Comprehensive Mutational Landscape of Yeast Mutator Strains Reveals the Genetic Basis of Mutational Signatures in Cancer

Yeast Spontaneous Mutation Rate and Spectrum Vary with Environment

Although mutation rates have been extensively studied, variation in mutation rates throughout the genome is poorly understood. To understand patterns of genetic variation, it is important to understand how mutation rates vary. Chromatin modifications may be an important factor in determining variation in mutation rates in eukaryotic genomes. To study variation in mutation rates, we performed a mutation accumulation (MA) experiment in the filamentous fungus Neurospora crassa and sequenced the genomes of the 40 MA lines that had been propagated asexually for approximately mitoses. We detected 1322 mutations in total and observed that the mutation rate was higher in regions of low GC, in domains of H3K9 trimethylation, in centromeric regions, and in domains of H3K27 trimethylation. The rate of single-nucleotide mutations in euchromatin was . In contrast, the mutation rate in H3K9me3 domains was 10-fold higher: 2.43 . We also observed that the spectrum of single-nucleotide mutations was different between H3K9me3 and euchromatic domains. Our statistical model of mutation rate variation predicted a moderate amount of extant genetic variation, suggesting that the mutation rate is an important factor in determining levels of natural genetic variation. Furthermore, we characterized mutation rates of structural variants, complex mutations, and the effect of local sequence context on the mutation rate. Our study highlights that chromatin modifications are associated with mutation rates, and accurate evolutionary inferences should take variation in mutation rates across the genome into account.

Decision letter: Limited role of generation time changes in driving the evolution of the mutation spectrum in humans

Editor's evaluation: Limited role of generation time changes in driving the evolution of the mutation spectrum in humans

Author response: Limited role of generation time changes in driving the evolution of the mutation spectrum in humans

Mutations are the ultimate source of genetic diversity on which natural selection and genetic drift act, playing a crucial role in evolution and long-term adaptation. At the molecular level, the spontaneous mutation rate (µ), defined as the number of mutations per base per generation, thus determines the adaptive potential of a species. Through a mutation accumulation experiment, we estimate the mutation rate and spectrum in Prasinoderma coloniale, a phytoplankton species from an early-branching lineage within the Archaeplastida, characterized by an unusually high genomic guanine-cytosine (GC) content (69.8%). We find that P. coloniale has a very low total mutation rate of µ = 2.00 × 10-10. The insertion-deletion mutation rate is almost 5 times lesser than the single nucleotide mutation rate with µID = 3.40 × 10-11 and µSNM = 1.62 × 10-10. Prasinoderma coloniale also exhibits an atypical mutational spectrum: While essentially all other eukaryotes show a bias toward GC to AT mutations, no evidence of this AT-bias is observed in P. coloniale. Since cytosine methylation is known to be mutagenic, we hypothesized that this may result from an absence of C-methylation. Surprisingly, we found high levels of C-methylation (14% in 5mC, 25% in 5mCG contexts). Methylated cytosines did not show increased mutation rates compared with unmethylated ones, not supporting the prevailing notion that C-methylation universally leads to higher mutation rates. Overall, P. coloniale combines a GC-rich genome with a low mutation rate and original mutation spectrum, suggesting the almost universal AT-bias may not have been present in the ancestor of the green lineage.

Mutational Signatures in Helicobacter pylori–induced Gastric Cancer: Lessons From New Sequencing Technologies

