• My NEB
  • Print
  • PDF
  • Endonuclease VIII

    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- methylhydanton, uracil glycol, 6-hydroxy-5, 6-dihydrothymine and methyltartronylurea (1,2). While Endonuclease VIII is similar to Endonuclease III, Endonuclease VIII has β and δ lyase activity while Endonuclease III has β lyase activity.


    Highlights

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

    Product Source

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

    Reagents Supplied

    The following reagents are supplied with this product:

    Store at (°C)Concentration
    Endonuclease VIII Reaction Buffer10X

    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 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.

    Reaction Conditions

    1X Endonuclease VIII Reaction Buffer
    Incubate at 37°C

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

    Storage Temperature

    -20°C

    Storage Conditions

    10 mM Tris-HCl
    250 mM NaCl
    0.1 mM EDTA
    50% Glycerol
    pH 8.0 @ 25°C

    Heat Inactivation

    75°C for 10 min

    Unit Assay Conditions

    1X Endonclease VIII Reaction Buffer containing 10 pmol of fluorescently labeled oligonucleotide duplex in a total reaction volume of 10 μl.

    Quality Control

    Quality Assurance Statement

    • Purified free of contaminating endonucleases and exonucleases.

    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.
    • 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.
    • Protein Purity (SDS-PAGE):
      The physical purity is assessed by comparing contaminating protein bands in a concentrated sample to the protein of interest band in a sample of known dilution. The purity is determined by SDS-PAGE.

    Notes

    1. Recommended Dilution for Comet Assay: 1:104 to 1:105 (3,4,5,8). For a protocol please visit: http://cometassay.com

    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.
    8. Marks, K. unpublished observation.

    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 VIII in NEBUffers?
    2. Does Endonuclease VIII cleave RNA?
    3. Will Endonuclease VIII be able to cleave at abasic sites that are located in short (1-5nt) single stranded 5' and 3' overhangs in DNA?
    4. Does treatment of DNA with Endonuclease VIII leave a 5' phosphate?
    5. What damaged bases does Endonuclease VIII recognize?

    Selection Tools

    Usage Guidelines & Tips