Determinants in nuclease specificity of Ape1 and Ape2, human homologues of Escherichia coli exonuclease III

J Mol Biol. 2002 Feb 22;316(3):853-66. doi: 10.1006/jmbi.2001.5382.


Abasic sites and non-conventional 3'-ends, e.g. 3'-oxidized fragments (including 3'-phosphate groups) and 3'-mismatched nucleotides, arise at significant frequency in the genome due to spontaneous decay, oxidation or replication errors. To avert the potentially mutagenic or cytotoxic effects of these chromosome modifications/intermediates, organisms are equipped with apurinic/apyrimidinic (AP) endonucleases and 3'-nucleases that initiate repair. Ape1, which shares homology with Escherichia coli exonuclease III (ExoIII), is the major abasic endonuclease in mammals and an important, yet selective, contributor to 3'-end processing. Mammals also possess a second protein (Ape2) with sequence homology to ExoIII, but this protein exhibits comparatively weak AP site-specific and 3'-nuclease activities. Prompted by homology modeling studies, we found that substitutions in the hydrophobic pocket of Ape1 (comprised of F266, W280 and L282) reduce abasic incision potency about fourfold to 450,000-fold, while introduction of an ExoIII-like pocket into Ape2 enhances its AP endonuclease function. We demonstrate that mutations at F266 and W280 of Ape1 increase 3' to 5' DNA exonuclease activity. These results, coupled with prior comparative sequence analysis, indicate that this active-site hydrophobic pocket influences the substrate specificity of a diverse set of sequence-related proteins possessing the conserved four-layered alpha/beta-fold. Lastly, we report that wild-type Ape1 excises 3'-mismatched nucleotides at a rate up to 374-fold higher than correctly base-paired nucleotides, depending greatly on the structure and sequence of the DNA substrate, suggesting a novel, selective role for the human protein in 3'-mismatch repair.

Publication types

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

MeSH terms

  • Amino Acid Sequence
  • Base Pair Mismatch / genetics
  • Base Sequence
  • Binding Sites
  • DNA / genetics
  • DNA / metabolism
  • DNA Repair
  • DNA-(Apurinic or Apyrimidinic Site) Lyase
  • Endonucleases
  • Escherichia coli / enzymology*
  • Exodeoxyribonucleases / chemistry*
  • Exodeoxyribonucleases / genetics
  • Exodeoxyribonucleases / metabolism*
  • Humans
  • Hydrophobic and Hydrophilic Interactions
  • Kinetics
  • Models, Molecular
  • Molecular Sequence Data
  • Multifunctional Enzymes
  • Mutation
  • Protein Structure, Secondary
  • Protein Structure, Tertiary
  • Sequence Alignment
  • Sequence Homology, Amino Acid*
  • Substrate Specificity


  • Multifunctional Enzymes
  • DNA
  • Endonucleases
  • Exodeoxyribonucleases
  • exodeoxyribonuclease III
  • APEX2 protein, human
  • DNA-(Apurinic or Apyrimidinic Site) Lyase