Purification and characterization of human endonucleases specific for damaged DNA. Analysis of lesions induced by ultraviolet or x-radiation.
1. An endonuclease activity from a cultured human lymphoblast cell line, CCRF-CEM, was further purified by chromatography on phosphocellulose to remove a nonspecific acid endonuclease. 2. The purified enzyme acted quantitatively on apurinic sites in the DNA of PM2 phage. It showed optimum activity over a broad range of pH (7.0--8.5), was absolutely dependent on Mg2+ (optimum concentration 0.5 mM) and did not have detectable activity against intact DNA. 3. This enzyme was used as a probe to estimate the number of apurinic or apyrimidinic lesions induced in PM2 DNA by either ultraviolet or X-ray irradiation. High doses of ultraviolet radiation (2500 to 5000 J/m2) immediately induced 0.2 to 0.4 endonuclease-susceptible lesions per molecule of DNA. The lesions continued to increase for several hours after irradiation, reaching a level more than double that found initially. By contrast, in DNA exposed to 5000 rads of X-ray irradiation, the number of endonuclease-susceptible sites reached a maximum of about 0.6 per molecule immediately after exposure and did not increase further. It thus appears that ultraviolet-irradiated (but not X-ray irradiated) DNA contains damaged bases that are lost spontaneously after irradiation. 4. A second endonuclease was purified and was shown to act on ultraviolet-induced lesions that are distinct from either apurinic or apyrimidinic sites. These new lesions occur about ten times more frequently than ultraviolet-induced apurinic or apyrimidinic sites.
D002460:Cell Line; D004247:DNA / Q000528:radiation effects*; D003851:Deoxyribonucleases / Q000302:isolation & purification / Q000378:metabolism*; D004307:Dose-Response Relationship, Radiation; D004720:Endonucleases / Q000302:isolation & purification / Q000378:metabolism*; D006801:Humans; D007700:Kinetics; D008274:Magnesium / Q000494:pharmacology; D011839:Radiation, Ionizing; D014466:Ultraviolet Rays; D014965:X-Rays
D004247:DNA; D003851:Deoxyribonucleases; D004720:Endonucleases; D008274:Magnesium