Defects in Replicative DNA Polymerases Linked to Cancer Predisposition and Tumour Development.

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Recent findings have highlighted the involvement of germline and somatic defects in replicative DNA polymerases (Pol-delta and Pol-epsilon) in contributing to the development of colorectal and endometrial cancers (reviewed in Rayner et al., Nat Rev Cancer 2016;16:71-8). Mutations affect the exonuclease domain responsible for proofreading, and some germline mutants show mutation rates comparable to catalytically dead (i.e. no 3'-5' exonuclease activity) polymerases. More intriguingly, some Pol-epsilon variants associated with hypermutated tumours have mutation rates an order of magnitude higher than exonuclease-defective polymerases, thus the mechanism of mutagenesis must involve some process other than simple loss of proofreading. This project will have the following components:1. Assess the pathogenicity of cancer-associated DNA polymerase epsilon variants by constructing the mutations in a model organism (S. pombe) and assessing the effect on replication fidelity. 2. Determine the mechanism of hypermutation seen with Pol-epsilon variants S297F and S459F. For this we will use a combination of biochemical and in vivo assays for polymerase function. This will clarify the relative contributions of replicative errors due to polymerase malfunction per se, and mutagenic repair pathways leading to genome instability.3. Study the mechanism that causes frequent C>T and G>T changes in Pol-epsilon exonuclease domain mutants, specifically whether this simply reflects a bias in uncorrected polymerization errors, or is a consequence of an error-prone repair/fork restart process. We will also study whether these base changes are random or associated with specific sequence contexts, such as 'hard-to-replicate' genomic regions.This work will aid genetic counselling for families with Pol-epsilon germline variants and clarify the tumour driver potential of somatic Pole mutations.