
Exonuclease III (ExoIII) exhibits robust enzymatic activities on single-stranded DNA, expanding its known functions beyond double-stranded DNA. The author argues that understanding these novel enzymatic activities sheds light on the biological roles of ExoIII in DNA repair and diagnostics.


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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 ( Escherichia coli ) 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.

### Competing Interest Statement

The authors have declared no competing interest.

Bacterial exonuclease III expands its enzymatic activities on single-stranded DNA

bacterial-exonuclease-iii-enzymatic-activities-on-single-stranded-dna


Bacterial Exonuclease III: Enzymatic Activities on Single-Stranded DNA
