Characterization of a catalytically slow AP lyase activity in DNA polymerase gamma and other family A DNA polymerases

J Biol Chem. 2000 Apr 28;275(17):12509-14. doi: 10.1074/jbc.275.17.12509.

Abstract

Mitochondrial DNA polymerase gamma (pol gamma) is active in base excision repair of AP (apurinic/apyrimidinic) sites in DNA. Usually AP site repair involves cleavage on the 5' side of the deoxyribose phosphate by AP endonuclease. Previous experiments suggested that DNA pol gamma acts to catalyze the removal of a 5'-deoxyribose phosphate (dRP) group in addition to playing the conventional role of a DNA polymerase. We confirm that DNA pol gamma is an active dRP lyase and show that other members of the family A of DNA polymerases including Escherichia coli DNA pol I also possess this activity. The dRP lyase reaction proceeds by formation of a covalent enzyme-DNA intermediate that is converted to an enzyme-dRP intermediate following elimination of the DNA. Both intermediates can be cross-linked with NaBH(4). For both DNA pol gamma and the Klenow fragment of pol I, the enzyme-dRP intermediate is extremely stable. This limits the overall catalytic rate of the dRP lyase, so that family A DNA polymerases, unlike pol beta, may only be able to act as dRP lyases in repair of AP sites when they occur at low frequency in DNA.

Publication types

  • Research Support, U.S. Gov't, P.H.S.

MeSH terms

  • Base Sequence
  • Carbon-Oxygen Lyases / metabolism*
  • Catalysis
  • Chromatography, High Pressure Liquid
  • Chromatography, Ion Exchange
  • DNA Polymerase I / metabolism
  • DNA Polymerase gamma
  • DNA Repair
  • DNA-(Apurinic or Apyrimidinic Site) Lyase
  • DNA-Directed DNA Polymerase / metabolism*
  • Deoxyribonuclease IV (Phage T4-Induced)
  • Escherichia coli Proteins*
  • Mitochondria / enzymology
  • Models, Chemical
  • Molecular Sequence Data
  • Moloney murine leukemia virus / enzymology
  • RNA-Directed DNA Polymerase / metabolism
  • Temperature
  • Time Factors

Substances

  • Escherichia coli Proteins
  • bacteriophage T7 induced DNA polymerase
  • RNA-Directed DNA Polymerase
  • DNA Polymerase I
  • DNA Polymerase gamma
  • DNA-Directed DNA Polymerase
  • Deoxyribonuclease IV (Phage T4-Induced)
  • endonuclease IV, E coli
  • Carbon-Oxygen Lyases
  • DNA-(Apurinic or Apyrimidinic Site) Lyase