The rarA gene as part of an expanded RecFOR recombination pathway: Negative epistasis and synthetic lethality with ruvB, recG, and recQ

The RarA protein, homologous to human WRNIP1 and yeast MgsA, is a AAA+ ATPase and one of the most highly conserved DNA repair proteins. With an apparent role in the repair of stalled or collapsed replication forks, the molecular function of this protein family remains obscure. Here, we demonstrate that RarA acts in late stages of recombinational DNA repair of post-replication gaps. A deletion of most of the rarA gene, when paired with a deletion of ruvB or ruvC, produces a growth defect, a strong synergistic increase in sensitivity to DNA damaging agents, cell elongation, and an increase in SOS induction. Except for SOS induction, these effects are all suppressed by inactivating recF, recO, or recJ, indicating that RarA, along with RuvB, acts downstream of RecA. SOS induction increases dramatically in a rarA ruvB recF/O triple mutant, suggesting the generation of large amounts of unrepaired ssDNA. The rarA ruvB defects are not suppressed (and in fact slightly increased) by recB inactivation, suggesting RarA acts primarily downstream of RecA in post-replication gaps rather than in double strand break repair. Inactivating rarA, ruvB and recG together is synthetically lethal, an outcome again suppressed by inactivation of recF, recO, or recJ. A rarA ruvB recQ triple deletion mutant is also inviable. Together, the results suggest the existence of multiple pathways, perhaps overlapping, for the resolution or reversal of recombination intermediates created by RecA protein in post-replication gaps within the broader RecF pathway. One of these paths involves RarA.

Joint effects of genes underlying a temperature specialization tradeoff in yeast

A central goal of evolutionary genetics is to understand, at the molecular level, how organisms adapt to their environments. For a given trait, the answer often involves the acquisition of variants at unlinked sites across the genome. Genomic methods have achieved landmark successes in pinpointing these adaptive loci. To figure out how a suite of adaptive alleles work together, and to what extent they can reconstitute the phenotype of interest, requires their transfer into an exogenous background. We studied the joint effect of adaptive, gain-of-function thermotolerance alleles at eight unlinked genes from Saccharomyces cerevisiae, when introduced into a thermosensitive sister species, S. paradoxus. Although the loci damped each other’s beneficial impact (that is, they were subject to negative epistasis), most boosted high-temperature growth alone and in combination, and none was deleterious. The complete set of eight genes was sufficient to confer ~15% of the S. cerevisiae thermotolerance phenotype in the S. paradoxus background. The same loci also contributed to a heretofore unknown advantage in cold growth by S. paradoxus. Together, our data establish temperature resistance in yeasts as a model case of a genetically complex evolutionary tradeoff, which can be partly reconstituted from the sequential assembly of unlinked underlying loci.

Inferring inter- and intrachromosomal Dobzhansky-Muller incompatibilities from imbalanced segregation of recombinant haplotypes in hybrid populations

Dobzhansky-Muller incompatibilities (DMIs) are a major component of reproductive isolation between species. DMIs imply negative epistasis, exposed when two diverged populations hybridize. Mapping the locations of DMIs has largely relied on classical genetic mapping, but these approaches are stymied by low power and the challenge of identifying DMI loci on the same chromosome, because strong initial linkage of parental haplotypes weakens statistical tests. Here, we propose new statistics to infer negative epistasis from haplotype frequencies in hybrid populations. When two divergent populations hybridize, the variance of two-locus heterozygosity decreases faster with time at DMI loci than at random pairs of loci. If two populations hybridize at near-even admixture proportions, the deviation of the observed variance from its expectation is negative, which enables us to detect signals of intermediate to strong negative epistasis both within and between chromosomes. When the initial proportion of the two parental populations is uneven, only strong DMIs can be detected with our method, unless migration reintroduces haplotypes from the minor parental population. We use the two new statistics to infer candidate DMIs from three hybrid populations of swordtail fish. We identify numerous new DMI candidates some of which are inferred to interact with several loci within and between chromosomes. Moreover, we discuss our results in the context of an expected enrichment in intrachromosomal over interchromosomal DMIs.

