Catalyzes the stepwise removal of mononucleotides from 3' -hydroxyl termini of duplex DNA . A limited number of nucleotides  are removed during each binding event, resulting in coordinated progressive deletions with in the population of DNA molecules.  Exonuclease III has also been reported to have RNase H, 3' -phosphatase and AP-endonuclease activities. 

The preferred substrates are blunt or recessed 3' -termini, although the enzyme also acts at nicks in duplex DNA to produce  single-strand gaps. 3' -protruding termini are resistant to cleavage; the degree of resistance depends on the length of the extension,with extensions 4 bases or longer being essentially resistant to cleavage.

Exonuclease III activity depends partially on helical structure and displays sequence dependence (C>A=T>G). Temperature, salt concentration and the ratio of enzyme to DNA greatly affect enzyme activity, requiring reaction conditions to be tailored to specific applications.

1. Enzyme activity is basically consistent with well-known domestic brands

Fig. A fluorescence constant temperature rapid amplification reaction was performed, and a fluorescence curve appeared,which was similar to the fluorescence curve of the competitor