Genome-Wide Estimates of Mutation Rates and Spectrum in Schizosaccharomyces pombe Indicate CpG Sites are Highly Mutagenic Despite the Absence of DNA Methylation

Abstract

We accumulated mutations for 1952 generations in 79 initially identical, haploid lines of the fission yeast Schizosaccharomyces pombe, and then performed whole-genome sequencing to determine the mutation rates and spectrum. We captured 696 spontaneous mutations across the 79 mutation accumulation (MA) lines. We compared the mutation spectrum and rate to a recently published equivalent experiment on the same species, and to another model ascomycetous yeast, the budding yeast Saccharomyces cerevisiae. While the two species are approximately 600 million years diverged from each other, they share similar life histories, genome size and genomic G/C content. We found that Sc. pombe and S. cerevisiae have similar mutation rates, but Sc. pombe exhibits a stronger insertion bias. Intriguingly, we observed an increased mutation rate at cytosine nucleotides, specifically CpG nucleotides, which is also seen in S. cerevisiae. However, the absence of methylation in Sc. pombe and the pattern of mutation at these sites, primarily C → A as opposed to C → T, strongly suggest that the increased mutation rate is not caused by deamination of methylated cytosines. This result implies that the high mutability of CpG dinucleotides in other species may be caused in part by a methylation-independent mechanism. Many of our findings mirror those seen in the recent study, despite the use of different passaging conditions, indicating that MA is a reliable method for estimating mutation rates and spectra.

Keywords: cytosine mutation; insertion bias; mutation accumulation.

Figure 1

Summary of mutations for each of six possible nucleotide changes for the ancestor (ATCC 26189) vs. the reference, the 79 mutation accumulation (MA) lines in this study, and the 96 Farlow et al. (2015) MA lines. Also shown are the mutation frequencies observed in the 145 diploid Saccharomyces cerevisiae MA lines (Zhu et al. 2014).

Figure 2

Summary of types of mutations identified across 79 MA lines. SNM, Single nucleotide mutation.

Figure 3

Histogram of SNM numbers per line across the 79 MA lines. The distribution is consistent with a negative binomial (P = 0.95, χ² test). Dashes represent the expected numbers for the best-fit negative binomial distribution (mean = 4.13, overdispersion = 2.06). The total number of SNMs across all 79 MA lines is 326.

Figure 4

Mutation rate is affected by context. Relative SNM rate adjusted by its trinucleotide context. Trinucleotide classes represent mutation rates of both strand orientations. For example, aCa trinucleotide class includes overall mutation rate at aCa and tGt sites. Mutation rate is shown relative to the average SNM rate across all sites (= 1.7 × 10⁻¹⁰ per base, per generation). The average, relative mutation rate of 1.8 at G:C bases shows clear overall elevation over the corresponding rate of 0.66 at A:T bases. The four black-filled points are those trinucleotides with a C in the central position with a G as the 3′ neighbor. * P ≤ 0.003 Bonferroni corrected t-test.

Figure 5

Summary of the locations and predicted functional consequences of the 326 SNMs (excluding SNMs in double and complex mutations), 288 small insertions and 47 small deletions accumulated in the MA lines. To facilitate comparison across the three mutational classes, frequencies are shown. NC, noncoding; UTR transcribed, untranslated region.