By altering the dynamics of DNA replication and repair, alternative ploidy states may experience different rates and types of new mutations, leading to divergent evolutionary outcomes. We report a direct comparison of the genome-wide spectrum of spontaneous mutations arising in haploids and diploids following a mutation-accumulation experiment in the budding yeast Saccharomyces cerevisiae Characterizing the number, types, locations, and effects of thousands of mutations revealed that haploids were more prone to single-nucleotide mutations (SNMs) and mitochondrial mutations, while larger structural changes were more common in diploids. Mutations were more likely to be detrimental in diploids, even after accounting for the large impact of structural changes, contrary to the prediction that mutations would have weaker effects, due to masking, in diploids. Haploidy is expected to reduce the opportunity for conservative DNA repair involving homologous chromosomes, increasing the insertion-deletion rate, but we found little support for this idea. Instead, haploids were more susceptible to SNMs in late-replicating genomic regions, resulting in a ploidy difference in the spectrum of substitutions. In diploids, we detect mutation rate variation among chromosomes in association with centromere location, a finding that is supported by published polymorphism data. Diploids are not simply doubled haploids; instead, our results predict that the spectrum of spontaneous mutations will substantially shape the dynamics of genome evolution in haploid and diploid populations.

Mutations provide the raw material for natural selection to act. Therefore, understanding the variety and relative frequency of different type of mutations is critical to understanding the nature of genetic diversity in a population. Mutation accumulation (MA) experiments have been used in this context to estimate parameters defining mutation rates, distribution of fitness effects (DFE), and spectrum of mutations. MA experiments can be performed with different effective population sizes. In MA experiments with bacteria, a single founder is grown to a size of a colony (~ 108). It is assumed that natural selection plays a minimal role in dictating the dynamics of colony growth. In this work, we simulate colony growth via a mathematical model, and use our model to mimic an MA experiment. We demonstrate that selection ensures that, in an MA experiment, fraction of all mutations that are beneficial is over-represented by a factor of almost two, and that the distribution of fitness effects of beneficial and deleterious mutations are inaccurately captured in an MA experiment. Given this, the estimate of mutation rates from MA experiments is non-trivial. We then perform an MA experiment with 160 lines of E. coli, and show that due to the effect of selection in a growing colony, the size and sector of a colony from which the experiment is propagated impacts the results. Overall, we demonstrate that the results of MA experiments need to be revisited taking into account the action of selection in a growing colony.

The mutation-accumulation (MA) experiment is a fixture of evolutionary biology, though its execution is laborious. MA experiments typically take between months and years to acquire sufficient mutations to measure DNA mutation rates and mutation spectra. MA experiments for many organisms rely on colony formation on agar plates and repetitive streaking, an environment which at first glance appears somewhat contrived, a poor imitation of actual environmental living conditions. We propose that a fully liquid-phase MA experiment may at times more accurately reflect the environment of an organism. We note also that whereas automation of streaking plates is a daunting prospect, automation of liquid handling, and serial dilution is already commonplace. In principle, this type of MA experiment can be automated so as to reduce the human capital requirements of measuring mutation rates. We demonstrate that a liquid MA recapitulates the mutation rate estimated for MMR-E. coli in liquid LB culture vs. plate Lysogeny Broth culture. We detect a modified mutation spectrum with a transition skew of 4.7:1 of vs. mutations, highlighting the potential role of tautomerization as a mechanism of DNA mutation.

Archaea are a major part of Earth's microbiota and extremely diverse. Yet, we know very little about the process of mutation that drives such diversification. To expand beyond previous work with the moderate halophilic archaeal species Haloferax volcanii, we performed a mutation-accumulation experiment followed by whole-genome sequencing in the extremely halophilic archaeon Halobacterium salinarum. Although Hfx. volcanii and Hbt. salinarum have different salt requirements, both species have highly polyploid genomes and similar GC content. We accumulated mutations for an average of 1250 generations in 67 mutation accumulation lines of Hbt. salinarum, and revealed 84 single-base substitutions and 10 insertion-deletion mutations. The estimated base-substitution mutation rate of 3.99 × 10-10 per site per generation or 1.0 × 10-3 per genome per generation in Hbt. salinarum is similar to that reported for Hfx. volcanii (1.2 × 10-3 per genome per generation), but the genome-wide insertion-deletion rate and spectrum of mutations are somewhat dissimilar in these archaeal species. The spectra of spontaneous mutations were AT biased in both archaea, but they differed in significant ways that may be related to differences in the fidelity of DNA replication/repair mechanisms or a simple result of the different salt concentrations.

