Exonuclease III (ExoIII) exhibits four catalytic activities. 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. A limited number of nucleotides are removed during each binding event, resulting in coordinated progressive deletions within the population of DNA molecules. 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 on single-stranded DNA, or on phosphorothioate-linked nucleotides. This property can be exploited to produce unidirectional deletions from a linear molecule with one resistant (3´-overhang) and one susceptible (blunt or 5´-overhang) terminus.

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. ExoIII apurinic/apyrimidinic (AP) endonuclease activity cleaves phosphodiester bonds at apurinic or apyrimidinic sites to produce 5\'-termini that are base-free deoxyribose 5\'-phosphate residues.

• Creation of unidirectional deletions in DNA fragments in conjunction with S1 Nuclease.
• Generation of a single-stranded template for dideoxy-sequencing of DNA.
• Site-directed mutagenesis.
• Cloning of PCR products.
• Preparation of strand-specific probes for dideoxy sequencing

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.

• Endonuclease III: 100,000 units/ml in 25 mM Tris-HCl, pH 8.0 @ 25°C, 50 mM KCl, 1.0 mM DTT, 0.1% mM EDTA, 50% Glycerol
• 10X Reaction Buffer: 100 mM Tris-HCl, pH 7.0 @ 25°C, 100 mM MgCl₂, 10 mM DTT.

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

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