Lethal mutagenesis of poliovirus mediated by a mutagenic pyrimidine analogue

Lethal mutagenesis is the mechanism of action of ribavirin against poliovirus (PV) and numerous other RNA viruses. However, there is still considerable debate regarding the mechanism of action of ribavirin against a variety of RNA viruses. Here we show by using T7 RNA polymerase-mediated production of PV genomic RNA, PV polymerase-catalyzed primer extension, and cell-free PV synthesis that a pyrimidine ribonucleoside triphosphate analogue (rPTP) with ambiguous base-pairing capacity is an efficient mutagen of the PV genome. The in vitro incorporation properties of rPTP are superior to ribavirin triphosphate. We observed a log-linear relationship between virus titer reduction and the number of rPMP molecules incorporated. A PV genome encoding a high-fidelity polymerase was more sensitive to rPMP incorporation, consistent with diminished mutational robustness of high-fidelity PV. The nucleoside (rP) did not exhibit antiviral activity in cell culture, owing to the inability of rP to be converted to rPMP by cellular nucleotide kinases. rP was also a poor substrate for herpes simplex virus thymidine kinase. The block to nucleoside phosphorylation could be bypassed by treatment with the P nucleobase, which exhibited both antiviral activity and mutagenesis, presumably a reflection of rP nucleotide formation by a nucleotide salvage pathway. These studies provide additional support for lethal mutagenesis as an antiviral strategy, suggest that rPMP prodrugs may be highly efficacious antiviral agents, and provide a new tool to determine the sensitivity of RNA virus genomes to mutagenesis as well as interrogation of the impact of mutational load on the population dynamics of these viruses.     

Avoiding Dangerous Missense: Thermophiles Display Especially Low Mutation Rates

Rates of spontaneous mutation have been estimated under optimal growth conditions for a variety of DNA-based microbes, including viruses, bacteria, and eukaryotes. When expressed as genomic mutation rates, most of the values were in the vicinity of 0.003–0.004 with a range of less than two-fold. Because the genome sizes varied by roughly 10⁴-fold, the mutation rates per average base pair varied inversely by a similar factor. Even though the commonality of the observed genomic rates remains unexplained, it implies that mutation rates in unstressed microbes reach values that can be finely tuned by evolution. An insight originating in the 1920s and maturing in the 1960s proposed that the genomic mutation rate would reflect a balance between the deleterious effect of the average mutation and the cost of further reducing the mutation rate. If this view is correct, then increasing the deleterious impact of the average mutation should be countered by reducing the genomic mutation rate. It is a common observation that many neutral or nearly neutral mutations become strongly deleterious at higher temperatures, in which case they are called temperature-sensitive mutations. Recently, the kinds and rates of spontaneous mutations were described for two microbial thermophiles, a bacterium and an archaeon. Using an updated method to extrapolate from mutation-reporter genes to whole genomes reveals that the rate of base substitutions is substantially lower in these two thermophiles than in mesophiles. This result provides the first experimental support for the concept of an evolved balance between the total genomic impact of mutations and the cost of further reducing the basal mutation rate.     

RNA mutations of prox1 detected in human esophageal cancer cells by the shifted termination assay

We have recently reported a novel finding that a candidate tumor suppressor gene prox1 suffered adenosine-to-inosine (A-to-I) RNA mutation without genomic mutation in a subset of human cancer cells and lost its function. Hence, screening of mutations in both cDNA and genomic DNA could be important in the analysis of causes for cancers. Here, we applied a sensitive, accurate, and simple method, called shifted termination assay (STA) for detection of an A-to-I RNA mutation (R334G) in prox1. We prepared PCR-amplified samples containing the target base of RNA mutation from cDNAs and genomic DNAs of various cell lines and clinical samples, to demonstrate that the STA method can be used to identify not only genomic mutations but also RNA mutations more effectively compared to sequencing. By means of STA, we found prox1 R334G RNA mutations but not genomic DNA mutations in 4 of 8 cases of esophageal cancers. This method can help us to detect RNA mutation effectively and progress research of a potential oncogenic principle.     