Negative linkage disequilibrium between amino acid changing variants reveals interference among deleterious mutations in the human genome

Evolutionary forces like Hill-Robertson interference and negative epistasis can lead to deleterious mutations being found on distinct haplotypes. However, the extent to which these forces depend on the selection and dominance coefficients of deleterious mutations and shape genome-wide patterns of linkage disequilibrium (LD) in natural populations with complex demographic histories has not been tested. In this study, we first used forward-in-time simulations to predict how negative selection impacts LD. Under models where deleterious mutations have additive effects on fitness, deleterious variants less than 10 kb apart tend to be carried on different haplotypes relative to pairs of synonymous SNPs. In contrast, for recessive mutations, there is no consistent ordering of how selection coefficients affect LD decay, due to the complex interplay of different evolutionary effects. We then examined empirical data of modern humans from the 1000 Genomes Project. LD between derived alleles at nonsynonymous SNPs is lower compared to pairs of derived synonymous variants, suggesting that nonsynonymous derived alleles tend to occur on different haplotypes more than synonymous variants. This result holds when controlling for potential confounding factors by matching SNPs for frequency in the sample (allele count), physical distance, magnitude of background selection, and genetic distance between pairs of variants. Lastly, we introduce a new statistic HR(j) which allows us to detect interference using unphased genotypes. Application of this approach to high-coverage human genome sequences confirms our finding that nonsynonymous derived alleles tend to be located on different haplotypes more often than are synonymous derived alleles. Our findings suggest that interference may play a pervasive role in shaping patterns of LD between deleterious variants in the human genome, and consequently influences genome-wide patterns of LD.

Multilocus adaptation to vaccination

Pathogen adaptation to public health interventions, such as vaccination, may take tortuous routes and involve a multitude of genetic mutations acting on distinct phenotypic traits. For example, pathogens can escape the vaccine-induced immune response, or adjust their virulence so as to increase transmission in vaccinated hosts. Despite its importance for public health and vaccine efficacy, how these two adaptations jointly evolve is poorly understood. Taking a trait-centered, rather than variant-centered perspective, here we elucidate the role played by epistasis and recombination, with an emphasis on the different protective effects of vaccination. Vaccines reducing transmission and/or increasing clearance generate positive epistasis between the vaccine-escape and virulence alleles, favouring strains that carry both mutations. Vaccines reducing virulence mortality generate negative epistasis, favouring strains that carry either mutation, but not both. If epistasis is positive, recombination can lead to the sequential fixation of the two mutations and prevent the transient build-up of more virulent escape strains. If epistasis is negative, recombination between loci can create an evolutionary bistability between alternative routes of adaptation, such that whichever adaptation is more accessible tends to be favoured in the long-term. More generally, our model provides a valuable framework for studying pathogen adaptation from a trait-centered view, shifting the focus away from variants.

Associative Overdominance and Negative Epistasis Shape Genome-Wide Ancestry Landscape in Supplemented Fish Populations

The interplay between recombination rate, genetic drift and selection modulates variation in genome-wide ancestry. Understanding the selective processes at play is of prime importance toward predicting potential beneficial or negative effects of supplementation with domestic strains (i.e., human-introduced strains). In a system of lacustrine populations supplemented with a single domestic strain, we documented how population genetic diversity and stocking intensity produced lake-specific patterns of domestic ancestry by taking the species’ local recombination rate into consideration. We used 552 Brook Charr (Salvelinus fontinalis) from 22 small lacustrine populations, genotyped at ~32,400 mapped SNPs. We observed highly variable patterns of domestic ancestry between each of the 22 populations without any consistency in introgression patterns of the domestic ancestry. Our results suggest that such lake-specific ancestry patterns were mainly due to variable associative overdominance (AOD) effects among populations (i.e., potential positive effects due to the masking of possible deleterious alleles in low recombining regions). Signatures of AOD effects were also emphasized by highly variable patterns of genetic diversity among and within lakes, potentially driven by predominant genetic drift in those small isolated populations. Local negative effects such as negative epistasis (i.e., potential genetic incompatibilities between the native and the introduced population) potentially reflecting precursory signs of outbreeding depression were also observed at a chromosomal scale. Consequently, in order to improve conservation practices and management strategies, it became necessary to assess the consequences of supplementation at the population level by taking into account both genetic diversity and stocking intensity when available.

