Characterisation of DNA polymerase δ exonuclease domain mutations associated with cancer predisposition using fission yeast

Heterozygous germline exonuclease/proofreading domain mutations (EDMs) in DNA polymerase delta (Pol δ) are linked predominantly to colorectal and endometrial cancers with microsatellite stability (MSS), such as POLD1 S478N. Pol δ synthesises the lagging strand during DNA replication and partakes in DNA repair. To assess the pathogenicity of more cancer-associated EDMs that affect highly conserved residues, POLD1 P327L, G321S, L474P and D316N variants were constructed in Schizosaccharomyces pombe (S. pombe) pol3 and mutation rate assays were carried out. Pol3(d)D316N had the highest mutation rate, comparable to Pol3 with no 3'-5' exonuclease activity (exo-null). Pol3(d)L474P had a moderate mutator phenotype, and pol3(d)P327L and pol3(d)G321S did not have increased mutation rates compared to wild type but showed hydroxyurea (HU) sensitivity, suggesting that DNA replication under stressful conditions is defective. Some Pol ε EDMs are hypermutating, but this is not seen with Pol δ EDMs. Interestingly, none of the variants had hyper-mutation rates in excess of Pol δ exo-null, unlike some Pol ε variants. To investigate this further, pol3(d)P327R, equivalent to the hypermutating pol2(e)P286R variant, was constructed but this had a low mutation rate. The HU-sensitivity phenotype of pol3P327L and pol3G321S was further investigated. A significant proportion of cells expressing pol3P327L displayed mitotic abnormalities during HU treatment. Pol3G321S cells took longer to complete S phase compared to the other mutants after HU block and release and had reduced viability in hydroxyurea. Both strains were synthetically sick with Rad3 inactivation, suggesting dependence on the DNA replication or repair checkpoint. Thirdly, a genome-wide systematic screen was carried out to identify any novel negative synthetic interactions with pol3L474P. The screen yielded 565 possible hits, of which 28 were designated for further characterisation. This identified fen1 and msh2, which have been previously reported as synthetically lethal with exo-null pol3.