Description
E.coli Endonuclease VIII functions as both an N-glycosylase by excising oxidative base lesions and generating an AP site, and AP-lyase, by subsequently cleaving the phosphodiester backbone and 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-dihydrymine and methyltartronylurea (2). This enzyme is supplied in 10 mM Tris-HCl, 250 mM NaCl, 0.1 mM EDTA and 50% glycerol; pH 8.0 at 25°C. At a temperature of 25°C and pH 8.0, the 10x buffer for Endonuclease Vlll comprises 100 mM Tris-HCl, 750 mM NaCl, and 10 mM EDTA.
Storage temperature: –25°C to –15°C Molecular weight: 29,845 Daltons Test Amount tested Specification Purity n/a >99% Specific activity n/a 770,000 U/mg Single-stranded exonuclease 10 U <1% released Double-stranded endonuclease 100 U <1% released E. coli DNA contamination 100 U <10 copies
The recombinant enzyme protein is produced by an E. coli strain which carries the cloned Endonuclease VIII gene. 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.
Usage Instructions Endonuclease VIII Reaction Buffer at 1x is recommended, but this enzyme is active in most molecular biology reaction buffers. Add 0.5 µL Endonuclease VIII to a reaction contains DNA substrate. Incubate at 37°C for 30 minutes. Heat Inactivation can be carried out at 75°C for 10 minutes. Note: Protocol can be modified for application specific usage. Quality Control Specific activity of Endonuclease VIII was measured using a two-fold serial dilution method. Dilutions of the enzyme were prepared in the enzyme’s glycerol storage solution and added to 10 µL reactions containing 2.0 µM of a FAM-labeled, 34-base, duplex oligonucleotide containing a single uracil. Note that the substrate was pre-treated for 2 minutes with one unit of UDG to create an abasic site. Reactions were incubated for 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 band's mass 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.
