T4 Endonuclease V
|Molecular Biology||Storage: -20°CShipping: Dry Ice|
Study of UV damage to DNA and its repair, including DNA damage in single cells. Detection of differential UV damage repair of transcribed sequences. Detection of UV mutational hotspots.
T4 Endonuclease V functions as part of a base excision repair pathway to recognize and remove
cyclobutane pyrimidine dimers. The enzyme binds to UV-irradiated DNA and processively
scans the DNA until a pyrimidine dimer is encountered. T4 Endonuclease V then cleaves
the glycosyl bond of the 5'-pyrimidine of the dimer and the 3' phosphodiester bond, resulting
in breakage of the DNA strand.1,2 Strand breakage is evident during subsequent electrophoresis
as a change in conformation (i.e., from covalent closed circular to relaxed circular molecules), or as the appearance of unique DNA fragments. The enzyme is a small protein that does not require divalent cations or other cofactors.1
gene denV in E. coli
One unit will change 1ug of UV-irradiated plasmid DNA from covalently closed circles (form I) to nicked closed circles (form II) in 30 minutes at 37°C.
The activity assay is performed in a 50ul reaction containing 50mM Tris-HCl pH 7.5) and 5mM using plasmid DNA irradiated for 1 minute at 254nm.
Contaminating Activity Assays:
T4 Endonuclease V is free of detectable exonuclease, non-T4 Endonuclease V-specific endonuclease and RNase activities as judged by electrophoresis after incubation of 1mg of various DNA substrates with 500-1000U of enzyme at 37°C for 16 hours.
50 mM Tris-HCl, pH 7.5, 100mM sodium chloride, 0.1mM EDTA, 1mM DTT, 0.1% Triton X-100, 50% glycerol.
Dilute the enzyme in 0.01M EDTA with 0.1mg/ml nuclease-free BSA, and buffer of choice (0.04M sodium phosphate or equivalant). The presence of EDTA is critical.
T4 Endonuclease V is free of detectable nonspecific exo-and endonuclease activities and RNase activity.
Storage and Stability:
Stable for one year at -20ºC.
Concentration: 20 U/uL
Form: Supplied as a frozen solution in 0.01M phosphate buffer, pH 6.5 containing 0.01M EDTA, 0.001M DTT and 20% glycerol.
Important Note: This product as supplied is intended for research use only, not for use in human, therapeutic or diagnostic applications without the expressed written authorization of United States Biological.
1. Friedberg, E.C. et al., (1995) DNA Repair and Mutagenesis, ASM Press, Washington D.C., 164.
2. Schrock, R.D. and Lloyd, R.S. (1993) J. Biol. Chem. 268, 880.