Exonuclease III, E. Coli

Description:
Exonuclease III is a 3′→ 5′ exonuclease which acts by digesting one strand of a dsDNA duplex at a time or digesting the RNA strand of an RNA-DNA heteroduplex^(1,2). Exonuclease III breaks phosphodiester bonds on the 5′ side of AP sites in both dsDNA and ssDNA⁽³⁾, removes 3′ terminal groups on dsDNA⁽³⁾, increases MutY turnover⁽⁴⁾, and efficiently degrades 3′ recessed but not 3′ protruding DNA ends (creating 5′ overhangs)⁽⁵⁾. Exo III removes a limited number of nucleotides per binding event, resulting in coordinated progressive deletions within the opulation of DNA molecules⁽¹⁾.

Specific Activity:
150,000U/mg

Applications:

• degrades excess single-stranded primer oligonucleotide from a reaction mixture containing double-stranded extension products

Source:
Purified from a strain of E. coli that expresses the recombinant Exonuclease III gene.

Supplied in:
25 mM Tris-HCl
50 mM KCl
1.0 mM DTT
0.1% MM EDTA
50% Glycerol
pH 8.0 @ 25°C

Supplied with:
10X Yellow Buffer

10X Yellow Buffer
100 mM Bis-Tris-Propane
100 mM MgCl2
10 mM dithiothreitol
pH 7.0 @ 25°C

Nuclease Contamination Tests

Endonuclease Activity:
A 50 µL reaction containing 0.5 µg of pBR322 DNA and 10 µL of enzyme solution incubated for 4 hours at 37°C resulted in no visually discernible conversion to nicked circular DNA as determined by agarose gel electrophoresis.

E.coli 16S rDNA Contamination Test:
Replicate 5 µL samples of enzyme solution were 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. The acceptance criterion for the test is the threshold cycle count (Ct) produced by the average of 3 replicate no template control samples. Based on the correlation between the no template control Ct values, and standard curve data, the detection limit of this assay is <10 copies genome/sample.

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.

Quality Control Analysis

Unit Characterization Assay:
Unit activity was measured using a 2-fold serial dilution method. Dilutions of enzyme were made in 1X reaction buffer ([ExoIII]f = 0.01-0.00008µg/µL) and added to 50 µL reactions containing a 1.1 kb trititated DNA fragment, and 1X Exo III Reaction Buffer. Reactions were incubated 10 minutes at 37°C, plunged on ice, and analyzed using a TCA-precipitation method.

Protein Concentration (OD280) Measurement:
A 2.0 µL sample of Exonuclease III was analyzed at OD280 using a Nanodrop ND-1000 spectrophotometer standardized using a 2.0 mg/ml BSA sample (Pierce Cat #23209), and blanked with Exonuclease III storage solution. The observed average measurement of 3 replicate samples was converted to mg/mL using an extinction coefficient of 38,690 and molecular weight of 30,969 Daltons. Acceptance for this assay is +/- 5% of reference sample.

SDS-Page (Physical Purity Assessment):
2.0 µL of concentrated enzyme solution was loaded on a denaturing 4-20% Tris-Glycine SDS-PAGE gel flanked by a broad-range MW marker and 2.0 µL of a 1:100 dilution of the sample. Following electrophoresis, the gel was stained and the samples compared to determine physical purity. The acceptance criteria for this test requires that the aggregate mass of contaminant bands in the concentrated sample do not exceed the mass of the protein of interest band in the dilute sample, confirming greater than 99% purity of the concentrated sample.

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 (22), 4572-4576.
3. Doetsch, P. W. (1990) Mutat. Res. 236 (2-3), 173-201.
4. Pope, M. A., et al. (2002) J. Biol. Chem. 277 (25), 22605-22615.
5. Henikoff, S. (1984) Gene 28, 351-359.