Genome-wide Mutation Rate Research Articles

MutLα suppresses error-prone DNA mismatch repair and preferentially protects noncoding DNA from mutations

The DNA mismatch repair (MMR) system promotes genome stability and protects humans from certain types of cancer. Its primary function is the correction of DNA polymerase errors. MutLα is an important eukaryotic MMR factor. We have examined the contributions of MutLα to maintaining genome stability. We show here that loss of MutLα in yeast increases the genome-wide mutation rate by ∼130-fold and generates a genome-wide mutation spectrum that consists of small indels and base substitutions. We also show that loss of yeast MutLα leads to error-prone MMR that produces T > C base substitutions in 5'-ATA-3' sequences. In agreement with this finding, our examination of human whole-genome DNA sequencing data has revealed that loss of MutLα in induced pluripotent stem cells triggers error-prone MMR that leads to the formation of T > C mutations in 5'-NTN-3' sequences. Our further analysis has shown that MutLα-independent MMR plays a role in suppressing base substitutions in N3 homopolymeric runs. In addition, we describe that MutLα preferentially protects noncoding DNA from mutations. Our study defines the contributions of MutLα-dependent and independent mechanisms to genome-wide MMR.

Background Selection From Unlinked Sites Causes Nonindependent Evolution of Deleterious Mutations.

Background selection describes the reduction in neutral diversity caused by selection against deleterious alleles at other loci. It is typically assumed that the purging of deleterious alleles affects linked neutral variants, and indeed simulations typically only treat a genomic window. However, background selection at unlinked loci also depresses neutral diversity. In agreement with previous analytical approximations, in our simulations of a human-like genome with a realistically high genome-wide deleterious mutation rate, the effects of unlinked background selection exceed those of linked background selection. Background selection reduces neutral genetic diversity by a factor that is independent of census population size. Outside of genic regions, the strength of background selection increases with the mean selection coefficient, contradicting the linked theory but in agreement with the unlinked theory. Neutral diversity within genic regions is fairly independent of the strength of selection. Deleterious genetic load among haploid individuals is underdispersed, indicating nonindependent evolution of deleterious mutations. Empirical evidence for underdispersion was previously interpreted as evidence for global epistasis, but we recover it from a non-epistatic model.

A mutation rate model at the basepair resolution identifies the mutagenic effect of polymerase III transcription.

De novo mutations occur at substantially different rates depending on genomic location, sequence context and DNA strand. The success of methods to estimate selection intensity, infer demographic history and map rare disease genes, depends strongly on assumptions about the local mutation rate. Here we present Roulette, a genome-wide mutation rate model at basepair resolution that incorporates known determinants of local mutation rate. Roulette is shown to be more accurate than existing models. We use Roulette to refine the estimates of population growth within Europe by incorporating the full range of human mutation rates. The analysis of significant deviations from the model predictions revealed a tenfold increase in mutation rate in nearly all genes transcribed by polymerase III (Pol III), suggesting a new mutagenic mechanism. We also detected an elevated mutation rate within transcription factor binding sites restricted to sites actively used in testis and residing in promoters.

Contribution of the SOS response and the DNA repair systems to norfloxacin induced mutations in E. coli

Antibiotic-resistant bacteria severely threaten human health. Besides spontaneous mutations generated by endogenous factors, the resistance might also originate from mutations induced by certain antibiotics, such as the fluoroquinolones. Such antibiotics increase the genome-wide mutation rate by introducing replication errors from the SOS response pathway or decreasing the efficiency of the DNA repair systems. However, the relative contributions of these molecular mechanisms remain unclear, hindering understanding of the generation of resistant pathogens. Here, using newly-accumulated mutations of wild-type and SOS-uninducible Escherichia coli strains, as well as those of the strains deficient for the mismatch repair (MMR) and the oxidative damage repair pathways, we find that the SOS response is the major mutagenesis contributor in mutation elevation, responsible for ~ 30–50% of the total base-pair substitution (BPS) mutation-rate elevation upon treatment with sublethal levels of norfloxacin (0 ~ 50 ng/mL). We further estimate the significance of the effects on other mutational features of these mechanisms (i.e., transversions, structural variations, and mutation spectrum) in E. coli using linear models. The SOS response plays a positive role in all three mutational features (mutation rates of BPSs, transversions, structural variations) and affects the mutational spectrum. The repair systems significantly reduce the BPS mutation rate and the transversion rate, regardless of whether antibiotics are present, while significantly increasing the structural variation rate in E. coli. Our results quantitatively disentangle the contributions of the SOS response and DNA repair systems in antibiotic-induced mutagenesis.

