Exonuclease III

• Excises bases from duplex DNA in 3ʹ--> 5ʹ direction

Exonuclease III is a 3ʹ→5ʹ exonuclease which acts by digesting one strand of a double-stranded DNA duplex at a time or digesting the RNA strand of an RNA:DNA heteroduplex (1). Exonuclease III breaks phosphodiester bonds on the 5ʹ side of AP sites in both double and single-stranded DNA (3), removes 3ʹ terminal groups on double-stranded DNA (3), increases MutY turnover (4), and efficiently degrades 3ʹ recessed but not 3ʹ protruding DNA ends (creating 5ʹ overhangs) (5). Exo III removes a limited number of nucleotides during each binding event, resulting in coordinated progressive deletions within the population of DNA molecules (1).

This enzme is supplied in 25 mM Tris-HCl, 50 mM KCl, 1.0 mM DTT, 0.1% mM EDTA, and 50% glycerol: pH 8.0 @ 25°C.

10X Yellow Buffer (cat. no. B0130) contains 100 mM Bis-Tris-Propane-HCl, 100 mM MgCl₂, 10 mM DTT; pH 7.0 at 25°C.

SDS available upon request.

Enzyme properties

• Storage temperature: –25°C to –15°C
• Molecular weight: 30,969 Daltons

Source of recombinant enzyme protein
The protein is produced by a strain of E. coli that expresses the recombinant Exonuclease III gene.

Unit definition: One unit is defined as the amount of enzyme required to produce 1 nmol of acid-soluble total nucleotide in 30 minutes at 37°C.

References

1. Linn, S.M. (1982) Nucleases, pp. 291-309, Cold Spring Harbor Laboratory Press.
2. Shida, T., et al. (1996) Nucl. Acids Res. 24:4572.
3. Doetsch, P.W. (1990) Mutat. Res. 236:173.
4. Pope, M.A., et al. (2002) J. Biol. Chem. 277:22605.
5. Henikoff, S. (1984) Gene 28:351.

Quality control analysis

Unit activity was measured using a twofold 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 Exonucleas III Yellow Buffer. Reactions were incubated 10 minutes at 37°C, placed on ice, and analyzed using a TCA-precipitation method.

Protein concentration is determined by OD₂₈₀ absorbance.

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

E. coli contamination is evaluated using 5 µl replicate samples of enzyme solution that are 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.

A 50 µl reaction containing 1 µg of tritiated uracil containing DNA and 10 µl enzyme solution incubated for 40 minutes at 37°C under standard UDG unit characterization conditions resulted in the measurement of less than 20 U/ml UDG activity as determined by liquid scintillation analysis.

This OEM by QIAGEN product is available for bulk purchase for the following commercial assay applications.

• Plasmid prep cleanup
• Generates single-stranded DNA from linear double-stranded DNA
• Removes oligonucleotide primers after PCR