Knowing mutation rates and the molecular spectrum of spontaneous mutations is important to understanding how the genetic composition of viral populations evolves. Previous studies have shown that the rate of spontaneous mutations for RNA viruses widely varies between 0.01 and 2 mutations per genome and generation, with plant RNA viruses always occupying the lower side of this range. However, this peculiarity of plant RNA viruses is based on a very limited number of studies. Here we analyze the spontaneous mutational spectrum and the mutation rate of Tobacco etch potyvirus, a model system of positive sense RNA viruses. Our experimental setup minimizes the action of purifying selection on the mutational spectrum, thus giving a picture of what types of mutations are produced by the viral replicase. As expected for a neutral target, we found that transitions and nonsynonymous (including a few stop codons and small deletions) mutations were the most abundant type. This spectrum was notably different from the one previously described for another plant virus. We have estimated that the spontaneous mutation rate for this virus was in the range 10(-6)-10(-5) mutations per site and generation. Our estimates are in the same biological ballpark that previous values reported for plant RNA viruses. This finding gives further support to the idea that plant RNA viruses may have lower mutation rates than their animal counterparts.

Spontaneous mutations play a central role in evolution. Despite their importance, mutation rates are some of the most elusive parameters to measure in evolutionary biology. The combination of mutation accumulation (MA) experiments and whole-genome sequencing now makes it possible to estimate mutation rates by directly observing new mutations at the molecular level across the whole genome. We performed an MA experiment with the social amoeba Dictyostelium discoideum and sequenced the genomes of three randomly chosen lines using high-throughput sequencing to estimate the spontaneous mutation rate in this model organism. The mitochondrial mutation rate of 6.76×10−9, with a Poisson confidence interval of 4.1×10−9 − 9.5×10−9, per nucleotide per generation is slightly lower than estimates for other taxa. The mutation rate estimate for the nuclear DNA of 2.9×10−11, with a Poisson confidence interval ranging from 7.4×10−13 to 1.6×10−10, is the lowest reported for any eukaryote. These results are consistent with low microsatellite mutation rates previously observed in D. discoideum and low levels of genetic variation observed in wild D. discoideum populations. In addition, D. discoideum has been shown to be quite resistant to DNA damage, which suggests an efficient DNA-repair mechanism that could be an adaptation to life in soil and frequent exposure to intracellular and extracellular mutagenic compounds. The social aspect of the life cycle of D. discoideum and a large portion of the genome under relaxed selection during vegetative growth could also select for a low mutation rate. This hypothesis is supported by a significantly lower mutation rate per cell division in multicellular eukaryotes compared with unicellular eukaryotes.

Transcription-associated mutagenesis in yeast is directly proportional to the level of gene expression and influenced by the direction of DNA replication

Spontaneous mutations are the ultimate source of novel genetic variation on which evolution operates. Although mutation rate is often discussed as a single parameter in evolution, it comprises multiple distinct types of changes at the level of DNA. Moreover, the rates of these distinct changes can be independently influenced by genomic background and environmental conditions. Using fluctuation tests, we characterized the spectrum of spontaneous mutations in Escherichia coli grown in low and high glucose environments. These conditions are known to affect the rate of spontaneous mutation in wild-type MG1655, but not in a ΔluxS deletant strain - a gene with roles in both quorum sensing and the recycling of methylation products used in E. coli's DNA repair process. We find an increase in AT>GC transitions in the low glucose environment, suggesting that processes relating to the production or repair of this mutation could drive the response of overall mutation rate to glucose concentration. Interestingly, this increase in AT>GC transitions is maintained by the glucose non-responsive ΔluxS deletant. Instead, an elevated rate of GC>TA transversions, more common in a high glucose environment, leads to a net non-responsiveness of overall mutation rate for this strain. Our results show how relatively subtle changes, such as the concentration of a carbon substrate or loss of a regulatory gene, can substantially influence the amount and nature of genetic variation available to selection.