Expression of the cancer-associatedDNA polymerase ε P286Rin fission yeast leads to translesion synthesis polymerase dependent hypermutation and defective DNA replication

Abstract

Somatic mutations in the proofreading domain of the replicative DNA polymerase ϵ ( 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 κ, partially suppresses pol2-P287R hypermutation, indicating that polymerase switching contributes to this phenotype.

A simple bypass assay for DNA polymerases shows hypermutating variants associated with cancer show mechanistic differences in vitro

“…A and G are both purines, and so this substitution would cause the minimum disruption to the double stranded structure produced. This substitution is also consistent with the observation that C to T transitions are commonly seen in POLE mutant tumours, particularly (21) in a CG context, in yeast exonuclease-defective POLE strains lacking MMR and in S. pombe expressing the equivalent mutation (in this case P287R) (25) . The choice to incorporate an A rather than any other nucleotide must be made at the level of the polymerase catalytic site since the same misincorporation is seen, not only for the wild-type POLE, but also the exonuclease dead and P286R.…”