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Exonuclease III, E.coli

#EN0191 Supplied with: 10X Reaction Buffer	4000 u (200 u/µl) 0.2 ml
Related Documents (in pdf, ~40-60 KB): Certificate of Analysis: #EN0191 MSDS (English) MSDS (English-USA) MSDS (German)

Description

The Exonuclease III, E.coli (ExoIII) exhibits four catalytic activities (1):

• 3'=>5' exodeoxyribonuclease activity specific for double-stranded DNA:
  ExoIII degrades dsDNA from blunt ends, 5-overhangs or nicks, releasing 5'-mononucleotides from the 3'-ends of DNA strands and producing stretches of single-stranded DNA. It is not active on 3'-protruding ends of DNA that are at least four bases long; on single-stranded DNA, or on phosphorothioate-linked nucleotides.

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

Source
E.coli cells carrying a cloned E.coli xth gene.

Molecular Weight
31 kDa monomer.

Applications

• Creation of unidirectional deletions in DNA fragments in conjunction with S1 Nuclease (2-3), see Protocol for Generation of Unidirectional Deletions in DNA Fragments and Protocol for Creation of Nested Deletions.
• Generation of a single-stranded template for dideoxy sequencing of DNA (4), see Protocol for Production of Single-stranded Circular DNA Molecules from Supercoiled Double-stranded Plasmids in vitro.
• Site-directed mutagenesis (5).
• Cloning of PCR products (6).
• Preparation of strand-specific probes.

Quality Control
The absence of endodeoxyribonucleases confirmed by appropriate quality test. Functionally tested for creation of unidirectional deletions in DNA fragments.

Concentration
200 u/µl

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.

Storage Buffer
The enzyme is supplied in: 50 mM Tris-HCl (pH 8.0), 50 mM KCl, 1 mM DTT and 50% (v/v) glycerol.

10X Reaction Buffer
660 mM Tris-HCl (pH 8.0 at 30°C), 6.6 mM MgCl₂.

Inhibition and Inactivation
Inactivated by heating at 70°C for 10 min.

Note
The rate of DNA digestion by ExoIII strongly depends upon temperature, salt concentration and the molar ratio of DNA to enzyme in a reaction mixture (4, 7). Optimal reaction conditions should be determined experimentally.

Activity in Buffers for Restriction Enzymes, PCR, RT

Related Products

S1 Nuclease Nb.Bpu10I Glycogen Water, nuclease-free

References

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

Updated vasario 05, 2008 15:16