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

最近的发现强调了复制DNA聚合酶(Pol-Delta和Pol-epsilon)中的生殖系和体细胞缺陷参与了结直肠癌和子宫内膜癌的发展(见Rayner等人，NAT Rev Cancer 2016；16：71-8)。突变会影响负责校对的核酸外切酶结构域，一些胚系突变体的突变率与催化死亡(即NO 3‘-5’核酸外切酶活性)聚合酶的突变率相当。更有趣的是，一些与高突变肿瘤相关的Pol-epsilon变体的突变率比核酸外切酶缺陷聚合酶高出一个数量级，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。该项目将包括以下部分：1.通过在模式生物(S.pombe)中构建突变并评估其对复制保真度的影响，来评估癌症相关DNA聚合酶epsilon变体的致病性。2.确定Pol-epsilon突变体S297F和S459F的高突变机制。为此，我们将使用生化和体内聚合酶功能的联合检测。这将阐明由聚合酶本身故障引起的复制错误的相对贡献，以及导致基因组不稳定性的突变修复途径。研究导致POL-epsilon外切酶结构域突变的C&gt；T和G&gt；T频繁变化的机制，特别是这是否仅仅反映了未纠正的聚合错误中的偏见，或者是容易出错的修复/分叉重新启动过程的结果。我们还将研究这些碱基变化是否是随机的，或者与特定的序列背景有关，例如难以复制的基因组区域。这项工作将有助于对具有Pol-epsilon生殖系变异的家庭进行遗传咨询，并澄清体细胞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.