DNA damage can occur naturally or through environmental factors, leading to mutations in DNA replication and genomic instability in cells. Normally, natural d-nucleotides were selected by DNA polymerases. The template l-thymidine (l-T) has been shown to be bypassed by several types of DNA polymerases. However, DNA replication fidelity of nucleotide incorporation opposite l-thymidine in vivo remains unknown. Here, we constructed plasmids containing a restriction enzyme (PstI) recognition site in which the l-T lesion was site-specifically located within the PstI recognition sequence (CTGCAG). Further, we assessed the efficiencies of nucleotide incorporation opposite the l-T site and l-T lesion bypass replication in vitro and in vivo. We found that recombinants containing the l-T lesion site inhibited DNA replication. In addition, A was incorporated opposite the l-T lesion by routine PCR assay, whereas preference for nucleotide incorporation opposite the l-T site was A (13%), T (22%), C (46%), and G (19%), and no nucleotide insertion and deletions were detected in E. coli cells. In particular, a novel restriction enzyme-mediated method for detection of the mutagenic properties of DNA lesion was established, which allows us to readily detect restriction–digestion of the l-T-bearing plasmids. The study provided significant insight into how mirror-image nucleosides perturb the fidelity of DNA replication in vivo and whether they elicit mutagenic effects, which may help to understand both how DNA damage interferes with the flow of genetic information during DNA replication and development of diseases caused by gene mutation.

中文翻译：

---

在大肠杆菌细胞中复制含有镜像胸苷的 DNA。

DNA 损伤可以自然发生，也可以通过环境因素发生，导致 DNA 复制突变和细胞基因组不稳定。通常，天然d-核苷酸是由 DNA 聚合酶选择的。已显示模板l-胸苷 ( l -T) 可被多种类型的 DNA 聚合酶绕过。然而，体内与l-胸苷相反的核苷酸掺入的 DNA 复制保真度仍然未知。在这里，我们构建了包含限制酶 ( Pst I) 识别位点的质粒，其中l -T 病变位点特异性地位于PstI 识别序列 (CTGCAG)。此外，我们在体外和体内评估了与l -T 位点相对的核苷酸掺入和l -T 病变旁路复制的效率。我们发现含有l -T 损伤位点的重组体抑制了 DNA 复制。此外，通过常规 PCR 测定，A 在l- T 病变对面掺入，而在l- T 位点对面掺入核苷酸的偏好是 A（13%）、T（22%）、C（46%）和 G（ 19%)，在大肠杆菌中未检测到核苷酸插入和缺失细胞。特别是，建立了一种新的限制性内切酶介导的检测 DNA 病变诱变特性的方法，这使我们能够轻松检测携带l -T 的质粒的限制性消化。该研究提供了关于镜像核苷如何扰乱体内 DNA 复制的保真度以及它们是否引起诱变效应的重要见解，这可能有助于了解 DNA 损伤如何干扰 DNA 复制过程中遗传信息的流动和引起疾病的发展。通过基因突变。