Saccharomyces cerevisiae mutants characterized by increased induced mutagenesis. II. Genetic analysis of mutants

Induction of forward adenine-dependent (Ade+----Ade-) mutations by HAP was used to analyse genetically yeast mutants with enhanced induced mutagenesis. Three mutations studied in detail segregated as a single mendelian trait and composed independent complementation groups (HIM1, HIM2, HIM3). the him1-1 mutation was centromere-linked, the him3-1 and him2-1 mutations being not. All three mutations did not show any cross-linkage. Uracil-DNA glycosylase activity was determined in crude cell extract from wild type strain and him mutants; no detectable differences were observed.     

Repair system for noncanonical purines in Escherichia coli

Exposure of Escherichia coli strains deficient in molybdopterin biosynthesis (moa) to the purine base N-6-hydroxylaminopurine (HAP) is mutagenic and toxic. We show that moa mutants exposed to HAP also exhibit elevated mutagenesis, a hyperrecombination phenotype, and increased SOS induction. The E. coli rdgB gene encodes a protein homologous to a deoxyribonucleotide triphosphate pyrophosphatase from Methanococcus jannaschii that shows a preference for purine base analogs. moa rdgB mutants are extremely sensitive to killing by HAP and exhibit increased mutagenesis, recombination, and SOS induction upon HAP exposure. Disruption of the endonuclease V gene, nfi, rescues the HAP sensitivity displayed by moa and moa rdgB mutants and reduces the level of recombination and SOS induction, but it increases the level of mutagenesis. Our results suggest that endonuclease V incision of DNA containing HAP leads to increased recombination and SOS induction and even cell death. Double-strand break repair mutants display an increase in HAP sensitivity, which can be reversed by an nfi mutation. This suggests that cell killing may result from an increase in double-strand breaks generated when replication forks encounter endonuclease V-nicked DNA. We propose a pathway for the removal of HAP from purine pools, from deoxynucleotide triphosphate pools, and from DNA, and we suggest a general model for excluding purine base analogs from DNA. The system for HAP removal consists of a molybdoenzyme, thought to detoxify HAP, a deoxyribonucleotide triphosphate pyrophosphatase that removes noncanonical deoxyribonucleotide triphosphates from replication precursor pools, and an endonuclease that initiates the removal of HAP from DNA.     

Genetic analysis of mitochondrial rho-mutability in Saccharomyces. I. Polygenic determination of spontaneous rho-mutability: gene-modifiers and srm mutation

The phenotypic trait "starry colony" in Saccharomyces is associated with a high spontaneous rho- petite mutability. Genetic analysis of this trait has shown the high rho- mutability to be caused by several modifying genes present together in the cell genome. Every single modifying gene only produces a relatively small enhancement in the rho- mutability. Mutations in four nuclear srm (spontaneous rho- mutability) loci were isolated after mutagenic treatment of highly rho- mutable haploid cells. In contrast to the modifying genes, each of these mutations has a pronounced effect on the spontaneous rho- mutability, causing significant decrease in it.     

Mutants of the phototrophic bacteria Rhodobacter sphaeroides sensitive to the mutagenic action of long-wave ultraviolet light

The mutagenic action of near ultraviolet (NUV, greater than or equal to 280) nm) on purple phototrophic soil bacteria Rhodobacter sphaeroides: wild strain 2R and 12 mutants obtained earlier sensitive to UV derivates (UVS) was investigated. The mutagenic action of NUV was measured by induction of resistance to tetracycline (Tet) and nalidixic acid (Nal) and reversion of pigment mutants to wild-type phenotype. The NUV light induces the mutations of resistance to Nal and Tet in wild-type strain 2R; the UVS mutants differed greatly in their NUV-induced mutability. Three UVS mutants were characterized by greatly increased mutability in all analysed loci; slight mutability was found in seven mutants. On the basis of the data obtained it has been concluded that the UVS mutants R. sphaeroides can be used as test organisms in estimation of mutagenic activity of NUV. The molecular mechanisms and genetic control of NUV-induced mutagenesis are discussed.     

Perspective on mutagenesis and repair: the standard model and alternate modes of mutagenesis

The basic ideas of replication, mutagenesis, and repair have outlined a picture of how point mutations occur that has provided a valuable framework for theory and experiment, much as the Standard Model of particle physics has done for our concept of fundamental particles. However, alternative modes of mutagenesis are being defined that are changing our perspective of the "Standard Model" of mutagenesis, requiring an expanded model. The genome is now envisioned as being in dynamic equilibrium between a multitude of forces for mutational change and forces that counteract such change. By maintaining a delicate balance between these forces, cells avoid unwanted or excessive mutations. Yet, cells allow mutagenesis to occur under certain conditions. We can define an emerging paradigm. Namely, mechanisms exist that can direct point mutations to specific designated genes or regions of genes. In some cases, this is achieved by specific enzymes, and in other cases high mutability is programmed into the sequence of certain genes to help generate diversity. In yet additional cases, general mutability is increased under stress, and selective forces allow the recovery of favorable mutants.     

