Clustered apurinic/apyrimidinic (AP) sites are more cytotoxic than isolated AP lesions because double strand breaks (DSB) can be formed during repair of closely positioned bistranded AP sites. Formation of DSB due to simultaneous cleavage of bistranded AP sites may be regulated by proteins specifically interacting with this complex lesion. A set of AP DNA duplexes containing AP sites in both strands in different mutual orientation (BS-AP DNAs) was used for search in the extracts of human cells proteins specifically recognizing clustered AP sites. A protein, which formed the Schiff-base-dependent covalent products having an apparent molecular mass of 50 kDa with the subset of BS-AP DNAs, was identified by mass spectrometry as apurinic/apyrimidinic endonuclease 1 (APE1). The identity of trapped protein was confirmed by Western blot analysis with anti-APE1 antibodies. Purified recombinant human APE1 is also capable of forming the 50 kDa-adducts with efficiency of BS-AP DNAs cross-linking to APE1 being dependent on the mutual orientation of AP sites.

In spite of formation of the Schiff-base-dependent intermediate, which is prerequisite for the β-elimination mechanism, APE1 is unable to cleave AP sites. APE1 lacking the first 34 amino acids at the N-terminus, unlike wild type enzyme, is unable to form cross-links with BS-AP DNAs that testifies to the involvement of disordered N-terminal extension, which is enriched in lysine residues, in the interaction with AP sites. The yield of APE1-AP DNA cross-links was found to correlate with the enzyme amount in the extracts estimated by the immunochemical approach; therefore the BS-AP DNA-probes can be useful for comparative analysis of APE1 content in cell extracts.

簇状的嘌呤/双嘧啶（AP）位点比单独的AP病变更具细胞毒性，因为在修复紧密定位的双链AP位点时会形成双链断裂（DSB）。由于双分裂AP位点的同时裂解而导致的DSB的形成可以通过与该复杂病变特异性相互作用的蛋白质来调节。一组AP DNA双链体在两条链中包含相互不同方向的AP位点（BS-AP DNA），用于搜索专门识别簇状AP位点的人细胞蛋白质的提取物。通过质谱法鉴定了与BS-AP DNA的子集形成表观分子量为50 kDa的席夫碱依赖性共价产物的蛋白质，其为嘌呤/嘧啶核糖核酸内切酶1（APE1）。通过用抗APE1抗体进行的蛋白质印迹分析确认了捕获的蛋白质的身份。纯化的重组人APE1还能够形成50 kDa加合物，其中BS-AP DNA与APE1交联的效率取决于AP位点的相互定向。

尽管形成了依赖Schiff碱的中间体，这是β消除机制的先决条件，但APE1无法裂解AP位点。与野生型酶不同，在N末端缺少前34个氨基酸的APE1无法与BS-AP DNA形成交联，从而证明了N末端无序延伸的参与，而N末端延伸富含赖氨酸残基。与AP网站的互动。发现APE1-AP DNA交联的产量与通过免疫化学方法估计的提取物中的酶含量相关。因此，BS-AP DNA探针可用于细胞提取物中APE1含量的比较分析。