E. coli Endonuclease VIII

Endonuclease VIII from E. coli acts as both an N-glycosylase and an AP-lyase. The N-glycosylase activity releases damaged pyrimidines from double-stranded DNA, generating an apurinic (AP site). The AP-lyase activity cleaves 3´ and 5´ to the AP site leaving a 5´ phosphate and a 3´ phosphate. Damaged bases recognized and removed by Endonuclease VIII include urea, 5, 6- dihydroxythymine, thymine glycol, 5-hydroxy-5- methylhydanton, uracil glycol, 6-hydroxy-5, 6-dihydrothymine and methyltartronylurea (1,2). While Endonuclease VIII is similar to Endonuclease III, Endonuclease VIII has β and δ lyase activity while Endonuclease III has β lyase activity.

• Single cell gel electrophoresis (Comet assay) (3,4,5)
• Alkaline elution (6)
• Alkaline unwinding (7)

One unit is defined as the amount of enzyme required to cleave 1 pmol of a 34 mer oligonucleotide duplex containing a single AP site* in a total reaction volume of 10 µl in 1 hour at 37°C in 1X Endonuclease VIII Reaction Buffer containing 10 pmol of fluorescently labeled oligonucleotide duplex.

*An AP site is created by treating 10 pmol of a 34 mer oligonucleotide duplex containing a single uracil residue with 1 unit of Uracil-DNA Glycosylase (UDG) for 2 minutes at 37°C.

• Endonuclease VIII: 10,000 U/ml in 10 mM Tris-HCl, pH 8.0 @ 25°C, 250 mM NaCl, 0.1 mM EDTA, 50% glycerol
• 10X Reaction Buffer: 100 mM Tris-HCl, pH 8.0 @ 25°C, 750 mM NaCl, 10 mM EDTA

-20 °C

1. Dizdaroglu, M., Laval, J. and Boiteux, S. (1993). Biochemistry . 32, 12105-12111.
2. Hatahet, Z. et al. (1994). J. Biol. Chem.. 269, 18814-18820.
3. Singh, N. et al. (1988). Exp. Cell. Res. . 175, 184-191.
4. Collins, A. et al. (1993). Carcinogenesis . 14, 1733-1735.
5. Collins, A. et al. (1996). Environ. Health Perspect . 104, 465-469.
6. Pflaum, M. et al. (1998). Free Rad. Res. . 29, 585-594.
7. Hartwig, A. et al. (1996). Toxicol. Lett.. 88, 85-90.
8. Marks, K. unpublished observation.