Expression of a putative catalytic domain of the human APEX nuclease (a major apurinic/apyrimidinic endonuclease) in Escherichia coli.
1. Sequence analyses of APEX nuclease, a mammalian major apurinic/apyrimidinic (AP) endonuclease homologous to Escherichia coli exonuclease III, suggested that APEX nuclease is organized into two domains, a Mr 6000 N-terminal domain containing nuclear location signals and a Mr 29,000 C-terminal catalytic domain. 2. In order to study the enzyme structure further, vectors expressing APEX nuclease (pTAPXH1) and the Mr 29,000 C-terminal region (pTAPXH61) were constructed using cDNA (APX cDNA) for the human APEX nuclease and pTrc99A plasmid. The constructs were introduced into BW2001 strain (xth-11, nfo-2) cells of E. coli to produce transformants designated as BW2001/pTAPXH1 and BW2001/pTAPXH61, respectively. Both the APEX nuclease expressed in BW2001/pTAPXH1 and the Mr 29,000 C-terminal peptide expressed in BW2001/pTAPXH61 were partially purified by column chromatography and highly purified by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. 3. The purified APEX nuclease and the Mr 29,000 C-terminal peptide both showed equally high AP endonuclease activity which indicates that the Mr 29,000 C-terminal region of the APEX nuclease is (or contains) the AP endonuclease domain.
Ono
Y
Y
Department of Neurological Surgery, Okayama University Medical School, Japan.
Seki
S
S
Akiyama
K
K
Watanabe
S
S
Furuta
T
T
Ohmoto
T
T
D000595:Amino Acid Sequence; D001483:Base Sequence; D001665:Binding Sites; D019757:Carbon-Oxygen Lyases; D002384:Catalysis; D043603:DNA-(Apurinic or Apyrimidinic Site) Lyase; D004591:Electrophoresis, Polyacrylamide Gel; D004706:Endodeoxyribonucleases / Q000737:chemistry / Q000235:genetics*; D004926:Escherichia coli / Q000201:enzymology* / Q000235:genetics; D005090:Exodeoxyribonucleases / Q000737:chemistry / Q000235:genetics*; D015870:Gene Expression; D006657:Histones / Q000737:chemistry; D006801:Humans; D008969:Molecular Sequence Data; D008970:Molecular Weight; D009097:Multienzyme Complexes / Q000737:chemistry / Q000235:genetics*; D010446:Peptide Fragments / Q000737:chemistry; D017386:Sequence Homology, Amino Acid
D006657:Histones; D009097:Multienzyme Complexes; D010446:Peptide Fragments; D004706:Endodeoxyribonucleases; D005090:Exodeoxyribonucleases; C020133:exodeoxyribonuclease III; D019757:Carbon-Oxygen Lyases; C085108:APEX1 protein, human; D043603:DNA-(Apurinic or Apyrimidinic Site) Lyase