Department of Veterinary Medicine, Zhejiang University College of Animal Sciences, Hangzhou, China.
Hainan Institute of Zhejiang University, Sanya, China.
Elife. 2024 Jul 3;13:RP95648. doi: 10.7554/eLife.95648.
Bacterial exonuclease III (ExoIII), widely acknowledged for specifically targeting double-stranded DNA (dsDNA), has been documented as a DNA repair-associated nuclease with apurinic/apyrimidinic (AP)-endonuclease and 3'→5' exonuclease activities. Due to these enzymatic properties, ExoIII has been broadly applied in molecular biosensors. Here, we demonstrate that ExoIII () possesses highly active enzymatic activities on ssDNA. By using a range of ssDNA fluorescence-quenching reporters and fluorophore-labeled probes coupled with mass spectrometry analysis, we found ExoIII cleaved the ssDNA at 5'-bond of phosphodiester from 3' to 5' end by both exonuclease and endonuclease activities. Additional point mutation analysis identified the critical residues for the ssDNase action of ExoIII and suggested the activity shared the same active center with the dsDNA-targeted activities of ExoIII. Notably, ExoIII could also digest the dsDNA structures containing 3'-end ssDNA. Considering most ExoIII-assisted molecular biosensors require the involvement of single-stranded DNA (ssDNA) or nucleic acid aptamer containing ssDNA, the activity will lead to low efficiency or false positive outcome. Our study revealed the multi-enzymatic activity and the underlying molecular mechanism of ExoIII on ssDNA, illuminating novel insights for understanding its biological roles in DNA repair and the rational design of ExoIII-ssDNA involved diagnostics.
细菌核酸外切酶 III（ExoIII），因其能够特异地靶向双链 DNA（dsDNA）而被广泛认可，现已被记录为一种与 DNA 修复相关的核酸内切酶，具有脱嘌呤/脱嘧啶（AP）内切酶和 3'→5' 外切酶活性。由于这些酶学特性，ExoIII 已被广泛应用于分子生物传感器。在这里，我们证明 ExoIII（）在 ssDNA 上具有高度活跃的酶活性。通过使用一系列 ssDNA 荧光猝灭报告子和荧光标记探针，并结合质谱分析，我们发现 ExoIII 通过外切酶和内切酶活性从 3' 端到 5' 端在 5'-磷酸二酯键处切割 ssDNA。进一步的点突变分析确定了 ExoIII 切割 ssDNA 所需的关键残基，并表明该活性与 ExoIII 靶向 dsDNA 的活性共享相同的活性中心。值得注意的是，ExoIII 还可以消化含有 3'-端 ssDNA 的 dsDNA 结构。考虑到大多数 ExoIII 辅助的分子生物传感器需要涉及单链 DNA（ssDNA）或含有 ssDNA 的核酸适体，该活性将导致低效率或假阳性结果。我们的研究揭示了 ExoIII 在 ssDNA 上的多种酶活性及其潜在的分子机制，为理解其在 DNA 修复中的生物学作用以及涉及 ssDNA 的 ExoIII 的合理设计提供了新的见解。