Isolation and characterization of the putative nuclear modifier gene MTO1 involved in the pathogenesis of deafness-associated mitochondrial 12 S rRNA A1555G mutation

The human mitochondrial 12 S rRNA A1555G mutation has been found to be associated with aminoglycoside-induced and non-syndromic deafness. However, putative nuclear modifier gene(s) have been proposed to regulate the phenotypic expression of this mutation. In yeast, the mutant alleles of MTO1, encoding a mitochondrial protein, manifest respiratory-deficient phenotype only when coupled with the mitochondrial 15 S rRNA P(R)454 mutation corresponding to human A1555G mutation. This suggests that the MTO1-like modifier gene may influence the phenotypic expression of human A1555G mutation. Here we report the identification of full-length cDNA and elucidation of genomic organization of the human MTO1 homolog. Human Mto1 is an evolutionarily conserved protein that implicates a role in the mitochondrial tRNA modification. Functional conservation of this protein is supported by the observation that isolated human MTO1 cDNA can complement the respiratory deficient phenotype of yeast mto1 cells carrying P(R)454 mutation. MTO1 is ubiquitously expressed in various tissues, but with a markedly elevated expression in tissues of high metabolic rates including cochlea. These observations suggest that human MTO1 is a structural and functional homolog of yeast MTO1. Thus, it may play an important role in the pathogenesis of deafness-associated A1555G mutation in 12 S rRNA gene or mutations in tRNA genes.     

Conditional coalescent trees with two mutation rates and their application to genomic instability

Humans have invested several genes in DNA repair and fidelity replication. To account for the disparity between the rarity of mutations in normal cells and the large number of mutations present in cancer, an hypothesis is that cancer cells must exhibit a mutator phenotype (genomic instability) during tumor progression, with the initiation of abnormal mutation rates caused by the loss of mismatch repair. In this study we introduce a stochastic model of mutation in tumor cells with the aim of estimating the amount of genomic instability due to the alteration of DNA repair genes. Our approach took into account the difficulties generated by sampling within tumoral clones and the fact that these clones must be difficult to isolate. We provide corrections to two classical statistics to obtain unbiased estimators of the raised mutation rate, and we show that large statistical errors may be associated with such estimators. The power of these new statistics to reject genomic instability is assessed and proved to increase with the intensity of mutation rates. In addition, we show that genomic instability cannot be detected unless the raised mutation rates exceed the normal rates by a factor of at least 1000.     

Non-B DNA-forming sequences and WRN deficiency independently increase the frequency of base substitution in human cells

Although alternative DNA secondary structures (non-B DNA) can induce genomic rearrangements, their associated mutational spectra remain largely unknown. The helicase activity of WRN, which is absent in the human progeroid Werner syndrome, is thought to counteract this genomic instability. We determined non-B DNA-induced mutation frequencies and spectra in human U2OS osteosarcoma cells and assessed the role of WRN in isogenic knockdown (WRN-KD) cells using a supF gene mutation reporter system flanked by triplex- or Z-DNA-forming sequences. Although both non-B DNA and WRN-KD served to increase the mutation frequency, the increase afforded by WRN-KD was independent of DNA structure despite the fact that purified WRN helicase was found to resolve these structures in vitro. In U2OS cells, ～70% of mutations comprised single-base substitutions, mostly at G·C base-pairs, with the remaining ～30% being microdeletions. The number of mutations at G·C base-pairs in the context of NGNN/NNCN sequences correlated well with predicted free energies of base stacking and ionization potentials, suggesting a possible origin via oxidation reactions involving electron loss and subsequent electron transfer (hole migration) between neighboring bases. A set of ～40,000 somatic mutations at G·C base pairs identified in a lung cancer genome exhibited similar correlations, implying that hole migration may also be involved. We conclude that alternative DNA conformations, WRN deficiency and lung tumorigenesis may all serve to increase the mutation rate by promoting, through diverse pathways, oxidation reactions that perturb the electron orbitals of neighboring bases. It follows that such "hole migration" is likely to play a much more widespread role in mutagenesis than previously anticipated.     

Resistance to extinction of low fitness virus subjected to plaque-to-plaque transfers: diversification by mutation clustering.

Plaque-to-plaque transfers of RNA viruses lead to accumulation of mutations and fitness decrease. To test whether continuing plaque-to-plaque transfers would lead to viral extinction, we have subjected several low fitness foot-and-mouth disease virus (FMDV) clones to up to 130 successive plaque transfers, and have analyzed the evolution of plaque titers and genomic nucleotide sequences. No case of viral extinction could be documented. Some low fitness clones that posses an internal poly(A) tract evaded extinction by modifying the length or base composition of the poly(A) tract. The comparison of entire genomic sequences of FMDV clones at increasing plaque transfer number revealed that mutations accumulated at a uniform rate, and that they were distributed unevenly along the genome. Clusters of mutations were identified at different genomic sites in two plaque transfer lineages. Mutation clustering appears to occur stochastically and could not be related to fixation of compensatory mutations. The results document resistance of viral clones to extinction, and suggest that mutation clustering may be a mechanism of genetic diversification of low fitness virus.     

