参与碱基切除修复的AP内切酶1的克隆和蛋白质纯化

Cloning and Purification of AP Endonuclease One of the Enzymes Participating in Base Excision Repair

碱基切除修复途径是去除氧化和甲基化碱基的最主要途径。在碱基切除修复过程中,多个蛋白质,诸如DNA糖基酶、APE1内切酶、DNA聚合酶beta和DNA连接酶在体内的精密调节下高度协调地作用,从而切除受损碱基,使DNA恢复正常序列。碱基切除修复对维持基因组的稳定及抑制肿瘤发生等生理过程有重要作用。为了进一步从分子水平阐明APE1的作用机制,我们从HeLa细胞的cDNA文库中克隆得到APE1基因,使APE1在大肠杆菌中得到表达,并用蛋白质纯化的快速液相层析法经过一系列层析柱纯化了重组APE1蛋白质,APE1的生物化学功能研究正在进行中。

The base excision repair pathway is critical for removal of oxidized and DNA methylated bases from the DNA. Several proteins including DNA glycosylases, the APE1 endonuclease, DNA polymerase beta and DNA ligase, act in a highly regulated and coordinated manner during base excision repair to excise the base adducts from the DNA and restore the normal DNA sequence. The base excision repair plays a vital role in maintenance of the genome stability. And many cases have implied that the base excision repair acts as a tumor suppressor mechanism in human. In order to elucidate the molecular mechanism of APE1, the APE1 was cloned from HeLa eDNA library, and APE1 protein was overexpressed from E. coli. Subsequent column chromatograph was carried out using a fast protein liquid chromatograph（FPLC） system. The biochemical activity of APE1 was under experiment.