https://orcid.org/0000-0002-0368-7843
Figures
Abstract
We constructed a panel of S. pombe strains expressing DNA polymerase ε variants associated with cancer, specifically POLES297F, POLEV411L, POLEL424V, POLES459F, and used these to compare mutation rates determined by canavanine resistance with other selective methods. Canavanine-resistance mutation rates are broadly similar to those seen with reversion of the ade-485 mutation to adenine prototrophy, but lower than 5-fluoroorotic acid (FOA)-resistance rates (inactivation of ura4+ or ura5+ genes). Inactivation of several genes has been associated with canavanine resistance in S. pombe but surprisingly whole genome sequencing showed that 8/8 spontaneous canavanine-resistant mutants have an R175C mutation in the any1/arn1 gene. This gene encodes an α-arrestin-like protein involved in mediating Pub1 ubiquitylation of target proteins, and the phenotypic resistance to canavanine by this single mutation is similar to that shown by the original “can1-1” strain, which also has the any1R175C mutation. Some of the spontaneous mutants have additional mutations in arginine transporters, suggesting that this may marginally increase resistance to canavanine. The any1R175C strain showed internalisation of the Cat1 arginine transporter as previously reported, explaining the canavanine-resistance phenotype.
Citation: Pai C-C, Heitzer E, Bertrand S, Toumazou S, Humphrey TC, Kearsey SE (2023) Using canavanine resistance to measure mutation rates in Schizosaccharomyces pombe. PLoS ONE 18(1): e0271016. https://doi.org/10.1371/journal.pone.0271016
Editor: Juan Mata, University of Cambridge, UNITED KINGDOM
Received: June 21, 2022; Accepted: December 19, 2022; Published: January 10, 2023
Copyright: © 2023 Pai et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.
Data Availability: All sequencing data are available now at the NCBI-SRA database, accession number PRJNA877936 (https://www.ncbi.nlm.nih.gov/sra/PRJNA877936).
Funding: This work was funded by the Medical Research Council (grant MR/L016591/1 to S.E.K.) The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.
Competing interests: The authors have declared that no competing interests exist.
Introduction
Measuring mutation rates using resistance to the toxic amino acid canavanine has been widely used in fluctuation analysis in Saccharomyces cerevisiae [1–3]. In this organism, the basis of the resistance is simple in that mutation of the arginine transporter CAN1 blocks canavanine import, and sequencing of mutations in the CAN1 gene can be used to determine the mutational spectra of different yeast strains [4]. In Schizosaccharomyces pombe, canavanine resistance has also been used in mutation rate analysis [5–7], but here the genetic basis of the resistance is less clear. An early study of a “can1-1” strain, identified can1 and canX as responsible for the canavanine resistance phenotype [8], but recent studies showed that the phenotype is due at least in part to an R175C missense mutation in the any1 gene, which is closely linked to can1+(now renamed vhc1+) [9,10]. However, loss of function mutations in a number of S. pombe genes, such as the arginine-transporter cat1+, the cyclin pas1+ [11] and the tsc1+/tsc2+ genes involved in TORC1 signalling [12,13] have also been reported to confer canavanine resistance, so it is not clear which genes are most commonly mutated in spontaneous canavanine-resistant mutants generated in fluctuation analysis experiments. This information would be useful as it would facilitate directed sequencing to determine mutation spectra and would clarify whether mutation rates determined are likely to be representative of genome-wide mutation rates.
We present here a simple protocol for carrying out mutation rate assays using fluctuation analysis in S. pombe. We generated a panel of S. pombe strains expressing DNA polymerase ε variants with mutations in the exonuclease domain of Pol2, which have been identified as cancer-associated variants, and compared mutation rates using canavanine resistance, reversion of an ade6-485 auxotrophic mutation, and resistance to 5-fluoroorotic acid (FOA) generated by loss-of-function mutations in ura4+ or ura5+ genes. The genomes of eight canavanine-resistant mutants were sequenced and surprisingly all had an any1R175C mutation. Comparison with strains with single mutations in amino-acid transporters showed that the any1R175C mutation confers a more pronounced resistance to canavanine.