Characterization of PitA and PitB from Escherichia coli

Escherichia coli contains two major systems for transporting inorganic phosphate (P(i)). The low-affinity P(i) transporter (pitA) is expressed constitutively and is dependent on the proton motive force, while the high-affinity Pst system (pstSCAB) is induced at low external P(i) concentrations by the pho regulon and is an ABC transporter. We isolated a third putative P(i) transport gene, pitB, from E. coli K-12 and present evidence that pitB encodes a functional P(i) transporter that may be repressed at low P(i) levels by the pho regulon. While a pitB(+) cosmid clone allowed growth on medium containing 500 microM P(i), E. coli with wild-type genomic pitB (pitA Delta pstC345 double mutant) was unable to grow under these conditions, making it indistinguishable from a pitA pitB Delta pstC345 triple mutant. The mutation Delta pstC345 constitutively activates the pho regulon, which is normally induced by phosphate starvation. Removal of pho regulation by deleting the phoB-phoR operon allowed the pitB(+) pitA Delta pstC345 strain to utilize P(i), with P(i) uptake rates significantly higher than background levels. In addition, the apparent K(m) of PitB decreased with increased levels of protein expression, suggesting that there is also regulation of the PitB protein. Strain K-10 contains a nonfunctional pitA gene and lacks Pit activity when the Pst system is mutated. The pitA mutation was identified as a single base change, causing an aspartic acid to replace glycine 220. This mutation greatly decreased the amount of PitA protein present in cell membranes, indicating that the aspartic acid substitution disrupts protein structure.     

Insertion of an extra codon for threonine is a cause of dihydropteridine reductase deficiency.

The mutation in a patient with dihydropteridine reductase deficiency has been located and characterized. Polymerase chain reaction (PCR) was used to amplify the coding sequence of human dihydropteridine reductase from the messenger RNA of skin fibroblasts. Chemical cleavage of mismatches indicated a mismatched thymine and cytosine at approximately 117 and 147 bases, respectively, from the end of the probe. Cloning and sequencing of the mutant PCR products revealed the insertion of the triplet ACT (threonine), after alanine 122 (base 390). Amplification of a small region around this mutation by using genomic DNA as the PCR target indicates that the mutation is completely within an exon. Unequal crossing-over at the second base in the preceding alanine codon and duplication of the bases CTA may be the mechanism of mutagenesis. The cleavage site 147 bases from the end of the probe corresponded to the conversion of guanine to adenine at base 420 (CTG to CTA) and does not alter the code for leucine. This change, which was also seen in another dihydropteridine reductase-deficient child and in a control subject probably represents a common neutral polymorphism.     

The role of water in the catalytic efficiency of triosephosphate isomerase

The structural basis for the effect of the S96P mutation in chicken triosephosphate isomerase (cTIM) has been analyzed using a combination of X-ray crystallography and Fourier transform infrared spectroscopy. The X-ray structure is that of the enzyme complexed with phosphoglycolohydroxamate (PGH), an intermediate analogue, solved at a resolution of 1.9 A. The S96P mutation was identified as a second-site reverent when catalytically crippled mutants, E165D and H95N, were subjected to random mutagenesis. The presence of the second mutation leads to enhanced activity over the single mutation. However, the effect of the S96P mutation alone is to decrease the catalytic efficiency of the enzyme. The crystal structures of the S96P double mutants show that this bulky proline side chain alters the water structure within the active-site cavity (E165D; ref 1) and prevents nonproductive binding conformations of the substrate (H95N; ref 2). Comparison of the S96P single mutant structure with those of the wild-type cTIM, those of the single mutants (E165D and H95N), and those of the double mutants (E165D/S96P and H95N/S96P) begins to address the role of the conserved serine residue at this position. The results indicate that the residue positions the catalytic base E165 optimally for polarization of the substrate carbonyl, thereby aiding in proton abstraction. In addition, this residue is involved in positioning critical water molecules, thereby affecting the way in which water structure influences activity.     

Determination of the rate of base-pair substitution and insertion mutations in retrovirus replication.

We recently described a protocol for determination of retrovirus mutation rates, that is, the mutation frequency in a single cycle of retrovirus replication (J.P. Dougherty and H.M. Temin, Mol. Cell. Biol. 6:4378-4395, 1987; J.P. Dougherty and H.M. Temin, p. 18-23, in J. H. Miller and M. P. Calos, ed., Gene Transfer Vectors for Mammalian Cells, 1987). We used this protocol to determine the mutation rates for defined mutations in a replicating retrovirus by using a spleen necrosis virus-based vector. We determined that the mutation rate for a single base pair substitution during replication of this avian retrovirus is 2 x 10(-5) per base pair per replication cycle and the insertion rate is 10(-7) per base pair per replication cycle. It will be possible to use this protocol to determine mutation rates for other retroviruses.     

