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Exonuclease I (Exo I)

Recombinant enzyme Thermal inactivation at 80°C in 15 min

Exonuclease I (ExoI) degrades single-stranded DNA in a 3'→5' direction, releasing deoxyribonucleoside 5'-monophosphates in a stepwise manner and leaving 5'-terminal dinucleotides intact.
  
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Thermo Scientific Exonuclease I (ExoI) degrades single-stranded DNA in a 3'=>5' direction. It releases deoxyribonucleoside 5'-monophosphates in a stepwise manner and leaves 5'-terminal dinucleotides intact.

It does not cleave DNA strands with terminal 3'-OH groups blocked by phosphoryl or acetyl groups (see Reference 1).

Highlights

  • Active in PCR buffers

Applications

  • Primer removal from PCR mixtures:
    • prior to PCR product sequencing (see Reference 2)
    • for one-tube "megaprimer" PCR mutagenesis (see​ Reference 3)
  • Removal of single-stranded DNA containing a 3'-hydroxyl terminus from nucleic acid mixtures
  • Assay for the presence of single-stranded DNA with a 3'-hydroxyl terminus (see​ Reference 4)

Note

The enzyme is not suitable for removing 3'-overhangs of dsDNA.

  
Storage Condition-20 C
HazardousNo
10X Reaction Buffer500 mM Tris-acetate (pH 7.5), 500 mM KCl, 10 mM EDTA, 0.5% (v/v) Triton X-100.
Definition of Activity Unit
  • One unit of the enzyme catalyzes the release of 10 nmol of acid soluble nucleotides in 30 min at 37°C.
  • Enzyme activity is assayed in the following mixture: 67 mM glycine-KOH (pH 9.5), 6.7 mM MgCl2, 1 mM DTT, and 0.17 mg/mL single-stranded [3H]-DNA.
InactivationInactivated by heating in boiling water bath for 10 min, preferably in the presence of EDTA.
InhibitionInhibitors: no specific inhibitors have been described.
Molecular Weight54.5 kDa monomer.
Quality ControlThe absence of other endo-, exodeoxyribonucleases, and ribonucleases confirmed by appropriate quality tests.
SourceE. coli cells with a cloned E. coli sbcB gene.
Storage BufferThe enzyme is supplied in:
20 mM Tris-HCl (pH 7.5), 0.1 mM EDTA, 1 mM DTT and 50% (v/v) glycerol.
Exonuclease I activity

Exonuclease I activity

Exonuclease I activity


References

  1. I. R. Lehman, A. L. Nussbaum, The deoxyribonucleases of Escherichia coli. V. On the specificity of exonuclease I (phosphodiesterase). J. Biol. Chem. 239, 2628-2636 (1964).
  2. E. Werle et al., Convenient single-step, one tube purification of PCR products for direct sequencing. Nucleic Acids Res. 22, 4354-4355 (1994).
  3. S. Nabavi, R. N. Nazar, Simplified one-tube “megaprimer” polymerase chain reaction mutagenesis. Anal. Biochem. 2, 346-348 (2005).
  4. J. Rosamond et al., Modulation of the action of the recBC enzyme of Escherichia coli K-12 by Ca2+. J. Biol. Chem. 254, 8646-8652 (1979).
  5. Y. Sasaki, D. Miyoshi, N.Sugimoto, Regulation of DNA nucleases by molecular crowding. Nucleic Acids Res. 35, 4086-4093 (2007).