A survey of pairwise epistasis supports an outside-in hierarchy of clade-specifying and function-defining residues in PSD95 PDZ3

Deep mutational scanning enables data-driven models of protein structure and function. Here, we adapted Saturated Programmable Insertion Engineering as an economical and programmable deep mutational scanning technique. We validate this approach with an existing single mutant dataset in the PSD95 PDZ3 domain, and further characterize most pairwise double mutants to study how a mutation’s phenotype depends on mutations at other sites, a phenomenon called epistasis. We observe wide-spread proximal negative epistasis, which we attribute to mutations affecting thermodynamic stability, and strong long-range positive epistasis, which is enriched in an evolutionarily conserved and function-defining network of ‘sector’ and clade-specifying residues. Conditional neutrality of mutations in clade-specifying residues compensates for deleterious mutations in sector positions. This suggests an outside-in hierarchy of interactions through which positive epistasis between clade-specifying residues and the PDZ sector facilitated the evolutionary expansion and specialization of PDZ domains.

Antagonism between substitutions in β-lactamase explains a path not taken in the evolution of bacterial drug resistance

CTX-M β-lactamases are widespread in Gram-negative bacterial pathogens and provide resistance to the cephalosporin cefotaxime but not to the related antibiotic ceftazidime. Nevertheless, variants have emerged that confer resistance to ceftazidime. Two natural mutations, causing P167S and D240G substitutions in the CTX-M enzyme, result in 10-fold increased hydrolysis of ceftazidime. Although the combination of these mutations would be predicted to increase ceftazidime hydrolysis further, the P167S/D240G combination has not been observed in a naturally occurring CTX-M variant. Here, using recombinantly expressed enzymes, minimum inhibitory concentration measurements, steady-state enzyme kinetics, and X-ray crystallography, we show that the P167S/D240G double mutant enzyme exhibits decreased ceftazidime hydrolysis, lower thermostability, and decreased protein expression levels compared with each of the single mutants, indicating negative epistasis. X-ray structures of mutant enzymes with covalently trapped ceftazidime suggested that a change of an active-site Ω-loop to an open conformation accommodates ceftazidime leading to enhanced catalysis. 10-μs molecular dynamics simulations further correlated Ω-loop opening with catalytic activity. We observed that the WT and P167S/D240G variant with acylated ceftazidime both favor a closed conformation not conducive for catalysis. In contrast, the single substitutions dramatically increased the probability of open conformations. We conclude that the antagonism is due to restricting the conformation of the Ω-loop. These results reveal the importance of conformational heterogeneity of active-site loops in controlling catalytic activity and directing evolutionary trajectories.

The Complexity Ratchet: Stronger than Selection, Stronger than Evolvability, Weaker than Robustness

Using the in silico experimental evolution platform Aevol, we have tested the existence of a complexity ratchet by evolving populations of digital organisms under environmental conditions in which simple organisms can very well thrive and reproduce. We observed that in most simulations, organisms become complex although such organisms are a lot less fit than simple ones and have no robustness or evolvability advantage. This excludes selection from the set of possible explanations for the evolution of complexity. However, complementary experiments showed that selection is nevertheless necessary for complexity to evolve, also excluding non-selective effects. Analyzing the long-term fate of complex organisms, we showed that complex organisms almost never switch back to simplicity despite the potential fitness benefit. On the contrary, they consistently accumulate complexity in the long term, meanwhile slowly increasing their fitness but never overtaking that of simple organisms. This suggests the existence of a complexity ratchet powered by negative epistasis: Mutations leading to simple solutions, which are favorable at the beginning of the simulation, become deleterious after other mutations—leading to complex solutions—have been fixed. This also suggests that this complexity ratchet cannot be beaten by selection, but that it can be overthrown by robustness because of the constraints it imposes on the coding capacity of the genome.