Genome-wide probing of eukaryotic nascent RNA structure elucidates cotranscriptional folding and its antimutagenic effect

The transcriptional intermediates of RNAs fold into secondary structures with multiple regulatory roles, yet the details of such cotranscriptional RNA folding are largely unresolved in eukaryotes. Here, we present eSPET-seq (Structural Probing of Elongating Transcripts in eukaryotes), a method to assess the cotranscriptional RNA folding in Saccharomyces cerevisiae. Our study reveals pervasive structural transitions during cotranscriptional folding and overall structural similarities between nascent and mature RNAs. Furthermore, a combined analysis with genome-wide R-loop and mutation rate approximations provides quantitative evidence for the antimutator effect of nascent RNA folding through competitive inhibition of the R-loops, known to facilitate transcription-associated mutagenesis. Taken together, we present an experimental evaluation of cotranscriptional folding in eukaryotes and demonstrate the antimutator effect of nascent RNA folding. These results suggest genome-wide coupling between the processing and transmission of genetic information through RNA folding.

Selective Pressure by Rifampicin Modulates Mutation Rates and Evolutionary Trajectories of Mycobacterial Genomes.

Resistance to the frontline antibiotic rifampicin constitutes a challenge to the treatment and control of tuberculosis. Here, we analyzed the mutational landscape of Mycobacterium smegmatis during long-term evolution with increasing concentrations of rifampicin, using a mutation accumulation assay combined with whole-genome sequencing. Antibiotic treatment enhanced the acquisition of mutations, doubling the genome-wide mutation rate of the wild-type cells. While antibiotic exposure led to extinction of almost all wild-type lines, the hypermutable phenotype of the ΔnucS mutant strain (noncanonical mismatch repair deficient) provided an efficient response to the antibiotic, leading to high rates of survival. This adaptative advantage resulted in the emergence of higher levels of rifampicin resistance, an accelerated acquisition of drug resistance mutations in rpoB (β RNA polymerase), and a wider diversity of evolutionary pathways that led to drug resistance. Finally, this approach revealed a subset of adaptive genes under positive selection with rifampicin that could be associated with the development of antibiotic resistance. IMPORTANCE Rifampicin is the most important first-line antibiotic against mycobacterial infections, including tuberculosis, one of the top causes of death worldwide. Acquisition of rifampicin resistance constitutes a major global public health problem that makes the control of the disease challenging. Here, we performed an experimental evolution assay under antibiotic selection to analyze the response and adaptation of mycobacteria, leading to the acquisition of rifampicin resistance. This approach explored the total number of mutations that arose in the mycobacterial genomes under long-term rifampicin exposure, using whole-genome sequencing. Our results revealed the effect of rifampicin at a genomic level, identifying different mechanisms and multiple pathways leading to rifampicin resistance in mycobacteria. Moreover, this study detected that an increase in the rate of mutations led to enhanced levels of drug resistance and survival. In summary, all of these results could be useful to understand and prevent the emergence of drug-resistant isolates in mycobacterial infections.

