Abasic site binding by the human apurinic endonuclease, Ape, and determination of the DNA contact sites.
The mutagenic and lethal effects of abasic sites in DNA are averted by repair initiated by 'class II' apurinic (AP) endonucleases, which cleave immediately 5'to abasic sites. We examined substrate binding by the human AP endonuclease, Ape protein (also called Hap1, Apex or Ref-1). In electrophoretic mobility-shift experiments, Ape bound synthetic DNA substrates containing single AP sites or tetrahydrofuran (F) residues. No complexes were detected with single-stranded substrates or unmodified duplex DNA. In EDTA, the concentration of Ape required to shift 50% of duplex F-DNA was approximately 50 nM, while the addition of 10 mM MgCl2 nearly eliminated detectable F-DNA@Ape complexes. Filter-binding studies demonstrated a half-life of approximately 50 s at 0 degrees C for F-DNA@Ape complexes in the presence of EDTA, and <15 s after the addition of Mg2+. The DNA recovered from F-DNA@Ape complexes was intact but was rapidly cleaved upon addition of Mg2+, which suggests that these protein-DNA complexes are on the catalytic pathway for incision. Methylation and ethylation interference experiments identified DNA contacts critical for Ape binding, and Cu-1, 10-phenanthroline footprinting suggested an Ape-induced structural distortion at the abasic site prior to cleavage.
Wilson
D M
DM
Department of Molecular and Cellular Toxicology, Harvard School of Public Health, Boston, MA 02115, USA. NY 11794, USA.
Takeshita
M
M
Demple
B
B
D001665:Binding Sites; D019757:Carbon-Oxygen Lyases; D004247:DNA / Q000378:metabolism*; D018983:DNA Footprinting; D043603:DNA-(Apurinic or Apyrimidinic Site) Lyase; D004720:Endonucleases / Q000378:metabolism*; D006801:Humans; D008274:Magnesium / Q000378:metabolism; D009687:Nuclear Proteins / Q000378:metabolism*; D013379:Substrate Specificity
D009687:Nuclear Proteins; D004247:DNA; D004720:Endonucleases; D019757:Carbon-Oxygen Lyases; C085108:APEX1 protein, human; D043603:DNA-(Apurinic or Apyrimidinic Site) Lyase; D008274:Magnesium