Author Correction: The rate and molecular spectrum of mutation are selectively maintained in yeast
Year: 2023
Type: erratum
Abstract: In the original version of this Article, some mutation accumulation (MA) lines did not have the correct MSH2 inserted; instead, the inserted MSH2 lacked the stop codon and encoded 24 extra amino acids at the 3’ end. Specifically, three of the 13 original second-order MA lines (MA28, MA33, and MA38) had the correct MSH2 inserted, while the remaining 10 had the MSH2 that lacked the stop codon. To correct this error, the wild-type MSH2 was inserted into all lines after the first round of MA, the inserted MSH2 sequence was confirmed by Sanger sequencing, and the downstream experiments were repeated, including fluctuation test, second round of MA, and whole-genome sequencing after the second round of MA. The newly generated data have been reanalyzed and presented in the corrected version of this Article. The following changes have been made: In the Results subsection ‘Mutagenesis frequently reduces μ’, the third and fourth paragraphs have been modified from: “Because the N e of the MA lines was about 10 (see “Methods”) while most mutations are expected to have a fitness effect on the order of 1% or smaller 21 , selection should be infrequent during the MA and was indeed the case (see “Methods”). To assess the impact of MA on μ, we first inserted MSH2 back to the MA lines, which was accomplished in 66 of the 93 lines (Fig. 1d). Using the classic fluctuation test based on the reporter gene CAN1 (see “Methods”), we successfully measured μ in the progenitor as well as 48 of the above 66 MA lines, all carrying an intact MSH2 (Fig. 1d). We found μ of the 48 MA lines to range from 0.01 to 27 times that of the progenitor (Fig. 2a, Supplementary Data 1), including 28 lines with significantly higher μ and 12 lines with significantly lower μ than the progenitor (see “Methods”). Furthermore, 10 of the 12 lines with significantly decreased μ had μ reduced by at least 50%, while the remaining two lines had μ reduced by 46% and 38%, respectively (Fig. 2a). That 30% of MA lines with significantly altered μ exhibit such drastic reductions in μ is inconsistent with the DBH, because when μ is near the drift barrier, mutations are expected to be strongly biased toward increasing μ and are not expected to cause such large reductions of μ so frequently (Fig. 1b). Our finding suggests that the progenitor’s μ is well above the drift barrier (Fig. 1c). We found no significant correlation between μ and the number of mutations accumulated during the MA or the growth rate of the MA line (Supplementary Fig. 2). Because the above estimation of μ was based on loss-of-function mutations in one gene, we attempted to verify these results by performing another round of MA followed by WGS in 13 of the above 48 MA lines as well as the progenitor, all with an intact MSH2 (Fig. 1d). 18–20 parallel lines were established from each strain, and on average 695 generations of MA were performed in the medium similar to that used in the fluctuation test (Supplementary Table 2, Supplementary Data 1, see “Methods”). The MA + WGS results were generally consistent with those from the fluctuation test. For instance, compared with the progenitor, all five lines with higher CAN1 -based μ exhibited higher MA+ WGS-based SNV (Fig. 2b) and indel (Fig. 2c) rates. Among the eight lines with lower CAN1 -based μ, four exhibited significantly lower MA + WGS-based SNV and/or indel rates; the reduced consistency here is likely due to larger errors of lower CAN1 -based μ estimates (Fig. 2a). CAN1 -based μ is significantly correlated with the MA + WGS-based SNV rate ( r = 0.64, P = 0.013; Fig. 2d) but not with the MA+ WGS-based indel rate ( r = 0.11, P = 0.70; Fig. 2e). This observation is not unexpected given that most loss-of-function mutations in CAN1 are SNVs instead of indels 22 .” to now read: “Because the N e of the MA lines was about 10 (see “Methods”) while most mutations are expected to have a fitness effect on the order of 1% or smaller 21 , selection should be infrequent during the MA and was indeed the case (see “Methods”). To assess the impact of MA on μ, we first inserted MSH2 back to the MA lines, which was accomplished in 60 of the 93 lines (Fig. 1d). Using the classic fluctuation test based on the reporter gene CAN1 (see “Methods”), we successfully measured μ in the progenitor as well as 49 of the above 60 MA lines, all carrying an intact MSH2 (Fig. 1d). We subsequently found that five of the 49 lines were likely diploidized upon the insertion of MSH2 (see the following paragraph) and excluded them (marked with stars in Fig. 2a) from all CAN1 -based analyses. We found μ of the 44 remaining MA lines to range from 0.01 to 26 times that of the progenitor (Fig. 2a, Supplementary Data 1), including 19 lines with significantly higher μ and 13 lines with significantly lower μ than the progenitor (see “Methods”). Furthermore, 10 of the 13 lines with significantly decreased μ had μ reduced by at least 50%, while the remaining three lines had μ reduced by 40% to 43% (Fig. 2a). That over 40% of MA lines with significantly altered μ exhibit such drastic reductions in μ is inconsistent with the DBH, because when μ is near the drift barrier, mutations are expected to be strongly biased toward increasing μ and are not expected to cause such large reductions of μ so frequently (Fig. 1b). Our finding suggests that the progenitor’s μ is well above the drift barrier (Fig. 1c). We found a significant positive correlation between μ and the number of mutations accumulated during the MA, but μ was not significantly correlated with the growth rate of the MA line (Supplementary Fig. 2). Because the above estimation of μ was based on loss-of-function mutations in one gene, we attempted to verify these results by performing another round of MA followed by WGS in 16 of the above 49 MA lines as well as the progenitor (and a diploid version of the progenitor), all with an intact MSH2 (Fig. 1d). 4–20 parallel lines were established from each strain, and on average 684 generations of MA were performed in the medium similar to that used in the fluctuation test (Supplementary Table 2, Supplementary Data 1, see “Methods”). Four of the 16 MA lines were apparently diploid, because the majority of the mutations observed in MA+WGS were in heterozygous state. Diploids should not produce mutant colonies in the fluctuation test. To be conservative in inferring mutation rate reductions in the first round of MA, we additionally regarded a line that was not subject to MA+WGS but had only two mutant colonies in the fluctuation test as putatively diploid (right most line marked with a star in Fig. 2a). We excluded these five diploid lines from all CAN1 -based analyses. Because haploid and diploid progenitors showed similar mutation rates (Fig. 2b, c), all 16 lines with MA+WGS were included in analyses based solely on MA+WGS. The MA + WGS results were generally consistent with those from the fluctuation test. For instance, compared with the progenitor, all eight lines with higher CAN1 -based μ exhibited higher MA+ WGS-based SNV (Fig. 2b) or indel (Fig. 2c) rates. Among the four lines with lower CAN1 -based μ, three exhibited significantly lower MA + WGS-based SNV or indel rates; the reduced consistency here is likely due to larger errors of lower CAN1 -based μ estimates (Fig. 2a). CAN1 -based μ is significantly correlated with both the MA + WGS-based SNV rate ( r = 0.88, P = 8.9×10 -5 ; Fig. 2d) and the MA+ WGS-based indel rate ( r = 0.78, P = 1.6×10 -3 ; Fig. 2e), although the latter correlation is weaker than the former. This observation is not unexpected given that most loss-of-function mutations in CAN1 are SNVs instead of indels 22 .” In the Results subsection ‘Stabilizing selection of μ’, the first paragraph sentence “To estimate V m that is comparable with V g , we used the CAN1 -based μ estimates from the 48 MA lines, but corrected for the increased mutagenesis in the MA induced by deleting MSH2 .” has been corrected to read ‘44 haploid’ in place of ‘48’. The second paragraph has been modified from: “We found V g /V m to be at least 800 times lower than the neutral expectation of 4 N e ≈ 4 × 10 7 (see “Methods”), regardless of the particular V m used (Table 1), indicating strong stabilizing selection of μ. This signal of stabilizing selection is not an artifact of the physical limits of μ, because the range of μ among the natural strains is even smaller than that of the MA lines (Fig. 2a). To investigate whether the stabilizing selection prohibits the evolution of higher μ, lower μ, or both, we separated V m into two components that respectively