Genome-wide contributions of the MutSα- and MutSβ-dependent DNA mismatch repair pathways to the maintenance of genetic stability in Saccharomyces cerevisiae

The DNA mismatch repair (MMR) system is a major DNA repair system that suppresses inherited and sporadic cancers in humans. In eukaryotes the MutSα-dependent and MutSβ-dependent MMR pathways correct DNA polymerase errors. Here, we investigated these two pathways on a whole-genome level in S. cerevisiae. We found that inactivation of MutSα-dependent MMR by deletion of the MSH6 gene increases the genome-wide mutation rate by ∼17-fold, and loss of MutSβ-dependent MMR via deletion of MSH3 elevates the genome-wide mutation rate by ∼4-fold. We also found that MutSα-dependent MMR does not show a preference for protecting coding or noncoding DNA from mutations, whereas MutSβ-dependent MMR preferentially protects noncoding DNA from mutations. The most frequent mutations in the msh6Δ strain are C>T transitions, whereas 1-6-bp deletions are the most common genetic alterations in the msh3Δ strain. Strikingly, MutSα-dependent MMR is more important than MutSβ-dependent MMR for protection from 1-bp insertions, while MutSβ-dependent MMR has a more critical role in the defense against 1-bp deletions and 2-6-bp indels. We also determined that a mutational signature of yeast MSH6 loss is similar to mutational signatures of human MMR deficiency. Furthermore, our analysis showed that compared to other 5'-NCN-3' trinucleotides, 5'-GCA-3' trinucleotides are at the highest risk of accumulating C>T transitions at the central position in the msh6Δ cells and that the presence of a G/A base at the -1 position is important for the efficient MutSα-dependent suppression of C>T transitions. Our results highlight key differences between the roles of the MutSα-dependent and MutSβ-dependent MMR pathways.

Estimating the genome-wide mutation rate from thousands of unrelated individuals

We provide a method for estimating the genome-wide mutation rate from sequence data on unrelated individuals by using segments of identity by descent (IBD). The length of an IBD segment indicates the time to shared ancestor of the segment, and mutations that have occurred since the shared ancestor result in discordances between the two IBD haplotypes. Previous methods for IBD-based estimation of mutation rate have required the use of family data for accurate phasing of the genotypes. This has limited the scope of application of IBD-based mutation rate estimation. Here, we develop an IBD-based method for mutation rate estimation from population data, and we apply it to whole-genome sequence data on 4,166 European American individuals from the TOPMed Framingham Heart Study, 2,996 European American individuals from the TOPMed My Life, Our Future study, and 1,586 African American individuals from the TOPMed Hypertension Genetic Epidemiology Network study. Although mutation rates may differ between populations as a result of genetic factors, demographic factors such as average parental age, and environmental exposures, our results are consistent with equal genome-wide average mutation rates across these three populations. Our overall estimate of the average genome-wide mutation rate per 108 base pairs per generation for single-nucleotide variants is 1.24 (95% CI 1.18-1.33).

Fragaria mitogenomes evolve rapidly in structure but slowly in sequence and incur frequent multinucleotide mutations mediated by microinversions.

Plant mitochondrial DNA has been described as evolving rapidly in structure but slowly in sequence. However, many of the noncoding portions of plant mitogenomes are not homologous among species, raising questions about the rate and spectrum of mutations in noncoding regions. Recent studies have suggested that the lack of homology in noncoding regions could be due to increased sequence divergence. We compared 30 kb of coding and 200 kb of noncoding DNA from 13 sequenced Fragaria mitogenomes, followed by analysis of the rate of sequence divergence, microinversion events and structural variations. Substitution rates in synonymous sites and nongenic sites are nearly identical, suggesting that the genome-wide point mutation rate is generally consistent. A surprisingly high number of large multinucleotide substitutions were detected in Fragaria mitogenomes, which may have resulted from microinversion events and could affect phylogenetic signal and local rate estimates. Fragaria mitogenomes preferentially accumulate deletions relative to insertions and substantial genomic arrangements, whereas mutation rates could positively associate with these sequence and structural changes among species. Together, these observations suggest that plant mitogenomes exhibit low point mutations genome-wide but exceptionally high structural variations, and our results favour a gain-and-loss model for the rapid loss of homology among plant mitogenomes.

