Compensatory Evolution to DNA Replication Stress is Robust to Nutrient Availability

Abstract

Evolutionary repair refers to the compensatory evolution that follows perturbations in cellular processes. While evolutionary trajectories are often reproducible, other studies suggest they are shaped by genotype-by-environment (GxE) interactions. Here, we test the predictability of evolutionary repair in response to DNA replication stress-a severe perturbation impairing the conserved mechanisms of DNA synthesis, resulting in genetic instability. We conducted high-throughput experimental evolution on Saccharomyces cerevisiae experiencing constitutive replication stress, grown under different glucose availabilities. We found that glucose levels impact the physiology and adaptation rate of replication stress mutants. However, the genetics of adaptation show remarkable robustness across environments. Recurrent mutations collectively recapitulated the fitness of evolved lines and are advantageous across macronutrient availability. We also identified a novel role of the mediator complex of RNA polymerase II in adaptation to replicative stress. Our results highlight the robustness and predictability of evolutionary repair mechanisms to DNA replication stress and provide new insights into the evolutionary aspects of genome stability, with potential implications for understanding cancer development.

Figure 1.. Glucose concentration impacts cell physiology in the presence of DNA replication stress.

(A) Population growth rates (min⁻¹) of ancestral WT (black) and ctf4∆ mutant (orange) across different glucose concentrations. Box plots show median, interquartile range (IQR), and whiskers extending to 1.5×IQR, with individual data points beyond whiskers as outliers. (B) Cell-cycle profiles of ancestral WT (left) and ctf4Δ (right) across glucose concentrations. Colors and line styles represent different glucose concentrations: solid line light blue (0.25%), solid line dark blue (0.5%), dash line light green (2%), and dash line dark green (8%). Bold lines indicate mean profiles, and shaded areas represent standard deviation (SD). 1C represents the DNA content of a cell in G1 and 2C of a cell in G2/M. (C) Time spent (minutes) in G1 phase for ancestral WT and ctf4Δ, across different glucose concentrations, estimated from DNA content and doubling times (see Materials and Methods). (D) Mode cell diameter of ancestral WT and ctf4Δ across different glucose concentrations. (E) Mean relative fitness of ancestral ctf4∆ relative to reference WT across different glucose concentrations. Colors represent glucose concentration. Error bars represent SD. Detailed statistical analysis and underlying data for this figure are provided in Supplementary File 1.

Figure 2.. Glucose availability impacts the dynamics of fitness recovery.

(A) Schematic of experimental layout. 48 isogenic clones of ancestral ctf4∆ and 48 WT clones were inoculated in either glucose starvation (0.25% and 0.5%) or abundance (2% and 8%) on deep 96-well plates. Clones were grown to saturation, and diluted daily until reaching 1000 generations. Bottleneck was adjusted to maintain Ne within the same order of magnitude throughout the experiment. (B) Relative fitness trajectories relative to a WT reference over 1000 generations. Solid and dashed lines indicate mean fitness of WT and ctf4∆ evolved populations (1000 generations), respectively. Shaded areas represent the standard error of the mean (SEM) across the 12 parallel populations. Colors represent glucose concentrations: light blue (0.25%), dark blue (0.5%), light green (2%), and dark green (8%). (C) Fitness gains (∆) at generation 1000 for evolved WT (upper panel, black) and ctf4∆ (lower panel, orange) populations. Box plots show median, IQR, and whiskers extending to 1.5×IQR, with individual data points beyond whiskers considered outliers. Fitness gains were calculated by subtracting ancestral relative fitness from evolved populations’ relative fitness, per glucose concentration. (D) Estimated rate of adaptation (parameter b) across glucose concentrations. Each datapoint represents the estimated rate of adaptation of a single evolved population of either WT or ctf4∆. Detailed statistical analysis and underlying data for this figure are provided in Supplementary File 2.

Figure 3.. The mutational profile is mainly influenced by the genotype.

(A) Total detected mutations in CDS per evolved WT (black) and ctf4∆ (orange) populations, at generation 1000. Box plots show median, IQR, and whiskers extending to 1.5×IQR, with individual data points beyond whiskers considered outliers. (B) Distribution of mutated read fractions across glucose concentrations for WT (left) and ctf4∆ (right) evolved populations, used as a proxy for clonality. Mutation fraction (%) was calculated as the fraction of reads from whole populations sequencing that contained a particular mutation in CDS. Colors represent glucose concentrations: light blue (0.25%), dark blue (0.5%), light green (2%), dark green (8%). Histograms are overlaid with a kernel density estimate (KDE, colored lines) to illustrate frequency distributions. Kolmogorov-Smirnov (KS) tests were used to compare read fraction distributions between glucose concentrations and genotypes. (C) Total mutations detected in CDS across glucose concentrations for WT (left) and ctf4∆ (right) populations at generation 1000. Detailed statistical analysis and underlying data for this figure are provided in Supplementary File 3.

