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    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 (1,2).

    Highlights

    • Isolated from a recombinant source
    • Supplied with 10X Reaction Buffer

    Product Source

    An E. coli strain which carries the cloned nth gene from Escherichia coli.

    Reagents Supplied

    The following reagents are supplied with this product:

    Store at (°C)Concentration
    Endonuclease III (Nth) Reaction Buffer-2010X

    Advantages and Features

    Applications

    • Single cell gel electrophoresis (Comet assay) (3,4,5)
    • Alkaline elution (6)
    • Alkaline unwinding (7)

    Properties and Usage

    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

    Heat Inactivation

    65°C for 20 min

    Unit Assay Conditions

    1X Endonuclease III (Nth) Reaction Buffer containing 10 pmol of fluorescently labeled oligonucleotide duples in a total reaction volume of 10 µl

    Quality Control

    Quality Control Assays

    The following Quality Control Tests are performed on each new lot and meet the specifications designated for the product. Individual lot data can be found on the Product Summary Sheet/Datacard or Manual which can be found in the Supporting Documents section of this page.
    • Endonuclease Activity (Nicking):
      The product is tested in a reaction containing a supercoiled DNA substrate. After incubation for 4 hours the percent converted to the nicked form is determined by agarose gel electrophoresis.
    • Exonuclease Activity (Radioactivity Release):
      The product is tested in a reaction containing a radiolabeled mixture of single and double-stranded DNA. After incubation for 4 hours the exonuclease activity is determined by the % release of radioactive nucleotides.
    • Non-Specific DNase Activity (16 hour):
      The product is tested for non-specific nuclease degradation in a reaction containing a DNA substrate. After incubation for 16 hours there is no detectable degradation of the DNA substrate as determined by agarose gel electrophoresis.

    References

    1. Dizdaroglu, M., Laval, J. and Boiteux, S. (1993). Biochemistry. 32, 12105-12111.
    2. Hatahet, Z. et al. (1994). J. Biol. Chem.. 269, 18814-18820.
    3. Singh, N. et al. (1988). Exp. Cell Res.. 175, 184-191.
    4. Collins, A. et al. (1993). Carcinogenesis. 14, 1733-1735.
    5. Collins, A. et al. (1996). Environ. Health Perspect . 104, 465-469.
    6. Pflaum, M. et al. (1998). Free Rad. Res.. 29, 585-594.
    7. Hartwig, A. et al. (1996). Toxicol. Lett. 88, 85-90.

    Supporting Documents

    Material Safety Datasheets

    The following is a list of Material Safety Data Sheets (MSDS) that apply to this product to help you use it safely. The following file naming structure is used to name these document files: [Product Name] MSDS. For international versions please contact us at info@neb.com.

    Datacards

    The Product Summary Sheet, or Data Card, includes details for how to use the product, as well as details of its formulation and quality controls. The following file naming structure is used to name the majority of these document files: [Catalog Number]Datasheet-Lot[Lot Number]. For those product lots not listed below, please contact NEB at info@neb.com or fill out the Technical Support Form for appropriate document.
    1. What is the activity of Endonuclease III in the NEBuffer 1-4?
    2. What is the molecular weight of Endonuclease III?
    3. What types of damaged bases are recognized and removed by Endonuclease III?
    4. Is Endonuclease III a tagged protein?
    5. What substrate is used to test Endonuclease III activity?
    1. Comet Assay - Modified for Detection of Oxidized Bases Using the Repair Endonucleases Fpg, hOGG1 and Endonuclease III (Nth)

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