Sarker A H, Watanabe S, Akiyama K, Nakagawa Y, Wakabayashi H, Tan Y, Seki S
Department of Molecular Biology, Okayama University Medical School, Japan.
Biochim Biophys Acta. 1995 Dec 14;1245(3):299-304. doi: 10.1016/0304-4165(95)00112-3.
Purification and characterization of a DNA repair enzyme having 5' apurinic/apyrimidinic (AP) endonuclease activity are reported. The enzyme extracted from mouse ascites sarcoma (SR-C3H/He) cells with 0.2 M potassium phosphate buffer (pH 7.5) was purified by successive chromatographies on phosphocellulose, DEAE-cellulose, phosphocellulose (a second time) and single-stranded DNA cellulose, and by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). The purified enzyme has 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 it 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-terminal amino acid and internal amino acid sequences are determined.
报道了一种具有5' 脱嘌呤/脱嘧啶（AP）内切核酸酶活性的DNA修复酶的纯化及特性研究。用0.2 M磷酸钾缓冲液（pH 7.5）从小鼠腹水肉瘤（SR-C3H/He）细胞中提取的该酶，先后经磷酸纤维素、DEAE-纤维素、磷酸纤维素（第二次）和单链DNA纤维素柱层析以及十二烷基硫酸钠-聚丙烯酰胺凝胶电泳（SDS-PAGE）进行纯化。经SDS-PAGE测定，纯化后的酶表观分子量为30 kDa。该酶对酸脱嘌呤的DNA具有切口活性，对完整DNA则无此活性，并且对酸脱嘌呤的DNA和博来霉素处理的DNA具有DNA聚合酶引发活性。结果表明，它是一种具有5' AP内切核酸酶和DNA 3' 修复二酯酶活性的多功能DNA修复酶。该酶活性依赖于二价阳离子如Mg2+ 的存在。测定了其氨基末端氨基酸序列和内部氨基酸序列。