Genome-Wide Mutation Avalanches Induced in Diploid Yeast Cells by a Base Analog or an APOBEC Deaminase

Genetic information should be accurately transmitted from cell to cell; conversely, the adaptation in evolution and disease is fueled by mutations. In the case of cancer development, multiple genetic changes happen in somatic diploid cells. Most classic studies of the molecular mechanisms of mutagenesis have been performed in haploids. We demonstrate that the parameters of the mutation process are different in diploid cell populations. The genomes of drug-resistant mutants induced in yeast diploids by base analog 6-hydroxylaminopurine (HAP) or AID/APOBEC cytosine deaminase PmCDA1 from lamprey carried a stunning load of thousands of unselected mutations. Haploid mutants contained almost an order of magnitude fewer mutations. To explain this, we propose that the distribution of induced mutation rates in the cell population is uneven. The mutants in diploids with coincidental mutations in the two copies of the reporter gene arise from a fraction of cells that are transiently hypersensitive to the mutagenic action of a given mutagen. The progeny of such cells were never recovered in haploids due to the lethality caused by the inactivation of single-copy essential genes in cells with too many induced mutations. In diploid cells, the progeny of hypersensitive cells survived, but their genomes were saturated by heterozygous mutations. The reason for the hypermutability of cells could be transient faults of the mutation prevention pathways, like sanitization of nucleotide pools for HAP or an elevated expression of the PmCDA1 gene or the temporary inability of the destruction of the deaminase. The hypothesis on spikes of mutability may explain the sudden acquisition of multiple mutational changes during evolution and carcinogenesis.     

Isolation and characterisation of nuclear mutants with enhanced mitochondrial mutability in the fission yeast Schizosaccharomyces pombe

In this paper we report the isolation and preliminary characterisation of nuclear mutants with increased mitochondrial mutability in fission yeast. Screening of about 2000 clones after nitrosoguanidine mutagenesis led to the isolation of ten mutator mutants. For one of them (mut-1) we show that the mutation is chromosomally encoded. The activity of the mutator is restricted to the mitochondrial genome, since it increases the mutation rate to mitochondrially encoded drug resistance considerably, whereas the mutability of nuclear genes is not altered.     

Studies on the ultraviolet-sensitive mutants of blue-green alga Synechocystis aquatilis Sanv.

Summary Several mutants of the unicellular blue-green alga Synechocystis aquatilis Sanv. were isolated. They differed from the wild type by the levels of sensitivity to ultraviolet (UV) irradiation. The most sensitive mutant is unable to carry out photoreactivation and shows increased resistance to mitomycin C, N-methyl-N-nitro-N-nitrosoguanidine and methyl methanesulfonate. This strain shows an enhanced rate of spontaneous and UV-induced mutagenesis. Another UV-sensitive mutant with normal level of X-ray sensitivity is characterized by a decreased mutability. The three other UV-sensitive mutants show simultaneous decrease of resistance to X-ray and alkylating agents. The existence of these cross-sensitive mutants indicates that a repair mechanism may operate in blue-green algae similar to dark repair systems of bacteria and yeast.     

Mutability of LYS2 gene in diploid Saccharomyces yeasts. II. Frequency of mutants induced by 6-N-hydroxylaminopurine and propiolactone

Chemical mutagens 6-N-hydroxylaminopurine (HAP) and propiolactone (PRO) induce Lys2 mutants with high frequency in diploid yeast Saccharomyces cerevisiae. HAP induces such mutants even in tetraploid strains. The genetic analysis of mutants was performed. It is shown that PRO induces mutants by means of "mutation-mitotic segregation" mechanism, while HAP induces mutants through novel mechanism "both allele mutation". Manifestation of such mechanism is the null fertility after meiosis of diploid mutants induced by HAP.     

Isolation of conditional lethal mutator mutants of Escherichia coli by localized mutagenesis.

By using localized mutagenesis with N-methyl-N'-nitro-N-nitrosoguanidine, we isolated 39 temperature-sensitive growth mutants that exhibited high mutability when the bacteria were grown at the permissive temperature. Two of the mutations, dnaQ186 and dnaQ231, were shown to be new alleles of the dnaQ gene by genetic mapping and complementation tests with the dnaQ49 mutation previously isolated. They shared common properties with the dnaQ49 strain, but their mutator activity was not temperature dependent. The dnaQ mutants exhibited increased sensitivity to inhibitors of DNA gyrase and to DNA intercalating and alkylating agents.     

Multiple mutants of Saccharomyces cerevisiae. IV. Mutability of yeast cultures at different growth stages]

Mutability at different stages of culture growth in liquid media of two yeast strains, which revealed a property of "multiple mutability" and one strain of wild type for this property, was studied. One strain that possessed the property of "multiple mutability" showed at stationary phase a very high frequency of mutability, which reached 3.5%. It was found that multiple mutants arose in both strains, that possessed the property under investigation, at lag- and log-growth phases, and only in one strain -- at the stationary phase of growth. The possible reasons of "multiple mutability" display are discussed.     

