TY - JOUR
T1 - Mapping the protein-DNA interface and the metal-binding site of the major human apurinic/apyrimidinic endonuclease
AU - Nguyen, Lam H.
AU - Barsky, Daniel
AU - Erzberger, Jan P.
AU - Wilson, David M.
N1 - Funding Information:
This work was carried out under the auspices of the US Department of Energy by Lawrence Livermore National Laboratory under contract number W-7405-ENG-48 and supported by an NIH grant (CA79056) to DMWIII. We thank Ms Tina Xi and Dr Harvey Mohrenweiser for sequencing support, and Dr Ian McConnell for critical inputs.
PY - 2000/5/5
Y1 - 2000/5/5
N2 - 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.
AB - 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.
KW - AP endonuclease
KW - Ape1
KW - Base excision repair
KW - DNA binding
KW - Metal coordination
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U2 - 10.1006/jmbi.2000.3653
DO - 10.1006/jmbi.2000.3653
M3 - Article
C2 - 10772862
AN - SCOPUS:0034607548
SN - 0022-2836
VL - 298
SP - 447
EP - 459
JO - Journal of Molecular Biology
JF - Journal of Molecular Biology
IS - 3
ER -