Characterisation of new substrate specificities of Escherichia coli and Saccharomyces cerevisiae AP endonucleases

Nucleic Acids Res. 2003 Nov 1;31(21):6344-53. doi: 10.1093/nar/gkg812.

Abstract

Despite the progress in understanding the base excision repair (BER) pathway it is still unclear why known mutants deficient in DNA glycosylases that remove oxidised bases are not sensitive to oxidising agents. One of the back-up repair pathways for oxidative DNA damage is the nucleotide incision repair (NIR) pathway initiated by two homologous AP endonucleases: the Nfo protein from Escherichia coli and Apn1 protein from Saccharomyces cerevisiae. These endonucleases nick oxidatively damaged DNA in a DNA glycosylase-independent manner, providing the correct ends for DNA synthesis coupled to repair of the remaining 5'-dangling nucleotide. NIR provides an advantage compared to DNA glycosylase-mediated BER, because AP sites, very toxic DNA glycosylase products, do not form. Here, for the first time, we have characterised the substrate specificity of the Apn1 protein towards 5,6-dihydropyrimidine, 5-hydroxy-2'-deoxyuridine and 2,6-diamino-4-hydroxy-5-N-methylformamidopyrimidine deoxynucleotide. Detailed kinetic comparisons of Nfo, Apn1 and various DNA glycosylases using different DNA substrates were made. The apparent K(m) and kcat/K(m) values of the reactions suggest that in vitro DNA glycosylase/AP lyase is somewhat more efficient than the AP endonuclease. However, in vivo, using cell-free extracts from paraquat-induced E.coli and from S.cerevisiae, we show that NIR is one of the major pathways for repair of oxidative DNA base damage.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Cell Extracts
  • DNA Damage / drug effects
  • DNA Repair*
  • DNA, Superhelical / drug effects
  • DNA, Superhelical / metabolism
  • DNA-(Apurinic or Apyrimidinic Site) Lyase / metabolism*
  • DNA-Binding Proteins / metabolism
  • Escherichia coli / enzymology*
  • Humans
  • Kinetics
  • Oligodeoxyribonucleotides / metabolism
  • Oxidants / pharmacology
  • Oxidation-Reduction / drug effects
  • Paraquat / pharmacology
  • Plasmids / drug effects
  • Plasmids / metabolism
  • Saccharomyces cerevisiae / enzymology*
  • Substrate Specificity

Substances

  • Cell Extracts
  • DNA, Superhelical
  • DNA-Binding Proteins
  • Oligodeoxyribonucleotides
  • Oxidants
  • DNA-(Apurinic or Apyrimidinic Site) Lyase
  • Paraquat