E. coli fpg

• Excises damaged purines from duplex DNA, cleaves AP sites leaving 3’ and 5’ phosphates

Fpg (also known as formamidopyrimidine DNA glycosylase, Mut M, FAPY DNA glycosylase, and 8-oxoguanine DNA glycosylase) participates in the base-excision (BER) pathway of DNA repair enzymes and acts both as a N-glycosylase and an AP-lyase. The N-glycosylase activity releases damaged purines from double-stranded DNA, generating an apurinic/apyrimidinic (AP site). The AP-lyase activity cleaves both the 3ʹ and 5′ phosphodiester bonds at the AP site, producing a 1-base gap in the DNA and 3ʹ and 5ʹ phosphate termini. Bases recognized and removed by fpg include 7, 8-dihydro-8-oxoguanine (8-oxoguanine), 8-oxoadenine, fapy-guanine, methy-fapy-guanine, fapy-adenine, aflatoxin B1-fapy-guanine, 5-hydroxy-cytosine and 5-hydroxy-uracil (1, 2).

This enzme is supplied in 20 mM Tris-HCl, 50 mM NaCl, 1.0 mM DTT, 0.1 mM EDTA and 50% glycerol; pH 8.0 at 25°C.

10X Yellow Buffer (cat. no. B0130) contains 100 mM Bis-Tris-Propane, 100 mM MgCl₂ and 10 mM DTT: pH 7.0 at 25°C.

SDS is availaboe upon request.

Enzyme properties

• Storage temperature: –25°C to –15°C
• Moleculare weight: 30,290 Daltons

Source of recombinant enzyme protein
The protein is produced by a recombinantE. coli strain carrying the cloned fpg gene.

Unit definition: One unit is defined as the amount of enzyme required to cleave 1 pmol of a 34mer oligo-nucleotide duplex containing an 8-oxoguanine base paired with a cysteine in 1 hour at 37°C in a total reaction volume of 10 µl in reaction buffer

References

1. Tchou, J., et al. (1994) Substrate specificity of Fpg protein. J. Biol. Chem., 269:15318.
2. Hatahet, Z., et al. (1994) New substrates for old enzymes. J. Biol. Chem., 269:18814.
3. Boiteux, S., O’Connor, T., and Laval, J. (1987) Formamidopyrimidine-DNA glycosylase of Escherichia coli: cloning and sequencing of the fpg structural gene and overproduction of the protein. EMBO J., 5:3177.
4. Singh, N., McCoy, M., Tice, R., and Schneider, L. (1988) A simple technique for quantitation of low levels of DNA damage in individual cells. Exp. Cell Res., 175:184.
5. Collins, A., Duthie, S., and Dobson, V. (1993) Direct enzymatic detection of endogenous oxidative base damage in human lymphocyte DNA. Carcinogenesis, 14:1733.
6. Collins, A., Dusinska, M., Gedik, C., and Stetina, R. (1996) Oxidative damage to DNA: do we have a reliable biomarker?. Environ. Health Perspect., 104:465
7. Pflaum, M., Will, O., Mahler, H.C., and Epe, B. (1998) DNA oxidation products determined with repair endonucleases in mammalian cells: types, basal levels and influence of cell proliferation. Free Rad. Res., 29:585.
8. Hartwig, A., Dally, H., and Schlepegrell, R. (1996) Sensitive analysis of oxidative DNA damage in mammalian cells: use of the bacterial Fpg protein in combination with alkaline unwinding. Toxicol. Lett., 88:85.
9. Czene, S., and Harms-Ringdahl, M. (1995) Detection of single strand breaks and formamidopyrimidine-DNA glycosylase-sensitive sites in DNA of cultured human fibroblasts. Mutat. Res., 336:235.

Protocol for Comet Assay

After lysis of cells/nuclei embedded in low melting temperature agarose:

1. Add 100 µl of 1X reaction buffer per slide and apply cover slip.
2. Equilibrate 5 minutes.
3. Remove cover slip, then tap slide on its side to remove excess reaction buffer.
4. Dilute fpg 82X in 1X Yellow Buffer.
5. Add 100 µl dilute fpg solution per slide and replace cover slip.
6. Incubate slide at 37°C for 30 minutes.
7. Proceed with further enzymatic manipulation or continue with alkali unwinding.

Notes
Fpg is a DNA repair enzyme which cleaves the phosphodiester bond at abasic sites, a common form of naturally occurring DNA damage. Following thorough characterization of the fpg enzyme in our nuclease quality control tests, both during and after purification, we have concluded the inherent presence of abasic sites in DNA substrates contributes to false positives in tests for exogenous endo- and exonuclease contaminants.

Quality control analysis

Specific activity was measured using a twofold serial dilution method. Dilutions of enzyme were made in 1X reaction buffer and added to 10 µl reactions containing 1X Yellow Buffer and a FAM-labeled duplex oligonucleotide, containing a single 8-oxoguanine site. Reactions were incubated 1 hour at 37°C, placed on ice, denatured with N-N-dimethylformamide and analyzed on a 15% TBE-urea acrylamide gel.

Protein concentration is determined by OD₂₈₀ absorbance.

Physical purity is evaluated by SDS-PAGE of concentrated and diluted enzyme solutions followed by silver-stain detection. Purity is assessed by comparing the aggregate mass of contaminant bands in the concentrated sample to the mass of the band corresponding to the protein of interest in the diluted sample.

Single-stranded exonuclease is determined in a 50 µl reaction containing a radiolabeled single-stranded DNA substrate and 10 µl of enzyme solution incubated for 4 hours at 37°C.

Double-stranded exonuclease activity is determined in a 50 µl reaction containing a radiolabeled double-stranded DNA substrate and 10 µl of enzyme solution incubated for 4 hours at 37°C.

E. coli contamination is evaluated using 5 µl replicate samples of enzyme solution that are denatured and screened in a TaqMan qPCR assay for the presence of contaminating E. coli genomic DNA using oligonucleotide primers corresponding to the 16S rRNA locus.

Non-specific RNAse contamination is assessed using the RNAse Alert kit, (Integrated DNA Technologies), following the manufacturer’s guidelines.

This OEM by QIAGEN product is available for bulk purchase for the following commercial assay applications.

• Excise damaged purines from duplex DNA
• Cleaves AP sites leaving 3’ and 5’ phosphates