Figure 1.
Two mechanisms of generation of mutants in diploids.
Mutation (red bar) in a gene (blue rectangle) occurring with frequency “m” will lead to a phenotypic change in diploid, either by a concomitant loss of heterozygosity (by recombination with frequency “r”) or deletion (“d”) or chromosome information loss/inactivation (“l”). The frequency of such events “f” should be the product of the frequencies of each independent event. Mutations can also occur independently in both alleles of a diploid but at different sites, and their frequency should be the square of “m”.
Figure 2.
Induction of mutations by HAP and PmCDA1.
A. Induction of G-C to A-T transitions by the mechanism of HAP misincorporation. B. Induction of A-T to G-C transition mutations by the mechanism of misincorporation opposite HAP. C. Induction of G-C to A-T mutations by cytosine deamination followed by incorporation of adenine opposite uracil. D. Numbers of SNVs induced by HAP in haploid (LAN201-1 - LAN201-4) and diploid (LAN211-1 – LAN211-10) strains. The proportions of substitution types are shown by color. E. Numbers of SNVs induced in haploid (LAN200-L1 - LAN200-L4) and diploid (LAN210-L1 – LAN210-L7), PmCDA1-induced mutants CANR mutants, and diploid PmCDA1-induced FOAR mutants (LAN210-FOA-1 and LAN210-FOA-2). Proportions of substitution types are shown by color.
Table 1.
Both HAP and PmCDA1 induce significantly more mutants than expected in diploid strains.
Table 2.
Summary of all detected mutations in HAP-treated clones.
Figure 3.
DNA sequence context of HAP- and PmCDA1-induced substitutions.
The spectra of mutations induced by HAP in genomes are from this study. Data for the URA3 reporter is from this work and [26] and for the LYS2 reporter from [43]. PmCDA1-induced mutation spectra in reporter genes and in lamprey VLRs are a combination of data from this work and [31].
Table 3.
Summary of all detected mutations in PmCDA1-treated clones.
Figure 4.
Dependence of mutational load on ploidy and mutant selection.
A. Number of mutations for different types of HAP-treated genomes. B. Number of mutations for different types of PmCDA1-treated genomes. Bars are median values. Note the logarithmic scale. p values for Mann-Whitney test are shown for significant differences. C. Viability of haploid spores obtained from different diploid clones. Spores resulting from dissecting tetrads produced from wild-type strains (LAN211 and LAN210), mutants induced by HAP (LAN211-5) and PmCDA1 (LAN210-L4), and non-mutant clones treated with HAP (LAN211-NM3) and PmCDA1 (LAN210-NM2) are shown. Each vertical row of four colonies represents the progeny of four haploid spores produced in a single meiosis.
Table 4.
Viability of haploid progeny of wild-type and mutant yeast strains.
Figure 5.
Predicted effects of SNVs on proteins in different types of clones.
Results for haploid and diploid mutant and diploid non-mutant clones are shown for HAP (A) and for haploid and diploid mutants for PmCDA1 (B). Numbers are mean values of percentages with 95% confidence limits in parentheses. PmCDA1-treated, non-mutant clones are excluded from the analysis due to low levels of mutations in their genomes.
Figure 6.
HAP induces mutants in duplicated genes when ploidy is more than one.
HAP solution was spotted on a disk in the center of plates and colonies of mutants appear as a circle around the place of application. A. Mutant colonies induced with HAP in diploid (left) and a haploid strain with a duplicated LYS2 reporter (right). B. HAP-induced mutants in a triploid strain (left) and diploid strains with duplication of LYS2 reporter in one homologous chromosome (right).
Figure 7.
Hypothetical distribution of cells with different induced mutation rates.
In the cartoon we used normal distribution as an example. The right side of distribution (highlighted by light green and by red rectangles) contains cells with a very high induced mutation rate. The fraction of hypermutable cells explains the observed increase of mutation frequencies in diploids. The selection of CanR driver mutants in diploids results in the recovery of thousands of passenger mutations. These mutants originated from a transiently hypermutable fraction of cells that survive an extremely high frequency of mutations. Such cells die in haploids (red zone) but survive in diploids. Only the hypermutators from the light green zone survive in haploids. The size of this fraction can be substantial, because mutation avalanches evidence for transient hypermutagenesis was even observed in the genomes of unselected HAP-treated non-mutant clones.