Exonuclease III catalyses the stepwise removal of mononucleotides from dsDNA starting from a 3'-OH at nicks, blunt ends, recessed ends and 3'-overhangs of less than 4 bases, yielding nucleoside 5'-phosphates.

This 3' - 5' exonuclease will also degrade DNA from 3'-phosphate ends due to its intrinsic 3'-phosphatase activity. In addition, the enzyme has apurinic endonuclease activity and ribonuclease H activity. The enzyme is used in conjunction with S1 nuclease for unidirectional deletion of sequences from the termini of DNA fragments. Exonuclease III is provided with a 10X Reaction Buffer consisting of 660 mM Tris-HCl (pH 8,0 at 25 °C), 6,6 mM MgCl₂.

Source: Recombinant E. coli strain.

Applications: Generating nested sets of deletions with double-stranded, linear DNA; preparation of single-stranded DNA.