Exonuclease III

Exonuclease III (ExoIII) is an enzyme that belongs to the exonuclease family. ExoIII catalyzes the stepwise removal of mononucleotides from 3´-hydroxyl termini of duplex 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].

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Function

The preferred substrates are blunt or recessed 3´-termini, although ExoIII also acts at nicks in duplex DNA to produce single-strand gaps. The enzyme is not active on single-stranded DNA, and thus 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. 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 ^[3].

ExoIII activity depends partially on the DNA helical structure ^[4] and displays sequence dependence (C>A=T>G) ^[5].

ExoIII has also been reported to have RNase H, 3´-phosphatase and AP-endonuclease activities ^[1].

Regulation

Temperature, salt concentration and the ratio of enzyme to DNA greatly affect enzyme activity, requiring reaction conditions to be tailored to specific applications.

References

1. Exonuclease III of Escherichia coli K-12, an AP endonuclease. Methods Enzymol. 1980; 65: 201-11.
2. Molecular Cloning: A Laboratory Manual. 1989, 2nd. Ed., 5.84-5.85.
3. Unidirectional digestion with exonuclease III creates targeted breakpoints for DNA sequencing. Gene. 1984; 28: 351-9 Abstract
4. A deoxyribonucleic acid phosphatase-exonuclease from Escherichia coli. II. Characterization of the exonuclease activity. J Biol Chem. 1964; 239: 251-8 Free text (PDF - 937KB)
5. Sequence specificity of exonuclease III from E. coli. Nucleic Acids Res. 1982; 10: 4845-59. Free text