Forward Mutation Rate Research Articles

Mismatch recognition function of Arabidopsis thaliana MutSγ

Genetic stability depends in part on an efficient DNA lesion recognition and correction by the DNA mismatch repair (MMR) system. In eukaryotes, MMR is initiated by the binding of heterodimeric MutS homologue (MSH) complexes, MSH2–MSH6 and MSH2–MSH3, which recognize and bind mismatches and unpaired nucleotides. Plants encode another mismatch recognition protein, named MSH7. MSH7 forms a heterodimer with MSH2 and the protein complex is designated MutSγ. We here report the effect the expression of Arabidopsis MSH2 and MSH7 alone or in combination exert on the genomic stability of Saccharomyces cerevisiae. AtMSH2 and AtMutSγ proteins failed to complement the hypermutator phenotype of an msh2 deficient strain. However, overexpressing AtMutSγ in MMR proficient strains generated a 4-fold increase in CAN1 forward mutation rate, when compared to wild-type strains. Canr mutation spectrum analysis of AtMutSγ overproducing strains revealed a substantial increase in the frequency of base substitution mutations, including an increased accumulation of base pair changes from G:C to A:T and T:A to C:G, G:C or A:T. Taken together, these results suggest that AtMutSγ affects yeast genomic stability by recognizing specific mismatches and preventing correction by yeast MutSα and MutSβ, with subsequent inability to interact with yeast downstream proteins needed to complete MMR.

The allele-frequency spectrum in a decoupled Moran model with mutation, drift, and directional selection, assuming small mutation rates

We analyze a decoupled Moran model with haploid population size N, a biallelic locus under mutation and drift with scaled forward and backward mutation rates θ1=μ1N and θ0=μ0N, and directional selection with scaled strength γ=sN. With small scaled mutation rates θ0 and θ1, which is appropriate for single nucleotide polymorphism data in highly recombining regions, we derive a simple approximate equilibrium distribution for polymorphic alleles with a constant of proportionality. We also put forth an even simpler model, where all mutations originate from monomorphic states. Using this model we derive the sojourn times, conditional on the ancestral and fixed allele, and under equilibrium the distributions of fixed and polymorphic alleles and fixation rates. Furthermore, we also derive the distribution of small samples in the diffusion limit and provide convenient recurrence relations for calculating this distribution. This enables us to give formulas analogous to the Ewens–Watterson estimator of θ for biased mutation rates and selection. We apply this theory to a polymorphism dataset of fourfold degenerate sites in Drosophila melanogaster.

Nature, Position, and Frequency of Mutations Made in a Single Cycle of HIV-1 Replication

There is considerable HIV-1 variation in patients. The extent of the variation is due to the high rate of viral replication, the high viral load, and the errors made during viral replication. Mutations can arise from errors made either by host DNA-dependent RNA polymerase II or by HIV-1 reverse transcriptase (RT), but the relative contributions of these two enzymes to the mutation rate are unknown. In addition, mutations in RT can affect its fidelity, but the effect of mutations in RT on the nature of the mutations that arise in vivo is poorly understood. We have developed an efficient system, based on existing technology, to analyze the mutations that arise in an HIV-1 vector in a single cycle of replication. A lacZalpha reporter gene is used to identify viral DNAs that contain mutations which are analyzed by DNA sequencing. The forward mutation rate in this system is 1.4 x 10(-5) mutations/bp/cycle, equivalent to the retroviral average. This rate is about 3-fold lower than previously reported for HIV-1 in vivo and is much lower than what has been reported for purified HIV-1 RT in vitro. Although the mutation rate was not affected by the orientation of lacZalpha, the sites favored for mutations (hot spots) in lacZalpha depended on which strand of lacZalpha was present in the viral RNA. The pattern of hot spots seen in lacZalpha in vivo did not match any of the published data obtained when purified RT was used to copy lacZalpha in vitro.

