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  • 5' Deadenylase

    Description

    Yeast 5´ Deadenylase is a member of the HIT (histidine triad) family proteins and specifically a member of the Hint branch. It is the yeast orthologue of aprataxin. Mutations in human aprataxin have been known to be involved in the neurological disorder known as ataxia oculomotor apraxia-1 (1). The human protein has been shown to resolve abortive ligation intermediates by removing the AMP at the 5´ end of DNA (AMP-DNA hydrolase activity) (2). It also repairs DNA damage at 3´ ends by removing 3´-phosphate and 3´-phosphoglycolate (3). Human aprataxin acts on small molecules as well, nucleotide polyphosphates such as diadenosine tetraphosphate (AppppA) as well as lysyl-AMP. 

    The 5´ Deadenylase is encoded by HNT3 gene of S. cerevisiae. NEB has shown this protein is capable of deadenylation from 5´ end of DNA and RNA, leaving the phosphate at 5´ end. It also cleaves AppppA into ATP and AMP. Its activity on lysyl-AMP is not detectable.

    Product Source

    Purified from an E. coli strain carrying a plasmid encoding 5´ Deadenylase from S. cerevisiae.

    Reagents Supplied

    The following reagents are supplied with this product:

    Store at (°C)Concentration
    NEBuffer 2-2010X

    Properties and Usage

    Unit Definition

    One unit is defined as the amount enzyme required to remove 10 pmoles of AMP from a 5'-adenylated DNA oligo in 10 minutes at 30°C.

    Reaction Conditions

    1X NEBuffer 2
    Incubate at 30°C

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

    Storage Temperature

    -20°C

    Storage Conditions

    10 mM Tris-HCl
    500 mM NaCl
    2 mM DTT
    0.1 mM EDTA
    50% Glycerol
    0.1% Triton® X-100
    pH 7.5 @ 25°C

    Heat Inactivation

    70°C for 20 min

    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.
    • Phosphatase Activity (Oligo):
      The product is tested in a reaction containing a 5'-phosphorylated oligo. After incubation there is no detectable dephosphorylation as determined by SDS-PAGE
    • RNase Activity (1 Hour Digestion):
      The product is tested in a reaction containing a RNA substrate.  After incubation for 1 hour there is no detectable degradation of the RNA substrate as determined by gel electrophoresis.

    Notes

    1. 5'-adenylated DNA could be dsDNA, ssDNA and nicked DNA with the adenylate residue at the 5' terminus of the nick. Deadenylation is more efficient with dsDNA and nicked DNA.

    References

    1. Le Ber, I. et al. (2003). Brain. 126, 2761.
    2. Ahel, I. et al. (2006). Nature. 443, 713.
    3. Takahashi, T. et al. (2007). Nucl. Acids Res. 35, 3797.

    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 5’ Deadenylase?
    2. Is 5’ Deadenylase a tagged protein?
    3. Can 5’ Deadenylase deadenylate ssDNA?
    1. A Typical Deadenylation Reaction (M0331)