Fig 1.
Mutation rates of DNA polymerase e variants.
(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.
Table 1.
DNA polymerase ε variants analysed in Fig 1B.
Table 2.
Genes mutated in spontaneous canavanine-resistant mutants.
Fig 2.
Location of the R175C mutation in Any1.
The lower panel shows an alignment of S. pombe Any1, Any2 and S. cerevisiae Art1, generated by Praline [29].
Fig 3.
Comparison of canavanine-resistance phenotype of strains mutated in transmembrane transporters and vhc1+.
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).
Fig 4.
Cat1-GFP localisation in WT and any1R175C cells.
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.