High‐throughput linkage analysis of Mutator insertion sites in maize

Insertional mutagenesis is a cornerstone of functional genomics. High-copy transposable element systems such as Mutator (Mu) in maize (Zea mays) afford the advantage of high forward mutation rates but pose a challenge for identifying the particular element responsible for a given mutation. Several large mutant collections have been generated in Mu-active genetic stocks, but current methods limit the ability to rapidly identify the causal Mu insertions. Here we present a method to rapidly assay Mu insertions that are genetically linked to a mutation of interest. The method combines elements of MuTAIL (thermal asymmetrically interlaced) and amplification of insertion mutagenized sites (AIMS) protocols and is applicable to the analysis of single mutants or to high-throughput analyses of mutant collections. Briefly, genomic DNA is digested with a restriction enzyme and adapters are ligated. Polymerase chain reaction is performed with TAIL cycling parameters, using a fluorescently labeled Mu primer, which results in the preferential amplification and labeling of Mu-containing genomic fragments. Products from a segregating line are analyzed on a capillary sequencer. To recover a fragment of interest, PCR products are cloned and sequenced. Sequences with lengths matching the size of a band that co-segregates with the mutant phenotype represent candidate linked insertion sites, which are then confirmed by PCR. We demonstrate the utility of the method by identifying Mu insertion sites linked to seed-lethal mutations with a preliminary success rate of nearly 50%.

Maize Mutator transposon

Transposable elements are widely distributed in eukaryotes. Due to its high copy numbers, high forward mutation rate and preferential insertion into low-copy DNA sequences, among others, the Mutator system has been widely used as a mutagen in genomic research. The discovery, classification, transposition specificity and epigenetic regulation of Mutator transposons were described. The application of Mutator tagging in plant genomic research was also presented. The role of Mu-like elements in genome evolution was briefly depicted. Moreover, the direction of Mutator transposon research in the future was discussed.

Radiation-Induced Genomic Instability in Tandem Repeat Sequences is not Predictive of Unique Sequence Instability

Tandem repeat sequences, classified as minisatellite sequences or partially duplicated genes, are inherently unstable. Radiation exposure can increase the instability of such repeat sequences, but the biological consequences of this elevated instability are not well characterized. To learn more about the characteristics of the instability at different sequences in the genome, we created mutant HT1080 cells bearing 8.4 kb of partially duplicated allele at the HPRT locus by gene targeting. The cells were then tested to determine whether repeat-sequence instability (assessed by elevated reversion rate caused by loss of one duplicated segment) accompanied increased forward mutation rates at the restored wild-type HPRT allele. After a 4-Gy X irradiation, 32 clones were selected (out of 500 clones, 6%) that showed elevated reversion rates even after many cell generations. These clones also showed general increases in the forward mutation rate, whereas the paired individual mutation rates did not correlate with each other. Furthermore, levels of intracellular reactive oxygen species (ROS) and nuclear gamma-H2AX foci, which are hallmarks for DNA damage responses, were also generally elevated, although the levels did not correlate with the individual reversion rates. It was concluded that repeat sequence instability is not predictive of unique sequence instability, probably because the instability is generated by multiple mechanisms after radiation exposure.

Mutator Phenotypes Caused by Substitution at a Conserved Motif A Residue in Eukaryotic DNA Polymerase δ

Eukaryotic DNA polymerase (Pol) delta replicates chromosomal DNA and is also involved in DNA repair and genetic recombination. Motif A in Pol delta, containing the sequence DXXXLYPSI, includes a catalytically essential aspartic acid as well as other conserved residues of unknown function. Here, we used site-directed mutagenesis to create all 19 amino acid substitutions for the conserved Leu(612) in Motif A of Saccharomyces cerevisiae Pol delta. We show that substitutions at Leu(612) differentially affect viability, sensitivity to genotoxic agents, cell cycle progression, and replication fidelity. The eight viable mutants contained Ile, Val, Thr, Met, Phe, Lys, Asn, or Gly substitutions. Individual substitutions varied greatly in the nature and extent of attendant phenotypic deficiencies, exhibiting mutation rates that ranged from near wild type to a 37-fold increase. The L612M mutant exhibited a 7-fold elevation of mutation rate but essentially no detectable effects on other phenotypes monitored; the L612T mutant showed a nearly wild type mutation rate together with marked hypersensitivity to genotoxic agents; and the L612G and L612N strains exhibited relatively high mutation rates and severe deficits overall. We compare our results with those for homologous substitutions in prokaryotic and eukaryotic DNA polymerases and discuss the implications of our findings for the role of Leu(612) in replication fidelity.

