GSE169231
Expression of the cancer-associated DNA polymerase epsilon P286R in fission yeast leads to translesion polymerase dependent hypermutation and defective DNA replication
Somatic mutations in the proofreading domain of the replicative DNA polymerase epsilon (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 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 also has a strong mutator effect in vivo, with 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 mutations are TCT > TAT transversions and, in contrast to human, TCG > TTG transitions are not elevated, likely due to the absence of cytosine methylation in fission yeast. The high mutation burden of pol2-P287R leads to increased sensitivity to elevated dNTP levels and DNA damaging agents and a phenotype that is exacerbated by depletion of the Pfh1 helicase. In addition, S phase is aberrant and RPA foci are elevated, suggestive of persistent ssDNA or DNA damage, and pol2-P287R shows synthetic lethality with rad3 deletion,  indicative of checkpoint activation. Significantly, deletion of translesion polymerases kappa and eta partially suppresses pol2-P287R hypermutation, indicating that both enzymes contribute to this phenotype. Overall design: DNA-Seq of 8 S. pombe strains at 2 different time generations (G0 and G200) to detect somatic mutations that are incorporated.