Exonuclease III is a 3'->5' exonuclease, releasing 5'-mononucleotides from the 3'-ends of DNA strands.
Description:
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The 3'->5' exonuclease specific towards double-stranded DNA.
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Contains DNA 3'-phosphatase, hydrolyzing 3'-terminal phosphomonoesters.
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Contains AP endonuclease, cleaving phosphodiester bonds at apurinic or apyrimidinic sites to produce 5'-termini that are base-free deoxyribose 5'-phosphate residues (1).
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The enzyme has ribonuclease H activity, preferentially degrading the RNA strand in a DNA-RNA hybrid duplex, presumably exonucleolytically (1).
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Exonuclease III digests duplex DNA at nicks producing single-stranded gaps.
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Will not degrade double-stranded DNA with 3’ overhang of at least 4 base pairs, single-stranded DNA or phosphorothioate-linked nucleotides.
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Ultrapure recombinant enzyme.
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Applications of the enzyme:
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construction of nested unidirectional deletions of DNA fragments (2)
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generation of a single-stranded template for dideoxy-sequencing of DNA (3)
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site-directed mutagenesis (4) and cloning of PCR products (5).
Unit Definition: One unit is defined as the amount of enzyme required to produce 1 nmol of acid-soluble radio-activity in 30 min at 37°C (6).
Storage Conditions: Store at –20°C.
Storage Buffer: 25 mM Tris-HCl (pH 8.0 at 22°C), 0.05 mM dithiothreitol and 50% glycerol.
Assay Conditions: 50 mM Tris-HCl (pH 7.6 at 22°C), 10 mM MgCl2, 1 mM dithiothreitol and 1.5 nM duplex [³H] lambda DNA. Incubation is at 37°C for 30 min in a reaction volume of 20 µl.
Quality Control: All preparations are assayed for contaminating endonuclease activity. Typical preparations are greater than 95% pure, as judged by SDS polyacrylamide gel electrophoresis.
References:
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Rogers, S.G. and Weiss, B., Exonuclease III of Escherichia coli K-12, an AP endonuclease, Methods Enzymol., 65, 201-211, 1980.
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Henikoff, S., Unidirectional digestion with exonuclease III creates targeted breakpoints for DNA sequencing, Gene, 28, 351-359, 1984.
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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.
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Vandeyar, M.A., et al., A simple and rapid method for the selection of oligodeoxynucleotide-directed mutants, Gene, 65, 129-133, 1988.
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Li, Ch., Evans, R.M., Ligation independent cloning irrespective of restriction site compatibility, Nucleic Acids Res., 25, 4165-4166, 1997.
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Richardson, C. C.,Lehman, I. R. and Kornberg, A. (1964) J. Biol. Chem.239,251-258.