Exonuclease III

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.