Exonuclease III is a 3'-->5' exonuclease specific for double-stranded DNA. The enzyme catalyzes the stepwise removal of mononucleotides starting from a 3'-OH at nicks, blunt ends, recessed ends and 3'-overhangs of less than 4 bases, yielding nucleoside 5'-phosphates. Exonuclease III 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. Exonuclease III is used in conjunction with S1 nuclease for unidirectional deletion of sequences from the termini of DNA fragments.
