Fpg (formamidopyrimidine [fapy]-DNA glycosylase) (also known as 8-oxoguanine DNA glycosylase), a key enzyme in the DNA base excision repair pathway (BER), catalyzes the excision of a broad spectrum of modified purines such as formamidopyrimidine and 8-oxoguanine. Fpg has both DNA glycosylase activity that removes the mutated base and AP-lyase activity that releases ribose, leaving both 5′-and 3′-phosphorylated ends in the DNA (1, 2). Fpg protein possesses a zinc finger motif at the C-terminus and this region is responsible for the DNA binding and AP-lyase activity. In addition, its N-terminal proline was found to act as a nucleophile to produce a Schiff base intermediate, which is essential for enzyme action (3).
• Single cell gel electrophoresis (Comet assay) (4, 5)
• DNA repair.
• DNA nicking and nick translation.
The physical purity of this enzyme is ≥98% as assessed by SDS-PAGE with Coomassie® blue staining (see figure below)
E. coli BL21 (DE3) strain expressing the cloned E. coli Fpg gene.
• 10x Fpg buffer
• 0.1 mg/ml BSA
1x Fpg reaction buffer
10 mM HEPES-KOH
100 mM KCl
10 mM MgCl2
1 mM DTT
(pH 7.4 @ 25°C)
50 mM Tris-HCl, 50 mM KCl, 1 mM DTT, 0.1 mM EDTA, 50% Glycerol, pH 7.5 @ 25ºC
65°C for 15 min
One unit is defined as the amount of enzyme required to cleave 1 pmol of a 50-mer oligonucleotide duplex containing a single 8-oxoguanine base paired with a cytosine in a total reaction volume of 10 μl at 37°C for 1 hr.
Quality Control assays
Fpg is free from detectable contaminating nuclease activities.
1. Tchou, J. et al. (1994). J. Biol. Chem. 269, 15318-15324.
2. Hatahet, Z. et al. (1994). J. Biol. Chem. 269, 18814-18820.
3. Zharkov, D. et al. (1997) J. Biol. Chem. 272, 5335-5341.
4. Singh, N. et al. (1961). Experimental Cell Research. 175, 184-191.
5. Collins, A. et al. (1993). Carcinogenesis. 14, 1733-1735.