APE 1

Description:
APE 1, also known as HAP 1 or Ref-1, acts as an AP lyase by hydrolyzing the phosphodiester backbone at the 5′ end of an apurinic (AP) site, generating a 1 base gap in the DNA duplex and leaving 3′-hydroxyl and 5′-deoxyribose phosphate termini. Evidence suggests that APE 1 may exhibit weak DNA 3′-diesterase, 3′ to 5′ exonuclease and RNase H activities ^(1-4).

Specific Activity:
2,000,000U/mg

Applications:

• Single cell gel electrophoresis (Comet assay)
• Alkaline elution
• Alkaline unwinding
• Modified nick translation

Source:
An E. coli strain which carries the cloned human APE 1 gene.

Supplied in:
10 mM Tris-HCl
50 mM NaCl
1 mM dithiothreitol
0.1 mM EDTA
50% glycerol
pH 8.0 @ 25°C

Supplied with:
10X Green Buffer

10X Green Buffer
200 mM Tris-Acetate
500 mM Potassium Acetate
10 mM Dithiothreitol
100 mM Magnesium Acetate
pH 7.9 @ 25°C

Unit Definition:
One unit is defined as the amount of enzyme required to cleave 20 pmol of a 34-mer oligonucleotide duplex contain-ing a single AP site in 1 hour at 37°C.

Single-Stranded Exonuclease Activity:
A 50 µL reaction containing 11,000 cpm of a radiolabeled single-stranded DNA substrate and 10 µL of enzyme solution incubated for 4 hours at 37°C resulted in less than 5.0% release of TCA-soluble counts.

Double-Stranded Exonuclease Activity:
A 50 µL reaction containing 5,000 cpm of a radiolabeled double-stranded DNA substrate and 10 µL of enzyme solution incubated for 4 hours at 37°C resulted in less than 0.5% release of TCA-soluble counts.

Endonuclease Activity:
A 50 µL reaction containing 0.5 µg of pBR322 DNA and 10 µL of enzyme solution incubated for 4 hours at 37°C resulted in 20% conversion to nicked circular DNA as determined by agarose gel electrophoresis.

E.coli 16S rDNA Contamination Test:              
Replicate 5 µL samples of enzyme solution were heat denatured and screened in a TaqMan qPCR assay for the presence of contaminating E.coli genomic DNA using primers for the 16S rRNA locus. The acceptance criterion for the test is the threshold cycle count (Ct) produced by the average of 3 replicate no template control samples. Based on no template control Ct values, the detection limit of this assay is <10 copies genome/sample.

Notes:
APE 1 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 APE 1 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.

Recommended Storage Condition:
 -20ºC  

References:

1. Demple, B. et al. (1991) Proc. Natl. Acad. Sci. USA, 88, 11450-11454.
2. Barzilay, G. et al. (1995) Nucl. Acids Res., 23, 1544-1550.
3. Barzilay, G. et al. (1995) Nature Struc. Biol., 2, 451-468.
4. Unpublished observations (See “Notes” Section)