Purification and characterization of a 39kDa apurinic/apyrimidinic endonuclease from mouse ascites sarcoma cells.
We purified an apurinic/apyrimidinic (AP) endonuclease from mouse ascites sarcoma (SR-C3H/He) cells. The enzyme showed nicking activity on acid-depurinated DNA but not on untreated, intact DNA. It also showed priming activity for DNA polymerase on both acid-depurinated and bleomycin-damaged DNA. The priming activity on bleomycin-damaged DNA was two times higher than that on an acid-depurinated DNA. The enzymatic properties indicate that the enzyme is a class II AP endonuclease having DNA 3' repair diesterase activity. The purified enzyme has a molecular weight of 39,000 as determined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The optimal pH for AP endonuclease activity was 8.0 in 50 mM Tris-HCl buffer. The AP endonuclease activity depended on divalent cation such as Mg2+ and Co2+ ions, and was inhibited by 2 mM EDTA with no addition of the divalent cation. An appropriate concentration of sodium or potassium salt stimulated the activity. Partial digestion of the AP endonuclease with Staphylococcus aureus V8 protease produced 4 major peptide fragments which may be used for protein sequencing.
Wakabayashi
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H
Department of Molecular Biology, Okayama University Medical School, Japan.
Tsuji
T
T
Seki
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S
D000143:Acids / Q000494:pharmacology; D000818:Animals; D001761:Bleomycin / Q000494:pharmacology; D004273:DNA, Neoplasm / Q000737:chemistry / Q000187:drug effects / Q000378:metabolism; D043603:DNA-(Apurinic or Apyrimidinic Site) Lyase; D043223:Deoxyribonuclease IV (Phage T4-Induced); D004355:Drug Stability; D004591:Electrophoresis, Polyacrylamide Gel; D008190:Lyases / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification*; D051379:Mice; D008970:Molecular Weight; D010449:Peptide Mapping; D011687:Purines / Q000032:analysis; D012513:Sarcoma, Experimental / Q000201:enzymology* / Q000473:pathology; D014407:Tumor Cells, Cultured
D000143:Acids; D004273:DNA, Neoplasm; D011687:Purines; D001761:Bleomycin; D043223:Deoxyribonuclease IV (Phage T4-Induced); D008190:Lyases; D043603:DNA-(Apurinic or Apyrimidinic Site) Lyase