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

最近的研究结果强调了生殖系和体细胞缺陷在复制DNA聚合酶（Pol-delta和Pol-epsilon）中参与结直肠癌和子宫内膜癌的发展（review in Rayner et al., Nat Rev Cancer 2016;16:71-8）。突变影响负责校对的外切酶结构域，一些种系突变显示出与催化死亡（即没有3‘-5’外切酶活性）聚合酶相当的突变率。更有趣的是，一些与高突变肿瘤相关的Pol-epsilon变异的突变率比外切酶缺陷聚合酶高一个数量级，因此突变的机制必须涉及一些过程，而不是简单的校对丢失。该项目将有以下组成部分：1。通过在模式生物（S. pombe）中构建突变并评估对复制保真度的影响，评估癌症相关DNA聚合酶epsilon变异的致病性。2. 确定Pol-epsilon变体S297F和S459F的超突变机制。为此，我们将使用生化和体内聚合酶功能测定相结合的方法。这将澄清由于聚合酶功能障碍本身引起的复制错误和导致基因组不稳定的诱变修复途径的相对贡献。研究Pol-epsilon外切酶结构域突变体中导致频繁的C>T和G>T变化的机制，特别是这是否仅仅反映了未纠正聚合错误的偏见，或者是容易出错的修复/叉重新启动过程的结果。我们还将研究这些碱基变化是随机的还是与特定的序列背景有关，例如“难以复制”的基因组区域。这项工作将有助于Pol-epsilon生殖系变异家庭的遗传咨询，并阐明体细胞Pole突变的肿瘤驱动潜力。

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.