A mouse DNA repair enzyme (APEX nuclease) having exonuclease and apurinic/apyrimidinic endonuclease activities: purification and characterization.
A mouse repair enzyme having priming activity on bleomycin-damaged DNA for DNA polymerase was purified to apparent homogeneity and characterized. The enzyme extracted from permeabilized 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), Sephadex G-100, single-stranded DNA cellulose and hydroxyapatite. The purified enzyme has an Mr of 34,000 as determined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Enzymatical studies indicated that it is a multifunctional enzyme having exonuclease, apurinic/apyrimidinic endonuclease and phosphatase activities, similar to Escherichia coli exonuclease III. This enzyme is tentatively designated as APEX nuclease for apurinic/apyrimidinic endonuclease and exonuclease activities. The amino acid composition, amino-terminal amino acid sequence and an internal amino acid sequence of APEX nuclease are determined.
Seki
S
S
Department of Molecular Biology, Okayama University Medical School, Japan.
Ikeda
S
S
Watanabe
S
S
Hatsushika
M
M
Tsutsui
K
K
Akiyama
K
K
Zhang
B
B
D000595:Amino Acid Sequence; D000818:Animals; D001761:Bleomycin / Q000633:toxicity; D019757:Carbon-Oxygen Lyases; D002853:Chromatography, Liquid; D004247:DNA / Q000187:drug effects; 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*; D029968:Escherichia coli Proteins; D005090:Exodeoxyribonucleases / Q000302:isolation & purification* / Q000378:metabolism; D005092:Exonucleases / Q000378:metabolism*; D051379:Mice; D008969:Molecular Sequence Data; D009097:Multienzyme Complexes / Q000302:isolation & purification* / Q000378:metabolism; D010744:Phosphoric Monoester Hydrolases / Q000378:metabolism; D012689:Sequence Homology, Nucleic Acid
D029968:Escherichia coli Proteins; D009097:Multienzyme Complexes; D001761:Bleomycin; D004247:DNA; D004706:Endodeoxyribonucleases; D005090:Exodeoxyribonucleases; D005092:Exonucleases; D043223:Deoxyribonuclease IV (Phage T4-Induced); C468972:endonuclease IV, E coli; D010744:Phosphoric Monoester Hydrolases; D019757:Carbon-Oxygen Lyases; C486216:Apex1 protein, mouse; D043603:DNA-(Apurinic or Apyrimidinic Site) Lyase