Absence of strand bias for deletion mutagenesis during chromosomal leading and lagging strand replication in Escherichia coli

Investigations were carried out to determine whether both DNA strands involved in Escherichia coli chromosomal DNA replication are replicated with similar accuracy. Experiments consisted of measuring the forward mutation rate from tonB(+) to tonB(-) in pairs of polA deficient strains in which the chromosomal target gene tonB was oriented in the two possible directions relative to the origin of replication, oriC. Within these pairs, the tonB sequence would be subjected to leading strand replication in one orientation and to lagging strand replication in the other. The most common tonB mutations in the polA1 strain were deletions followed by frameshifts. Among the deletions, a strong hotspot site with a 13-base deletion in the polA1 strains accounted for 18 of the 33 deletions in the one orientation, and 31 of the 58 deletions in the other. The results suggested that the two strands were replicated with equal or similar accuracy for deletion formation.

A new cell-based assay for measuring the forward mutation rate of HIV-1

Over 20 years into the ever-worsening AIDS pandemic, genetic variation remains the greatest obstacle for treating and preventing HIV-1 infection. Mutation rate assays for HIV-1 have been reported; however, none measure directly the forward mutation rate during replication of the virus in cell culture while still retaining the ability to propagate and further study mutant proviruses. Therefore, the objective of the current study was to develop such a phenotypic cell-based assay for measuring the forward mutation rate of HIV-1. Conventional recombinant DNA techniques and polymerase chain reaction were used to create a replication defective HIV-1 vector, pNL4-3Δ+cass, which is based on the NL4-3 strain and contains the thymidine kinase gene from human herpes virus type 1 as the mutational target. A series of transfection and infection steps were used to introduce the vector into 143B cells, which are negative for thymidine kinase function, and produce vector virus for a single cycle of replication. Viral titers were measured by counting the number of drug resistant colonies on the assay plates, and forward mutation rates were calculated from the viral titers. Mutant proviruses were sequenced to determine the types of genetic alterations that occurred. The average forward mutation rate for HIV-1 was 2.2 × 10 −5 mutations/base/cycle. The majority of mutations were base substitutions, including high frequencies of C → U and G → A transitions. Single adenosine insertions were also observed frequently. The new assay is economical and provides a direct measurement of the mutation rate during a single cycle of viral replication. Target cells containing mutant proviruses survive the drug selection process and may be propagated for further analysis. The new assay is novel and has many advantages over previous mutation rate assays and thus will be very useful in future studies on genetic variation of HIV-1.

Activator mutagenesis of the pink scutellum1/viviparous7 locus of maize.

The transposable elements Activator/Dissociation (Ac/Ds) were first discovered in maize, yet they have not been used extensively in their native host for gene-tagging experiments. This can be attributed largely to the low forward mutation rate and the propensity for closely linked transpositions associated with Ac and its nonautonomous deletion derivative Ds. To overcome these limitations, we are developing a series of nearly isogenic maize lines, each with a single active Ac element positioned at a well-defined location. These Ac elements are distributed at 10- to 20-centimorgan intervals throughout the genome for use in regional mutagenesis. Here, we demonstrate the utility of this Ac-based gene-tagging approach through the targeted mutagenesis of the pink scutellum1/viviparous7 (ps1/vp7) locus. Using a novel PCR-based technique, the Ps1 gene was cloned and Ac elements positioned precisely in each of the seven alleles recovered. The Ps1 gene is predicted to encode lycopene beta-cyclase and is necessary for the accumulation of both abscisic acid and the carotenoid zeaxanthin in mature maize embryos. This study demonstrates the utility of an Ac mutagenesis program to efficiently generate allelic diversity at closely linked loci in maize.

