DNA repair in Saccharomyces cerevisiae: purification and characterization of apurinic endonucleases

Abstract

Five chromatographically distinct apurinic endonucleases (D1, D2, D3, D4, and E) were purified from Saccharomyces cerevisiae 234, 122, 1,000, 4,550, and 5,490-fold, respectively. All appeared to be class II apurinic endonucleases and were not contaminated with exonuclease or nonspecific endonuclease activities under the reaction conditions used. All had similar pH optima, but endonucleases D4 and E showed higher salt requirements and endonuclease D4 had a lower Mg2+ requirement for optimal activity than the other endonucleases. Endonuclease D4 also nicked OsO4-treated DNA. The molecular weights of the apurinic endonucleases as determined by glycerol gradient sedimentation analysis were 37,000, 49,000, and 10,000, for endonucleases E, D4, and D2, respectively. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis of samples of radioiodinated endonuclease E showed the presence of two proteins.