PURIFICATION AND CHARACTERIZATION OF AN AP ENDONUCLEASE DNA 3'REPAIR DIESTERASE FROM MOUSE ASCITES SARCOMA-CELLS

Purification and characterization of a DNA repair enzyme having 5' apu rinic/apyrimidinic (AP) endonuclease activity are reported. The enzyme extracted from mouse ascites sarcoma (SR-C3H/He) cells with 0.2 M pot assium phosphate buffer (pH 7.5) was purified by successive chromatogr aphies on phosphocellulose, DEAE-cellulose, phosphocellulose (a second time) and single-stranded DNA cellulose, and by sodium dodecyl sulfat e-polyacrylamide gel electrophoresis (SDS-PAGE). The purified enzyme h as an apparent molecular mass of 30 kDa as determined by SDS-PAGE. It was shown to have nicking activity on acid-depurinated DNA but not on intact DNA, and to have priming activities for DNA polymerase on acid- depurinated DNA and bleomycin-treated DNA. The results indicate that i t is a multifunctional DNA repair enzyme having 5' AP endonuclease and DNA 3' repair diesterase activities. The enzyme activity is dependent upon the presence of a divalent cation such as Mg2+. Its amino-termin al amino acid acid internal amino acid sequences are determined.