T4 Endonuclease V

Applications

• Study of UV damage to DNA¹ and its repair, including DNA damage in single cells.²
• Detection of differential UV damage repair of transcribed sequences.^3-5
• Detection of UV mutational hotspots.^6,7

T4 Endonuclease V has N-glycosylase and apurinic/apyrimidinic lyase (AP lyase) activities. Ultraviolet (UV) light produces covalent photoproducts in DNA, the most prevalent being a cis-syn cyclobutane pyrimidine dimer. T4 Endonuclease V locates and binds to pyrimidine dimers in dsDNA. The enzyme then cleaves the N-glycosylic bond of the 5´ pyrimidine of the dimer and breaks the phosphodiester bond 3´ to the resulting abasic site.¹

Unit Definition: One unit of T4 Endonuclease V converts 1 µg of UV-irradiated plasmid DNA from covalently closed circles to nicked form in 30 minutes at 37°C.

Storage Buffer: 50% glycerol containing 50 mM Tris-HCl (pH 7.5), 100 mM NaCl, 0.1 mM EDTA, 1 mM DTT, and 0.1% Triton® X-100.

Quality Control: T4 Endonuclease V is free of detectable nonspecific exo- and endonuclease activities and RNase activity.

Figure 1. Mechanism of pyrimidine dimer cleavage by T4 Endonuclease V. (Adapted with permission from Friedburg, E.C. et al. (1995) DNA Repair and Mutagenesis, ASM Press.)

Figure 2. Use of T4 Endonuclease V to detect DNA damage caused by UV exposure. DNA encoding a 1.2-kb DNA fragment was isolated with (UV⁺) and without (UV^-) exposure to a UV transilluminator and digested with T4 Endonuclease V (Endo V). Lane 1, 1-kb ladder.