Materials and methods
S. pombe methods
Standard media and genetic techniques were used as previously described [14]. Cultures were grown in either rich medium (YE3S), EMM or PMG plates supplemented with the appropriate supplements and incubated at 30°C. Nitrogen starvation was carried out using EMM lacking NH4Cl. The relevant genotypes and source of the strains used in this study are listed in S1 Table. Oligos used for strain constructions and genotyping are listed in S2 Table.
Spontaneous canavanine-resistant mutants were derived by setting up separate cultures starting at 104 cells/ml, growing to saturation, and then spotting 150 ul of each culture onto PMG+canavanine plates (80μg/ml) and incubating for 1 week at 30°C. Only one canavanine-resistant colony per spot was selected. The level of canavanine-resistance was determined by spot testing, and strains were only selected for sequencing if resistance was comparable to the original “can1-1” strain (3647).
For spot testing, cells were grown overnight to exponential phase and serial dilutions (1/10) of cells were spotted on agar plates using a replica plater (Sigma R2383). Plates were photographed after 2–5 days at 30°C.
An AxioPlan 2 microscope was used for fluorescence microscopy as previously described [5] and pictures were taken using a Hamamatsu ORCA E camera system with Micro-Manager 1.3 software.
Construction of S. pombe strains
The pol2L425V strain was constructed by amplifying pol2+ segments with primer pairs; 1083 and 1108 and 1084 and 1109. The products were purified, annealed and amplified using primers 1083 and 1084. The product was digested with BglII and AscI and inserted into BamHI, AscI-cleaved pFA6a-kanMX6 [15]. The pol2V412L strain was similarly constructed using primer combinations:1083 and1137, 1084 and 1138; and for the pol2S298F strain the oligos 1083 and 1144, 1084 and 1145 were used. Plasmids were integrated into the pol2+ locus after linearization with BamHI and selecting for G418 resistance. Constructs were verified by Sanger sequencing.
The vhc1Δ::hphMX6 strain was constructed using oligos 1382 and 1383, using plasmid pFA6ahphMX6 as template. Vhc1Δ strains were identified using colony PCR with oligos 1432 and 1433.
Strains were genotyped for the any1R175C mutation by colony PCR (94°C 5 min, then 30 cycles of 94°C 30 sec, 62°C 15 sec, 68°C 15 sec) using oligos 1420 and 1421, which gives a product with the wild-type strain, and oligos 1419 and 1421 which gives a product with the any1R175C strain (product size = 600 bp). Alternatively, oligos 1436 and 1437 were used to amplify a segment of the any1 gene, and this was subsequently digested with HpyCH4IV. The any1R175C mutation destroys a HpyCH4IV site, and gives fragment sizes of 373 and 289 bp, whereas the WT gene gives fragment sizes of 289, 260 and 113 bp.
Determination of mutation rates by fluctuation analysis
The protocol for mutation rate assays was derived from [2]. Briefly, for each assay a culture (104 cells/ml) was aliquoted into 12 wells of a 96-well microtitre plate (0.25 ml/well) and allowed to grow to saturation. Dilutions were plated onto YES plates to determine the cell concentration and, for canavanine-resistance, 0.15 ml of each culture was plated out onto pre-dried PMG plates (EMM-G, [8]) containing 80 μg/ml canavanine. We found that reducing the pH of the PMG medium to 4.5 reduced the time necessary to count canavanine-resistant colonies (S1 Fig), which were counted after 5–10 days at 30°C. For FOA resistance, cells were plated onto EMM medium containing 1 g/l FOA (Formedium). Since background growth on the primary FOA-containing plates made it difficult to score FOA-resistant colonies, the primary plates were replica plated onto fresh FOA media after 3 days and scored after an additional 3 days. For mutation rate determination based on reversion of the ade6-485 allele, cells were plated onto EMM-adenine plates. Each assay was repeated at least three times from independent clones.
Mutation rates were calculated using the Ma-Sandri-Sarkar maximum likelihood estimator [16], implemented in rSalvador [17].
