title: Mapping the protein-DNA interface and the metal-binding site of the major human apurinic/apyrimidinic endonuclease.
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 Mg(2+) or in the presence of DNA polymerase beta (polbeta), 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 polbeta on the Ape1-DNA interaction, and reveal new insights into the Ape1 active site and overall protein dynamics.
authors.affiliation: Molecular and Structural Biology Division, Lawrence Livermore National Laboratory, Livermore, CA, 94551, USA.
authors.forename: L H
authors.initials: LH
authors.lastname: Nguyen
authors.forename: D
authors.initials: D
authors.lastname: Barsky
authors.forename: J P
authors.initials: JP
authors.lastname: Erzberger
authors.forename: D M
authors.initials: DM
authors.lastname: Wilson
mesh_terms: D019943:Amino Acid Substitution / Q000235:genetics; D001483:Base Sequence; D001665:Binding Sites; D019757:Carbon-Oxygen Lyases / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D002384:Catalysis / Q000187:drug effects; D003198:Computer Simulation; D018360:Crystallography, X-Ray; D004247:DNA / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D018983:DNA Footprinting; D019951:DNA Polymerase beta / Q000378:metabolism / Q000494:pharmacology; D043603:DNA-(Apurinic or Apyrimidinic Site) Lyase; D004268:DNA-Binding Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D043223:Deoxyribonuclease IV (Phage T4-Induced); D006801:Humans; D008274:Magnesium / Q000378:metabolism / Q000494:pharmacology; D008670:Metals / Q000378:metabolism* / Q000494:pharmacology; D008958:Models, Molecular; D009154:Mutation / Q000235:genetics; D009690:Nucleic Acid Conformation / Q000187:drug effects; D018583:Pliability / Q000187:drug effects; D011485:Protein Binding; D011487:Protein Conformation / Q000187:drug effects; D013329:Structure-Activity Relationship; D013816:Thermodynamics
chemical_list: D004268:DNA-Binding Proteins; D008670:Metals; D004247:DNA; D019951:DNA Polymerase beta; D043223:Deoxyribonuclease IV (Phage T4-Induced); D019757:Carbon-Oxygen Lyases; C085108:APEX1 protein, human; D043603:DNA-(Apurinic or Apyrimidinic Site) Lyase; D008274:Magnesium