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Endonuclease IV, E.coli (Endo IV)

Recombinant enzyme Thermal inactivation at 80°C in 15 min

Enzyme acting on oxidative damaged DNA which recognizes apurinic/apyrimidinic (AP) sites of dsDNA and cleaves the phosphodiester bond 5' to the lesion generating a hydroxyl group at the 3'-terminus.
  
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Thermo Scientific Endonuclease IV (Endo IV) recognizes apurinic/apyrimidinic (AP) sites of dsDNA and cleaves the phosphodiester bond 5' to the lesion generating a hydroxyl group at the 3'-terminus (see Figure 1 in Supporting Data). The enzyme can also act as a 3'-diesterase that is able to release 3'-phosphoglycolate or 3'-phosphate from the damaged ends of dsDNA (see Reference 1). Endo IV possesses also a 3'=>5' exonuclease activity. Its progression on substrates is sensitive to ionic strength, metal ions, EDTA, and reducing conditions. Substrates with 3'-recessed ends are preferred substrates for the 3'=>5' exonuclease activity (see​ Reference 2).

The enzyme has no requirement for Mg2+ and is fully active in the presence of EDTA in moderate concentrations.

Applications

  • Studies of DNA damage and repair (see​ References 3, 4, 5)
  • Single cell electrophoresis (comet assay) (see​ Reference 6)
  • Antitumor drug research (see​ Reference 4)
  • DNA structure research (see​ References 5, 7)
  • SNP analysis (see​ Reference 8)
  
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.
Concentration2 U/µL
Definition of Activity Unit
  • One unit of the enzyme relaxes 1 µg of partially depurinated, covalently closed supercoiled plasmid DNA in 30 min at 37°C.
  • Enzyme activity is assayed in the following mixture: 50 mM Tris-acetate (pH 7.5), 50 mM KCl, 1 mM EDTA, 0.05% (v/v) Triton X-100, and 2 µg of partially depurinated pUC19 DNA.
InactivationInactivated by heating at 80°C for 15 min.
InhibitionInhibitors: although the enzyme is fairly resistant to EDTA during reaction, it becomes sensitive to even submillimolar quantities of chelators when no DNA substrate is present.
Molecular Weight31.6 kDa monomer
Quality ControlThe absence of other endo-, exodeoxyribonucleases, and ribonucleases confirmed by appropriate quality tests.
SourceE. coli cells with a cloned nfo gene
Storage BufferThe enzyme is supplied in:
50 mM Tris-acetate (pH 7.7), 50 mM KCl, 1 mM DTT, 0.05% (v/v) Triton X-100, and 50% (v/v) glycerol.
Endonuclease IV activity

Endonuclease IV activity

Endonuclease IV activity


References

  1. J. D. Levin, Homogeneous Escherichia coli endonuclease IV. Characterization of an enzyme that recognizes oxidative damage in DNA. J. Biol. Chem. 263, 8066-8071 (1988).
  2. S. M. Kerins, et al., Characterization of an endonuclease IV 3’-5’ exonuclease activity. J. Biol Chem. 278(5), 3048-3054 (31 January 2003).
  3. B. Demple, L. Harrison, Repair of oxidative damage to DNA: enzymology and biology. Annu. Rev. Biochem. 63, 915-948 (1994).
  4. J. D. Levin, B. Demple, In vitro detection of endonuclease IV – specific DNA damage formed by bleomycin in vivo. Nucleic Acids Res. 24, 885-889 (1996).
  5. J. N. Patro, et al., Probing the configurations of formamidopyrimidine lesions Fapy.dA and Fapy.dG in DNA using endonuclease IV. Biochemistry. 43, 13397-13403 (2004).
  6. A. R. Collins, The comet assay for DNA damage and repair: principles, applications, and limitations.Molec. Biotechnol. 26, 249-261 (2004).
  7. D.J. Hosfield et al., Structure of the DNA repair enzyme endonuclease IV and its DNA complex: double-nucleotide flipping at abasic sites and three-metal-ion catalysis. Cell. 98, 397-408 (1999).
  8. I. V. Kutyavin et al., A novel endonuclease IV post-PCR genotyping system. Nucleic Acids Res. 29, 1-9 (2006).

Protocols and Product Manuals

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