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

Apurinic/apyrimidinic (AP) endonuclease Ape1 is a key enzyme in the mammali an base excision repair pathway that corrects AP sites in the genome. Ape1 cleaves the phosphodiester bond immediately 5' to AP sites through a hydrol ytic reaction involving a divalent metal, co-factor. Here, site-directed mu tagenesis, chemical footprinting techniques, and molecular dynamics simulat ions 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 (pol beta), 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. Experim ental evidence is provided that amino acid residues E96, D70, and D308 of A pe1 are involved in metal coordination. Molecular dynamics simulations, sta rting from the active site of the Ape1 crystal structure, suggest that D70 and E96 bind directly to the metal, while D308 coordinates the cation throu gh the first hydration shell. These studies define the Ape1-AP DNA interfac e, determine the effect of pol beta on the Ape1-DNA interaction, and reveal new insights into the Ape1 active site and overall protein dynamics. (C) 2 000 Academic Press.