Partial purification of endonuclease activity from human lymphoblasts. Separation of activities for depurinated DNA and DNA irradiated with ultraviolet light.
Crude extracts of cultured human lymphoblasts (CCRF-CEM) contain endonuclease activity that cleaves ultraviolet-irradiated DNA in preference to untreated DNA. Purification of this activity was carried out using ultraviolet-irradiated PM2 phage DNA (5000 ergs/mm2) as substrate in the enzyme assay. Since endonuclease specific for depurinated or depyrimidinated DNA might account for the apparent ultraviolet-irradiated DNA-specific activity, fractions derived during purification were also assayed with partially depurinated DNA. Chromatography of a 45-60% (NH4)2SO4 fraction on a Sephadex G-100 column yielded a peak of activity (35 000 daltons) highly active against depurinated DNA but also active for ultraviolet-irradiated DNA. Further purification by DEAE-cellulose chromatography resolved two activities. One was highly specific for depurinated DNA with only minor activity for ultraviolet-irradiated DNA, and was strongly stimulated by Mg2+. The other was non-specifically active against ultraviolet-irradiated or untreated DNA and was independent of Mg2+. Additional studies suggest that neither of these two activities but a third enzyme is responsible for the ultraviolet-irradiated DNA-specific endonuclease activity observed in crude cell extracts.
D002460:Cell Line; D004279:DNA, Viral / Q000302:isolation & purification / Q000528:radiation effects*; D003851:Deoxyribonucleases / Q000302:isolation & purification* / Q000378:metabolism; D004492:Edetic Acid / Q000494:pharmacology; D004720:Endonucleases / Q000302:isolation & purification* / Q000378:metabolism; D008274:Magnesium / Q000494:pharmacology; D011830:Radiation Effects; D014466:Ultraviolet Rays
D004279:DNA, Viral; D004492:Edetic Acid; D003851:Deoxyribonucleases; D004720:Endonucleases; D008274:Magnesium