Genome-wide Mutation Rate Modeling Identifies Novel Driver Mutations.

Driver mutations were identified in cryptic splice regions, 5' UTRs, and rarely mutated genes.

Base-substitution mutation rate across the nuclear genome of Alpheus snapping shrimp and the timing of isolation by the Isthmus of Panama

BackgroundThe formation of the Isthmus of Panama and final closure of the Central American Seaway (CAS) provides an independent calibration point for examining the rate of DNA substitutions. This vicariant event has been widely used to estimate the substitution rate across mitochondrial genomes and to date evolutionary events in other taxonomic groups. Nuclear sequence data is increasingly being used to complement mitochondrial datasets for phylogenetic and evolutionary investigations; these studies would benefit from information regarding the rate and pattern of DNA substitutions derived from the nuclear genome.ResultsTo estimate the genome-wide neutral mutation rate (µ), genotype-by-sequencing (GBS) datasets were generated for three transisthmian species pairs in Alpheus snapping shrimp. A range of bioinformatic filtering parameters were evaluated in order to minimize potential bias in mutation rate estimates that may result from SNP filtering. Using a Bayesian coalescent approach (G-PhoCS) applied to 44,960 GBS loci, we estimated µ to be 2.64E−9 substitutions/site/year, when calibrated with the closure of the CAS at 3 Ma. Post-divergence gene flow was detected in one species pair. Failure to account for this post-split migration inflates our substitution rate estimates, emphasizing the importance of demographic methods that can accommodate gene flow.ConclusionsResults from our study, both parameter estimates and bioinformatic explorations, have broad-ranging implications for phylogeographic studies in other non-model taxa using reduced representation datasets. Our best estimate of µ that accounts for coalescent and demographic processes is remarkably similar to experimentally derived mutation rates in model arthropod systems. These results contradicted recent suggestions that the closure of the Isthmus was completed much earlier (around 10 Ma), as mutation rates based on an early calibration resulted in uncharacteristically low genomic mutation rates. Also, stricter filtering parameters resulted in biased datasets that generated lower mutation rate estimates and influenced demographic parameters, serving as a cautionary tale for the adherence to conservative bioinformatic strategies when generating reduced-representation datasets at the species level. To our knowledge this is the first use of transisthmian species pairs to calibrate the rate of molecular evolution from GBS data.

Streptomycin and nalidixic acid elevate the spontaneous genome-wide mutation rate in Escherichia coli.

Since antibiotic resistance is a growing public health problem worldwide, it is important to understand how antibiotics and spontaneous mutations cooperate and shape the genome-wide mutation rate and spectrum. Here, we quantitatively evaluate genome-wide mutational profiles of Escherichia coli after long-term subinhibitory exposure to a broad-spectrum (streptomycin) and a narrow-spectrum antibiotic (nalidixic acid), using a mutation accumulation design combined with whole-genome resequencing of replicate lines as a mutagenicity test. We determined that, while the genome-wide mutation rate is slightly higher in the streptomycin-treated lines compared to the control lines, there is a significant increase in the nalidixic acid-treated lines. Our findings suggest that both broad and narrow-spectrum antibiotics may elevate the mutation rates in E. coli, but mechanisms of action may affect the consequence, thus contribute to accelerating the rate of adaptation and conferring antibiotic resistance.

Rate volatility and asymmetric segregation diversify mutation burden in cells with mutator alleles

Mutations that compromise mismatch repair (MMR) or DNA polymerase ε or δ exonuclease domains produce mutator phenotypes capable of fueling cancer evolution. Here, we investigate how combined defects in these pathways expands genetic heterogeneity in cells of the budding yeast, Saccharomyces cerevisiae, using a single-cell resolution approach that tallies all mutations arising from individual divisions. The distribution of replication errors present in mother cells after the initial S-phase was broader than expected for a single uniform mutation rate across all cell divisions, consistent with volatility of the mutator phenotype. The number of mismatches that then segregated to the mother and daughter cells co-varied, suggesting that each division is governed by a different underlying genome-wide mutation rate. The distribution of mutations that individual cells inherit after the second S-phase is further broadened by the sequential actions of semiconservative replication and mitotic segregation of chromosomes. Modeling suggests that this asymmetric segregation may diversify mutation burden in mutator-driven tumors.

