL homologues, Mlh1 seems to play the major role in this process, while Pms1 and Mlh3 are partially redundant and appear to substitute for each other. These data suggest that MMR proteins, particularly MutS homologues, are involved in the control of mutability in stationary-phase yeast cells.

Manganese mutagenesis in yeast. A practical application of manganese for the induction of mitochondrial antibiotic-resistant mutations.

When yeast cells were incubated for 4 to 8 h in yeast extract-peptone-glucose medium, pH 6, containing 8 mM-manganese, and then plated on selective media, there was a strong induction of antibiotic-resistant mutations. Indirect evidence suggests that practically all resistant mutants selected were of independent origin. The analysis of manganese-induced resistant mutants showed that most were extranuclear, while those tested showed recombination with known mitochondrial markers. Our results suggest that manganese can be considered as a mutagen which specifically induces mitochondrial mutations in Saccharomyces cerevisiae.

Effects of the CDC2 gene on adaptive mutation in the yeast Saccharomyces cerevisiae

We have studied the influence of a temperature-sensitive cdc2-1 mutation in DNA polymerase δ on the selection-induced mutation occurring at the LYS-2 locus in the yeast Saccharomyces cerevisiae. It was found that in cells plated on synthetic complete medium lacking only lysine, the numbers of Lys+ revertant colonies accumulated in a time-dependent manner in the absence of any detectable increase in cell number. When cdc2-1 mutant cells, after selective plating, were incubated at the restrictive temperature of 37°C for 5 h daily for 7 days, the frequency of an adaptive reversion of lys- →Lys+ was significantly higher than the frequency in cells incubated only at the permissive temperature, or in wild-type cells incubated either at 23°C or 37°C. Therefore, when the proof-reading activity of DNA polymerase δ is impaired under restrictive conditions, the frequency of adaptive mutations is markedly enhanced.

Manganese mutagenesis in yeast. II. Conditions of induction and characteristics of mitochondrial respiratory deficient Saccharomyces cerevisiae mutants induced with manganese and cobalt.

Manganese and cobalt are capable of inducing ρ − mutations* in non-growing cells of Saccharomyces cerevisiae , but their mutagenic action is much stronger in growing cells. At a given concentration cobalt and manganese can be either strongly mutagenic or non-mutagenic, depending on the cell density. Most of the ρ − mutants induced with manganese and a considerable proportion of those induced with cobalt are suppressive and/or transmit drug resistance markers, so they must still carry mitochondrial DNA. Cobalt can decrease suppressiveness with low efficiency and eliminate drug resistance markers from established ρ − clones.

Involvement of the RE V3 gene in the methylated base-excision repair system. Co-operation of two DNA polymerases, δ and Rev3p, in the repair of MMS-induced lesions in the DNA of Saccharomyces cerevisiae

The ability of four yeast DNA polymerase mutant strains to carry out the repair of DNA treated with MMS was studied. Mutation in DNA polymerase Rev3, as well as the already known mutation in the catalytic subunit of DNA polymerase δ, were both found to lead to the accumulation of single-strand breaks, which indicates defective repair. A double-mutant strain carrying mutations in DNA polymerase δ and a deletion in the REV3 gene had a complete repair defect, both at permissive (23°C) and restrictive (38°C) temperatures, which was not observed in other pairwise combinations of tested polymerase mutants. Other polymerases are not involved in the repair of exogenous DNA methylation damage, since neither mutation in the DNA polymerase ɛ, nor deletion in the DNA polymerase IV (β70) gene, caused defective repair. The data obtained suggest that DNA polymerases δ and Rev3p are both necessary to perform repair synthesis in the base-excision repair of methylation damage. The results are discussed in the light of current concepts on the role of DNA polymerase Rev3 in mutagenesis.

Mitochondrial mutagenesis in Saccharomyces cerevisiae : V. Frequencies of different mit (-) mutants and loss of their mit (+) alleles in rho (-) clones.

SummaryFour types of mit− mutations induced with manganese are found in the following relative proportions: oxi3− > cob-box− > oxi2− ⩾ oxi1−1. The frequences of loss of their respective mit+ alleles in manganese-induced rho−] primary and secondary clones follow the same order. The possible interdependence between these two sets of data is discussed.

On the specificity of caffeine effects

SummaryCaffeine in 0.1% or higher concentration reversibly inhibits E. coli and yeast growth. It inhibits RNA and protein synthesis (Tables 1+3) within a few minutes after being added to the incubation medium (Fig. 2). The suggestion is made that these effects of caffeine, as well as its synergism with some mutagens, are due to its ability to costack with free purines and to form complexes with single-stranded nucleic acids.

DNA polymerase III is required for DNA repair in Saccharomyces cerevisiae

We have studied the role of DNA polymerase III, encoded in S. cerevisiae by the CDC2 gene, in the repair of yeast nuclear DNA. It was found that the repair of MMS-induced single-strand breaks is defective in the DNA polymerase III temperature-sensitive mutant cdc2-1 at the restrictive temperature (37 °C), but is not affected at the permissive temperature (23 °C). Under conditions where only a small number of lesions was introduced into DNA (80% survival), the repair of MMS-induced damage could also be observed in the mutant at the restrictive temperature, although with low efficiency. When the quantity of lesions increased (50% survival or less), the repair of single-strand breaks was blocked. At the same time we observed a high rate of reversion in the meth, his and trp loci of the cdc2-1 mutant under restrictive conditions. The results presented suggest that DNA polymerase III is involved in the repair of MMS-induced lesions in yeast DNA and that the cdc2-1 mutation affects the proofreading activity of this polymerase.