Uracil Cleavage System

• Two-enzyme system, Uracil DNA Glycosylase (UDG) and Endonuclease VIII
• UDG catalyzes the excision of the uracil base, creating an abasic site with an intact phosphodiester backbone
• Endonuclease VIII breaks the phosphodiester backbone both 3’ and 5’ to the abasic site, liberating the deoxyribose sugar

The Uracil Cleavage System provides two enzymes, which, when added sequentially to a reaction containing a synthetic DNA fragment containing a deoxy-uracil, generate a single nucleotide gap at the location of the uracil residue. The two individual enzyme components, Uracil DNA Glycosylase (UDG, cat. no. G5010-1140) and Endonuclease VIII (cat. no. Y9080-62500), provided at a 10X concentration, are to be added to a reaction containing a uracil-containing polynucleotide sequence. UDG catalyzes the excision of the uracil base, creating an abasic site with an intact phosphodiester backbone (1, 2). The lyase activity of Endonuclease VIII breaks the phosphodiester backbone both 3ʹ and 5ʹ to the abasic site, liberating the deoxyribose sugar (3,4).

UDG (cat. no. G5010) is supplied in 10 mM Tris-HCl, 50 mM NaCl, 1 mM DTT, 0.1 mM EDTA and 50% glycerol; pH 7.5 at 25°C.

Endonuclease VIII ( cat. no. Y9080) is supplied in 10 mM Tris-HCl, 250 mM NaCl, 0.1 mM EDTA and 50% glycerol; pH 8.0 at 25°C.

SDS available upon request.

Enzyme properties

• Storage temperature: –25°C to –15°C

Source of recombinant enzyme protein
The proteins are produced by and purified separately from E. coli strains containing recombinant Endonuclease VIII and Uracil-DNA Glycosylase genes.

Unit Definition, UDG: 1 unit is defined as the amount of enzyme that catalyzes the release of 1.8 nmol of uracil in 30 minutes from double-stranded, tritiated, uracil-containing DNA at 37°C in 1X UDG Reaction Buffer.

Unit Definition, Endonuclease Vlll: One unit is defined as the amount of enzyme required to cleave 1 pmol of an oligonucleotide duplex containing a single AP site in 1 hour at 37°C.

References

1. Lindhal, T., et al. (1977) J. Biol. Chem. 252:3286.
2. Lindhal, T. (1982) Annu. Rev. Biochem. 51:61.
3. Melamede, R.J., et al. (1994) Biochemistry 33:1255.
4. Jiang, D., et al. (1997) J. Biol. Chem. 272:32230.

PROTOCOL

The following instructions are for a reaction volume of 10 µl. The volume can be increased or decreased according to application requirements by maintaining the ratios of components described below.

1. Prepare uracil-containing DNA (e.g., PCR amplification product). The Uracil Cleavage System enzymes are active in most buffers used for molecular biology reaction, and it is unlikely you will need to change buffer prior to assembling the cleavage reaction.
2. Combine and mix the following components in a sterile tube:

   Amount	Description	Final concentration
   1–10 µl	Uracil-containing DNA (up to 3 pmol)	Up to 300 nM
   to 10 µl	Type I Water	N/A
   10 µl	Total volume
3. To the tube containing the DNA, add 0.5 µl UDG (ordered separately with cat. no. G5010).
4. Add 0.5 µl Endonuclease VIII (ordered separately with cat. no. Y9080) to the tube containing the DNA.
5. Incubate at 37°C for 30 minutes.
6. Following the incubation period, the reaction can be stopped (by adding a gel-loading stop solution in preparation for electrophoretic analysis) or prepared for transformation. In the case of transformation, the fragment can be ligated into a cloning vector using T4 DNA Ligase (cat. no. L6030-HC-L).

Quality control analysis

Unit Characterization Assay, UDG
Specific activity was measured using a twofold serial dilution method. Dilutions of enzyme were made in 1X reaction buffer and added to 50 µl reactions containing1X UDG Reaction Buffer and a PCR product a 3H-dUTP. Reactions were incubated for 10 minutes at 37°C, placed on ice, and analyzed using a TCA-precipitation method.

Unit Characterization Assay, Endonuclease VIII
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 2.0 µM of a FAM-labeled, 34-base duplex oligonucleotide, which contained a single uracil. Note that the was substrate pre-treated for 2 minutes with UDG to create an abasic site. Reactions were incubated 60 minutes at 37°C, placed on ice, denatured with N-N-dimethyl -formamide and analyzed on a 15% TBE-urea acrylamide gel.

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 for each enzyme separately 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 for each enzyme separately 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.

Double-stranded endonuclease activity v in a 50 µl reaction containing 0.5 µg of plasmid DNA 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.

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

• Cloning