Nakamura J, Walker V E, Upton P B, Chiang S Y, Kow Y W, Swenberg J A
Department of Environmental Sciences and Engineering, The University of North Carolina, Chapel Hill 27599-7400, USA.
Cancer Res. 1998 Jan 15;58(2):222-5.
One of the most prevalent lesions in DNA is the apurinic/apyrimidinic (AP) site, which is derived from the cleavage of the N-glycosyl bond by DNA glycosylase or by spontaneous depurination. AP sites are repaired by AP endonucleases during the process of base excision repair; however, an imbalance in this DNA repair system may cause mutations as well as cell death. We have established a sensitive and convenient slot-blot method to detect AP sites in genomic DNA using a novel aldehyde reactive probe (ARP), which reacts with the aldehydic group of ring-opened AP sites. The reaction of 1 mM of ARP with 15 microg of genomic DNA containing AP sites at 37 degrees C was completed within 1 min. The AP site-ARP complex was remarkably stable during incubation in TE buffer, even at 100 degrees C for 60 min. The sensitivity of this assay enables detection of 2.4 AP sites per 10(7) bases. By using this ARP-slot-blot assay, the rate of spontaneous depurination of calf thymus DNA was determined. Under physiological conditions, AP sites were increased at 1.54 AP sites/10(6) nucleotides/day (9000 AP sites/cell/day). This highly sensitive assay allows us to determine the endogenous level of AP sites in genomic DNA, as well as to investigate whether DNA-damaging agents cause imbalances of base excision/AP endonuclease repair in vivo and in vitro.
DNA中最常见的损伤之一是无嘌呤/无嘧啶（AP）位点，它是由DNA糖基化酶切断N-糖基键或自发脱嘌呤作用产生的。在碱基切除修复过程中，AP位点由AP核酸内切酶修复；然而，这种DNA修复系统的失衡可能导致突变以及细胞死亡。我们建立了一种灵敏且便捷的狭缝杂交方法，使用一种新型醛反应探针（ARP）来检测基因组DNA中的AP位点，该探针可与开环AP位点的醛基发生反应。1 mM的ARP与15 μg含有AP位点的基因组DNA在37℃下反应1分钟内即可完成。AP位点-ARP复合物在TE缓冲液中孵育时非常稳定，即使在100℃下孵育60分钟也是如此。该检测方法的灵敏度能够检测到每10^7个碱基中有2.4个AP位点。通过使用这种ARP-狭缝杂交检测法，测定了小牛胸腺DNA的自发脱嘌呤率。在生理条件下，AP位点以1.54个AP位点/10^6个核苷酸/天（9000个AP位点/细胞/天）的速度增加。这种高灵敏度检测法使我们能够确定基因组DNA中AP位点的内源性水平，以及研究DNA损伤剂是否会在体内和体外导致碱基切除/AP核酸内切酶修复失衡。
