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). 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 population of DNA molecules (1).
Source of Protein
Purified from a strain of E. coli that expresses the recombinant Exonuclease III gene.
25 mM Tris-HCl
50 mM KCl
1.0 mM DTT
0.1% mM EDTA
pH 8.0 @ 25°C
B0130 (10X Yellow Buffer)
10X Yellow Buffer (B0130)
100 mM Bis-Tris-Propane-HCl
100 mM MgCl2
10 mM DTT
pH 7.0 @ 25°C
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.
Unit Characterization Assay
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 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 280 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.
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.
Double-Stranded 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.
UDG Contamination Test
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.
|Unit Size||50,000 U|
|SDS||Available on request|
|Storage Temperature||-25 to -15°C|
|Specific Activity||100,000 U/mg|
|DS Endonuclease||1000 U = No conversion|
|E.coli DNA Contamination||1000 U <10 copies|
|UDG Activity||< 20U/mL activity|
* For a detailed summary of assay conditions and data, refer to the Quality Controls Analysis section.
Limitations of Use
This product was developed, manufactured, and sold for in vitro use only. The product is not suitable for administration to humans or animals. SDS sheets relevant to this product are available upon request.
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