A rapid and simple method for the determination of base substitution and frameshift specificity of mutagens

A rapid method for the determination of mutagenic specificity has been developed which makes use of the ochre mutation (TAA) in the his-4 gene of Escherichiacoli. Reversion to His⁺ may occur by suppressor mutation (Type I) or by mutation within the his-4 gene (Type II). The Type I mutations may be further subdivided with respect to the type of suppressor mutation by their ability to suppress nonsense mutants of bacteriophage T4, thus allowing the identification of the responsible base substitution (Kato et al., 1980). The system has the ability to identify mutagens which produce A:T → G:C transitions since only Type II mutants can arise through this base substitution; and in fact, the system confirms the A:T → G:C specificity of the mutagen, N⁴-hydroxycytidine (Janion and Glickman, 1980) since only Type II mutants were induced by treatment with this base analogue.When this system was further tested with several additional mutagens, the results indicate that ethyl methanesulphonate, methyl nitrosourea and ethyl nitrosourea produce primarily Type I revertants which were primarily G:C → A:T transitions. UV-light, γ-rays, 4NQO and methyl methanesulphonate produced all types of base substitutions. The tester strain was further improved by introducing a series of sequenced trp^? frameshift mutations, thus allowing the simultaneous monitoring of frameshift and base-substitution mutations.     

The evolution of mutation rates: separating causes from consequences

Natural selection can adjust the rate of mutation in a population by acting on allelic variation affecting processes of DNA replication and repair. Because mutation is the ultimate source of the genetic variation required for adaptation, it can be appealing to suppose that the genomic mutation rate is adjusted to a level that best promotes adaptation. Most mutations with phenotypic effects are harmful, however, and thus there is relentless selection within populations for lower genomic mutation rates. Selection on beneficial mutations can counter this effect by favoring alleles that raise the mutation rate, but the effect of beneficial mutations on the genomic mutation rate is extremely sensitive to recombination and is unlikely to be important in sexual populations. In contrast, high genomic mutation rates can evolve in asexual populations under the influence of beneficial mutations, but this phenomenon is probably of limited adaptive significance and represents, at best, a temporary reprieve from the continual selection pressure to reduce mutation. The physiological cost of reducing mutation below the low level observed in most populations may be the most important factor in setting the genomic mutation rate in sexual and asexual systems, regardless of the benefits of mutation in producing new adaptive variation. Maintenance of mutation rates higher than the minimum set by this "cost of fidelity" is likely only under special circumstances.     

Germ-line origins of mutation in families with hemophilia B: the sex ratio varies with the type of mutation.

Previous epidemiological and biochemical studies have generated conflicting estimates of the sex ratio of mutation. Direct genomic sequencing in combination with haplotype analysis extends previous analyses by allowing the precise mutation to be determined in a given family. From analysis of the factor IX gene of 260 consecutive families with hemophilia B, we report the germ-line origin of mutation in 25 families. When combined with 14 origins of mutation reported by others and with 4 origins previously reported by us, a total of 25 occur in the female germ line, and 18 occur in the male germ line. The excess of germ-line origins in females does not imply an overall excess mutation rate per base pair in the female germ line. Bayesian analysis of the data indicates that the sex ratio varies with the type of mutation. The aggregate of single-base substitutions shows a male predominance of germ-line mutations (P < .002). The maximum-likelihood estimate of the male predominance is 3.5-fold. Of the single-base substitutions, transitions at the dinucleotide CpG show the largest male predominance (11-fold). In contrast to single-base substitutions, deletions display a sex ratio of unity. Analysis of the parental age at transmission of a new mutation suggests that germ-line mutations are associated with a small increase in parental age in females but little, if any, increase in males. Although direct genomic sequencing offers a general method for defining the origin of mutation in specific families, accurate estimates of the sex ratios of different mutational classes require large sample sizes and careful correction for multiple biases of ascertainment. The biases in the present data result in an underestimate of the enhancement of mutation in males.     