Variation of the adaptive substitution rate between species and within genomes

The importance of adaptive mutations in molecular evolution is extensively debated. Recent developments in population genomics allow inferring rates of adaptive mutations by fitting a distribution of fitness effects to the observed patterns of polymorphism and divergence at sites under selection and sites assumed to evolve neutrally. Here, we summarize the current state-of-the-art of these methods and review the factors that affect the molecular rate of adaptation. Several studies have reported extensive cross-species variation in the proportion of adaptive amino-acid substitutions (α) and predicted that species with larger effective population sizes undergo less genetic drift and higher rates of adaptation. Disentangling the rates of positive and negative selection, however, revealed that mutations with deleterious effects are the main driver of this population size effect and that adaptive substitution rates vary comparatively little across species. Conversely, rates of adaptive substitution have been documented to vary substantially within genomes. On a genome-wide scale, gene density, recombination and mutation rate were observed to play a role in shaping molecular rates of adaptation, as predicted under models of linked selection. At the gene level, it has been reported that the gene functional category and the macromolecular structure substantially impact the rate of adaptive mutations. Here, we deliver a comprehensive review of methods used to infer the molecular adaptive rate, the potential drivers of adaptive evolution and how positive selection shapes molecular evolution within genes, across genes within species and between species.

Selection on mutators is not frequency-dependent.

The evolutionary fate of mutator mutations - genetic variants that raise the genome-wide mutation rate - in asexual populations is often described as being frequency (or number) dependent. Mutators can invade a population by hitchhiking with a sweeping beneficial mutation, but motivated by earlier experiments results, it has been repeatedly suggested that mutators must be sufficiently frequent to produce such a driver mutation before non-mutators do. Here, we use stochastic, agent-based simulations to show that neither the strength nor the sign of selection on mutators depend on their initial frequency, and while the overall probability of hitchhiking increases predictably with frequency, the per-capita probability of fixation remains unchanged.

CG hypomethylation leads to complex changes in DNA methylation and transpositional burst of diverse transposable elements in callus cultures of rice.

CG methylation (m CG) is essential for preserving genome stability in mammals, but this link remains obscure in plants. OsMET1-2, a major rice DNA methyltransferase, plays critical roles in maintaining m CG in rice. Null mutation of OsMET1-2 causes massive CG hypomethylation, rendering the mutant suitable to address the role of m CG in maintaining genome integrity in plants. Here, we analyzed m CG dynamics and genome stability in tissue cultures of OsMET1-2 homozygous (-/-) and heterozygous (+/-) mutants, and isogenic wild-type (WT). We found m CG levels in cultures of -/- were substantially lower than in those of WT and +/-, as expected. Unexpectedly, m CG levels in 1- and 3-year cultures of -/- were 77.6% and 48.7% higher, respectively, than in shoot, from which the cultures were initiated, suggesting substantial regain of m CG in -/- cultures, which contrasts to the general trend of m CG loss in all WT plant tissue cultures hitherto studied. Transpositional burst of diverse transposable elements (TEs) occurred only in -/- cultures, although no elevation of genome-wide mutation rate in the form of single nucleotide polymorphisms was detected. Altogether, our results establish an essential role of m CG in retaining TE immobility and hence genome stability in rice and likely in plants in general.

Contribution of retrotransposition to developmental disorders

Mobile genetic Elements (MEs) are segments of DNA which can copy themselves and other transcribed sequences through the process of retrotransposition (RT). In humans several disorders have been attributed to RT, but the role of RT in severe developmental disorders (DD) has not yet been explored. Here we identify RT-derived events in 9738 exome sequenced trios with DD-affected probands. We ascertain 9 de novo MEs, 4 of which are likely causative of the patient’s symptoms (0.04%), as well as 2 de novo gene retroduplications. Beyond identifying likely diagnostic RT events, we estimate genome-wide germline ME mutation rate and selective constraint and demonstrate that coding RT events have signatures of purifying selection equivalent to those of truncating mutations. Overall, our analysis represents a comprehensive interrogation of the impact of retrotransposition on protein coding genes and a framework for future evolutionary and disease studies.

