Isolation of bacteriophage T4 DNA polymerase mutator mutants

More than 20 new bacteriophage T4 DNA polymerase mutants have been isolated by a procedure designed to select mutants with high spontaneous mutation rates. Some of the mutants produce the highest mutation frequencies that have been observed in T4 thus far. The design of the selection procedure allows for the isolation of mutator mutants that preferentially induce certain types of replication errors, and some of the mutator mutants have mutational specificities different from wild-type. The new mutants are clustered at just two sites in the DNA polymerase gene, and this result confirms an earlier observation.     

Oenothera chloroplast DNA polymorphisms associated with plastome mutator activity

Summary Oenothera plants homozygous for a recessive allele at the plastome mutator (pm) locus show non-Mendelian mutation frequencies that are 1000-fold higher than spontaneous levels. Chloroplast DNA (cpDNA) was isolated from nine mutants and two green isolates of the plastome mutator line. cpDNA restriction patterns were compared to cpDNA from a representative of the progenitor Johansen strain, and cpDNAs from all eleven plastome mutator lines show changes of fragment mobility due to deletion events at five discrete regions of the plastome. Most of the mutants have cpDNA restriction patterns identical to that of one of the green isolates from the plastome mutator line, and therefore, most of the differences in fragment length are probably not responsible for the mutant phenotypes. In contrast to the plastome mutator line, cpDNA from several populations of a closely related wild-type Oenothera species have few restriction fragment length polymorphisms. This suggests that both mutation frequencies and site-specific cpDNA deletions are elevated in the plastome mutator line, and implicates a defect in the cpDNA repair or replication machinery.     

Isolation and characteristics of new mutants of Saccharomyces cerevisiae with increased spontaneous mutability]

To isolate some new genes controlling the process of spontaneous mutagenesis, a collection of 16 yeast strains with enhanced rate of spontaneous canavanine resistant mutations was obtained. Genetical analysis allowed to define that the mutator phenotype of these strains is due to a single nuclear mutation. Such mutations were called hsm (high spontaneous mutagenesis). Recombinational test showed that 5 mutants under study carried 5 nonallelic mutations. It was revealed that the mutation hsm3-1 is a nonspecific mutator elevating the rate of both spontaneous canavanine resistant mutations and the frequency of reversions in mutations lys1-1 and his1-7. Genetical analysis revealed that mutation hsm3-1 is recessive. The study of cross sensitivity of mutator strains to physical and chemical mutagens demonstrated that 12 of 16 hsm mutants were resistant to the lethal action of UV, gamma rays and methylmethanesulfonate, and 4 mutants were only sensitive to these factors. Possible nature of hsm mutations is discussed.     

Rates of spontaneous mutation in an archaeon from geothermal environments.

To estimate the efficacy of mechanisms which may prevent or repair thermal damage to DNA in thermophilic archaea, a quantitative assay of forward mutation at extremely high temperature was developed for Sulfolobus acidocaldarius, based on the selection of pyrimidine-requiring mutants resistant to 5-fluoro-orotic acid. Maximum-likelihood analysis of spontaneous mutant distributions in wild-type cultures yielded maximal estimates of (2.8 +/- 0.7) x 10(-7) and (1.5 +/- 0.6) x 10(-7) mutational events per cell per division cycle for the pyrE and pyrF loci, respectively. To our knowledge, these results provide the first accurate measurement of the genetic fidelity maintained by archaea that populate geothermal environments. The measured rates of forward mutation at the pyrE and pyrF loci in S. acidocaldarius are close to corresponding rates reported for protein-encoding genes of Escherichia coli. The normal rate of spontaneous mutation in E. coli at 37 degrees C is known to require the functioning of several enzyme systems that repair spontaneous damage in DNA. Our results provide indirect evidence that S. acidocaldarius has cellular mechanisms, as yet unidentified, which effectively compensate for the higher chemical instability of DNA at the temperatures and pHs that prevail within growing Sulfolobus cells.     

Mutations in uvrD induce the SOS response in Escherichia coli.

