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Exonuclease III (Exo III)

B buffer for 100% activity Recombinant enzyme Thermal inactivation at 70°C in 10 min

Exonuclease III is a 3'–>5' exonuclease specific for double-stranded DNA or DNA-RNA hybrids. Exhibits also 3' phosphatase and purinic/apyrimidinic-endonuclease activities.
  
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Thermo Scientific Exonuclease III (ExoIII) exhibits four catalytic activities (see Reference1). The 3'=>5' exodeoxyribonuclease activity of ExoIII is specific for double-stranded DNA. ExoIII degrades dsDNA from blunt ends, 5'-overhangs or nicks, releases 5'-mononucleotides from the 3'-ends of DNA strands and produces stretches of single-stranded DNA. It is not active on 3'-overhang ends of DNA that are at least four-bases long and do not carry a 3'-terminal C-residue (see Reference 2) on single-stranded DNA, or on phosphorothioate-linked nucleotides.

ExoIII 3'-phosphatase activity removes the 3'-terminal phosphate, generating a 3'-OH group. ExoIII Rnase H activity exonucleolytically degrades the RNA strand in RNA-DNA hybrids. ExpOOO apurinic/apyrimidinic-endonuclease activity cleaves phosphodiester bonds at apurinic or apyrimidinic sites to produce 5'-termini that are base-free deoxyribose 5'-phosphate residues.

Highlights

  • Active in restriction enzyme buffers

Applications

  • Creation of unidirectional deletions in DNA fragments in conjunction with S1 Nuclease (see References 2, 3)
  • Generation of a single-stranded template for dideoxy-sequencing of DNA (see Reference 4)
  • Site-directed mutagenesis (see Reference 5)
  • Cloning of PCR products (see Reference 6)
  • Preparation of strand-specific probes

Note

The rate of DNA digestion by ExoIII depends upon temperature, salt concentration, and the molar ratio of DNA to enzyme in the reaction mixture (see References 4, 8). Optimal reaction conditions should be determined experimentally.

  
Storage Condition-20 C
HazardousNo
10X Reaction Buffer660 mM Tris-HCl (pH 8.0 at 30°C), 6.6 mM MgCl2.
Definition of Activity Unit
  • One unit of the enzyme catalyzes the release of 1 nmol of acid soluble reaction products from E. coli [3H]-DNA in 30 min at 37°C.
  • Enzyme activity is assayed in the following mixture: 50 mM Tris-HCl (pH 8.0), 5 mM MgCl2, 1 mM DTT, and 0.05 mM sonicated E. coli [3H]-DNA.
InactivationInactivated by heating at 70°C for 10 min.
InhibitionInhibitors: metal chelators, p-chloromercuri benzoate (50-90% inhibitory at 0.1 mM) (see Reference 7).
Molecular Weight31 kDa monomer.
Quality Control
  • The absence of endodeoxyribonucleases confirmed by appropriate quality test.
  • Functionally tested for creation of unidirectional deletions in DNA fragments.
SourceE. coli cells with a cloned E. colixth gene.
Storage BufferThe enzyme is supplied in:
50 mM Tris-HCl (pH 8.0), 50 mM KCl, 1 mM DTT and 50% (v/v) glycerol.
Exonuclease III activity

Exonuclease III activity

Exonuclease III activity


References

  1. S. G. Rogers, B. Weiss, Exonuclease III of Escherichia coli K-12, an AP endonuclease. Methods Enzymol. 65, 201-211 (1980).
  2. J. D. Hoheisel, On the Activities of Escherichia coli Exonuclease III. Anal. Biochem. 209, 238-246 (1993).
  3. S. Henikoff, Unidirectional digestion with exonuclease III creates targeted breakpoints for DNA sequencing. Gene. 28, 351-359 (1984).
  4. L-H. Guo, R. Wu, New rapid methods for DNA sequencing based on exonuclease III digestion followed by repair synthesis. Nucleic Acids Res. 10, 2065-2084 (1982).
  5. M. A. Vandeyar et al., A simple and rapid method for the selection of oligodeoxynucleotide-directed mutants. Gene. 65, 129-133 (1988).
  6. C. Li, R. M. Evans, Ligation independent cloning irrespective of restriction site compatibility. Nucleic Acids Res. 25, 4165-4166 (1997).
  7. H. M. Eun, Enzymology Primer for Recombinant DNA Technology (Academic Press Inc., San Diego, California, 1996).
  8. J. F. Tomb, G. J. Barcak, Regulating the 3’-5’ activity of exonuclease III by varying the sodium chloride concentration. BioTechniques. 7, 932-933 (1989).

Protocols and Product Manuals

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