measure the variance of μ created by mutations decreasing μ ( V mL ) and increasing μ ( V mH ). If there is no selection against a reduction in μ, V g should be at least as large as 4 N e V mL . However, we found V g to be at least 700 times lower than 4 N e V mL (Table 1), indicating the action of selection prohibiting a reduction of μ in evolution. Similarly, V g was at least 150 times lower than 4 N e V mH (Table 1), indicating the action of selection prohibiting a rise of μ in evolution. In the above tests, the smallest difference observed between V g and a neutral expectation was 150 times, based on V m2 that corresponds to a conservative test. Therefore, it is exceedingly unlikely that our test results are due to confounding factors such as mutation spectrum differences between wild-type and MSH2 -lacking strains or the inaccuracy of yeast’s N e estimate (see “Methods”). Together, the above results demonstrate that μ has been selectively maintained at an intermediate level in S. cerevisiae .” to now read: “We found V g /V m to be at least 540 times lower than the neutral expectation of 4 N e ≈ 4 × 10 7 (see “Methods”), regardless of the particular V m used (Table 1), indicating strong stabilizing selection of μ. This signal of stabilizing selection is not an artifact of the physical limits of μ, because the range of μ among the natural strains is even smaller than that of the MA lines (Fig. 2a). To investigate whether the stabilizing selection prohibits the evolution of higher μ, lower μ, or both, we separated V m into two components that respectively measure the variance of μ created by mutations decreasing μ ( V mL ) and increasing μ ( V mH ). If there is no selection against a reduction in μ, V g should be at least as large as 4 N e V mL . However, we found V g to be at least 300 times lower than 4 N e V mL (Table 1), indicating the action of selection prohibiting a reduction of μ in evolution. Similarly, V g was at least 230 times lower than 4 N e V mH (Table 1), indicating the action of selection prohibiting a rise of μ in evolution. In the above tests, the smallest difference observed between V g and a neutral expectation was 230 times, based on V m2 that corresponds to a conservative test. Therefore, it is exceedingly unlikely that our test results are due to confounding factors such as mutation spectrum differences between wild-type and MSH2 -lacking strains or the inaccuracies of V m , V g , and N e estimates (see “Methods”). Together, the above results demonstrate that μ has been selectively maintained at an intermediate level in S. cerevisiae . Note that the selective forces to increase and to suppress μ are not equally strong, because the mean μ of the MA lines is higher than the progenitor ( P = 4.3 × 10 -3 for CAN1 -based μ, t -test; P = 5.7 × 10 -7 for WGS-based SNV rate, t -test).” The third paragraph sentences: “We obtained V m based on the 13 MA lines with MA+ WGS-based estimates of SNV rates and corrected the impact of deleting MSH2 as in the above analysis. We found D 2 / V m to be at least 10 4 times smaller than T (Table 1). We also respectively estimated V mL and V mH using the 13 MA lines with MA+ WGS-based estimates of μ. Again, we found D 2 / V mL to be at least 300 times smaller and D 2 / V mH at least 6000 times smaller than T (Table 1), demonstrating selection against lowering as well as increasing μ in the divergence of Saccharomyces species.” have been modified to now read: “We obtained V m based on the 16 MA lines with MA+ WGS-based estimates of SNV rates and corrected the impact of deleting MSH2 as in the above analysis. We found D 2 / V m to be at least 4000 times smaller than T (Table 1). We also respectively estimated V mL and V mH using the 16 MA lines with MA+ WGS-based estimates of μ. Again, we found D 2 / V mL to be at least 400 times smaller and D 2 / V mH at least 2400 times smaller than T (Table 1), demonstrating selection against lowering as well as increasing μ in the divergence of Saccharomyces species.” In the Results subsection ‘Discovery of PSP2 as a mutator gene’, the first paragraph sentences: “To this end, we first identified candidate mutator genes by screening genes that were more frequently mutated in low-μ lines than in high-μ lines among the 48 MA lines with CAN1 -based μ estimates (Supplementary Table 5). We then picked four candidates ( RAD9 , YFL013W-A , PSP2 , and MSH4 ) based on their ranks from the screening and annotated functions for a follow-up study.” have been modified to now read: “We originally identified candidate mutator genes by screening genes that were more frequently mutated in low-μ lines than in high-μ lines among MA lines with CAN1 -based μ estimates (Supplementary Table 5), and picked four candidates ( RAD9 , YFL013W-A , PSP2 , and MSH4 ) based on their ranks from the screening and annotated functions for a follow-up study. We subsequently found that some MA lines had erroneous MSH2 , so reinserted MSH2 followed by re-estimation of CAN1 -based μ. Based on these new estimates of μ (Fig. 2a), the four genes are ranked 1362, 911, 58, and 76, respectively.” In the Results subsection ‘Mutation spectrum has been shaped by selection’, the first paragraph sentences: “To investigate the potential role of natural selection in shaping yeast’s mutation spectrum, we compared the variance ( V g ) in a component of the mutation spectrum among five divergent natural yeast strains having published MA+WGS data (Supplementary Fig. 3), with the corresponding mutational variance per generation estimated from the 13 MA lines with MA+WGS data. For instance, even under the most generous calculation, V g / V m (4.48 × 10 4 ) of the proportion of mutations that are SNVs is orders of magnitude smaller than the neutral expectation of 4 × 10 7 (Supplementary Table 6). In fact, the variance of the proportion of SNVs is smaller among the five natural strains than among the 13 MA lines (Fig. 4a), despite that the numbers of generations separating the natural strains are much greater than those separating the MA lines even after the correction for the increased mutagenesis of MA lines induced by deleting MSH2 . Similar results were found regarding the proportion of insertions (maximal V g / V m =7.84 × 103) and that of deletions (maximal V g / V m =5.12 × 10 4 ) (Fig. 4a, Supplementary Table 6).” have been modified to now read: “To investigate the potential role of natural selection in shaping yeast’s mutation spectrum, we compared the variance ( V g ) in a component of the mutation spectrum among five divergent natural yeast strains having published MA+WGS data (Supplementary Fig. 3), with the corresponding mutational variance per generation estimated from the 16 MA lines with MA+WGS data. Because haploid and diploid progenitors show similar mutational spectrums (Fig. 4), we analyze the MA lines and natural strains regardless of their ploidy. Even under the most generous calculation, V g / V m (3.07 × 10 4 ) of the proportion of mutations that are SNVs is orders of magnitude smaller than the neutral expectation of 4 × 10 7 (Supplementary Table 6). In fact, the variance of the proportion of SNVs is smaller among the five natural strains than among the 16 MA lines (Fig. 4a), despite that the numbers of generations separating the natural strains are much greater than those separating the MA lines even after the correction for the increased mutagenesis of MA lines induced by deleting MSH2 . Similar results were found regarding the proportion of insertions (maximal V g / V m = 3.57 × 103) and that of deletions (maximal V g / V m = 3.27 × 10 4 ) (Fig. 4a, Supplementary Table 6).” The second paragraph sentence: “There are six different types of SNVs (Fig. 4b) and we found evidence for stabilizing selections on each of the six fractions (maximal V g / V m ranging between 1.51 × 10 4 and 1.31 × 10 6 ; Supplementary Table 6).” has been corrected to read ‘3.03 × 10 4 ’ in place of ‘1.51 × 10 4 ’ and ‘1.35 × 10 6 ’ in place of ‘1.31 × 10 6 ’. The third paragraph sentences: “We found evidence for stabilizing selection of Ts/Tv (Fig. 4c); the maximal V g / V m equals 4.11 × 10 4 (Supplementary Table 6). In particular, Ts/Tv is higher than that of the progenitor in 10 of the 13 MA lines, and Ts/Tv is increased in all 7 MA lines with a significantly altered Ts/Tv (P = 0.016, two-tailed binomial test) (Fig. 4c).” have been modified to now read: “We found evidence for stabilizing selection of Ts/Tv (Fig. 4c); the maximal V g / V m equals 9.13 × 10 4 (Supplementary Table 6). In particular, Ts/Tv is significantly higher (or lower) than that of the progenitor in two (or zero) MA lines (Fig. 4c).” The fourth paragraph sentences: “We found that the ratio