| Summary: | <p>We constructed a panel of <em>S</em>. <em>pombe</em> 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 <em>ade-485</em> mutation to adenine prototrophy, but lower than 5-fluoroorotic acid (FOA)-resistance rates (inactivation of <em>ura4</em><sup>+</sup> or <em>ura5</em><sup>+</sup> genes). Inactivation of several genes has been associated with canavanine resistance in <em>S</em>. <em>pombe</em> but surprisingly whole genome sequencing showed that 8/8 spontaneous canavanine-resistant mutants have an <em>R175C</em> mutation in the <em>any1/arn1</em> 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 “<em>can1-1</em>” strain, which also has the <em>any1R175C</em> mutation. Some of the spontaneous mutants have additional mutations in arginine transporters, suggesting that this may marginally increase resistance to canavanine. The <em>any1R175C</em> strain showed internalisation of the Cat1 arginine transporter as previously reported, explaining the canavanine-resistance phenotype.</p>
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