Substitution of Asp-210 in HAP1 (APE/Ref-1) eliminates endonuclease activity but stabilises substrate binding

D. G. Rothwell, B. Hang, M. A. Gorman, P. S. Freemont, B. Singer, I. D. Hickson

Research output: Contribution to journalArticlepeer-review

Abstract

HAP1, also known as APE/Ref-1, is the major apurinic/apyrimidinic (AP) endonuclease in human cells. Previous structural studies have suggested a possible role for the Asp-210 residue of HAP1 in the enzymatic function of this enzyme. Here, we demonstrate that substitution of Asp-210 by Asn or Ala eliminates the AP endonuclease activity of HAP1, while substitution by Glu reduces specific activity ~500-fold. Nevertheless, these mutant proteins still bind efficiently to oligonucleotides containing either AP sites or the chemically unrelated bulky p-benzoquinone (pBQ) derivatives of dC, dA and dG, all of which are substrates for HAP1. These results indicate that Asp-210 is required for catalysis, but not substrate recognition, consistent with enzyme kinetic data indicating that the HAP1-D210E protein has a 3000-fold reduced K(cat) for AP site cleavage, but an unchanged K(m). Through analysis of the binding of Asp-210 substitution mutants to oligonucleotides containing either an AP site or a pBQ adduct, we conclude that the absence of Asp-210 allows the formation of a stable HAP1-substrate complex that exists only transiently during the catalytic cycle of wild-type HAP1 protein. We interpret these data in the context of the structure of the HAP1 active site and the recently determined co-crystal structure of HAP1 bound to DNA substrates.
Original languageEnglish
Pages (from-to)2207-2213
Number of pages6
JournalNucleic acids research.
Volume28
Issue number11
Publication statusPublished - 1 Jun 2000

Keywords

  • metabolism: Benzoquinones
  • Binding Sites
  • chemistry: Carbon-Oxygen Lyases
  • DNA-(Apurinic or Apyrimidinic Site) Lyase
  • genetics: DNA-Binding Proteins
  • Escherichia coli
  • Humans
  • Hydrogen Bonding
  • Kinetics
  • Models, Molecular
  • Mutation
  • metabolism: Oligodeoxyribonucleotides
  • Protein Structure, Secondary
  • Research Support, Non-U.S. Gov't
  • Research Support, U.S. Gov't, P.H.S.
  • Substrate Specificity

Fingerprint

Dive into the research topics of 'Substitution of Asp-210 in HAP1 (APE/Ref-1) eliminates endonuclease activity but stabilises substrate binding'. Together they form a unique fingerprint.

Cite this