Wilson D M, Takeshita M, Demple B
Department of Molecular and Cellular Toxicology, Harvard School of Public Health, Boston, MA 02115, USA. NY 11794, USA.
Nucleic Acids Res. 1997 Mar 1;25(5):933-9. doi: 10.1093/nar/25.5.933.
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.
DNA中无碱基位点的诱变和致死效应可通过“II类”脱嘌呤（AP）内切核酸酶启动的修复得以避免，这类酶在无碱基位点的5'端立即切割。我们研究了人AP内切核酸酶Ape蛋白（也称为Hap1、Apex或Ref-1）与底物的结合情况。在电泳迁移率变动实验中，Ape与含有单个AP位点或四氢呋喃（F）残基的合成DNA底物结合。未检测到与单链底物或未修饰双链DNA形成的复合物。在EDTA存在下，使50%的双链F-DNA发生迁移所需的Ape浓度约为50 nM，而添加10 mM MgCl2几乎消除了可检测到的F-DNA@Ape复合物。滤膜结合研究表明，在0℃时，EDTA存在下F-DNA@Ape复合物的半衰期约为50 s，添加Mg2+后半衰期小于15 s。从F-DNA@Ape复合物中回收的DNA是完整的，但添加Mg2+后会迅速被切割，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对Ape结合至关重要的DNA接触位点，铜-1,10-菲咯啉足迹分析表明，在切割之前，Ape在无碱基位点诱导了结构畸变。
