biostudies-literatureWang HNational Program on Key Research Project of ChinaKey research and Development Program of HangzhouZhejiang Provincial Natural Science Foundation of ChinaScientific Research Fund of Zhejiang Provincial Education DepartmentZhejiang Provincial Key R&amp;D ProgramHorizon 2020 - Research and Innovation Framework ProgrammeZhejiang Provincial Key R&D ProgramRP95648https://www.ebi.ac.uk/biostudies/studies/S-EPMC11221836biostudies-literatureUnknown13Bacterial 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 (<i>Escherichia coli</i>) 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 aceLifeBacterial exonuclease III expands its enzymatic activities on single-stranded DNA.PMC112218362019YFE0103900LZ24C180002202203A082022YFC2604201861917-SAFFIY2022508722021C020082023C03045Jiang ZLi YYe CZhou CZhao GJiang CYue MWang HZhang CLu QLi NfalseBacterial exonuclease III expands its enzymatic activities on single-stranded DNA.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 (<i>Escherichia coli</i>) 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 ac2024-01-01T00:00:00Z2024 Jul2026-06-02T21:18:39.759Z2025-04-04T12:21:38.49ZS-EPMC112218363895906210.7554/eLife.95648