of the number of GC→AT mutations to the number of AT→GC mutations is significantly different in 7 of the 13 MA lines when compared with the progenitor, including 6 lines that increased the bias and one line that reversed the bias to the opposite direction (Fig. 4d). Clearly, the AT mutational bias is subject to genetic control and is not a chemical necessity. Furthermore, V g / V m for the AT mutational bias is at least 700 times lower than the neutral expectation (Supplementary Table 6), indicating that the bias has been maintained by stabilizing selection.” have been modified to now read: “We found that, in one MA line, the ratio of the number of GC→AT mutations to the number of AT→GC mutations is significantly different from that in the progenitor, and is reversed from >1 to <1 (Fig. 4d). Clearly, the AT mutational bias is subject to genetic control and is not a chemical necessity. Furthermore, V g / V m for the AT mutational bias is at least 120 times lower than the neutral expectation (Supplementary Table 6), indicating that the bias has been maintained by stabilizing selection.” In the Methods subsection ‘Strains and genetic manipulations’, the first paragraph sentences: “Transformation was performed in each MA line for up to three times. Restoration of MSH2 was successful in only 66 of the 93 MA lines, probably due to reduced transformation efficiencies in the MA lines.” have been modified to now read: “Transformation was performed in each MA line for up to three times and was confirmed by Sanger sequencing of the reinserted locus. Restoration of MSH2 was successful in only 60 of the 93 MA lines, probably due to reduced transformation efficiencies in the MA lines.” In the Methods subsection ‘MA and whole-genome sequencing’, the second paragraph sentences: “A total of 14 strains, including 13 of the above 93 MA lines and BY4741, all with intact MSH2 , were subject to the second round of MA. Eighteen to 20 replicate lines were established for each strain. Cells were propagated at 30 °C on SC (synthetic complete) plates, similar to that used in the fluctuation test. The total time in the second round of MA for all lines was kept at ~100 days and the number of generations between bottlenecks was kept at ~20. The between-bottleneck duration was different among these 14 strains because of their different generation times. It was 48 h in BY4741, MA28, MA29, and MA38, 72 h in MA13, MA23, MA25, MA33, MA77, MA78, and MA85, and 96 h in MA45, MA59, and MA95. The genomes of 272 MA lines at the end of the second round of MA and their 14 ancestral strains were sequenced.” have been modified to now read: “A total of 18 strains, including 16 of the above 93 MA lines, BY4741 (haploid), and BY4743 (diploid), all with intact MSH2 , were subject to the second round of MA. Four to 20 replicate lines were established for each strain. Cells were propagated at 30 °C on SC (synthetic complete) plates, similar to that used in the fluctuation test. The total time in the second round of MA for all lines was kept at ~100 days and the number of generations between bottlenecks was kept at ~20. The between-bottleneck duration was different among these 18 strains because of their different generation times. It was 48 h in BY4741, BY4743, MA28, and MA38, 72 h in MA15, MA21, MA23, MA25, MA29, MA33, MA44, MA63, and MA92, and 96 h in MA45, MA51, MA56, MA64, and MA94. The genomes of 209 MA lines at the end of the second round of MA and their 18 ancestral strains were sequenced.” In the Methods subsection ‘Fluctuation test’, the first paragraph sentence: “We subjected all 66 MA lines with reinserted MSH2 to the fluctuation test, but only 48 of them grew in the medium.” has been corrected to read ‘60’ in place of ‘66’ and ‘49’ in place of ‘48’. The second paragraph sentence: “No CAN1 mutant was observed in 4 MA lines, and their μ values were calculated by assuming the observation of one mutant colony to allow plotting μ in a logarithmic scale.” has been corrected to read ‘two’ in place of ‘4’. In the Methods subsection ‘Estimation of V m , V g , and D 2 ’, a new paragraph was appended to the end:
Source: Nature Communications
Authors: Haoxuan Liu, Jianzhi Zhang
Institution: University of Michigan
Language: English
Subfield: Genetics
Domain: Life Sciences
Open Access status: gold
Estimated APC paid: $6,290
OpenAlex ID: W4362602675