Thermo Scientific 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. 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.

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-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
• Generation of a single-stranded template for dideoxy-sequencing of DNA
• Site-directed mutagenesis
• Cloning of PCR products
• 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 . Optimal reaction conditions should be determined experimentally.