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  • Exonuclease III (E. coli)


    Catalyzes the stepwise removal of mononucleotides from 3´-hydroxyl termini of duplex DNA (1). A limited number of nucleotides are removed during each binding event, resulting in coordinated progressive deletions within the population of DNA molecules (2).

    The preferred substrates are blunt or recessed 3´-termini, although the enzyme also acts at nicks in duplex DNA to produce single-strand gaps. The enzyme is not active on single-stranded DNA, and thus 3´-protruding termini are resistant to cleavage. The degree of resistance depends on the length of the extension, with extensions 4 bases or longer being essentially resistant to cleavage. This property can be exploited to produce unidirectional deletions from a linear molecule with one resistant (3´-overhang) and one susceptible (blunt or 5´-overhang) terminus (3).

    Exonuclease III activity depends partially on helical structure (4) and displays sequence dependence (C>A=T>G; ref. 5). Temperature, salt concentration and the ratio of enzyme to DNA greatly affect enzyme activity, requiring reaction conditions to be tailored to specific applications. 

    Exonuclease III has also been reported to have RNase H, 3´-phosphatase and AP-endonuclease activities (1).


    • Isolated from a recombinant source
    • 3'->5' exonuclease
    • Produces unidirectional nested deletions
    • Site-directed mutagenesis
    • Supplied with 10X Reaction Buffer

    Product Source

    Purified from E.coli K-12, BE257/pSGR3 strain (kindly supplied by B. Weiss)

    Reagents Supplied

    The following reagents are supplied with this product:

    Store at (°C)Concentration
    NEBuffer 1-2010X

    Advantages and Features


    • Unidirectional nested deletions (3)
    • Site-directed mutagenesis (6)
    • Preparation of strand-specific probes (2)
    • Preparation of single-stranded substrates for dideoxy sequencing (7)

    Properties and Usage

    Unit Definition

    One unit is defined as the amount of enzyme required to produce 1 nmol of acid-soluble total nucleotide in a total reaction volume of 50 μl in 30 minutes at 37°C in 1X NEBuffer 1 with 0.15 mM sonicated duplex [3H]-DNA.

    Reaction Conditions

    1X NEBuffer 1
    Incubate at 37°C

    1X NEBuffer 1:
    10 mM Bis-Tris-Propane-HCl
    10 mM MgCl2
    1 mM DTT
    pH 7 @ 25°C

    Storage Temperature


    Storage Conditions

    5 mM KPO4
    200 mM KCl
    5 mM β-ME
    0.05 mM EDTA
    200 μg/ml BSA
    50% Glycerol
    pH 6.5 @ 25°C

    Heat Inactivation

    70°C for 20 min

    Specific Activity

    150,000 units/mg

    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.


    1. Phosphorothioate linkages are not cleaved by Exonuclease III. Unidirectional deletions can also be created by protecting one terminus by incorporation of α-phosphorothioate-containing nucleotide (8).


    1. Rogers, G.S. and Weiss, B. (1980). L. Grossman and K. Moldave(Ed.), Methods Enzymol.. 65, 201-211. New York: Academic Press.
    2. Sambrook, J., Fritsch, E.F. and Maniatis, T. (1989). Molecular Cloning: A Laboratory Manual (2nd. Ed.). 5.84-5.85.
    3. Henikoff, S. (1984). Gene. 28, 351-359.
    4. Richardson, C.C. et al. (1964). J. Biol. Chem. 239, 251-258.
    5. Linxweiler, W. and Horz, W. (1982). Nucl. Acids Res. 10, 4845-4859.
    6. Vandeyar, M.A. (1988). Gene. 65, 129-133.
    7. Guo, L.H. and Wu, R. (1982). Nucl. Acids Res.. 10, 2065-2084.
    8. Putney, S., Benkovic, S. and Schimmel, P. (1981). Proc. Natl. Acad. Sci. USA. 78, 7350-7354.

    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.


    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. Can Exonuclease I be used with a double stranded exonuclease to clean up plasmid preparations?
    2. How does T5 Exonuclease differ from Exonuclease III (NEB# M0206)?
    3. How Does T7 Exonuclease differ from Exonuclease III (NEB# M0206)?

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