Fragile DNA Motifs Trigger Mutagenesis at Distant Chromosomal Loci in <i>Saccharomyces cerevisiae</i>

<div><p>DNA sequences capable of adopting non-canonical secondary structures have been associated with gross-chromosomal rearrangements in humans and model organisms. Previously, we have shown that long inverted repeats that form hairpin and cruciform structures and triplex-forming GAA/TTC repeats induce the formation of double-strand breaks which trigger genome instability in yeast. In this study, we demonstrate that breakage at both inverted repeats and GAA/TTC repeats is augmented by defects in DNA replication. Increased fragility is associated with increased mutation levels in the reporter genes located as far as 8 kb from both sides of the repeats. The increase in mutations was dependent on the presence of inverted or GAA/TTC repeats and activity of the translesion polymerase Polζ. Mutagenesis induced by inverted repeats also required Sae2 which opens hairpin-capped breaks and initiates end resection. The amount of breakage at the repeats is an important determinant of mutations as a perfect palindromic sequence with inherently increased fragility was also found to elevate mutation rates even in replication-proficient strains. We hypothesize that the underlying mechanism for mutagenesis induced by fragile motifs involves the formation of long single-stranded regions in the broken chromosome, invasion of the undamaged sister chromatid for repair, and faulty DNA synthesis employing Polζ. These data demonstrate that repeat-mediated breaks pose a dual threat to eukaryotic genome integrity by inducing chromosomal aberrations as well as mutations in flanking genes.</p></div

Inverted repeat and GAA/TTC-induced DSB detection in wild-type and mutant strains.

<p>Upper panel depicts the relative positions of the inverted repeats and the probe (open rectangle) used. For the detection of inverted repeat-mediated breaks <i>Δsae2</i> strains were used as in these mutants the hairpin-capped breaks are not opened and resection is abolished <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1003551#pgen.1003551-Lobachev1" target="_blank">[21]</a>. As a consequence, inverted dimer molecules accumulate in <i>Δsae2</i> mutants as previously demonstrated. Contour-clamped homogeneous electric field gel electrophoresis and Southern hybridization were used to highlight the intact chromosome V and the broken fragment. Lanes 1, 2 and 3 depict the <i>Alu-</i>IR, <i>pol3-P664L Alu-</i>IR, and <i>IS50-</i>IR strains respectively. Lanes 4 and 5 depict GAA/TTC₍₂₃₀₎ and TET-<i>POL3</i> GAA/TTC₍₂₃₀₎ strains respectively. Intact chromosome V, DSB fragments and inverted dimers (in the case of inverted repeats) are indicated.</p

Mutagenesis by fragile <i>Alu</i>-IRs depends on the distance of reporter from the DSB site.

a<p>TP denotes telomere-proximal location of <i>URA3</i> with respect to the <i>Alu</i>-IRs.</p>b<p>TD denotes telomere-distal location of <i>URA3</i> with respect to the <i>Alu</i>-IRs.</p>c<p>Numbers in parentheses indicate 95% confidence intervals.</p>*<p>Depicts mutation rates significantly higher than the wild-type strain at the respective loci (P<0.01).</p>#<p>Depicts mutation rates in <i>Δrev3</i> and <i>Δsae2</i> strains significantly lower than the <i>pol3-P664L</i> strain at the respective loci (P<0.01).</p><p>ND - not determined.</p

Experimental system to study fragile motif-induced mutagenesis.

<p><i>Alu</i>-quasi-palindrome, <i>IS50-</i>palindrome or GAA/TTC repeats were inserted into <i>LYS2</i> gene on the left arm of chromosome V. Positions of <i>CAN1</i> and <i>URA3</i> reporters located telomere-proximal (TP) or telomere-distal (TD) to the repeat insertion are shown. The position of the <i>ARS507</i> and the direction of replication fork migrating through the repeat region are indicated. Breakage at the location of secondary-structure-adopting repeats can lead to loss of 43 kb telomere-proximal deletion resulting in red-colored Can^RAde⁻ clones. Mutations in <i>CAN1</i> reporter will yield white-colored Can^RAde⁺ isolates. Mutations in <i>URA3</i> gene will give rise to colonies resistant to medium containing 5-fluoorotic acid (5-FOA^R).</p

Unisexual and Heterosexual Meiotic Reproduction Generate Aneuploidy and Phenotypic Diversity <i>De Novo</i> in the Yeast <i>Cryptococcus neoformans</i>

