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.

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