Identification and characterization of mitochondrial abasic (AP)-endonuclease in mammalian cells

Nucleic Acids Res. 2006 Apr 14;34(7):2067-76. doi: 10.1093/nar/gkl177. Print 2006.

Abstract

Abasic (AP)-endonuclease (APE) is responsible for repair of AP sites, and single-strand DNA breaks with 3' blocking groups that are generated either spontaneously or during repair of damaged or abnormal bases via the DNA base excision repair (BER) pathway in both nucleus and mitochondria. Mammalian cells express only one nuclear APE, 36 kDa APE1, which is essential for survival. Mammalian mitochondrial (mt) BER enzymes other than mtAPE have been characterized. In order to identify and characterize mtAPE, we purified the APE activity from beef liver mitochondria to near homogeneity, and showed that the mtAPE which has 3-fold higher specific activity relative to APE1 is derived from the latter with deletion of 33 N-terminal residues which contain the nuclear localization signal. The mtAPE-sized product could be generated by incubating 35S-labeled APE1 with crude mitochondrial extract, but not with cytosolic or nuclear extract, suggesting that cleavage of APE1 by a specific mitochondria-associated N-terminal peptidase is a prerequisite for mitochondrial import. The low abundance of mtAPE, particularly in cultured cells might be the reason for its earlier lack of detection by western analysis.

Publication types

  • Research Support, N.I.H., Extramural

MeSH terms

  • Amino Acid Sequence
  • Animals
  • Cattle
  • DNA-(Apurinic or Apyrimidinic Site) Lyase / genetics
  • DNA-(Apurinic or Apyrimidinic Site) Lyase / isolation & purification
  • DNA-(Apurinic or Apyrimidinic Site) Lyase / metabolism*
  • Enzyme Stability
  • Humans
  • Kinetics
  • Mice
  • Mitochondria / enzymology*
  • Mitochondria, Liver / enzymology
  • Mitochondrial Proteins / genetics
  • Mitochondrial Proteins / isolation & purification
  • Mitochondrial Proteins / metabolism*
  • Molecular Sequence Data
  • Peptide Hydrolases / metabolism
  • Recombinant Proteins / metabolism
  • Sequence Deletion

Substances

  • Mitochondrial Proteins
  • Recombinant Proteins
  • Peptide Hydrolases
  • APEX1 protein, human
  • DNA-(Apurinic or Apyrimidinic Site) Lyase