Mapping the protein-DNA interface and the metal-binding site of the major human apurinic/apyrimidinic endonuclease

Lam H. Nguyen, Daniel Barsky, Jan P. Erzberger, David M. Wilson

Research output: Contribution to journalArticle

50 Scopus citations

Abstract

Apurinic/apyrimidinic (AP) endonuclease Ape1 is a key enzyme in the mammalian base excision repair pathway that corrects AP sites in the genome. Ape1 cleaves the phosphodiester bond immediately 5' to AP sites through a hydrolytic reaction involving a divalent metal co-factor. Here, site-directed mutagenesis, chemical footprinting techniques, and molecular dynamics simulations were employed to gain insights into how Ape1 interacts with its metal cation and AP DNA. It was found that Ape1 binds predominantly to the minor groove of AP DNA, and that residues R156 and Y128 contribute to protein-DNA complex stability. Furthermore, the Ape1-AP DNA footprint does not change along its reaction pathway upon active-site coordination of Mg2+ or in the presence of DNA polymerase beta (polβ), an interactive protein partner in AP site repair. The DNA region immediately 5' to the abasic residue was determined to be in close proximity to the Ape1 metal-binding site. Experimental evidence is provided that amino acid residues E96, D70, and D308 of Ape1 are involved in metal coordination. Molecular dynamics simulations, starting from the active site of the Ape1 crystal structure, suggest that D70 and E96 bind directly to the metal, while D308 coordinates the cation through the first hydration shell. These studies define the Ape1-AP DNA interface, determine the effect of polβ on the Ape1-DNA interaction, and reveal new insights into the Ape1 active site and overall protein dynamics. (C) 2000 Academic Press.

Original languageEnglish (US)
Pages (from-to)447-459
Number of pages13
JournalJournal of Molecular Biology
Volume298
Issue number3
DOIs
StatePublished - May 5 2000

Keywords

  • AP endonuclease
  • Ape1
  • Base excision repair
  • DNA binding
  • Metal coordination

ASJC Scopus subject areas

  • Structural Biology
  • Molecular Biology

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