Substitutions of Phe61 Located in the Vicinity of Template 5′-Overhang Influence Polymerase Fidelity and Nucleoside Analog Sensitivity of HIV-1 Reverse Transcriptase

Human immunodeficiency virus type 1 reverse transcriptase (RT) is an error-prone DNA polymerase. Structural determinants of its fidelity are incompletely understood. RT/template primer contacts have been shown to influence its fidelity and sensitivity to nucleoside analog inhibitors. The Phe(61) residue, located within the beta 3 sheet of the finger subdomain, is highly conserved among retroviral RTs. The crystal structure of a ternary complex revealed that Phe(61) contacts the first and second bases of the 5'-template overhang. To determine whether such contacts influence the dNTP-binding pocket, we performed a limited vertical scanning mutagenesis (Phe --> Ala, Leu, Trp, or Tyr) at Phe(61). The F61A mutant displayed the highest increase in fidelity, followed by the F61L and F61W variants, which had intermediate phenotypes. F61Y RT had a minimal effect. The increase in fidelity of the F61A mutant was corroborated by a 12-fold decrease in its forward mutation rate. The Phe(61) mutant RTs also displayed large reductions in sensitivity to 2',3'-dideoxythymidine triphosphate and 2',3'-dideoxy,2'3'-didehydrothymidine triphosphate. Mutants displaying the largest increase in fidelity (F61A and F61L) were also the most resistant. These results suggest that contacts between the finger subdomain of human immunodeficiency virus type 1 RT and the template 5'-overhang are important determinants of the geometry of the dNTP-binding pocket.

Pol32, a Subunit of Saccharomyces cerevisiae DNA Polymerase δ, Suppresses Genomic Deletions and Is Involved in the Mutagenic Bypass Pathway

Abstract The Pol32 subunit of S. cerevisiae DNA polymerase (Pol) δ plays an important role in replication and mutagenesis. Here, by measuring the CAN1 forward mutation rate, we found that either POL32 or REV3 (which encodes the Pol ζ catalytic subunit) inactivation produces overlapping antimutator effects against rad mutators belonging to three epistasis groups. In contrast, the msh2Δ pol32Δ double mutant exhibits a synergistic mutator phenotype. Canr mutation spectrum analysis of pol32Δ strains revealed a substantial increase in the frequency of deletions and duplications (primarily deletions) of sequences flanked by short direct repeats, which appears to be RAD52 and RAD10 independent. To better understand the pol32Δ and rev3Δ antimutator effects in rad backgrounds and the pol32Δ mutator effect in a msh2Δ background, we determined Canr mutation spectra for rad5Δ, rad5Δ pol32Δ, rad5Δ rev3Δ, msh2Δ, msh2Δ pol32Δ, and msh2Δ rev3Δ strains. Both rad5Δ pol32Δ and rad5Δ rev3Δ mutants exhibit a reduction in frameshifts and base substitutions, attributable to antimutator effects conferred by the pol32Δ and rev3Δ mutations. In contrast, an increase in these two types of alterations is attributable to a synergistic mutator effect between the pol32Δ and msh2Δ mutations. Taken together, these observations indicate that Pol32 is important in ensuring genome stability and in mutagenesis.

Frequency of direct repeat deletion in a human immunodeficiency virus type 1 vector during reverse transcription in human cells.

Retroviral genetic rearrangements can result from reverse transcriptase template switching. Most published data suggest that errors such as base misincorporation occur at similar frequencies for HIV-1 and for simple retroviruses such as spleen necrosis virus (SNV) and murine leukemia virus (MuLV). However, previous reports have suggested that template switch-mediated recombination is much more frequent for HIV-1 than for simple retroviruses. In this report, direct repeat deletion vectors similar to those previously used for measuring template switching events for SNV and MuLV were developed for HIV-1. Forward mutation rates and the frequency of template switching during a single cycle of HIV-1 replication were determined. The frequency of HIV-1-mediated repeat deletion was measured for three separate internal repeats in lacZ and was compared to rates observed with identical repeats for MuLV. The results indicated that the error rate and the frequency of repeat deletion of HIV-1 were similar to those of MuLV.

Genetic fidelity under harsh conditions: analysis of spontaneous mutation in the thermoacidophilic archaeon Sulfolobus acidocaldarius.

