Evidence for a common active site for cleavage of an AP site and the benzene-derived exocyclic adduct, 3,N4-benzetheno-dC, in the major human AP endonuclease

B. Hang, D. G. Rothwell, J. Sagi, I. D. Hickson, B. Singer

Research output: Contribution to journalArticlepeer-review


We have previously reported that the 3,N4-benzetheno-dC (p-BQ-dC) endonuclease activity found in HeLa cells is a novel function of the major human AP endonuclease (HAP1) [Hang et al. (1996) Proc. Natl. Acad. Sci U.S.A. 93, 13737-13741]. In this study, we compare the enzymatic and biochemical properties of the enzyme toward p-BQ-dC and an AP site in a defined oligonucleotide. A comparative analysis of the specificity constants (K(cat.)/K(m)) for p-BQ-dC and an AP site indicates that the AP site is the preferred substrate. The enzyme does not cleave other structurally related exocylic adducts and modified nucleosides such as 1,N6-etheno-dA, 3,N4- etheno-dC, 1,N2-etheno-dG, 1,N2-propano-dG, 8-oxo-dG, and thymine glycol. The p-BQ-dC activity requires a double-stranded DNA substrate and is affected by the base in the opposite strand, with maximal activity for a p-BQ-dC·G pair and minimal activity for a p-BQ-dC·C pair. The p-BQ-dC activity also requires Mg2+, Mn2+, or Zn2+ with optimal concentration spectra similar to those for the AP function. The optimal pH ranges for these two functions are also similar to each other (5.5-6.5). Six mutant HAP1 proteins containing single amino acid substitutions were assayed in parallel for comparison of their activities toward p-BQ-dC and the AP site. These mutants either concomitantly lost (N212A, D210N) or had reduced (D219A, E96A, and N212Q) or unchanged (H116N) p-BQ-dC and AP activities. This parallelism strongly supports the hypothesis that cleavage of p-BQ-dC requires the same catalytic active site as that proposed for the AP function. This dual activity toward two structurally unrelated substrates, an AP site and a bulky exocyclic adduct, has implications for substrate recognition. The AP site and p-BQ-dC cause different changes in the local conformation around the lesion as it is visualized by molecular modeling.
Original languageEnglish
Pages (from-to)15411-15418
Number of pages7
Issue number49
Publication statusPublished - 9 Dec 1997


  • Binding Sites
  • metabolism: Carbon-Oxygen Lyases
  • chemistry: DNA Adducts
  • DNA-(Apurinic or Apyrimidinic Site) Lyase
  • analogs & derivatives: Deoxycytidine Monophosphate
  • Deoxyribonuclease IV (Phage T4-Induced)
  • Hela Cells
  • Humans
  • Hydrolysis
  • Molecular Structure
  • 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.


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