Description
Endonuclease III (Nth) protein from E. coli acts as both N-glycosylase and a AP-lyase. The N-glycosylase activity releases damaged pyrimidines from double-stranded DNA, generating a basic (AP site). The AP-lyase activity of the enzyme cleaves 3´ to the AP site leaving a 5´ phosphate and a 3´ ring opened sugar. Some of the damaged bases recognized and removed by Endouclease III include urea, 5, 6 dihydroxythymine, thymine glycol, 5-hydroxy-5 methylhydanton, uracil glycol, 6-hydroxy-5, 6-dihdrothimine and methyltartronylurea.
Applications
Single cell gel electrophoresis (Comet assay)
Alkaline elution
Alkaline unwinding
Source
E. coli
Purity
> 95 % as determined by SDS-PAGE
Storage
-80 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 III 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.
Reaction Conditions
1X Endonuclease III (Nth) Reaction Buffer
Incubate at 37°C
1X Endonuclease III (Nth) Reaction Buffer
20 mM Tris-HCl
1 mM EDTA
1 mM DTT
(pH 8 @ 25°C)
Usage Concentration
10,000 units/ml
Storage Temperature
-20°C
Storage Conditions
10 mM Tris-HCl, 250 mM NaCl, 1 mM DTT, 0.1 mM EDTA, 200 µg/ml BSA, 50% Glycerol, pH 7.4
Heat Inactivation
65°C for 20 min