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

By Ignacio Soriano, Enrique Vazquez, Nagore de Leon, Sibyl Betrand, Ellen Heitzer, Sophia Toumazou, Claire Palles, Chen-Chun Pai, Timothy Humphrey, Ian Tomlinson, Sue Cotterill, Stephen Kearsey

Posted 06 Apr 2021
bioRxiv DOI: 10.1101/2021.04.06.438567

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 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 T C T > T A T transversions and, in contrast to human mutations, T C G > T T G 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.

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