<div><p>Aneuploidy is known to be deleterious and underlies several common human diseases, including cancer and genetic disorders such as trisomy 21 in Down's syndrome. In contrast, aneuploidy can also be advantageous and in fungi confers antifungal drug resistance and enables rapid adaptive evolution. We report here that sexual reproduction generates phenotypic and genotypic diversity in the human pathogenic yeast <i>Cryptococcus neoformans</i>, which is globally distributed and commonly infects individuals with compromised immunity, such as HIV/AIDS patients, causing life-threatening meningoencephalitis. <i>C. neoformans</i> has a defined <b>a</b>-α opposite sexual cycle; however, >99% of isolates are of the α mating type. Interestingly, α cells can undergo α-α unisexual reproduction, even involving genotypically identical cells. A central question is: Why would cells mate with themselves given that sex is costly and typically serves to admix preexisting genetic diversity from genetically divergent parents? In this study, we demonstrate that α-α unisexual reproduction frequently generates phenotypic diversity, and the majority of these variant progeny are aneuploid. Aneuploidy is responsible for the observed phenotypic changes, as chromosome loss restoring euploidy results in a wild-type phenotype. Other genetic changes, including diploidization, chromosome length polymorphisms, SNPs, and indels, were also generated. Phenotypic/genotypic changes were not observed following asexual mitotic reproduction. Aneuploidy was also detected in progeny from <b>a</b>-α opposite-sex congenic mating; thus, both homothallic and heterothallic sexual reproduction can generate phenotypic diversity <i>de novo</i>. Our study suggests that the ability to undergo unisexual reproduction may be an evolutionary strategy for eukaryotic microbial pathogens, enabling <i>de novo</i> genotypic and phenotypic plasticity and facilitating rapid adaptation to novel environments.</p></div

Polζ- and Sae2-dependent mutagenesis by <i>IS50-</i>perfect palindrome.

a<p>Numbers in parentheses correspond to the 95% confidence interval.</p>*<p>Depicts mutation rates significantly higher than the wild-type strain with <i>Alu-</i>IR (see <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1003551#pgen-1003551-t001" target="_blank">Table 1</a>) (P<0.05).</p>#<p>Depicts mutation rates in <i>Δrev3</i> and <i>Δsae2</i> strains significantly lower than the wild-type strain (P<0.01).</p

Polζ- and Sae2-dependent mutagenesis by <i>Alu-</i>quasi-palindrome in replication mutants.

a<p>Numbers in parentheses correspond to the 95% confidence interval.</p>*<p>Depicts mutation rates significantly higher than the wild-type strain (P<0.05).</p>#<p>Depicts mutation rates in <i>Δrev3</i> and <i>Δsae2</i> strains significantly lower than corresponding replication-deficient strains (P<0.01).</p

Genome comparison of XL280 and JEC21.

<p>(A) CGH of XL280 versus JEC21. Coloring indicates gene dosage as follows: gray, no significant change; red, more abundant; green, less abundant. (B) Coverage analysis of XL280 Chr 5 and Chr 8 with JEC21 as the reference. The red circle indicates that XL280 lacks ∼28 kb near the right end of Chr 5 compared to JEC21. The blue circle indicates that XL280 has one copy of the 63 kb region that is duplicated in JEC21 and located on the left ends of Chr 8 and Chr 12. (C) Chromosome configuration of XL280 versus JEC21. Black arrows indicate the size differences of Chr 5, 8/9, and 13 between the two strains. (D) Two probes located before and after the break in Chr 8 of JEC21 hybridized to XL280 Chr 9 and 12, respectively. (E) Chr 9 and 12 of XL280 versus JEC21.</p

Unisexual reproduction generates phenotypic diversity.

<p>(A) Progeny produced by α-α unisexual reproduction of strain XL280 in solo cultures were grown in 10-fold serial dilution assays and under the following conditions: YPD at 30°C for 2 d; YPD at 37°C for 2 d; YPD plus 1 µg/mL FK506 at 30°C for 2 d; YPD plus 8 µg/mL fluconazole (FLC) at 30°C for 6 d; NS (melanin-inducing media) at 30°C for 3 d; and V8 pH = 7 (mating-inducing media) at room temperature for 11 d. (B) Diploid progeny were generated infrequently following unisexual reproduction. Flow cytometry profiles of cells stained with the fluorescent dye propidium iodide. JEC21 (1n, haploid control); XL143 (2n, diploid control); XL280 (1n); MN27 (2n, the only diploid strain identified among the 90 XL280 α-α unisexual reproduction progeny (1.1%). All other XL280 α-α unisexual progeny were haploid (or aneuploid) (e.g., MN55). Nuclear DNA content is indicated by 1n (haploid) and 2n (diploid). The <i>x</i>-axis indicates fluorescence intensity reflecting DNA content, and the <i>y</i>-axis indicates cell counts. (C) Three SNPs identified by NGS from strains MN7 and MN55 and were confirmed by Sanger sequencing. (D) The WT allele of <i>HSC20</i> was cloned in two independent plasmids, and these were used to transform strain MN7 and independent transformants were analyzed. This complementation test shows that the TS phenotype of MN7 is attributable to the recessive <i>hsc20-1</i> mutation.</p

Aneuploidy causes the observed phenotypic changes.

<p>Strain MN77, which contains an extra copy of Chr 10 and has increased melanin production, was grown on YPD at 37°C for 3 d to promote aneuploidy loss. The genotypes and phenotypes of 22 resulting mitotic progeny were analyzed. (A) Melanin production of 22 mitotic progeny on NS media at 30°C for 2 d. (B) Multiplex PCR of 22 progeny detected an extra Chr 10 in all of the strains with the increased melanin phenotype and loss of the extra copy of Chr 10 in all progeny with a wild-type phenotype. (C) Phenotypic analysis of 22 mitotic progeny at high-temperature growth (37°C for 2 d) and with drug treatment (FLC and FK506). XL280 (X) and MN77 (77) are the WT and aneuploid controls, respectively.</p