We have isolated three new mutations in uvrD that increase expression of the Escherichia coli SOS response in the absence of DNA damage. Like other uvrD (DNA helicase II) mutants, these strains are sensitive to UV irradiation and have high spontaneous mutation frequencies. Complementation studies with uvrD+ showed that UV sensitivity and spontaneous mutator activity were recessive in these new mutants. The SOS-induction phenotype, however, was not completely complemented, which indicated that the mutant proteins were functioning in some capacity. The viability of one of the mutants in combination with rep-5 suggests that the protein is functional in DNA replication. We suggest that these mutant proteins are deficient in DNA repair activities (since UV sensitivity is complemented) but are able to participate in DNA replication. We believe that defective DNA replication in these mutants increases SOS expression.     

Mutagen sensitivities and mutator effects of MMS-sensitive mutants in Neurospora

7 mus (mutagen-sensitive) mutants of Neurospora crassa, which are more sensitive to the toxic effects of MMS (methyl methanesulfonate) than wild-type, were investigated for cross-sensitivities to other mutagens and inhibitors. These mutants have recently been mapped in 5 new genes, mus-7 to mus-11, and mutant alleles from each gene were checked for their effects on mutation frequencies. It was found that mutants in 3 of these 5 genes showed radiation-induced mutation frequencies similar to wild-type. These included 2 alleles of the gene mus-10, which were cross-sensitive only to UV and were the only mutants that produced some viable ascospores in homozygous crosses. The mutant of the second gene, mus-8, was especially sensitive to UV and mitomycin C and produced slightly reduced frequencies of spontaneous mutation. In contrast, the mutant of the third gene, mus-7, was not UV-sensitive but showed some cross-sensitivity to X-rays; mus-7 was highly sensitive to MMS and also to histidine, which inhibits various repair-defective mutants at concentrations well below those that reduce wild-type growth. None of these mus resemble mutants previously found in Neurospora, nor do they conform clearly to mutant types identified in E. coli or yeast. On the other hand mutants in 2 further genes, mus-11, and especially 2 alleles of mus-9, are very similar to uvs-3 of Neurospora and generally resemble mutants that are considered to be defective in "error-prone" repair. They were UV- as well as X-ray-sensitive, and showed strong spontaneous mutator effects but almost no increase in recessive lethal frequencies in heterokaryons after UV-treatments.     

Isolation and characterization of mutator mutants from cultured mouse FM3A cells

A method to select mutator mutants was developed and 3 mutants were isolated from cultured mouse FM3A cells. Fluctuation analyses revealed that these mutator mutants have increased rates of spontaneous mutation at 3 genetic loci tested (resistance to ouabain, blasticidin S and tunicamycin). None of the 3 mutator mutants showed altered sensitivity to aphidicolin or arabinofuranosylcytosine, and so they differed from the mammalian mutator mutants reported previously. Also, all the mutator mutants had the same sensitivity as wild-type to UV or other DNA-damaging agents. Thus, these mutator mutants do not seem to have any deficiency in the DNA-repair process.

To determine whether the mutator activity was due to the intracellular dNTP pool imbalance, 4 dNTPs in these mutator mutants were determined by high-pressure liquid chromatography and compared to that of the wild-type cells. The results show that there is no large dNTP pool imbalance in these mutator mutants. Since the mutator activity is not associated with the dNTP pool imbalance, these mutants may have altered protein(s) directly involved in DNA replication.     

An improved method for isolation of mutator mutants from mouse FM3A cells and their characterization

An improved method to select mutator mutants was developed. By this new method, mutator mutants were isolated efficiently, and 7 mutants were obtained from cultured mouse FM3A cells. These mutator mutants have an elevated rate of spontaneous mutation at 3 genetic loci (resistance to ouabain, blasticidin S, and tunicamycin). The sensitivity of these mutants to aphidicolin and arabinofuranosylcytosine was the same as in the wild-type cells. Determination of the size of the cellular dNTP pool revealed that there was no large imbalance in the precursor pool in the mutator mutants. These results suggested that the mutator character may be due to alteration in some factor(s) correlated directly to DNA replication. Also, there was no change in the sensitivity of all these mutator mutants to DNA damaging agents.     

Isolation and characterization of mutator strains of Escherichia coli K-12.

