| T4 Pyrimidine DNA Glycosylase Bifunctional DNA Glycosylase with DNA N-glycosylase and AP Lyase Activities. Useful in studies 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 Reagents Supplied: The following reagents are supplied with this product: Component Name Store at (C) Amount Concentration T4 PDG (T4 Endonuclease V) -20 1 x 0.2ml 10,000U/ml T4 PDG Reaction Buffer -20 1 x 1.5ml 10X BSA -20 1x0.25ml 10mg/ml Source: gene denV in E. coli Concentration: 10u/ul Unit Definition: One unit is defined as the amount of enzyme that catalyzes the conversion of 0.5ug of UV irradiated supercoiled pUC19 DNA to > 95% nicked plasmid in a total reaction volume of 20ul in 30 minutes at 37C. Nicking is assessed by agarose gel electrophoresis. Irradiated plasmid contains an average of 3-5 pyrimidine dimers. Reaction Conditions: 1X T4 PDG Reaction Buffer Supplement with 100X Purified BSA Incubate at 37C 1X T4 PDG Reaction Buffer: 100mM NaCl 1mM DTT 1mM EDTA 25mM Na2HPO4 (pH 7.2 25C) Storage Buffer: 10mM Tris-HCl 250mM NaCl 1mM DTT 0.1mM EDTA 50% Glycerol 0.15% Triton X-100 pH 7.4 25C Heat Inactivation: No Unit Assay Conditions: 1X T4 PDG Reaction Buffer containing 0.5ug of UV irradiated supercoiled pUC19 DNA, supplemented with 100 µg/ml BSA in a 20ul reaction. Quality Control: T4 Endonuclease V is free of detectable nonspecific exo-and endonuclease activities and RNase activity. Storage and Stability: Stable for one year at -20C. |
