Endonuclease VIII, E. coli

Description
Endonuclease VIII from E. coli acts as both an N-glycosylase and an AP-lyase. The N-glycosylase activity releases damaged pyrimidines from double-stranded DNA, generating an apurinic (AP site). The AP-lyase activity cleaves 3´ and 5´ to the AP site leaving a 5´ phosphate and a 3´ phosphate. Damaged bases recognized and removed by Endonuclease VIII include urea, 5, 6- dihydroxythymine, thymine glycol, 5-hydroxy-5- methylhydantoin, uracil glycol, 6-hydroxy-5, 6-dihydrothymine and methyltartronylurea. While Endonuclease VIII is similar to Endonuclease III, Endonuclease VIII has β and δ lyase activity while Endonuclease III has only β lyase activity.

Applications

Single cell gel electrophoresis (Comet assay)

Alkaline elution

Alkaline unwinding

Source
An E. coli strain which carries the cloned nei gene.

Purity
> 95 % as determined by SDS-PAGE

1X Endonuclease VIII Reaction Buffer
10 mM Tris-HCl, 75 mM NaCl, 1 mM EDTA, pH 8 @ 25°C

Storage Buffer
10 mM Tris-HCl, 250 mM NaCl, 0.1 mM EDTA, 50% Glycerol, 200mg/ml BSA, pH 8 @ 25°C

Heat Inactivation
75°C for 10 min

Storage
-20℃ Avoid repeated freeze-thaw cycles.

Unit Definition
One unit is defined as the amount of enzyme required to cleave 1 pmol of a 34 mer oligonucleotide duplex containing a single AP site* in a total reaction volume of 10 µl in 1 hour at 37°C in 1X Endonuclease VIII Reaction Buffer containing 10 pmol of fluorescently labeled oligonucleotide duplex.

* An AP site is created by treating 10 pmol of a 34 mer oligonucleotide duplex containing a single uracil residue with 1 unit of Uracil-DNA Glycosylase (UDG) for 2 minutes at 37°C.