Mutation identification of the DSPP in a Chinese family with DGI-II and an up-to-date bioinformatic analysis

In this study, through linkage analysis of a four-generation Chinese family with multiple members afflicted with DGI (type II), we identified a novel missense mutation in DSPP. The mutation was located in exon 2 at the second nucleotide position of the last codon and resulted in a substitution of a proline with a leucine residue (c.50C>T, p.P17L, g.50C>T). To assess the potential effects of this novel mutation, we utilized various bioinformatics analysis programs. The results indicate that the mutation likely affects protein cleavage/trafficking. We also analyzed previously reported mutations of DSPP. In summary, our finding supports that the genomic sequence that corresponds to the P17 residue of DSPP is a mutational hotspot and P17 may be critical for the function of DSPP.     

Bystander effects may modulate ultraviolet A and B radiation-induced delayed mutagenesis

Ultraviolet irradiation of cells can induce a state of genomic instability that can persist for several cell generations after irradiation. However, questions regarding the time course of formation, relative abundance for different types of ultraviolet radiation, and mechanism of induction of delayed mutations remain to be answered. In this paper, we have tried to address these questions using the hypoxanthine phosphoribosyl transferase (HPRT) mutation assay in V79 Chinese hamster cells irradiated with ultraviolet A or B radiation. Delayed HPRT(-) mutations, which are indications of genomic instability, were detected by incubating the cells in medium containing aminopterin, selectively killing HPRT(-) mutants, and then treating the cells with medium containing 6-thioguanine, which selectively killed non-mutant cells. Remarkably, the delayed mutation frequencies found here were much higher than reported previously using a cloning method. Cloning of cells immediately after irradiation prevents contact between individual cell clones. In contrast, with the present method, the cells are in contact and are mixed several times during the experiment. Thus the higher delayed mutation frequency measured by the present method may be explained by a bystander effect. This hypothesis is supported by an experiment with an inhibitor of gap junctional intercellular communication, which reduced the delayed mutation frequency. In conclusion, the results suggest that a bystander effect is involved in ultraviolet-radiation-induced genomic instability and that it may be mediated in part by gap junctional intercellular communication.     

A stable and sensitive genotoxic testing system based on DNA damage induced gene expression in Saccharomyces cerevisiae

To evaluate the rpsL transgenic zebrafish (Brachydanio rerio) mutation assay, we treated the embryos with benzo[a]pyrene (B[a]P) (10 microg/ml) or 2-amino-3,8-dimethylimidazo[4,5-f]quinoxaline (MeIQx) (300 microg/ml) for 16h and determined the mutation spectra. These treatments were previously reported to induce mutant frequencies that were 4.3 and 2.4 times the control value, respectively. In the B[a]P-treated group, half of the mutations were single base substitutions, 74% of which occurred at G:C base pairs. Among G:C base pair substitutions, G:C to T:A and G: C to C:G transversions were predominant, suggesting that B[a]P induced mutations in zebrafish embryos by mechanisms previously described in mammalian tissues. In the MeIQx-treated group, about 60% of the mutations were deletions. Some specific mutations were found, but the compound primarily amplified the background mutation level; improvement in the conditions of treatment may be required for elucidating MeIQx-mutagenesis in this system. This study showed that transgenic zebrafish may be a useful tool for detecting mutagens in aquatic environments and for elucidating mutagenic mechanisms.     

Molecular characterization of genetic mutations in human lactate dehydrogenase (LDH) B (H) variant

Summary We have previously detected a single base substitution of G by A at the Arg codon CGC in exon 4 of the mutant lactate dehydrogenase (LDH) gene, an unstable LDH-B variant (case 1). Here, we use the polymerase chain reaction (PCR) to amplify genomic DNA of two cases (the original case 1 and a new patient, case 2). We were able to confirm that case 1 is homozygous for the mutation, causing a replacement of the conserved Arg by His at residue 173. The resulting LDH-B variant subunit is unstable in vivo. Whereas the mutation in exon 4 was not observed in case 2, a different single base substitution of A by C was detected at the Ser codon AGT in exon 3. This mutation causes a replacement of the conserved Ser by Arg at residue 131. Genomic analysis of the family of case 2 by mismatched PCR showed that the missense mutation was consistent with their biochemical phenotypes. The replacement results in a conformational change of the residues near the Ser, probably because the side chain of Arg is much more bulky than that of Ser. The change may affect the arrangement of the cofactor binding site and result in the loss of enzyme activity. The experimental observations are consistent with computer graphics analyses.     

To Wobble or Not to Wobble: Modified Bases Incorporated into DNA

Abstract

The ambivalent base analogue P was incorporated in the d(CGCGPG) hexamer to investigate the G.P base pair geometry by X-ray diffraction. Both Watson-Crick and wobble geometries have been found for the crystallographic independent G.P base pairs.