Izumi T, Hazra T K, Boldogh I, Tomkinson A E, Park M S, Ikeda S, Mitra S
Sealy Center for Molecular Science and Department of Human Biological Chemistry and Genetics, University of Texas Medical Branch, Galveston, TX 77555-1079, USA.
Carcinogenesis. 2000 Jul;21(7):1329-34.
The major mammalian apurinic/apyrimidinic (AP) endonuclease (APE1) plays a central role in the DNA base excision repair pathway (BER) in two distinct ways. As an AP endonuclease, it initiates repair of AP sites in DNA produced either spontaneously or after removal of uracil and alkylated bases in DNA by monofunctional DNA glycosylases. Alternatively, by acting as a 3'-phosphoesterase, it initiates repair of DNA strand breaks with 3'-blocking damage, which are produced either directly by reactive oxygen species (ROS) or indirectly through the AP lyase reaction of damage-specific DNA glycosylases. The endonuclease activity of APE1, however, is much more efficient than its DNA 3'-phosphoesterase activity. Using whole extracts from human HeLa and lymphoblastoid TK6 cells, we have investigated whether these two activities differentially affect BER efficiency. The repair of ROS-induced DNA strand breaks was significantly stimulated by supplementing the reaction with purified APE1. This enhancement was linearly dependent on the amount of APE1 added, while addition of other BER enzymes, such as DNA ligase I and FEN1, had no effect. Moreover, depletion of endogenous APE1 from the extract significantly reduced the repair activity, suggesting that APE1 is essential for repairing such DNA damage and is limiting in extracts of human cells. In contrast, when uracil-containing DNA was used as the substrate, the efficiency of repair was not affected by exogenous APE1, presumably because the AP endonuclease activity was not limiting. These results indicate that the cellular level of APE1 may differentially affect repair efficiency for DNA strand breaks but not for uracil and AP sites in DNA.
主要的哺乳动物脱嘌呤/脱嘧啶（AP）内切核酸酶（APE1）在DNA碱基切除修复途径（BER）中以两种不同方式发挥核心作用。作为一种AP内切核酸酶，它启动对DNA中自发产生的AP位点的修复，或者在单功能DNA糖基化酶去除DNA中的尿嘧啶和烷基化碱基后启动修复。另外，通过作为一种3'-磷酸二酯酶，它启动对具有3'-阻断损伤的DNA链断裂的修复，这种损伤要么由活性氧（ROS）直接产生，要么通过损伤特异性DNA糖基化酶的AP裂解酶反应间接产生。然而，APE1的内切核酸酶活性比其DNA 3'-磷酸二酯酶活性高效得多。我们使用来自人HeLa细胞和淋巴母细胞TK6细胞的全细胞提取物，研究了这两种活性是否对BER效率有不同影响。通过用纯化的APE1补充反应，显著刺激了ROS诱导的DNA链断裂的修复。这种增强与添加的APE1量呈线性相关，而添加其他BER酶，如DNA连接酶I和FEN1，则没有效果。此外，从提取物中耗尽内源性APE1显著降低了修复活性，这表明APE1对于修复此类DNA损伤至关重要，并且在人细胞提取物中是有限的。相反，当使用含尿嘧啶的DNA作为底物时，修复效率不受外源APE1的影响，推测是因为AP内切核酸酶活性不是限制因素。这些结果表明，APE1的细胞水平可能对DNA链断裂的修复效率有不同影响，但对DNA中的尿嘧啶和AP位点没有影响。
