Purification and characterization of an AP endonuclease/DNA 3' repair diesterase from mouse ascites sarcoma cells

Biochim Biophys Acta. 1995 Dec 14;1245(3):299-304. doi: 10.1016/0304-4165(95)00112-3.

Abstract

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.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Animals
  • Ascites / enzymology*
  • Chromatography, Ion Exchange
  • DNA Repair*
  • DNA-(Apurinic or Apyrimidinic Site) Lyase
  • Deoxyribonuclease IV (Phage T4-Induced)
  • Electrophoresis, Polyacrylamide Gel
  • Endodeoxyribonucleases / isolation & purification*
  • Endodeoxyribonucleases / metabolism
  • Lyases / isolation & purification*
  • Lyases / metabolism
  • Mice
  • Sarcoma, Experimental / enzymology*
  • Tumor Cells, Cultured

Substances

  • Endodeoxyribonucleases
  • Deoxyribonuclease IV (Phage T4-Induced)
  • 3'-phosphoglycoaldehyde diesterase
  • Lyases
  • DNA-(Apurinic or Apyrimidinic Site) Lyase