A selection procedure was devised to select for mutants of Escherichia coli K-12 with enhanced rates of spontaneous frameshift mutation. Three types of mutants were isolated. Two of the mutations apparently represent alleles of previously isolated mutL13 and mutS3. The third type of mutation, represented by two alleles, lies between lysA and thyA, and has been designated mutR. mutR increases the rate of spontaneous frameshift mutation and also the rate of base substitution mutations. The mutator phenotype is recessive. Reversion of a lac amber mutation located on an episome is increased in the presence of the mutator, indicating that mutR can act in trans. No change in sensitivity to ultraviolet irradiation or mitomycin C could be found when mutR34 was compared to the isogenic mutR+ strain. The mutator's activity was little affected by the type of medium in which the strain was grown. Deoxyribonucleoside triphosphate pools were normal in mutR34. Intergenic recombination frequencies were the same in mutR and mutR and mutR+ strains, but a two- to threefold increase in intragenic recombination was observed in Hfr times Fminus crosses when the recipeint was mutR34 as compared with mutR+. This increase appeared independent of the distance between the two markers within the gene in which the crossover took place.     

Isolation and characterization of mutants with deletions in dnaQ, the gene for the editing subunit of DNA polymerase III in Salmonella typhimurium.

dnaQ (mutD) encodes the editing exonuclease subunit (epsilon) of DNA polymerase III. Previously described mutations in dnaQ include dominant and recessive mutator alleles as well as leaky temperature-sensitive alleles. We describe the properties of strains bearing null mutations (deletion-substitution alleles) of this gene. Null mutants exhibited a growth defect as well as elevated spontaneous mutation. As a consequence of the poor growth of dnaQ mutants and their high mutation rate, these strains were replaced within single colonies by derivatives carrying an extragenic suppressor mutation that compensated the growth defect but apparently not the mutator effect. Sixteen independently derived suppressors mapped in the vicinity of dnaE, the gene for the polymerization subunit (alpha) of DNA polymerase III, and one suppressor that was sequenced encoded an altered alpha polypeptide. Partially purified DNA polymerase III containing this altered alpha subunit was active in polymerization assays. In addition to their dependence on a suppressor mutation affecting alpha, dnaQ mutants strictly required DNA polymerase I for viability. We argue from these data that in the absence of epsilon, DNA replication falters unless secondary mechanisms, including genetically coded alteration in the intrinsic replication capacity of alpha and increased use of DNA polymerase I, come into play. Thus, epsilon plays a role in DNA replication distinct from its known role in controlling spontaneous mutation frequency.     

Synergistic effect of an Escherichia coli mutator gene on mutagenesis by ultraviolet radiation and by alkylating agents

E. coli strains differing in a gene responsible for high spontaneous mutability (mut HI) were compared for their mutability by UV radiation and by the alkylating agents ethyl methanesulfonate and methyl methanesulfonate. All three exogenous mutagenic agents induced significantly higher frequencies of mutants with impaired carbohydrate-fermenting ability when the mutator allele rather than the wild-type allele was present. Thus the mut HI gene product possibly increases the probability of replication error due to alterations in the structure of the template strand of DNA. An attempt to detect an synergistic effect for UV-induced suppressor mutations was unsuccessful. The failure may have been due to the particular method used for scoring this type of mutation.     

Autosomal recessive lethal mutations in two mutator stocks of Drosophila ananassae.

The frequency of recessive lethals in the 2nd chromosome was examined in two mutator stocks of Drosophila ananassae, ca and ca; px. They are characterized respectively by possessing an extrachromosomal clastogenic mutator in males, and by the retrotransposon "tom", which induces Om mutability only in females. The frequencies of recessive lethal mutations in the 2nd chromosome among progenies from males and females of the ca; px stock are 0.35 and 0.34 percent, respectively. Similarity of these frequencies indicates that tom does not induce recessive lethals in females. In contrast to the ca; px stock, the frequency of recessive lethals in males of the ca mutator stock was estimated to be 1.54 percent for the 2nd chromosome. No visible mutants except Minutes were recovered. Some recessive lethals derived from ca stock males were associated with chromosomal rearrangements. Being consistent with its high rate of Minute mutation it was demonstrated that the ca clastogenic mutator also induced recessive lethals.     

