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  • hOGG1

    Description

    hOGG1 (α isoform) is an 8-oxoguanine DNA glycosylase which acts both as a N-glycosylase and an AP-lyase. The N-glycosylase activity releases damaged purines from double stranded DNA, generating an apurinic (AP) site. The AP-lyase activity cleaves 3´ to the AP site leaving a 5´ phosphate and a 3´-phospho-α, β-unsaturated aldehyde.

    Some of the damaged bases recognized and removed by hOGG1 include 7, 8-dihydro-8-oxoguanine (8-oxoguanine) when base paired with cytosine, 8-oxoadenine when base paired with cytosine, foramidopyrimidine (fapy)-guanine and methy-fapy-guanine (1,2).


    Highlights

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

    Product Source

    An E. coli strain that carries the cloned human ogg1 gene (3).

    Reagents Supplied

    The following reagents are supplied with this product:

    Store at (°C)Concentration
    NEBuffer 2-2010X
    BSA-2010 mg/ml

    Advantages and Features

    Applications

    • Single cell gel electrophoresis (Comet assay) (4,5,6)
    • Alkaline elution (7)
    • Alkaline unwinding (8)

    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 8-oxoguanine base paired with a cytosine in 10 µl of 1X NEBuffer 2 containing 10 pmol of substrate, supplemented with 100 µg/ml BSA in 1 hour at 37°C.

    Reaction Conditions

    1X NEBuffer 2
    Supplement with 100 μg/ml BSA
    Incubate at 37°C

    1X NEBuffer 2:
    50 mM NaCl
    10 mM Tris-HCl
    10 mM MgCl2
    1 mM DTT
    pH 7.9 @ 25°C

    Dilution for Comet Assay

    1:102 to 1:103 (4,5,6,9). For a protocol please visit: http://cometassay.com

    Storage Temperature

    -20°C

    Storage Conditions

    20 mM Tris-HCl
    50 mM NaCl
    1 mM EDTA
    200 μg/ml BSA
    50% Glycerol
    pH 8.0 @ 25°C

    Heat Inactivation

    65°C for 15 min

    Unit Assay Conditions

    1X NEBuffer 2 containing 10 pmol of fluorescently labeled oligonucleotide duplex, supplemented with 100 µg/ml BSA.

    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, DNA):
      The DNA is tested in a reaction under standard reaction conditions. After incubation for 16 hours there is no detectable degradation of the DNA 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.

    References

    1. Bjoras, M. et al. (1997). Opposite base-dependent reactions of a human base excision repair enzyme on DNA containing 7, 8-dihydro-8-oxoguanine and abasic sites. EMBO J. . 16, 6314-6322.
    2. Boiteux, S. and Radicella, J. (1999). Base excision repair of 8-hydroxyguanine protects DNA from endogenous oxidative stress. Biochimie . 81, 59-67.
    3. Radicella, J., Dherin, C., Desmze, C., Fox, M. and Boiteux, S. (1997). Cloning and characterization of <em>hOGG1</em>, a human homolog of the <em>OGG1</em> gene of <em>Saccharomyces cerevisiae</em>. Proc. Natl. Acad. Sci USA. 94, 8018-8015.
    4. Singh, N., McCoy, M., Tice, R. and Schneider, L. (1988). A simple technique for quantitation of low levels of DNA damage in individual cells. Exp. Cell. Res. 175, 184-191.
    5. Collins, A., Duthie, S. and Dobson, V. (1993). Direct enzymatic detection of endogenous oxidative base damage in human lymphocyte DNA. Carcinogenesis . 14, 1733-1735.
    6. Collins, A., Dusinska, M., Gedik, C. and Stetina, R. (1996). Oxidative damage to DNA: do we have a reliable biomarker?. Environ. Health Perspect. . 104, 465-469.
    7. Pflaum, M., Will, O., Mahler, H.-C. and Epe, B. (1998). DNA oxidation products determined with repair endonucleases in mammalian cells: types, basal levels and influence of cell proliferation. Free Rad. Res. 29, 585-594.
    8. Hartwig, A., Dally, H. and Schlepegrell, R. (1996). Sensitive analysis of oxidative DNA damage in mammalian cells: use of the bacterial Fpg protein in combination with alkaline unwinding. Toxicol. Lett. . 88, 85-90.
    9. Gutherie, E. 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 difference between hOGG1 and FPG?
    2. What is the activity of hOGG1 in the NEBuffers?
    3. What substrate is used to test hOGG1?
    4. What is the molecular weight of hOGG1?
    5. Is hOGG1 a tagged protein?
    1. Comet Assay - Modified for Detection of Oxidized Bases Using the Repair Endonucleases Fpg, hOGG1 and Endonuclease III (Nth)

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