Whole genome DNA sequencing
S. pombe DNA was extracted from cells grown to log phase at 32°C using a YeaStar genomic DNA purification kit (Zymo Research). Equal quantities of DNA from four spontaneous canavanine-resistant clones derived from strain 2299 were combined; similarly, DNA from four canavanine-resistant clones from strain 3938 were combined. DNA from strain 3647 (“can1-1”) was sequenced as a unique sample. Genomic DNA sent to Novogen (Cambridge, UK) Company Limited for whole genome sequencing (Illumina PE150). The resulting reads were aligned to the reference genome Schizosaccharomyces pombe ASM294v2 using BWA software (parameters: mem -t 4 -k 32 -M). Duplicates were removed by SAMTOOLS. The average sequencing depth was 68–107. Variants were called using GATK HaplotypeCaller. Sequence data are available from the NCBI-SRA database:
Results and discussion
We generated a panel of S. pombe strains expressing DNA polymerase ε variants with mutations in the exonuclease domain of the largest subunit Pol2/Cdc20 (Table 1, Fig 1A). These variants are either germline or somatic mutations associated with predisposition to colorectal and endometrial cancers [18]. In addition, we used an S. pombe Pol2D276AE278A mutant where the 3’-5’ exonuclease activity required for proofreading is inactivated. Mutation rates of these strains were determined by fluctuation analysis using canavanine resistance. In parallel, mutation rate assays were carried out selecting for Ade+ reversion of the ade6-485 allele, which requires acquisition of missense mutation in a stop codon, and selection for FOA resistance, which is caused by mutations inactivating the ura4+ or ura5+ genes.
(A) DNA polymerase e variants used in this study showing alignment of exonuclease domain. Top panel shows a schematic representation of the human catalytic subunit of Pol ε. The 2286 amino acid long subunit contains the two catalytic activities of Pol ε: 3’-5’exonuclease and 5’-3’ polymerase activity represented by the exo domain and polymerase domain, respectively. The exonuclease domain possesses five highly conserved domains called Exo motifs (I-V), highlighted in black. Lower panel shows an amino acid alignment of Pol ε and T4 polymerase sequences using T-Coffee [19] (default parameters; no matrix). Somatic and germline/somatic exonuclease domain mutations are highlighted in red, and violet, respectively, while the two catalytic sites are highlighted in green. Further details of the human clinical variants are shown in Table 1. Hs: human; Mm: mouse; Xl: Xenopus laevis; Dm: Drosophilia; Dr: Danio rerio; At: Arabidopsis thalinana; Sp: Schizosaccharomyces pombe; Sc: Saccharomyces cerevisiae; T4Pol: T4 DNA polymerase. (B) Mutation rates determined by fluctuation analysis of five DNA polymerase variants modelled in S. pombe, using ade6-485 reversion, resistance to canavanine or resistance to FOA. Y axis values represent: mutation rate of mutant/mutation rate of wt. Error bars show the range of three independent experiments. Correlation coefficients to the canavanine resistance data are 0.67 (Ade+ reversion) and 0.86 (FOA resistance). Absolute mutation rates are given in S3 Table.
The mutation rates determined by canavanine-resistance were lower than those determined by FOA resistance but showed a similar trend, and were quite comparable to those determined by ade6-485 reversion (Fig 1B). Interestingly two of the variants, S298F and S460F showed hypermutation, which we define as having a mutation rate higher than that of a strain with simple loss of exonuclease activity (i.e. Pol2D276AE278A). This suggests that the mechanism of mutagenesis must involve something other than a defect in proofreading, which has also been shown for a yeast strain expressing the equivalent mutation to the POLE P286R variant [5,24]. Also of interest is that the V412L variant shows a mutation rate lower than the Pol2D276AE278A strain, while the equivalent human mutation V411L causes hypermutation [20]. A similar low mutation rate was shown using S. cerevisiae to model the mutation [25], suggesting that this amino-acid substitution may affect the yeast and human polymerases differently.
