Exonuclease III, E.coli digests duplex DNA in the 3´→5´ direction from nicked DNA, blunt end DNA, 3´-recessed ends, or 3´-overhangs of less than four bases, and yields nucleoside 5´-phosphates. The DNA degradation proceeds at a uniform rate and produces stretches of ssDNA on the opposite strand. Under defined reaction conditions, the reaction can yield predictable and reproducible digestion results. Conditions such as temperature, ionic strength, template DNA sequence and the Exonuclease III to DNA ratio need to be optimized to suit specific applications to achieve the desired excision rate. Exonuclease III, E.coli is also capable of degrading DNA from 3´-phosphate ends due to intrinsic 3´-phosphatase activity. The enzyme also has apurinic DNA endonuclease activity as well as RNase H activity.