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' 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.
Department of Molecular Biology, Okayama University Medical School, Japan.
A H
AH
Sarker
S
S
Watanabe
K
K
Akiyama
Y
Y
Nakagawa
H
H
Wakabayashi
Y
Y
Tan
S
S
Seki
D000818:Animals; D001201:Ascites / Q000201:enzymology*; D002852:Chromatography, Ion Exchange; D004260:DNA Repair; D043603:DNA-(Apurinic or Apyrimidinic Site) Lyase; D043223:Deoxyribonuclease IV (Phage T4-Induced); D004591:Electrophoresis, Polyacrylamide Gel; D004706:Endodeoxyribonucleases / Q000302:isolation & purification* / Q000378:metabolism; D008190:Lyases / Q000302:isolation & purification* / Q000378:metabolism; D051379:Mice; D012513:Sarcoma, Experimental / Q000201:enzymology*; D014407:Tumor Cells, Cultured
D004706:Endodeoxyribonucleases; D043223:Deoxyribonuclease IV (Phage T4-Induced); C057799:3'-phosphoglycoaldehyde diesterase; D008190:Lyases; D043603:DNA-(Apurinic or Apyrimidinic Site) Lyase