Figure 4.. Environment impacts the genetic basis of adaptation of WT but not replication stress mutant.

(A) Venn diagram of putative adaptive genes mutated in evolved WT populations across glucose concentrations (upper panel). Colors represent glucose concentrations: light blue (0.25%), dark blue (0.5%), light green (2%), and dark green (8%). Numbers represent counts of putative adaptive genes (excluding zero counts). Gene names in the center are shared across conditions. GO-term enrichment across glucose concentrations in WT (bottom panel). Heatmap illustrates the total number of gene hits for a significant GO-terms. Fisher’s Exact Test was used to assess significance between pairwise glucose conditions. (B) Venn diagram of putative adaptive genes mutated in evolved ctf4∆ populations across glucose concentrations (upper panel) and corresponding GO-term enrichment heatmap (bottom panel). (C) Simplified interaction network of mutations detected in ctf4∆ evolved populations. Dark grey lines represent known genetic and physical interactions from the literature (https://string-db.org). Node diameter is proportional to the number of populations with mutations in that gene. Nodes are color-coded: blue for mutations in low glucose (0.25% and 0.5%), green for high glucose (2% and 8%), light orange for both high and low glucose, and dark orange for all conditions. Nodes with a bold outline indicate putative adaptive genes. Shaded clusters represent GO term enrichment for biological processes obtained using STRING. Network was curated in Cytoscape. Detailed statistical analysis and underlying data for this figure are provided in Supplementary File 3.

Figure 5.. Adaptive fitness and structural insights of Med14 mutation in replication stress mutants.

(A) Schematic representation of the prevalence of the med14-H919P point mutation in evolved ctf4∆ populations across glucose conditions. (B) Mean relative fitness of ctf4∆ ancestor (orange) and ctf4∆ carrying reconstructed mutation med14-H919P (pink). Error bars represent SD. (C) Composite model of the transcription pre-initiation complex of RNA pol II with mediator complex forming a dimer to act on a distal promoter (Gal4-activated) (PDB: 7UIO). Tail components, RNA pol II, transcription factors (TFs), DNA, and regulatory Gal4 are color-coded. Med14 is highlighted in pink, with secondary structures shown for the C-terminal domain (705–1082 aa). Right panel: top view of HIS 919 (blue) and surrounding amino acids, with a simulation of the HIS to Pro substitution at site 919 using Chimera X. Structural clashes were identified using Chimera X (affected residues in yellow). (D) Volcano plot of transcriptional changes after degron-removal of Med14 C-terminal (Warfield et al., 2022). Purple dashed lines indicate the significance threshold (p-value ≤ 0.01). Differentially expressed genes were defined by an adjusted p-value < 0.01 and an absolute log2 fold change > 1 (equivalent to a 2-fold change). GO enrichment analysis of downregulated genes shows enrichment in carbohydrate (light pink) and nucleotide (magenta) metabolic processes. Detailed statistical analysis and underlying data for this figure are provided in Supplementary File 4 and 5.

Figure 6.. Robustness in core genetics of adaptation to replication stress.

(A) Correlation between the fraction of populations carrying specific adaptive mutations and associated fitness benefits (∆) under replication stress. Each point represents a mutation, color-coded by gene: ixr1Δ (blue), rad9Δ (red), med14-H919P (purple), and 2xSCC2 (gray). The x-axis shows the fraction of evolved populations with each mutation, while the y-axis shows the conferred fitness advantage in the ancestral ctf4∆ background (%) for each glucose concentration. A positive correlation (R² = 0.88) is observed, with the shaded area representing the 95% confidence interval of the linear regression. (B) Comparison of ancestral, evolved, and computed fitness for ctf4∆ lines. For each evolved population, if a reconstructed gene was found mutated, its fitness effect in the respective glucose concentration was added to the ctf4∆ ancestor to calculate computed fitness (blue). Relative fitness of evolved ctf4∆ populations (orange) and ancestral ctf4∆ (dark orange) are shown. Error bars represent SD. (C) Mean relative fitness of ctf4∆ and reconstructed strain (ctf4∆ ixr1∆ rad9∆ 2xSCC2). Error bars represent SD. Each point indicates the mean fitness for a given nutrient and concentration (low or high), with marker shapes differentiating the genotypes (squares for ctf4∆ and inverted triangle for reconstructed) and colors representing concentration levels (light blue for low and dark green for high). Detailed statistical analysis and underlying data for this figure are provided in Supplementary File 5.