In prokaryotes and yeasts, DNA polymerase proofreading (PPR) and DNA mismatch repair (MMR) cooperatively counteracts replication errors leading to repeat sequence destabilization (i.e. insertions/deletions of repeat units). However, PPR has not thus far been regarded as a mechanism stabilizing repeat sequences in higher eukaryotic cells. In a human cancer cell line, DLD-1, which carries mutations in both MSH6 and the Exo domain of POLD1, we previously observed that mononucleotide microsatellites were markedly destabilized whereas being stable in the simple MMR-defective backgrounds. In this study, we introduced the Exo domain mutation found in DLD-1 cells into MSH2-null HeLa cell clones, using CRISPR/Cas9 system. In the established Exo-/MMR-mutated HeLa clones, mononucleotide repeat sequences were remarkably destabilized as in DLD-1 cells. In contrast, dinucleotide microsatellites were readily destabilized in the parental MMR-deficient backgrounds, and the instability was not notably increased in the genome-edited HeLa clones. Here, we show an involvement of the Exo domain functions of DNA polymerase delta in mononucleotide repeat stabilization in human cells, which also suggests a possible role division between DNA polymerase and MMR in repeat maintenance in the human genome.

在原核生物和酵母中，DNA 聚合酶校对 (PPR) 和 DNA 错配修复 (MMR) 共同抵消导致重复序列不稳定的复制错误（即重复单元的插入/缺失）。然而，迄今为止，PPR 还没有被认为是一种稳定高等真核细胞中重复序列的机制。在人类癌细胞系 DLD-1 中，它在 MSH6 和 POLD1 的 Exo 结构域中都携带突变，我们之前观察到单核苷酸微卫星明显不稳定，而在简单的 MMR 缺陷背景中稳定。在这项研究中，我们将在 DLD-1 细胞中发现的 Exo 结构域突变引入MSH2-null HeLa 细胞克隆，使用 CRISPR/Cas9 系统。在已建立的 Exo-/MMR-突变 HeLa 克隆中，单核苷酸重复序列与 DLD-1 细胞一样明显不稳定。相比之下，二核苷酸微卫星在亲本 MMR 缺陷背景中很容易不稳定，并且在基因组编辑的 HeLa 克隆中不稳定性没有显着增加。在这里，我们展示了 DNA 聚合酶 delta 的 Exo 结构域功能在人类细胞中单核苷酸重复稳定中的作用，这也表明 DNA 聚合酶和 MMR 在人类基因组的重复维持中可能存在角色划分。