Using canavanine resistance to measure mutation rates in Schizosaccharomyces pombe
Chen-Chun Pai1, Ellen Heitzer2, Sibyl Bertrand3
1Department of Oncology, CRUK-MRC Oxford Institute for Radiation Oncology, University of Oxford, ORCRB, Oxford, United Kingdom.
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.
Insights
DNA polymerase ε variants were studied using canavanine resistance in S. pombe. Surprisingly, all spontaneous mutants revealed a specific mutation in the any1/arn1 gene, explaining canavanine resistance.
Area of Science:
- Molecular Biology
- Genetics
- Biochemistry
Background:
- DNA polymerase ε (Pol ε) is crucial for DNA replication and its variants are linked to cancer.
- Canavanine resistance is a common method to screen for mutations in Schizosaccharomyces pombe (S. pombe).
- Previous studies have identified various genetic alterations conferring canavanine resistance.
Purpose of the Study:
- To compare mutation rates using canavanine resistance with other selective methods in S. pombe strains expressing cancer-associated DNA polymerase ε variants.
- To identify the specific genetic mutations responsible for spontaneous canavanine resistance in these strains.
Main Methods:
- Construction of S. pombe strains expressing specific DNA polymerase ε variants (POLES297F, POLEV411L, POLEL424V, POLES459F).
- Determination of mutation rates using canavanine resistance and comparison with adenine prototrophy reversion and 5-fluoroorotic acid (FOA) resistance assays.
- Whole genome sequencing of spontaneous canavanine-resistant mutants to identify causative mutations.
Main Results:
- Canavanine-resistance mutation rates were similar to ade-485 reversion rates but lower than FOA-resistance rates.
- Whole genome sequencing revealed that all eight spontaneous canavanine-resistant mutants possessed an R175C mutation in the any1/arn1 gene.
- The any1R175C mutation confers phenotypic resistance to canavanine, similar to the original "can1-1" strain, and is associated with the internalization of the Cat1 arginine transporter.
Conclusions:
- The any1/arn1 gene, encoding an α-arrestin-like protein, is the primary determinant of spontaneous canavanine resistance in S. pombe.
- The R175C mutation in any1/arn1 is sufficient to cause canavanine resistance by affecting arginine transporter localization.
- Canavanine resistance assays in S. pombe can be a sensitive tool for detecting specific genetic alterations, particularly in the any1/arn1 gene.
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