To determine the genes mutated in the canavanine fluctuation analysis, the genomes of eight canavanine-resistant mutants were sequenced. Mutants were selected only if their canavanine-resistant phenotype was at least as pronounced as the original “can1-1” mutant (3647). We initially sequenced the vhc1+ gene, previously called can1+, of the canavanine-resistant mutants by Sanger sequencing, but none showed a mutation. Whole genome sequencing showed that surprisingly all eight mutants had the any1R175C mutation (Table 2), recently also identified in the “can1-1” mutant [9,10]. Three canavanine-resistant strains additionally had a missense mutation in the transmembrane transporters cat1 or aat1. These mutations affected residues that are conserved between Cat1 and Aat1 (S2 Fig). We additionally sequenced the original “can1-1” strain and this showed the any1R175C mutation as recently reported [9,10]. In addition, there was a frameshift mutation in the cyclin pas1+ gene. Since inactivation of this gene has been shown to cause canavanine resistance [11], this may contribute to the phenotype of this strain, and may correspond to ‘canX’ in the analysis of Fantes and Creanor [8]. We also found that independent canavanine-resistant clones from the hypermutating strain pol2S298F had the any1R175C mutation (S3 Fig).
The R175C mutation occurs in the arrestin-fold domain of Any1 (Fig 2), which interacts with transmembrane transporters such as Aat1 and probably Cat1, allowing ubiquitylation by the Pub1 ubiquitin ligase [26,27]. R175 is conserved in the S. pombe Any1 homologue Any2/Arn2 (Fig 2), where it is close to K163 which has been identified as a ubiquitylation site [28], and the S. cerevisiae orthologue Art1. However the only ubiquitylation site identified in Any1 is K263 [26].
The lower panel shows an alignment of S. pombe Any1, Any2 and S. cerevisiae Art1, generated by Praline [29].
To compare the canavanine resistance of an anyR175C strain with mutants defective in amino-acid transporters, we carried out spot assays on PMG+canavanine plates (Fig 3). The any1R175R strain showed similar growth to the original “can1-1” strain. Strains with single deletions of the vhc1+ (originally can1+), cat1+, aat1+ and SPBPB2B2.01 genes showed no growth on canavanine. A strain deleted for both aat1+ and cat1+ genes showed some growth, although to a slightly lesser extent that with the any1R175C strain. Consistent with these findings, Aspuria and Tamanoi [12] referred to unpublished data showing that in the presence of ammonium Cat1 is the major route for uptake of canavanine but in the absence of ammonium, cat1Δ cells are no longer canavanine resistant, but that a double cat1Δ aat1Δ mutant shows resistance.
Serial (1/10) dilutions of strains were spotted on the YES and PMG+canavanine plates (80 μg/ml) and incubated at 30°C for 2–5 days. Strains used were: 2299 (wt); 3647 (can1-1); 4089 (any1R175C); 3791 (vhc1Δ); 4059 (cat1Δ); 3790 (aat1Δ); 3796 (SPBPB2B2.01Δ); 3797 (aat1Δ cat1Δ).
Mechanism of canavanine resistance conferred by Any1R175C
Any1/Arn1 is an arrestin of the HECT-type E3 ubiquitin ligase Pub1 and regulates amino-acid transporters Aat1 and probably Cat1 by ubiquitylation [26,27]. In S. pombe, ubiquitination of these amino acid transporters causes them to be transported away from the plasma membrane to the Golgi under nutrient-rich conditions [26,27]. Loss of Any1/Arn1 leads to canavanine sensitivity as Cat1 cannot be relocalised from the plasma membrane. Thus we found that wild-type Cat1-GFP cells growing in nitrogen-starvation conditions is localised to the plasma membrane, particularly at the growing cell ends, as previously reported [12,26]. Interestingly, in an any1R175C background the fluorescence is internalised as recently reported [10], presumably to the Golgi, resulting in a canavanine-resistant phenotype (Fig 4). Any1/Arn1 over-expression causes a similar phenotype [26,27], so we assume that the any1R175C is a change-of-function mutation that causes hyperactivation of the Pub1-Any1 ubiquitin ligase. Modelling of the R175C mutation suggests that it enables Any1 to interact more strongly with Cat1, thus presumably promoting Cat1 ubiquitylation [9]. Other targets of Pub1 have been identified, such as Cdc25 [30], and it would be interesting to see if Any1R175C affects their ubiquitylation.
Wild-type cells show Cat1 externalised to the plasma membrane, particularly to the growing tip ends, but in any1R175C cells Cat1 is internalised, presumably to the Golgi. Cells were grown to log phase in EMM+NH4Cl, washed and then transferred to EMM-NH4Cl for 60 min before imaging as live cells. Scale bar = 10μm.
