The role of Mg2+ and specific amino acid residues in the catalytic reaction of the major human abasic endonuclease: New insights from EDTA-resistant incision of acyclic abasic site analogs and site-directed mutagenesis

Jan P. Erzberger, David M. Wilson

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Apel, the major protein responsible for excising apurinic/apyrimidinic (AP) sites from DNA, cleaves 5' to natural AP sites via a hydrolytic reaction involving Mg2+. We report here that while Apel incision of the AP site analog tetrahydrofuran (F-DNA) was ~ 7300-fold reduced in 4 mM EDTA relative to Mg2+, cleavage of ethane (E-DNA) and proyane (P-DNA) acyclic abasic site analogs was only 20 and 30-fold lower, respectively, in EDTA compared to Mg2+. This finding suggests that the primary role of the metal ion is to promote a conformational change in the ring-containing abasic DNA, priming it for enzyme-mediated hydrolysis. Mutating the proposed metal-coordinating residue E96 to A or Q resulted in a ~ 600-fold reduced incision activity for both P and F-DNA in Mg2+ compared to wild-type. These mutants, while retaining full binding activity for acyclic P-DNA, were unable to incise this substrate in EDTA, pointing to an alternative or an additional function for E96 besides Mg2+-coordination. Other residues proposed to be involved in metal coordination were mutated (D70A, D70R, D308A and D308S), but displayed a relatively minor loss of incision activity for F and P-DNA in Mg2+, indicating a non-essential function for these amino acid residues. Mutations at Y171 resulted in a 5000-fold reduced incision activity. A Y171H mutant was fourfold less active than a Y171F mutant, providing evidence that Y171 does not operate as the proton donor in catalysis and that the additional role of E96 may be in establishing the appropriate active site environment via a hydrogen-bonding network involving Y171. D210A and D210N mutant proteins exhibited a ~ 25,000-fold reduced incision activity, indicating a critical role for this residue in the catalytic reaction. A D210H mutant was 15 to 20-fold more active than the mutants D210A or D210N, establishing that D210 likely operates as the leaving group proton donor.

Original languageEnglish (US)
Pages (from-to)447-457
Number of pages11
JournalJournal of Molecular Biology
Issue number2
StatePublished - Jul 9 1999



  • Ape1
  • DNA repair
  • Hap1
  • Nuclease
  • Phosphate hydrolysis

ASJC Scopus subject areas

  • Structural Biology
  • Molecular Biology

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