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Abasic Site Binding by the Human Apurinic Endonuclease, Ape, and Determination of the DNA Contact Sites

1997; Oxford University Press; Volume: 25; Issue: 5 Linguagem: Inglês

10.1093/nar/25.5.933

1362-4962

David M. Wilson, Masaru Takeshita, Bruce Demple,

CRISPR and Genetic Engineering

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 Hapl, 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 ∼50 nM, while the addition of 10 mM MgCl 2 nearly eliminated detectable F-DNA·Ape complexes. Filter-binding studies demonstrated a half-life of ∼50 s at 0°C for F-DNA·Ape complexes in the presence of EDTA, and <15 s after the addition of Mg 2+ . The DNA recovered from F-DNA·Apecomplexes was intact but was rapidly cleaved upon addition of Mg 2+ , 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.