Molecular characteristics of spontaneous deletions in the hyperthermophilic archaeon Sulfolobus acidocaldarius

Prokaryotic genomes acquire and eliminate blocks of DNA sequence by lateral gene transfer and spontaneous deletion, respectively. The basic parameters of spontaneous deletion, which are expected to influence the course of genome evolution, have not been determined for any hyperthermophilic archaeon. We therefore screened a number of independent pyrimidine auxotrophs of Sulfolobus acidocaldarius for deletions and sequenced those detected. Deletions accounted for only 0.4% of spontaneous pyrE mutations, corresponding to a frequency of about 10(-8) per cell. Nucleotide sequence analysis of five independent deletions showed no significant association of the endpoints with short direct repeats, despite the fact that several such repeats occur within the pyrE gene and that duplication mutations in pyrE reverted at high frequencies. Endpoints of the spontaneous deletions did not coincide with short inverted repeats or potential stem-loop structures. No consensus sequence common to all the deletions could be identified, although two deletions showed the potential of being stabilized by octanucleotide sequences elsewhere in pyrE, and another pair of deletions shared an octanucleotide at their 3' ends. The unusually low frequency and low sequence dependence of spontaneous deletions in the S. acidocaldarius pyrE gene compared to other genetic systems could not be explained in terms of possible constraints imposed by the 5-fluoroorotate selection.     

A Major Role for Bacteriophage T4 DNA Polymerase in Frameshift Mutagenesis

T4 DNA polymerase strongly influences the frequency and specificity of frameshift mutagenesis. Fifteen of 19 temperature-sensitive alleles of the DNA polymerase gene substantially influenced the reversion frequencies of frameshift mutations measured in the T4 rII genes. Most polymerase mutants increased frameshift frequencies, but a few alleles (previously noted as antimutators for base substitution mutations) decreased the frequencies of certain frameshifts while increasing the frequencies of others. The various patterns of enhanced or decreased frameshift mutation frequencies suggest that T4 DNA polymerase is likely to play a variety of roles in the metabolic events leading to frameshift mutation. A detailed genetic study of the specificity of the mutator properties of three DNA polymerase alleles (tsL56, tsL98 and tsL88) demonstrated that each produces a distinctive frameshift spectrum. Differences in frameshift frequencies at similar DNA sequences within the rII genes, the influence of mutant polymerase alleles on these frequencies, and the presence or absence of the dinucleotide sequence associated with initiation of Okazaki pieces at the frameshift site has led us to suggest that the discontinuities associated with discontinuous DNA replication may contribute to spontaneous frameshift mutation frequencies in T4.     

Evidence for joint genic control of spontaneous mutation and genetic recombination during mitosis in Saccharomyces.

Summary A procedure for detection of mutants exhibiting either enhanced or reduced spontaneous mutation during mitosis and/or meiosis has been developed to probe the joint genic control of spontaneous mutation and recombination in yeast. A semidominant mutator,rem1-1, recovered by this technique, exhibits enhanced spontaneous mutation, intragenic recombination, and intergenic recombination during mitosis. Diploids homozygous forrem1-1 exhibit normal levels of meiotic intragenic and intergenic recombination and diminished ascospore viability.     

Yeast MPH1 gene functions in an error-free DNA damage bypass pathway that requires genes from Homologous recombination, but not from postreplicative repair

The MPH1 gene from Saccharomyces cerevisiae, encoding a member of the DEAH family of proteins, had been identified by virtue of the spontaneous mutator phenotype of respective deletion mutants. Genetic analysis suggested that MPH1 functions in a previously uncharacterized DNA repair pathway that protects the cells from damage-induced mutations. We have now analyzed genetic interactions of mph1 with a variety of mutants from different repair systems with respect to spontaneous mutation rates and sensitivities to different DNA-damaging agents. The dependence of the mph1 mutator phenotype on REV3 and REV1 and the synergy with mutations in base and nucleotide excision repair suggest an involvement of MPH1 in error-free bypass of lesions. However, although we observed an unexpected partial suppression of the mph1 mutator phenotype by rad5, genetic interactions with other mutations in postreplicative repair imply that MPH1 does not belong to this pathway. Instead, mutations from the homologous recombination pathway were found to be epistatic to mph1 with respect to both spontaneous mutation rates and damage sensitivities. Determination of spontaneous mitotic recombination rates demonstrated that mph1 mutants are not deficient in homologous recombination. On the contrary, in an sgs1 background we found a pronounced hyperrecombination phenotype. Thus, we propose that MPH1 is involved in a branch of homologous recombination that is specifically dedicated to error-free bypass.     