Microbes whose genomes are encoded by DNA and for which adequate information is available display similar genomic mutation rates (average 0.0034 mutations per chromosome replication, range 0.0025 to 0.0046). However, this value currently is based on only a few well characterized microbes reproducing within a narrow range of environmental conditions. In particular, no genomic mutation rate has been determined either for a microbe whose natural growth conditions may extensively damage DNA or for any member of the archaea, a prokaryotic lineage deeply diverged from both bacteria and eukaryotes. Both of these conditions are met by the extreme thermoacidophile Sulfolobus acidocaldarius. We determined the genomic mutation rate for this species when growing at pH 3.5 and 75 degrees C based on the rate of forward mutation at the pyrE gene and the nucleotide changes identified in 101 independent mutants. The observed value of about 0.0018 extends the range of DNA-based microbes with rates close to the standard rate simultaneously to an archaeon and to an extremophile whose cytoplasmic pH and normal growth temperature greatly accelerate the spontaneous decomposition of DNA. The mutations include base pair substitutions (BPSs) and additions and deletions of various sizes, but the S. acidocaldarius spectrum differs from those of other DNA-based organisms in being relatively poor in BPSs. The paucity of BPSs cannot yet be explained by known properties of DNA replication or repair enzymes of Sulfolobus spp. It suggests, however, that molecular evolution per genome replication may proceed more slowly in S. acidocaldarius than in other DNA-based organisms examined to date.

Differential Influence of Nucleoside Analog-resistance Mutations K65R and L74V on the Overall Mutation Rate and Error Specificity of Human Immunodeficiency Virus Type 1 Reverse Transcriptase

Differential Influence of Nucleoside Analog-resistance Mutations K65R and L74V on the Overall Mutation Rate and Error Specificity of Human Immunodeficiency Virus Type 1 Reverse Transcriptase

Differential influence of nucleoside analog-resistance mutations K65R and L74V on the overall mutation rate and error specificity of human immunodeficiency virus type 1 reverse transcriptase.

Human immunodeficiency virus type 1 (HIV-1) reverse transcriptase (RT) variants with the K65R or L74V substitution display resistance to several nucleoside analogs. An in vitro dNTP exclusion assay revealed an increased fidelity for K65R RT compared with wild-type RT, but little change for L74V RT. When the forward mutation rates were measured via a gap-filling assay, the K65R variant displayed an 8-fold decrease in the overall mutation rate (1.0 x 10(-3) versus 8.6 x 10(-3) for wild-type HIV-1 RT), whereas the rate for the L74V variant was closer to that for wild-type RT (5.0 x 10(-3)). The increase in overall fidelity observed for K65R RT is the largest reported for any drug-resistant HIV-1 RT variant. Nucleotide sequence analysis of lacZalpha mutants generated by variant RTs indicated that K65R RT displays uniform reduction in most types of errors, whereas L74V RT does not. Modeling the substitutions into the x-ray structure of the ternary complex revealed that the major influence of Leu(74) in stabilizing the templating base is unaffected by Val substitution, whereas the K65R substitution appears to increase the stringency of dNTP binding. It is speculated that the increased fidelity of K65R RT is due to an altered interaction with the dNTP substrate.

Characterization of Mutations Induced by Ethyl Methanesulfonate, UV, and Trimethylpsoralen in the Nematode Caenorhabditis elegans

The genome project of the nematode Caenorhabditis elegans is completed. It is important and useful to disrupt nematode genes to know their function. We treated wild-type animals with potential candidates for mutagens for reverse genetics, EMS (ethyl methanesulfonate), short-wavelength UV, and long-wavelength UV in the presence of TMP (trimethylpsoralen). We estimated forward mutation rates by counting the occurrence of a marker unc-22 mutation. We found that the forward mutation rate by TMP/UV could be comparable with EMS by improving the frequency one order higher than before. We next isolated mutants of another marker gene ben-1 and examined the probability for the deletion mutations by PCR and sequencing. Deletion mutations were found only by TMP/UV method, which suggested TMP/UV is the choice for deletion mutagenesis among these methods. As a pilot experiment, we could isolate actual deletion mutations at a much higher frequency than previously.

No mutagenic or recombinogenic effects of mobile phone fields at 900 MHz detected in the yeast Saccharomyces cerevisiae.

