DescriptionExonuclease V, a RecBCD complex from E. coli has several different enzyme activities, including an ATP-dependent single-stranded DNA endonuclease activity, ss- and ds- DNA exonuclease activity. The hydrolysis in each case is bi-directional (from both the 3´ and 5´ ends) and processive, producing oligonucleotides (1,2,3). All Exonuclease V activities have divalent cation requirements. Mg2+ is required for the exonuclease activity, while Ca2+ inhibits the exonuclease activity and allows double-stranded DNA unwinding (helicase activity) without hydrolysis (4,5).
Product SourceAn E. coli strain containing plasmids for expressing the three subunits of E. coli Exonuclease V: RecB, RecC and RecD.
The following reagents are supplied with this product:
|Adenosine 5'-Triphosphate (ATP)||10 mM|
Advantages and Features
ApplicationsDegradation of linear ssDNA and dsDNA while preserving nicked and supercoiled plasmid DNA.
Properties and Usage
Unit DefinitionOne unit is defined as the amount of enzyme required to produce 1 nmol of acidsoluble deoxyribonucleotide from double-stranded DNA in 30 minutes at 37°C in a total reaction volume of 50 μl.
1X NEBuffer 4
Supplement with 1 mM ATP
Incubate at 37°C
1X NEBuffer 4:
50 mM Potassium Acetate
20 mM Tris-acetate
10 mM Magnesium Acetate
1 mM DTT
pH 7.9 @ 25°C
50 mM Tris-HCl
100 mM NaCl
1 mM DTT
0.1 mM EDTA
0.1% Triton® X-100
pH 7.5 @ 25°C
Heat Inactivation70°C for 30 min
Unit Assay Conditions1X NEBuffer 4, 1 mM ATP with 0.15 mM sonicated duplex [3H]-DNA.
Quality Control AssaysThe following Quality Control Tests are performed on each new lot and meet the specifications designated for the product. Individual lot data can be found on the Product Summary Sheet/Datacard or Manual which can be found in the Supporting Documents section of this page.
- Endonuclease Activity (Nicked Double Stranded DNA):
The product is tested in a reaction containing a nicked DNA substrate. After incubation for 4 hours the percent cleaved is determined by agarose gel electrophoresis.
- Endonuclease Activity (Nicking):
The product is tested in a reaction containing a supercoiled DNA substrate. After incubation for 4 hours the percent converted to the nicked form is determined by agarose gel electrophoresis.
- Protein Purity (SDS-PAGE):
The physical purity is assessed by comparing contaminating protein bands in a concentrated sample to the protein of interest band in a sample of known dilution. The purity is determined by SDS-PAGE.
- RNase Activity (2 Hour Digestion):
The product is tested in a reaction containing a RNA substrate. After incubation for 2 hours there is no detectable degradation of the RNA substrate as determined by gel electrophoresis.
Material Safety Datasheets
- Eichler, D.C. et al. (1977). J.Biol. Chem. 252, 499-503.
- Tayler, A.F. et al. (1985). J. Mol. Biol. 185, 431-443.
- Amundsen, S.K. et al. (1986). Proc. Natl. Acad. Sci. 83, 5558-5562.
- Palas, K.M. et al. (1990). J. Biol. Chem. 265, 3447-3454.
- Dillingham, M.S. et al. (2008). Microbiology and Mol. Biol. Review. 72, 642-671.