MCS, St George's University London, UK.
Department of Biology, University of Oxford, UK.
FEBS J. 2023 Dec;290(24):5744-5758. doi: 10.1111/febs.16936. Epub 2023 Aug 29.
Errors made by DNA polymerases contribute to both natural variation and, in extreme cases, genome instability and its associated diseases. Recently, the importance of polymerase misincorporation in disease has been highlighted by the identification of cancer-associated polymerase variants with mutations in the exonuclease domain. A subgroup of these variants have a hypermutation phenotype in tumours, and when modelled in yeast, they show mutation rates in excess of that seen with polymerase with simple loss of proofreading activity. We have developed a bypass assay to rapidly determine the tendency of a polymerase to misincorporate in vitro. We have used the assay to compare misincorporation by wild-type, exonuclease-defective and two hypermutating human DNA polymerase ε variants, P286R and V411L. The assay clearly distinguished between the misincorporation rates of wild-type, exonuclease dead and P286R polymerases. However, the V411L polymerase showed misincorporation rate comparable to the exonuclease dead enzyme rather than P286R, suggesting that there may be some differences in the way that these variants cause hypermutation. Using this assay, misincorporation opposite a templated C nucleotide was consistently higher than for other nucleotides, and this caused predominantly C-to-T transitions. This is consistent with the observation that C-to-T transitions are commonly seen in DNA polymerase ε mutant tumours.
DNA 聚合酶的错误导致了自然变异，在极端情况下，还会导致基因组不稳定及其相关疾病。最近，聚合酶错误掺入在疾病中的重要性已经通过鉴定具有外切酶结构域突变的癌症相关聚合酶变体得到了强调。这些变体中的一个亚组在肿瘤中具有高突变表型，并且在酵母中建模时，它们的突变率超过了简单丧失校对活性的聚合酶的突变率。我们开发了一种旁路测定法来快速确定聚合酶在体外错误掺入的趋势。我们使用该测定法比较了野生型、外切酶缺陷型和两种高突变人类 DNA 聚合酶 ε 变体 P286R 和 V411L 的错误掺入。该测定法清楚地区分了野生型、外切酶失活型和 P286R 聚合酶的错误掺入率。然而，V411L 聚合酶的错误掺入率与外切酶失活酶相当，而不是 P286R，这表明这些变体引起高突变的方式可能存在一些差异。使用该测定法，与模板化 C 核苷酸相对的错误掺入率始终高于其他核苷酸，并且这主要导致 C 到 T 的转换。这与 C 到 T 转换通常在 DNA 聚合酶 ε 突变肿瘤中观察到的观察结果一致。