As noted, on PMG plates, deletion of both the aat1+ and cat1+ genes gives some resistance to canavanine, and the any1R175C mutation causes not only Cat1to be internalised but probably Aat1as well [27], thus a single mutation can inactivate at least two arginine transporters. Since two of the spontaneous any1R175C mutants, and indeed the original “can1-1” strain had additional mutations in genes implicated in canavanine resistance, it is likely that these additional mutations slightly enhance the canavanine-resistance phenotype of the any1R175C mutation. It is also of interest to consider why a mutation equivalent to R175C in the S. cerevisiae Any1-orthologue Art1/Ldb19 has not been described to confer canavanine resistance. This may reflect the fact that probability of a knock-out mutation in the single arginine transporter CAN1 is higher than a specific point mutation in ART1/LDB19, whereas in S. pombe two arginine transporters need to be inactivated to provide canavanine resistance and this is less likely than the any1R175C mutation.
Implications for use of canavanine in mutation rate assays in fission yeast
Although canavanine resistance is a simple selection for mutation rate determination in fission yeast and, from the panel of strains tested here, the mutation rate trends are broadly in line with FOA-resistance and ade6-485 reversion, the restriction of most mutations to AnyR175C (C523T) means that this selectable mutation is not so useful for obtaining information about mutation spectra. Using different media, e.g. containing ammonium, might allow a wider selection of mutations to be selected for, for instance in the cat1+ gene.
Supporting information
S1 Fig. Effect of reducing the pH of PMG medium on canavanine-resistant colony appearance.
(A) 150 μl of a culture of strain 4355 (OD600 = 1) was spotted onto PMG + 80 μg/ml canavanine plates adjusted to the pHs shown. Plates were photographed after 5 days at 30°C. (B) As (A) except WT, any1R175C and pol2S298F strains were used. For the any1R175C strain approximately 5 cells were plated.
https://doi.org/10.1371/journal.pone.0271016.s001
(PDF)
S2 Fig. Location of missense mutations in the amino-acid transporters Cat1 and Aat1.
Sequences aligned are S. pombe Cat1 and Aat1. The alignment shows that the missense mutations detected in some of the canavanine-resistant strains are conserved between Cat1 and Aat1. The alignment was generated by Praline [29].
https://doi.org/10.1371/journal.pone.0271016.s002
(PDF)
S3 Fig. Canavanine-resistant clones isolated from the hypermutation strain pol2S298F (3221) also have the any1R175C mutation.
Strains were genotyped by amplifying an any1 gene fragment using oligos 1436 and 1437, followed by HpyCH4IV digestion. Lane 1, any1R175C control (strain 3647); lane 2, WT control (strain 2299); lanes 3–7, independent canavanine-resistant clones derived from hypermutating strain pol2S298F (3221).
https://doi.org/10.1371/journal.pone.0271016.s003
(PDF)
S3 Table. Absolute mutation rates of Pol2 strains.
https://doi.org/10.1371/journal.pone.0271016.s006
(PDF)
Acknowledgments
We are very grateful to Drs Akio Nakashima, Kaoru Takegawa and Oliver Fleck for S. pombe strains. The original “can1-1” strain was provided by the YGRC/NBRP, Japan. We thank Peter Fantes and Li-Lin Du for discussions.
References
- 1. Lang GI, Murray AW. Mutation rates across budding yeast chromosome VI are correlated with replication timing. Genome Biol Evol. 2011;3:799–811. Epub 2011/06/15. pmid:21666225; PubMed Central PMCID: PMC3170098.
- 2. Lang GI, Murray AW. Estimating the per-base-pair mutation rate in the yeast Saccharomyces cerevisiae. Genetics. 2008;178(1):67–82. Epub 2008/01/19. pmid:18202359; PubMed Central PMCID: PMC2206112.
- 3. Simon M, Giot L, Faye G. The 3’ to 5’ exonuclease activity located in the DNA polymerase delta subunit of Saccharomyces cerevisiae is required for accurate replication. EMBO J. 1991;10(8):2165–70. Epub 1991/08/01. pmid:1648480; PubMed Central PMCID: PMC452904.
