Endonuclease Vlll

Description:
E.coli Endonuclease VIII functions as both an N-glycosylase (by excising oxidative base lesions) and an AP lyase (by subsequently cleaving the phosphodiester backbone), leaving terminal phosphates at the 5′ and 3′ ends.⁽¹⁾  Damaged bases removed by Endonuclease VIII include: urea, 5, 6- dihydroxythymine, thymine glycol, 5-hydroxy-5- methylhydanton, uracil glycol, 6-hydroxy-5, 6-dihydrothymine and methyltartronylurea⁽²⁾.

Specific Activity:
770,000U/mg

Applications:

• Single cell gel electrophoresis (Comet assay)
• Alkaline elution
• Alkaline unwinding

Source:
An E. coli strain which carries the cloned Endonuclease VIII gene.

Supplied in:
10 mM Tris-HCl
250 mM NaCl
0.1 mM EDTA
50% glycerol
pH 8.0 @ 25°C

Supplied with:
10X Endonulease Vlll Buffer

10X Endonuclease Vlll Buffer
100 mM Tris-HCl
750 mM NaCl
10 mM EDTA
pH 8.0 @ 25°C

Unit Definition:
One unit is defined as the amount of enzyme required to cleave 1 pmol of an oligonucleotide duplex containing a single AP site in 1 hour at 37°C.

Nuclease Contamination Tests

Single-Stranded Exonuclease Activity:
A 50 µL reaction containing 15,000 cpm of a radiolabeled single-stranded DNA substrate and 10 µL of enzyme solution incubated for 4 hours at 37°C resulted in less than 0.1% release of TCA-soluble counts.

Double-Stranded Exonuclease Activity:
A 50 µL reaction containing 15,000 cpm of a radiolabeled double-stranded DNA substrate and 10 µL of enzyme solution incubated for 4 hours at 37°C resulted in less than 0.1% release of TCA-soluble counts.

Endonuclease Activity:
A 50 µL reaction containing 1 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 heat denatured and screened in a TaqMan qPCR assay for the presence of contaminating E.coli genomic DNA using primers for the 16S rRNA locus. Based on no template control Ct values, the detection limit of this assay is <10 copies genome/sample.

Quality Control Analysis

Unit Characterization Assay:
Specific activity was measured using a 2-fold serial dilution method. Dilutions of enzyme batch were made in Endo VIII glycerol storage solution and added to 10 µL reactions containing 2.0 µM of an FAM-labeled, 34-base, duplex oligonucleotide, containing a single Uracil. [Note: substrate pre-treated for 2 minutes with 1 unit of UDG to create an abasic site] Reactions were incubated 15 minutes at 37ºC, plunged on ice, denatured with N-N-dimethylformamide and analyzed by running and exposing to short-wave UV a 15% TBE-Urea acrylamide gel.

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. Dizdaroglu, M., et al., (1993) Biochemistry, 32, 12105-12111.
2. Hatahet, Z. et al. (1994) J. Biol. Chem., 269, 18814-18820.