Estimating the Genome-wide Mutation Rate with Three-Way Identity by Descent

The two primary methods for estimating the genome-wide mutation rate have been counting de novo mutations in parent-offspring trios and comparing sequence data between closely related species. With parent-offspring trio analysis it is difficult to control for genotype error, and resolution is limited because each trio provides information from only two meioses. Inter-species comparison is difficult to calibrate due to uncertainty in the number of meioses separating species, and it can be biased by selection and by changing mutation rates over time. An alternative class of approaches for estimating mutation rates that avoids these limitations is based on identity by descent (IBD) segments that arise from common ancestry within the past few thousand years. Existing IBD-based methods are limited to highly inbred samples, or lack robustness to genotype error and error in the estimated demographic history. We present an IBD-based method that uses sharing of IBD segments among sets of three individuals to estimate the mutation rate. Our method is applicable to accurately phased genotype data, such as parent-offspring trio data phased using Mendelian rules of inheritance. Unlike standard parent-offspring analysis, our method utilizes distant relationships and is robust to genotype error. We apply our method to data from 1,307 European-ancestry individuals in the Framingham Heart Study sequenced by the NHLBI TOPMed project. We obtain an estimate of 1.29× 10-8 mutations per base pair per meiosis with a 95% confidence interval of [1.02× 10-8, 1.56× 10-8].

Low genetic variation is associated with low mutation rate in the giant duckweed

Mutation rate and effective population size (Ne) jointly determine intraspecific genetic diversity, but the role of mutation rate is often ignored. Here we investigate genetic diversity, spontaneous mutation rate and Ne in the giant duckweed (Spirodela polyrhiza). Despite its large census population size, whole-genome sequencing of 68 globally sampled individuals reveals extremely low intraspecific genetic diversity. Assessed under natural conditions, the genome-wide spontaneous mutation rate is at least seven times lower than estimates made for other multicellular eukaryotes, whereas Ne is large. These results demonstrate that low genetic diversity can be associated with large-Ne species, where selection can reduce mutation rates to very low levels. This study also highlights that accurate estimates of mutation rate can help to explain seemingly unexpected patterns of genome-wide variation.

Biological Roles of Protein-Coding Tandem Repeats in the Yeast Candida Albicans.

Tandem repeat (TR) DNA mutates faster than other DNA by insertion and deletion of repeats. Large parts of eukaryotic proteomes are encoded by ORFs containing protein-coding TRs (TR-ORFs, pcTRs) with largely unknown biological consequences. We explored these in the yeast Candida albicans, an opportunistic human pathogen. We found that almost half of C. albicans’ proteins are encoded by TR-ORFs. pcTR frequency differed only moderately between different gene (GO) categories. Bioinformatic predictions of genome-wide mutation rates and clade-specific differences in pcTR allele frequencies indicated that pcTRs (i) significantly increase the genome-wide mutation rate; (ii) significantly impact on fitness and (iii) allow the evolution of selectively advantageous clade-specific protein variants. Synonymous mutations reduced the repetitiveness of many amino acid repeat-encoding pcTRs. A survey, in 58 strains, revealed that in some pcTR regions in which repetitiveness was not significantly diminished by synonymous mutations the habitat predicted which alleles were present, suggesting roles of pcTR mutation in short-term adaptation and pathogenesis. In C. albicans pcTR mutation apparently is an important mechanism for mutational advance and possibly also rapid adaptation, with synonymous mutations providing a mechanism for adjusting mutation rates of individual pcTRs. Analyses of Arabidopsis and human pcTRs showed that the latter also occurs in other eukaryotes.