- 4. Jiang P, Ollodart AR, Sudhesh V, Herr AJ, Dunham MJ, Harris K. A modified fluctuation assay reveals a natural mutator phenotype that drives mutation spectrum variation within Saccharomyces cerevisiae. Elife. 2021;10. Epub 2021/09/16. pmid:34523420; PubMed Central PMCID: PMC8497059.
- 5. Soriano I, Vazquez E, De Leon N, Bertrand S, Heitzer E, Toumazou S, et al. Expression of the cancer-associated DNA polymerase epsilon P286R in fission yeast leads to translesion synthesis polymerase dependent hypermutation and defective DNA replication. PLoS Genet. 2021;17(7):e1009526. Epub 2021/07/07. pmid:34228709; PubMed Central PMCID: PMC8284607.
- 6. Kaur B, Fraser JL, Freyer GA, Davey S, Doetsch PW. A Uve1p-mediated mismatch repair pathway in Schizosaccharomyces pombe. Mol Cell Biol. 1999;19(7):4703–10. Epub 1999/06/22. pmid:10373519; PubMed Central PMCID: PMC84268.
- 7. Fraser JL, Neill E, Davey S. Fission yeast Uve1 and Apn2 function in distinct oxidative damage repair pathways in vivo. DNA Repair (Amst). 2003;2(11):1253–67. Epub 2003/11/06. pmid:14599746.
- 8. Fantes PA, Creanor J. Canavanine resistance and the mechanism of arginine uptake in the fission yeast Schizosaccharomyces pombe. J Gen Microbiol. 1984;130(12):3265–73. Epub 1984/12/01. pmid:18357653.
- 9. Yang YS, Ning SK, Lyu XH, Suo F, Jia GS, Li W, et al. Canavanine resistance mutation can1-1 in Schizosaccharomyces pombe is a missense mutation in the ubiquitin ligase adaptor gene any1. MicroPubl Biol. 2022;2022. Epub 2022/03/19. pmid:35300005; PubMed Central PMCID: PMC8922049.
- 10. Ait Saada A, Costa AB, Lobachev KS. Characterization of canavanine-resistance of cat1 and vhc1 deletions and a dominant any1 mutation in fission yeast. PLoS One. 2022;17(5):e0269276. Epub 2022/06/01. pmid:35639710.
- 11. van Slegtenhorst M, Mustafa A, Henske EP. Pas1, a G1 cyclin, regulates amino acid uptake and rescues a delay in G1 arrest in Tsc1 and Tsc2 mutants in Schizosaccharomyces pombe. Hum Mol Genet. 2005;14(19):2851–8. Epub 2005/08/24. pmid:16115814.
- 12. Aspuria PJ, Tamanoi F. The Tsc/Rheb signaling pathway controls basic amino acid uptake via the Cat1 permease in fission yeast. Mol Genet Genomics. 2008;279(5):441–50. Epub 2008/01/26. pmid:18219492; PubMed Central PMCID: PMC2670428.
- 13. van Slegtenhorst M, Carr E, Stoyanova R, Kruger WD, Henske EP. Tsc1+ and tsc2+ regulate arginine uptake and metabolism in Schizosaccharomyces pombe. J Biol Chem. 2004;279(13):12706–13. Epub 2004/01/14. pmid:14718525.
- 14. Moreno S, Klar A, Nurse P. Molecular genetic analysis of fission yeast Schizosaccharomyces pombe. Methods Enzymol. 1991;194:795–823. Epub 1991/01/01. pmid:2005825.
- 15. Bahler J, Wu JQ, Longtine MS, Shah NG, McKenzie A 3rd, Steever AB, et al. Heterologous modules for efficient and versatile PCR-based gene targeting in Schizosaccharomyces pombe. Yeast. 1998;14(10):943–51. Epub 1998/08/26. pmid:9717240.
- 16. Sarkar S, Ma WT, Sandri GH. On fluctuation analysis: a new, simple and efficient method for computing the expected number of mutants. Genetica. 1992;85(2):173–9. Epub 1992/01/01. pmid:1624139.
- 17. Zheng Q. rSalvador: An R Package for the Fluctuation Experiment. G3 (Bethesda). 2017;7(12):3849–56. Epub 2017/11/01. pmid:29084818; PubMed Central PMCID: PMC5714482.
