Toumazou, S. | Search bioRxiv

bioRxiv2022

Using canavanine resistance to measure mutation rates in Schizosaccharomyces pombe

We constructed a panel of S. pombe strains expressing DNA polymerase {varepsilon} 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

genetics↗

bioRxiv2021

Expression of the cancer-associated DNA polymerase ϵ P286R in fission yeast leads to translesion synthesis polymerase dependent hypermutation and defective DNA replication

Somatic mutations in the proofreading domain of the replicative DNA polymerase {varepsilon} (POLE-exonuclease domain mutations, POLE-EDMs) are frequently found in colorectal and endometrial cancers and, occasionally, in other tumours. POLE-associated cancers typically display hypermutation, microsatellite stability and a unique mutational signature, with a predominance of C > A transversions in the context TCT. To understand better the contribution of hypermutagenesis to tumour development, we have modelled the most recurrent POLE-EDM (POLE-P286R) in Schizosaccharomyces pombe. Whole-genome sequencing analysis revealed that the corresponding pol2-P287R allele also has a strong mutator effect in vivo, with a high frequency of base substitutions and relatively few frameshift mutations. The mutations are equally distributed across different genomic regions, but they occur within an AT-rich context. The most abundant base-pair changes are TCT > TAT transversions and, in contrast to human mutations, TCG > TTG transitions are not elevated, likely due to the absence of cytosine methylation in fission yeast. The pol2-P287R variant has an increased sensitivity to elevated dNTP levels and DNA damaging agents, and shows reduced viability on depletion of the Pfh1 helicase. In addition, S phase is aberrant and RPA foci are elevated, suggestive of persistent ssDNA or DNA damage, and the pol2-P287R mutation is synthetically lethal with rad3 inactivation, indicative of checkpoint activation. Significantly, deletion of genes encoding some translesion synthesis polymerases, most notably Pol {kappa}, partially suppresses pol2-P287R hypermutation, indicating that polymerase switching contributes to this phenotype. AUTHOR SUMMARYCancer is a genetic disease caused by mutations that lead to uncontrolled cell proliferation and other tumour properties. Defects in DNA repair or replication can lead to cancer development by increasing the likelihood that cancer-causing mutations will happen. Here we look at a pathogenic variant of a polymerase involved in genome replication (DNA polymerase POLE-P286R). This variant is associated with highly mutated cancer genomes. By introducing this mutation into the polymerase gene of a model organism, fission yeast, we show that it causes a large increase in single base substitutions, scattered throughout the genome. The sequence context of mutations is similar in fission yeast and humans, suggesting that the yeast model is useful for understanding how POLE-P286R causes such a high mutation rate. Yeast POLE-P286R cells show slow chromosome replication, suggesting that the polymerase has difficulty in copying certain chromosomal regions. Yeast POLE-P286R cells become inviable when the concentration of dNTP building blocks for DNA synthesis is increased, probably because the mutation rate is pushed to an intolerable level. Interestingly, we find that specialised polymerases that are tolerant of DNA damage contribute to the high mutation rate caused by POLE-P286R. These findings have implications for the therapy of POLE-P286R tumours.

cancer biology↗