Mutational landscape of yeast mutator strains

Abstract

The acquisition of mutations is relevant to every aspect of genetics, including cancer and evolution of species on Darwinian selection. Genome variations arise from rare stochastic imperfections of cellular metabolism and deficiencies in maintenance genes. Here, we established the genome-wide spectrum of mutations that accumulate in a WT and in nine Saccharomyces cerevisiae mutator strains deficient for distinct genome maintenance processes: pol32Δ and rad27Δ (replication), msh2Δ (mismatch repair), tsa1Δ (oxidative stress), mre11Δ (recombination), mec1Δ tel1Δ (DNA damage/S-phase checkpoints), pif1Δ (maintenance of mitochondrial genome and telomere length), cac1Δ cac3Δ (nucleosome deposition), and clb5Δ (cell cycle progression). This study reveals the diversity, complexity, and ultimate unique nature of each mutational spectrum, composed of punctual mutations, chromosomal structural variations, and/or aneuploidies. The mutations produced in clb5Δ/CCNB1, mec1Δ/ATR, tel1Δ/ATM, and rad27Δ/FEN1 strains extensively reshape the genome, following a trajectory dependent on previous events. It comprises the transmission of unstable genomes that lead to colony mosaicisms. This comprehensive analytical approach of mutator defects provides a model to understand how genome variations might accumulate during clonal evolution of somatic cell populations, including tumor cells.

Keywords: MUTome; genetic instability; genome drift; mutation accumulation lines; mutation profile.

Fig. 1.

Experimental strategy. The haploid BY4741 (MATa) WT strain was transformed to delete the listed mutator gene(s) (Table 1). Next, four parallel long-term mutation accumulation (MA) lines were derived from each parental strain, by performing up to 100 single cell bottleneck passages (G₁₀₀), on growth on a nonselective rich medium [yeast extract/peptone/dextrose (YPD) plate] at 30 °C. The spots present in the G_25-100 lines symbolically represent the acquired mutations. Next-generation sequencing (NGS) of the WT and final passaged strains was performed (Fig. S1).

Fig. 2.

Mutational profiles. (A) Overview of the spectrum of mutations accumulated in each of the sequenced haploid MA lines. The total number of mutations per strain is shown at the top of the panels. (B) Type of InDels. (C) Type of large SV mutations. In B and C, only the mutator strains that accumulated more than eight events are depicted.

Fig. 3.

Dynamics of mutation accumulation. (A) mec1Δ tel1Δ O line. Ratio of aCGH signal on chromosome II from cells at G₂₅ (red; ratio of 1.4) and two independent colonies at G₂₆ (green; 1.8 ratio and blue;1.0 ratio). (B–F) G₁₀₀ rad27Δ Q line. (B) FACS analysis to determine the evolution of cell ploidy. The haploid (1C) rad27Δ Q line becomes diploid (2C) between G₂₆ to G₅₀ by endomitotic diploidization. (C) Evolution of yeast chromosome sizes visualized by PFGE through the bottleneck passages. Arrows indicate the chromosome bands of altered length. (D) Band CGH microarray analysis of the altered chromosome bands 1 and 2 excised from PFGE lane G₁₀₀ in C. A genomic microarray reveals a reciprocal translocation between chromosomes III and X (ratio of hybridization of the probes located on chromosomes III (blue) and X (red). The structure of the reciprocal translocation mediated by Ty1 retrotransposon elements is described below (for more details, see Dataset S7). (E) NGS read coverage of the G₁₀₀ line that became diploid. The twofold decrease of coverage at the end of chromosome VI and in the center of chromosome III reveals a heterozygous 60-kb terminal deletion (chromosome band 3) and a 17-kb interstitial deletion, respectively. Red and green points indicate the read coverage calculated in a 1-kb window size on forward and reverse strands, respectively. Black bars indicate multialigned regions. (F) Diagram indicating the chronological order in which chromosomal rearrangements accumulated in the haploid G₁₀₀ rad27Δ Q line.