Endonuclease III (Nth)

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