Endonuclease VIII

• Excises damaged pyrimidines from duplex DNA
• Cleaves AP sites leaving terminal phosphates at the 5ʹ and 3ʹ ends

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 5ʹ and 3ʹ 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).

This enzme is supplied in 10 mM Tris-HCl, 250 mM NaCl, 0.1 mM EDTA and 50% glycerol; pH 8.0 at 25°C.

10X Endonuclease Vlll Buffer (cat. no. B9080) contains 100 mM Tris-HCl, 750 mM NaCl and 10 mM EDTA; pH 8.0 at 25°C

SDS available upon request.

Enzyme properties

• Storage temperature: –25°C to –15°C
• Molecular weight: 29,845 Daltons

Source of recombinant enzyme protein
The protein is produced by an E. coli strain which carries the cloned Endonuclease VIII gene.

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.

References

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

Quality control analysis

Specific activity was measured using a twofold serial dilution method. Dilutions of enzyme were prepared 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 that the substrate was pre-treated for 2 minutes with 1 unit of UDG to create an abasic site. Reactions were incubated 60 minutes at 37°C, placed on ice, denatured with N-N-dimethylformamide and analyzed by running and exposing to short-wave UV a 15% TBE-Urea acrylamide gel.

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.

Single-stranded exonuclease is determined in a 50 µl reaction containing a radiolabeled single-stranded DNA substrate and 10 µl of enzyme solution incubated for 4 hours at 37°C.

Double-stranded exonuclease activity is determined in a 50 µl reaction containing a radiolabeled double-stranded DNA substrate and 10 µl of enzyme solution incubated for 4 hours at 37°C.

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.

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

• Excise damaged pyrimidines from duplex DNA
• Cleaves AP sites leaving 3ʹ and 5ʹ phosphates