| Source: |
Purified from E. coli containing a recombinant plasmid harboring the E. coli nth gene. |
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| Unit Definition: |
The amount of enzyme required to cleave 1 pmole of a 32P-labeled oligonucleotide probe containing an AP site within an oligonucleotide duplex in one hour at 37°C.
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| Assay Conditions: |
1X REC™ Buffer 4 (10 mM HEPES-KOH
(pH 7.4), 100 mM KCl, and 10 mM EDTA), 4 pmoles of AP site oligonucleotide
(Cat # 3851-100-01) labeled with 32P, 4 pmoles of Oligo Complement
B (Cat # 3849-100-02), and serial dilutions of enzyme in a 20
µl reaction volume are incubated for 1 hour at 37°C.
For analysis, 5 µl of REC™ 5X Loading Buffer (10
mM HEPES-KOH (pH 7.4)), 10 mM EDTA, 6 M urea, 50% glycerol,
and 0.2% bromophenol blue) are added, and the tubes are heated
at 95°C for 5 min then fast-cooled to 4°C. Cleavage
products are resolved by 20% denaturing polyacrylamide gel electrophoresis.
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| Applications: |
CometAssayTM/FLARETM
Supercoiled DNA relaxation assay
Alkaline elution assay
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| Storage: |
Freeze in working aliquots at -20°C in a manual defrost freezer to avoid repeated freeze-thawing. The enzyme is stored in 10 mM HEPES-KOH (pH 7.4), 100 mM KCl, 0.1 mg/ml BSA, and 50% glycerol. |
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| Substrate Specificity: |
Endonuclease III catalyzes the excision of the following forms of DNA damage:
Cis- and trans-thymine glycol, 5,6-dihydrothymine, 5,6-dihydroxydihydrothymine, urea 5-hydroxy-5-methylhydantoin, methyltartronylurea, 6-hydroxy-5,6-dihydropyrimidines, 5-hydroxycytosine and 5-hydroxyuracil, 5-hydroxy-6-hydrothymine, 5,6-dihydrouracil alloxan, uracil glycol, 5-hydroxy-6-hydrouracil, AP sites
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