Exonuclease III

• Exonuclease action excises bases from duplex DNA in 3’→5’ direction
• Multifunctional enzyme with phosphatase, exonuclease, AP endonuclease and RNase H activities

Exonuclease III is a 3′→5′ exonuclease that acts by digesting one strand of a dsDNA duplex at a time or digesting the RNA strand of an RNA-DNA heteroduplex. Exonuclease III removes a limited number of nucleotides per binding event, resulting in coordinated progressive deletions within the population of DNA molecules. Exonuclease III removes 3′ terminal groups on dsDNA, efficiently degrades 3′ recessed but not 3′ protruding DNA ends (creating 5′ overhangs), breaks phosphodiester bonds on the 5′ side of AP sites in both dsDNA and ssDNA and increases turnover of the DNA glycosylase MutY.

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.

The enzyme is supplied with 10X Yellow Buffer (B0130) containing 100 mM Bis-Tris-Propane-HCl, 100 mM MgCl₂, 10 mM DTT; pH 7.0 at 25°C.

The 3'→5' exonuclease can be inactivated by heating at 65°C for 5 minutes.

Most endonucleases and exonucleases have evolved as separate enzymes, but some enzymes have evolved with both activities. E. coli Exonuclease III is a multifunctional nuclease that performs both AP site-specific endonuclease and 3′→5′ exonuclease activities.

The 3'→5' exodeoxyribonuclease activity of Exonuclease III is specific for double-stranded DNA. Exonuclease III 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. The enzyme is not active on phosphorothioate-linked nucleotides.

Exonuclease III RNase H activity exonucleolytically degrades the RNA strand in RNA-DNA hybrids. Exonuclease III 3'-phosphatase activity removes the 3'-terminal phosphate, generating a 3'-OH group.

Instructions for using Exonuclease III are provided in the corresponding kit protocol in the resources below.

Quality Control

Exonuclease III Unit activity was measured using a 2-fold serial dilution method. Dilutions of enzyme were made in 1X reaction buffer and added to 50 μL reactions containing a tritiated DNA fragment and 1X Exo III Yellow Buffer. Reactions were incubated for 10 minutes at 37°C, plunged on ice, and analyzed using a TCA-precipitation method.

Protein concentration of Exonuclease III (OD₂₈₀) was determined by OD₂₈₀ absorbance.

Exonuclease III physical purity was evaluated by SDS-PAGE of concentrated and diluted enzyme solutions followed by silver stain detection. Purity was assessed by comparing the aggregate mass of contaminant bands in the concentrated sample to the mass of the protein of interest band in the diluted sample.

Double-stranded endonuclease activity in Exonuclease III enzyme was determined in a 50 μL reaction containing 0.5 μg of plasmid DNA and 10 μL of enzyme solution incubated for 4 hours at 37°C.

E. coli contamination of Exonuclease III was evaluated using 5 μl replicate samples of enzyme solution denatured and screened in a TaqMan qPCR assay for the presence of contaminating E. coli genomic DNA using oligonucleotide primers corresponding to the 16S rRNA locus.

Uracil DNA glycosylase (UDG) contamination of Exonuclease III was assessed in a 50 μL reaction containing tritiated uracil DNA and 10 μL enzyme solution incubated for 60 minutes at 37°C under standard UDG unit characterization conditions.

This product is available for molecular biology applications such as:

• DNA-protein interaction analysis by Exo III DNA footprinting
• Production of nested deletions in double-stranded DNA
• Enzymatic homology-based DNA assembly via SENAX (Stellar ExoNuclease Assembly mix)
• Cloning of PCR products
• Preparation of single-stranded DNA for dideoxy sequencing
• Site-directed mutagenesis
• Preparation of strand-specific probes