Mutants of Saccharomyces cerevisiae characterized by increased level of induced mutagenesis. I. Isolation and preliminary characterization of mutants

6 mutants with enhanced nitrous acid-induced reversibility of the ade2-42 allele were isolated and designated hm (high mutagenesis). Apart from sensitivity to the mutagenic exposure to nitrous acid, hm mutants were also spontaneous mutators and hypermutable under the action of UV-light and 6-N-hydroxyaminopurine. All these effects were detected not only when analysing reversibility of the ade2-42 allele, but also when scoring forward mutations in the ADE1, ADF2 genes. Gamma-mutagenesis, however, was not affected by hm mutations.     

Inheritance of disease lesion mimic leaf trait in groundnut

In groundnut, two identical mutants with disease lesion mimic leaf trait were isolated independently from two different parents through induced mutagenesis and in vitro culture technique. The leaf chlorophyll content in both the mutants was found to be drastically reduced. The segregation pattern in the F(2) and F(3) generations for normal and mutant traits fitted a 13:3 ratio, indicating that the disease lesion mimic trait in the mutants was due to suppressive gene action. Both mutants were allelic for the disease mimic trait.     

Perspective on Mutagenesis and Repair: The Standard Model and Alternate Modes of Mutagenesis

ABSTRACT

The basic ideas of replication, mutagenesis, and repair have outlined a picture of how point mutations occur that has provided a valuable framework for theory and experiment, much as the Standard Model of particle physics has done for our concept of fundamental particles. However, alternative modes of mutagenesis are being defined that are changing our perspective of the “Standard Model” of mutagenesis, requiring an expanded model. The genome is now envisioned as being in dynamic equilibrium between a multitude of forces for mutational change and forces that counteract such change. By maintaining a delicate balance between these forces, cells avoid unwanted or excessive mutations. Yet, cells allow mutagenesis to occur under certain conditions. We can define an emerging paradigm. Namely, mechanisms exist that can direct point mutations to specific designated genes or regions of genes. In some cases, this is achieved by specific enzymes, and in other cases high mutability is programmed into the sequence of certain genes to help generate diversity. In yet additional cases, general mutability is increased under stress, and selective forces allow the recovery of favorable mutants.     

The base analog 6-N-hydroxylaminopurine (HAP) mutagenesis is dependent on the integrity of the uvsE, uvsF and uvsB genes in Aspergillus nidulans

Most of the available data in lower eukaryotes are consistent with the idea that base analogs-induced mutagenesis is due to the mis-pairing properties of these compounds, which, in turn, is due to a shift in the tautomeric equilibrium of the molecule. A tautomeric shift may in fact lead to mismatches which, at least in Escherichia coli, can be repaired by genes involved in the post-replicative mismatch repair whose activity is necessary to control spontaneous mutagenesis. In filamentous fungi, such as Aspergillus nidulans, nothing is known about the repair of base pairing mistakes after base analogs treatment. For this reason, we have decided to screen UV-sensitive Aspergillus nidulans mutants for their mutagenic response to 6-N-hydroxylaminopurine (HAP). We have shown that three mutations (uvsB, uvsC and uvsE), which enhance the UV-sensitivity of germinating conidia, cause a lower mutagenic response to HAP. On the other hand, the uvsH mutation, has no effect on HAP-induced mutagenesis.     

Uvm mutants of Escherichia coli K 12 deficient in UV mutagenesis

Summary Selection for defective reversion induction, after UV treatment of E. coli K 12, yielded uvm mutants. These mutants exhibited highly reduced or no UV mutability for all loci tested although they were moderately and normally mutable by X-rays and EMS, respectively. Uvm mutations confer only a slight sensitivity to killing by UV and X-rays and no clear sensitivity to the lethal effect of HN2, EMS or MMS. Growth and viability of untreated uvm cells were normal. The properties of uvm mutants are discussed in relation to those of other relevant mutant types and to some actual problems of induced mutagenesis.     

Molecular Genetic Studies of the Cdc7 Protein Kinase and Induced Mutagenesis in Yeast

The Saccharomyces cerevisiae CDC7 gene encodes a protein kinase that functions in DNA replication, repair, and meiotic recombination. The sequence of several temperature-sensitive (ts) cdc7 mutations was determined and correlated with protein kinase consensus domain structure. The positions of these ts alleles suggests some general principles for predicting ts protein kinase mutations. Pedigree segregation lag analysis demonstrated that all of the mutant proteins are less active or less stable than wild-type Cdc7p. Two new mutations were constructed, one by site-directed and the other by insertional mutagenesis. All of the cdc7 mutants were assayed for induced mutagenesis in response to mutagenic agents at the permissive temperature. Some cdc7 mutants were found to be hypomutable, while others are hypermutable. The differences in mutability are observed most clearly when log phase cells are used. Both hypo- and hypermutability are recessive to wild type. Cdc7p may participate in DNA repair by phosphorylating repair enzymes or by altering chromatin structure to allow accessibility to DNA lesions.