Wilson D M, Takeshita M, Grollman A P, Demple B
Harvard University School of Public Health, Department of Molecular and Cellular Toxicology, Boston, Massachusetts 02115, USA.
J Biol Chem. 1995 Jul 7;270(27):16002-7. doi: 10.1074/jbc.270.27.16002.
The major apurinic/apyrimidinic (AP) endonuclease of human cells, the Ape protein, incises DNA adjacent to abasic sites to initiate DNA repair and counteract the cytotoxic and mutagenic effects of AP sites. Here we address the determinants of Ape AP endonuclease activity using duplex DNA substrates that contain synthetic analogs of AP sites: tetrahydrofuranyl (F), propanediol (P), ethanediol (E), or 2-(aminobutyl)-1,3-propanediol (Q). The last of these, a branched abasic structure, was a poor substrate for which Ape had kcat > 1000-fold lower than for F. In contrast, the specificity constant (kcat/Km) for E or P of Ape purified from HeLa cells was only 5-8-fold lower than for F. Positioning a phosphorothioate ester immediately 5' to F inhibited Ape incision activity 20-fold (Rp isomer) or > 10,000-fold (Sp isomer). Although Ape did not have detectable endonuclease activity toward single-stranded substrates or unmodified double-stranded DNA, the enzyme displayed a low level of 3'-exonuclease activity for duplex DNA (< 0.03% of its AP endonuclease activity), which was influenced by the reaction conditions. The base positioned opposite F did not dramatically affect the cleavage efficiency of Ape, but an F:F arrangement was cleaved at approximately one-third of the efficiency of F:C. A 3'-mismatch diminished P and E cleavage only slightly and F not at all. A 5'-mismatch reduced the Ape cleavage rate 4-10-fold for F and approximately 100-fold for P and E. A series of substrates with F at different positions along the oligonucleotide showed that Ape requires > or = 4 base pairs 5' to the abasic site and > or = 3 base pairs on the 3'-side. The implications of these results for substrate recognition by Ape are discussed.
人类细胞中的主要脱嘌呤/脱嘧啶（AP）内切核酸酶，即Ape蛋白，在无碱基位点附近切割DNA，以启动DNA修复并对抗AP位点的细胞毒性和诱变作用。在此，我们使用包含AP位点合成类似物的双链DNA底物来研究Ape AP内切核酸酶活性的决定因素：四氢呋喃基（F）、丙二醇（P）、乙二醇（E）或2-（氨基丁基）-1,3-丙二醇（Q）。其中最后一种，即分支无碱基结构，是一种较差的底物，Ape对其的催化常数（kcat）比对F的低1000倍以上。相比之下，从HeLa细胞中纯化的Ape对E或P的特异性常数（kcat/Km）仅比对F的低5-8倍。在F的紧邻5'端定位一个硫代磷酸酯会使Ape的切割活性降低20倍（Rp异构体）或>10000倍（Sp异构体）。尽管Ape对单链底物或未修饰的双链DNA没有可检测到的内切核酸酶活性，但该酶对双链DNA表现出低水平的3'-外切核酸酶活性（<其AP内切核酸酶活性的0.03%），这受反应条件影响。与F相对的碱基对Ape的切割效率没有显著影响，但F:F排列的切割效率约为F:C的三分之一。3'-错配仅略微降低P和E的切割，对F则无影响。5'-错配使F的Ape切割速率降低4-10倍，使P和E的降低约100倍。一系列在寡核苷酸上不同位置带有F的底物表明，Ape在无碱基位点的5'端需要≥4个碱基对，在3'端需要≥3个碱基对。讨论了这些结果对Ape底物识别的意义。