The rate and character of spontaneous mutation in Thermus thermophilus

Selection of spontaneous, loss-of-function mutations at two chromosomal loci (pyrF and pyrE) enabled the first molecular-level analysis of replication fidelity in the extremely thermophilic bacterium Thermus thermophilus. Two different methods yielded similar mutation rates, and mutational spectra determined by sequencing of independent mutants revealed a variety of replication errors distributed throughout the target genes. The genomic mutation rate estimated from these targets, 0.00097 +/- 0.00052 per replication, was lower than corresponding estimates from mesophilic microorganisms, primarily because of a low rate of base substitution. However, both the rate and spectrum of spontaneous mutations in T. thermophilus resembled those of the thermoacidophilic archaeon Sulfolobus acidocaldarius, despite important molecular differences between these two thermophiles and their genomes.     

The yield of visible mutations, spontaneous and N-nitroso-N-ethylurea-induced, in the Drosophila mei-9 11 strain defective for excision repair

The increase of spontaneous and induced frequencies in mature sperm by N-nitroso-N-ethylurea visible mutation in the stock mei-9(11) of Drosophila melanogaster blocking the excision reparation has been shown. It is suggested that the mutation mei-9(11) blocks the correct way of reparation that is shown in mutator effect of this mutation.     

Mutator Factor in Neisseria meningitidis Associated with Increased Sensitivity to Ultraviolet Light and Defective Transformation

A variant of Neisseria meningitidis was found to carry a mutator factor which endowed the bacteria with generalized genetic instability. The reversion frequencies of several biochemical mutants were increased up to 1,000-fold when the factor was introduced. The factor is not unidirectional in preference, since the mutator induced mutants generally reverted with increased frequency in its presence. There could be found no indication of insufficient synthesis of nucleic acid precursors. Attempts to demonstrate an unusual, mutagenic base incorporated in deoxyribonucleic acid (DNA) were negative. Strains carrying the mutator factor had significantly increased sensitivity to ultraviolet light. A mutation to a more ultraviolet-resistant type coincided with a disappearance of the mutator property. The presence of the mutator factor in a competent strain resulted in a reduction of the transformation frequency to between 0.5 and 5% of that in the parental strain. A mutation to the more ultraviolet-resistant type resulted in simultaneous loss of the mutator property and reestablishment of a normal transformation efficiency. It has been suggested that this mutator factor may represent a defect in the DNA repair mechanism, which is also of importance for genetic recombination. The mutator factor showed cotransformation with the locus for streptomycin resistance, but a true linkage could not be proved.     

Mutational specificity of the dnaE173 mutator associated with a defect in the catalytic subunit of DNA polymerase III of Escherichia coli.

We developed a system to examine forward mutations that occurred in the rpsL gene of Escherichia coli placed on a multicopy plasmid. Using this system we determined the mutational specificity for a dnaE173 mutator strain in which the editing function of DNA polymerase III is impeded. The frequency of rpsL- mutations increased 32,000-fold, due to the dnaE173 mutator, and 87 independent rpsL- mutations in the mutator strain were analyzed by DNA sequencing, together with 100 mutants recovered from dnaE+ strain, as the control. While half the number of mutations that occurred in the wild-type strain were caused by insertion elements, no such mutations were recovered from the mutator strain. A novel class of mutation, named "sequence substitution" was present in mutants raised in the dnaE173 strain; seven sequence substitutions induced in the mutator strain occurred at six sites, and all were located in quasipalindromic sequences, carrying the GTG or CAC sequence at one or both endpoints. While other types of mutation were found in both strains, single-base frameshifts were the most frequent events in the mutator strain. Thus, the mutator effect on this class of mutation was 175,000-fold. A total of 95% of the single-base frameshifts in the mutator strain were additions, most of which occurred at runs of A or C bases so as to increase the number of identical residues. Base substitutions, the frequency of which was enhanced 25,000-fold by the mutator effect, occurred primarily at several hotspots in the mutator strain, whereas those induced in the wild-type strain were more randomly distributed throughout the rpsL sequence. The dnaE173 mutator also increased the frequency of duplications 28,000-fold. Of the three duplications recovered from the mutator strain, one was a simple duplication, the region of which was flanked by direct repeats. The other duplications were complex, one half part of which was in the inverted orientation of a region containing two sets of inverted repeats. The same duplications were also recovered from the wild-type strain. The present data suggest that dnaE173 is a novel class of mutator that sharply induces sequence-directed mutagenesis, yielding high frequencies of single base frameshifts, duplications with inversions, sequence substitutions and base substitutions at hotspots.