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  • Endonuclease V

    Description

    Endonuclease V is a repair enzyme found in E. coli that recognizes deoxyinosine, a deamination product of deoxyadenosine in DNA. Endonuclease V, often called deoxyinosine 3´ endonuclease, recognizes DNA containing deoxyinosines (paired or not) on double-stranded DNA, single-stranded DNA with deoxyinosines and to a lesser degree, DNA containing abasic sites (ap) or urea, base mismatches, insertion/deletion mismatches, hairpin or unpaired loops, flaps and pseudo-Y structures. It is believed that Endonuclease V needs another protein to repair the DNA, as it does not remove the deoxyinsoine or the damaged bases (1,2,3).

    Endonuclease V cleaves the second and third phosphodiester bonds 3´ to the mismatch of deoxyinosine with a 95% efficiency for the second bond and a 5% efficiency for the third bond (2), leaving a nick with 3´-hydroxyl and 5´-phosphate (4).

    Figure 1: 5´ fluorescently labeled oligonucleotides contain a 6-carbon phosphodiester spacer that have a structure that may be similar to a urea moiety. Removal of the 5´ fluorescent label from an oligonucleotide by Endo V has been observed (6).

    Highlights

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

    Product Source

    An E. coli strain containing a gene fusion of the Endo V gene and the gene coding for the maltose binding protein (MBP). The fusion protein is purified to near homogeneity and is active as a fusion. The protein contains 223 amino acids and has a molecular weight of 24.9 kDa (5).

    Reagents Supplied

    The following reagents are supplied with this product:

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

    Advantages and Features

    Applications

    • Mismatch Cleavage
    • Cleavage of oligonucleotides containing deoxyinosines and a weaker affinity for oligonucleotides containing base mismatches (5)

    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 deoxyinosine site* in a total reaction volume of 10 μl in 1 hour at 37°C.

    * A deoxyinosine site is synthetically prepared with a dl in the middle of one strand of a 34 mer oligonucleotide duplex.

    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
    300 mM NaCl
    1 mM DTT
    1 mM EDTA
    50% Glycerol
    0.15% Triton® X-100
    pH 8.0 @ 25°C

    Heat Inactivation

    65°C for 20 min

    Unit Assay Conditions

    1X NEBuffer 4 containing 10 pmol of fluorescently labeled oligonucleotide duplex in a total 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.
    • 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.

    References

    1. Yao, M. and Kow, Y.W. (1996). J. Biol. Chem. 271, 30672-30673.
    2. Yao, M. and Kow, Y.W. (1995). J. Biol. Chem.. 270, 28609-28616.
    3. Yao, M. et. al. (1994). J. Biol. Chem.. 269, 31390-31396.
    4. He, B., Qing, H. and Kow, Y.W. (2000). Mutat. Res.. 459, 109-114.
    5. Yao, M. and Kow, Y.W. (1994). J. Biol. Chem.. 269, 31390-31396.
    6. Marks, K. and Landry D., New England Biolabs, Inc. 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 molecular weight of Endonuclease V?
    2. What substrate is used to test Endonuclease V activity?
    3. What is the activity of Endonuclease V in the different NEBuffers?
    4. How well does Endonuclease V cleave at U:A sites?
    5. Can Endonuclease V cleave the loop in a stem-loop DNA structure?
    6. Can a nick generated by Endonuclease V activity be repaired with DNA ligase?
    7. Can Endonuclease V recognize and nick mismatches?

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