Exonuclease III (50000 U)

From QIAGEN, this enzyme is a 3′→5′ exonuclease used for directional digestion of nucleic acids and analysis of DNA-protein interactions. It digests one strand of a dsDNA duplex at a time or the RNA strand of an RNA-DNA heteroduplex, removing a limited number of nucleotides per binding event and producing coordinated progressive deletions within the population of DNA molecules. The enzyme removes 3′ terminal groups on dsDNA, efficiently degrades 3′ recessed ends (creating 5′ overhangs) but not 3′ protruding ends, breaks phosphodiester bonds on the 5′ side of AP sites in both dsDNA and ssDNA, and increases turnover of the DNA glycosylase MutY.

The 3′→5′ exodeoxyribonuclease activity is specific for double-stranded DNA, degrading 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. The enzyme 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, and is not active on phosphorothioate-linked nucleotides. The enzyme also exhibits RNase H activity, exonucleolytically degrading the RNA strand in RNA-DNA hybrids, and 3′-phosphatase activity that removes the 3′-terminal phosphate, generating a 3′-OH group. It can be inactivated by heating at 65°C for 5 minutes.

Quality specifications include purity >99%, specific activity 100,000 U/mg, double-stranded endonuclease activity at 1000 U showing no conversion, E. coli DNA contamination at <10 copies per 1000 U, and UDG activity at <20 U/mL. The enzyme is supplied in 25 mM Tris-HCl, 50 mM KCl, 1 mM DTT, 0.1 mM EDTA, 50% glycerol; pH 8.0 at 25°C, together with 10X Yellow Buffer containing 100 mM Bis-Tris-Propane-HCl, 100 mM MgCl2, 10 mM DTT; pH 7.0 at 25°C. According to the third-party listing, the activity is 50000 U per vial with typical uses including deletion construction, end polishing, DNA trimming, deletion mapping, generation of blunt ends and controlled excision in cloning workflows; storage is at -20°C with minimisation of freeze–thaw cycles.