- 18. Rayner E, van Gool IC, Palles C, Kearsey SE, Bosse T, Tomlinson I, et al. A panoply of errors: polymerase proofreading domain mutations in cancer. Nat Rev Cancer. 2016;16(2):71–81. Epub 2016/01/30. pmid:26822575.
- 19. Notredame C, Higgins DG, Heringa J. T-Coffee: A novel method for fast and accurate multiple sequence alignment. J Mol Biol. 2000;302(1):205–17. Epub 2000/08/31. pmid:10964570.
- 20. Church DN, Briggs SE, Palles C, Domingo E, Kearsey SJ, Grimes JM, et al. DNA polymerase epsilon and delta exonuclease domain mutations in endometrial cancer. Hum Mol Genet. 2013;22(14):2820–8. Epub 2013/03/27. pmid:23528559; PubMed Central PMCID: PMC3690967.
- 21. Wimmer K, Beilken A, Nustede R, Ripperger T, Lamottke B, Ure B, et al. A novel germline POLE mutation causes an early onset cancer prone syndrome mimicking constitutional mismatch repair deficiency. Fam Cancer. 2017;16(1):67–71. Epub 2016/08/31. pmid:27573199; PubMed Central PMCID: PMC5243902.
- 22. Palles C, Cazier JB, Howarth KM, Domingo E, Jones AM, Broderick P, et al. Germline mutations affecting the proofreading domains of POLE and POLD1 predispose to colorectal adenomas and carcinomas. Nat Genet. 2013;45(2):136–44. Epub 2012/12/25. pmid:23263490; PubMed Central PMCID: PMC3785128.
- 23. Kawai T, Nyuya A, Mori Y, Tanaka T, Tanioka H, Yasui K, et al. Clinical and epigenetic features of colorectal cancer patients with somatic POLE proofreading mutations. Clin Epigenetics. 2021;13(1):117. Epub 2021/05/27. pmid:34034807; PubMed Central PMCID: PMC8146650.
- 24. Kane DP, Shcherbakova PV. A common cancer-associated DNA polymerase epsilon mutation causes an exceptionally strong mutator phenotype, indicating fidelity defects distinct from loss of proofreading. Cancer Res. 2014;74(7):1895–901. Epub 2014/02/15. pmid:24525744; PubMed Central PMCID: PMC4310866.
- 25. Barbari SR, Kane DP, Moore EA, Shcherbakova PV. Functional Analysis of Cancer-Associated DNA Polymerase epsilon Variants in Saccharomyces cerevisiae. G3 (Bethesda). 2018;8(3):1019–29. Epub 2018/01/21. pmid:29352080; PubMed Central PMCID: PMC5844290.
- 26. Nakashima A, Kamada S, Tamanoi F, Kikkawa U. Fission yeast arrestin-related trafficking adaptor, Arn1/Any1, is ubiquitinated by Pub1 E3 ligase and regulates endocytosis of Cat1 amino acid transporter. Biol Open. 2014;3(6):542–52. Epub 2014/05/31. pmid:24876389; PubMed Central PMCID: PMC4058089.
- 27. Nakase Y, Nakase M, Kashiwazaki J, Murai T, Otsubo Y, Mabuchi I, et al. The fission yeast beta-arrestin-like protein Any1 is involved in TSC-Rheb signaling and the regulation of amino acid transporters. J Cell Sci. 2013;126(Pt 17):3972–81. Epub 2013/07/03. pmid:23813957.
- 28. Beckley JR, Chen JS, Yang Y, Peng J, Gould KL. A Degenerate Cohort of Yeast Membrane Trafficking DUBs Mediates Cell Polarity and Survival. Mol Cell Proteomics. 2015;14(12):3132–41. Epub 2015/09/29. pmid:26412298; PubMed Central PMCID: PMC4762628.
- 29. Bawono P, Heringa J. PRALINE: a versatile multiple sequence alignment toolkit. Methods Mol Biol. 2014;1079:245–62. Epub 2013/10/31. pmid:24170407.
- 30. Nefsky B, Beach D. Pub1 acts as an E6-AP-like protein ubiquitiin ligase in the degradation of cdc25. EMBO J. 1996;15(6):1301–12. Epub 1996/03/15. pmid:8635463; PubMed Central PMCID: PMC450033.