Both actively growing and resting cells of the yeast Saccharomyces cerevisiae were exposed to 900-MHz fields that closely matched the Global System for Mobile Communication (GSM) pulsed modulation format signals for mobile phones at specific absorption rates (SAR) of 0.13 and 1.3 W/kg. Two identical anechoic test chambers were constructed to perform concurrent control and test experiments under well-controlled exposure conditions. Using specific test strains, we examined the genotoxic potential of mobile phone fields, alone and in combination, with a known genotoxic compound, the alkylating agent methyl methansulfonate. Mutation rates were monitored by two test systems, a widely used gene-specific forward mutation assay at CAN1 and a wide-range assay measuring the induction of respiration-deficient (petite) clones that have lost their mitochondrial function. In addition, two further assays measured the recombinogenic effect of mobile phone fields to detect possible effects on genomic stability: First, an intrachromosomal, deletion-formation assay previously developed for genotoxic screening; and second, an intragenic recombination assay in the ADE2 gene. Fluctuation tests failed to detect any significant effect of mobile phone fields on forward mutation rates at CAN1, on the frequency of petite formation, on rates of intrachromosomal deletion formation, or on rates of intragenic recombination in the absence or presence of the genotoxic agent methyl methansulfonate.

The impact of multidideoxynucleoside resistance-conferring mutations in human immunodeficiency virus type 1 reverse transcriptase on polymerase fidelity and error specificity.

Variants of human immunodeficiency virus type 1 (HIV-1) that are highly resistant to a number of nucleoside analog drugs have been shown to develop in some patients receiving 2',3'-dideoxy-3'-azidothymidine therapy in combination with 2',3'-dideoxycytidine or 2',3'-dideoxyinosine. The appearance, in the reverse transcriptase (RT), of the Q151M mutation in such variants precedes the sequential appearance of three or four additional mutations, resulting in a highly resistant virus. Three of the affected residues are proposed to lie in the vicinity of the template-primer in the three-dimensional structure of the HIV-1 RT-double-stranded DNA complex. The amino acid residue Q151 is thought to be very near the templating base. The nucleoside analog resistance mutations in the beta9-beta10 (M184V) and the beta5a (E89G) strands of HIV-1 RT were previously shown to increase the fidelity of deoxynucleoside triphosphate insertion. Therefore, we have examined wild-type HIV-1BH10 RT and two nucleoside analog-resistant variants, the Q151M and A62V/V75I/F77L/F116Y/Q151M (VILYM) RTs, for their overall forward mutation rates in an M13 gapped-duplex assay that utilizes lacZ alpha as a reporter. The overall error rates for the wild-type, the Q151M, and the VILYM RTs were 4.5 x 10(-5), 4.0 x 10(-5), and 2.3 x 10(-5) per nucleotide, respectively. Although the mutant RTs displayed minimal decreases in the overall error rates compared to wild-type RT, the error specificities of both mutant RTs were altered. The Q151M RT mutant generated new hot spots, which were not observed for wild-type HIV-1 RT previously. The VILYM RT showed a marked reduction in error rate at two of the predominant mutational hot spots that have been observed for wild-type HIV-1 RT.

Escherichia coli DNA polymerase II catalyzes chromosomal and episomal DNA synthesis in vivo.

We have investigated a role for Escherichia coli DNA polymerase II (Pol II) in copying chromosomal and episomal DNA in dividing cells in vivo. Forward mutation frequencies and rates were measured at two chromosomal loci, rpoB and gyrA, and base substitution and frameshift mutation frequencies were measured on an F'(lacZ) episome. To amplify any differences in polymerase error rates, methyl-directed mismatch repair was inactivated. When wild-type Pol II (polB+) was replaced on the chromosome by a proofreading-defective Pol II exo- (polBex1), there was a significant increase in mutation frequencies to rifampicin resistance (RifR) (rpoB) and nalidixic acid resistance (NalR) (gyrA). This increased mutagenesis occurred in the presence of an antimutator allele of E. coli DNA polymerase III (Pol III) (dnaE915), but not in the presence of wild-type Pol III (dnaE+), suggesting that Pol II can compete effectively with DnaE915 but not with DnaE+. Sequencing the RifR mutants revealed a G --> A hot spot highly specific to Pol II exo-. Pol II exo- caused a significant increase in the frequency of base substitution and frameshift mutations on F' episomes, even in dnaE+ cells, suggesting that Pol II is able to compete with Pol III for DNA synthesis on F episomes.