Abasic site binding by the human apurinic endonuclease, Ape, and determination of the DNA contact sites.
Abasic site binding by the human apurinic endonuclease, Ape, and determination of the DNA contact sites.
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DOI：
10.1093/nar/25.5.933
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发表时间：
1997-03
影响因子：
14.9
通讯作者：
David M. Wilson;Masaru Takeshita;B. Demple
中科院分区：
文献类型：
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作者：
David M. Wilson;Masaru Takeshita;B. Demple
DNA中脱碱基位点的致突变和致死效应通过由“II类”脱嘌呤（AP）核酸内切酶启动的修复来避免，所述酶立即切割5 '到脱碱基位点。我们检查了人AP内切核酸酶Ape蛋白（也称为Hap 1、Apex或Ref-1）的底物结合。在电泳迁移率变化实验中，Ape结合含有单个AP位点或四氢呋喃（F）残基的合成DNA底物。没有检测到与单链底物或未修饰的双链体DNA的复合物。在EDTA中，使双链体F-DNA移位50%所需的Ape浓度约为50 nM，而加入10 mM MgCl 2几乎消除了可检测的F-DNA@Ape复合物。滤膜结合研究表明，在EDTA存在下，F-DNA@Ape复合物在0 ℃下的半衰期约为50 s，加入Mg 2+后<15 s。从F-DNA@Ape复合物中回收的DNA是完整的，但在加入Mg 2+后迅速裂解，这表明这些蛋白质-DNA复合物处于切割的催化途径上。甲基化和乙基化干扰实验确定了Ape结合的关键DNA接触，Cu-1，10-菲咯啉足迹法表明Ape诱导的结构畸变在无碱基位点切割前。
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.