Exonuclease III digests duplex DNA in a 3´ to ;5´ direction from a nick, a blunt end, or 3´-recessed end, producing stretches of ssDNA on the opposite strand. ^5,6 Under defined reaction conditions, DNA degradation by Exonuclease III proceeds at a uniform rate yielding predictable and reproducible digestion results.

Applications

• Production of intermediates for site-directed mutagenesis. ^1-3
• Production of strand-specific radiolabelled probes. ⁴

Because the rate of exonucleolytic excision of deoxyribonucleotides by Exonuclease III is dependent upon reaction factors including temperature, ionic strength, template sequence, and enzyme-to-DNA ratios, ^4,7 each template must be optimised using sample digestions to achieve the desired excision rate.

Exonuclease III is not active on 3´-protruding ends of four bases or more in length, ssDNA, or on thioester-linked nucleotides. ² The enzyme also has intrinsic RNase H, 3´-DNA phosphatase, and apurinic DNA endonuclease activities. ^1,6

References

1. Sambrook, J. et al. (1989) in: Molecular Cloning: A Laboratory Manual (2nd ed.), Cold Spring Harbor Laboratory Press, New York.
2. Vandeyar, M.A. (1988) Gene 65, 129.
3. Luckow, B. et al. (1987) Nucleic Acids Res. 15, 417.
4. Richardson, C.C. et al. (1964) J. Biol. Chem. 239, 251.
5. Weiss, B. (1976) J. Biol. Chem. 251, 1896.
6. Rogers, S.G. and Weiss, B. (1980) Meth. Enzymol. 65, 201.
7. Guo, L.H. and Wu, R. (1982) Nucleic Acids Res. 10, 2065.