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  • APE 1

    Description

    Human apurinic/apyrimidinic (AP) endonuclease, APE 1, also known as HAP 1 or Ref-1, shares homology with Escherichia coli exonuclease III protein. APE 1 cleaves the phosphodiester backbone immediately 5´ to an AP site, via hydrolytic mechanism, to generate a single-strand DNA break leaving a 3´-hydroxyl and 5´-deoxyribose phosphate terminus. Besides AP endonuclease activity, APE 1 has also been reported to have weak DNA 3´ -diesterase, 3´ to 5´ exonuclease and RNase H activities (3-5).

    In addition to DNA repair activity, APE 1 is also capable of regulating the DNA binding activity of many transcription factors in vitro by a redox mechanism (Ref-1). As part of this process, APE 1 has been shown to stimulate the DNA binding activity of Fos-Jun heterodimers, Jun-Jun homodimers and Hela cell AP-1 proteins as well as that of several other transcription factors including NF-kB, Myb and members of the ATF/CREB family (7-9).


    Highlights

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

    Product Source

    An E. coli strain which carries the cloned human APE 1 gene.

    Reagents Supplied

    The following reagents are supplied with this product:

    Store at (°C)Concentration
    NEBuffer 4-2010X

    Advantages and Features

    Applications

    • Single cell gel electrophoresis (Comet assay)
    • Alkaline elution
    • Alkaline unwinding
    • Modified nick translation

    Properties and Usage

    Unit Definition

    One unit is defined as the amount of enzyme required to cleave 20 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.

    *An AP site is created by treating 20 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 NEBuffer 4
    Incubate at 37°C

    1X NEBuffer 4:
    50 mM Potassium Acetate
    20 mM Tris-acetate
    10 mM Magnesium Acetate
    1 mM DTT
    pH 7.9 @ 25°C

    Storage Temperature

    -20°C

    Storage Conditions

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

    Heat Inactivation

    65°C for 20 min

    Unit Assay Conditions

    1X NEBuffer 4 containing 20 pmol of fluorescently labled oligonucleotide duplex 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.
    • 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 the Comet Assay: 1:103. For a protocol please visit: http://cometassay.com

    References

    1. Robson, C.N. and Hickson, D.I. (1991). Nucl. Acids Res.. 19, 5519-5523.
    2. Demple, B. et al. (1991). Proc. Natl. Acad. Sci. USA. 88, 11450-11454.
    3. Barzilay, G. et al. (1995). Nucl. Acids Res.. 23, 1544-1550.
    4. Barzilay, G. et al. (1995). Nature Struc. Biol.. 2, 451-468.
    5. Gorman, M.A. et al (1997). EMBO J.. 16, 6548-6558.
    6. Xanthoudakis, S. et al. (1992). EMBO J.. 11, 3323-3335.
    7. Walker, L.J. et al. (1993). Mol. Cell. Biol.. 13, 5370-5376.
    8. Vidal, A.E. (2001). EMBO J.. 20, 6530-6539.
    9. Wilson, D.M. III et al. (1995). J. Biol. Chem.. 270, 16002-16007.
    10. Flaherty, D.M. (2001). Am. J. Respir. Cell. Mol. Biol.. 25, 664-667.

    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 APE 1 in NEBuffers 1-4?
    2. What is the molecular weight of APE 1?
    3. Is APE 1 a tagged protein?
    4. What substrate is used to test APE 1 activity?

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