Abasic site binding by the human apurinic endonuclease, Ape, and determination of the DNA contact sites

被引:87
作者
Wilson, DM
Takeshita, M
Demple, B
机构
[1] HARVARD UNIV, SCH PUBL HLTH, DEPT MOL & CELLULAR TOXICOL, BOSTON, MA 02115 USA
[2] SUNY STONY BROOK, DEPT PHARMACOL SCI, STONY BROOK, NY 11794 USA
关键词
D O I
10.1093/nar/25.5.933
中图分类号
Q5 [生物化学]; Q7 [分子生物学];
学科分类号
071010 ; 081704 ;
摘要
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 similar to 50 nM, while the addition of 10 mM MgCl2 nearly eliminated detectable F-DNA . Ape complexes, Filter-binding studies demonstrated a half-life of similar to 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.
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页码:933 / 939
页数:7
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