ExoIII catalyzes the stepwise removal of mononucleotides from 3´-hydroxyl termini of double-stranded DNA.

[1] A limited number of nucleotides are removed during each binding event, resulting in coordinated progressive deletions within the population of DNA molecules.

[2] The preferred substrates are blunt or recessed 3´-termini, although ExoIII also acts at nicks in duplex DNA to produce single-strand gaps.

This property is used to produce unidirectional deletions from a linear molecule with one resistant (3´-overhang) and one susceptible (blunt or 5´-overhang) terminus.

Exonucleases evolved early in the history of life and have vital biological roles.