Schizosaccharomyces pombe Raw sequence reads. Schizosaccharomyces pombe

We constructed a panel of S. pombe strains expressing DNA polymerase epsilon variants associated with cancer, specifically POLES297F, POLEV411L, POLEL424V, POLES4459F 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-resistance mutants have an R175C mutation in the any1/arn1 gene. This gene encodes a beta-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, explaining the canavanine-resistance phenotype. In addition, any1R175C cells were elongated compared to wild type. The mitotic activator Cdc25 has been identified as a Pub1 ubiquitylation target, and since Wee1 inhibition of Cdc2 is counteracted by Cdc25, it is likely that Cdc25 ubiquitylation and downregulation by Any1R175C-Pub1 delays mitotic entry.

本研究构建了一组表达与癌症相关的DNA聚合酶ε（DNA polymerase ε）变异体的粟酒裂殖酵母（Schizosaccharomyces pombe，S. pombe）菌株，具体包括POLES297F、POLEV411L、POLEL424V及POLES4459F，并利用这些菌株对比了通过刀豆氨酸抗性（canavanine resistance）测定的突变率与其他筛选方法所得结果。刀豆氨酸抗性突变率与ade-485突变回复为腺嘌呤原养型的突变率大体相近，但低于5-氟乳清酸（5-fluoroorotic acid，FOA）抗性对应的突变率（后者涉及ura4+或ura5+基因的失活）。在粟酒裂殖酵母中，已有多个基因的失活被证实与刀豆氨酸抗性相关，但令人意外的是，全基因组测序（whole genome sequencing）结果显示，8株自发刀豆氨酸抗性突变体均携带any1/arn1基因的R175C突变。该基因编码一种参与介导靶蛋白Pub1泛素化（Pub1 ubiquitylation）的β抑制蛋白样蛋白，且这一单突变所产生的刀豆氨酸抗性表型，与同样携带any1R175C突变的原始can1-1菌株所表现出的抗性水平相似。部分自发突变体还在精氨酸转运蛋白编码基因上携带额外突变，这提示该类突变或许可小幅提升对刀豆氨酸的抗性。any1R175C菌株出现了Cat1精氨酸转运蛋白（Cat1 arginine transporter）的内化现象，这为其刀豆氨酸抗性表型提供了解释。此外，any1R175C细胞相较于野生型细胞更为伸长。有丝分裂激活因子Cdc25已被证实为Pub1泛素化的靶标，且由于Wee1对Cdc2的抑制作用可被Cdc25所抵消，因此Any1R175C-Pub1介导的Cdc25泛素化与下调，大概率会延迟有丝分裂进入。