Development of a novel method for gene-specific DNA damage detection

DNA损伤的产生和修复在整个基因组中并不均匀，而是受到核的动态状态如转录和复制的影响。在这项研究中，我们已经开发出基本的方法来监测DNA损伤的染色体DNA的单个基因中产生的醛反应性探针（阿普）的方法，损伤特异性DNA糖基化酶，和传统的DNA阵列相结合。为了高灵敏度检测ARP标记的DNA，化学发光检测试剂被用作过氧化物酶底物代替常规显色底物。当采用化学发光检测时，阿普测定的检测限为每10^6个核苷酸1-2个脱碱基位点。为了通过阿普方法检测碱基损伤，需要通过DNA糖基化酶将它们定量转化为脱碱基位点或3 ′-切口脱碱基位点。为此目的，通过芬顿反应氧化DNA，并用不同量的Endo III或hOGG 1处理，其分别从DNA中去除氧化的嘧啶和嘌呤损伤。通过阿普测定对所得的3 '-切口脱碱基位点进行定量。基于这些数据，确定定量转化所需的Endo III或hOGG 1的量。还在各种条件下检查了探针杂交期间ARP标记的DNA的稳定性。最后，用阿普标记含有脱碱基位点的c-myc DNA，并在优化的条件下与膜上的模型DNA阵列杂交。将膜洗涤，与抗生物素蛋白-生物素-辣根过氧化物酶复合物孵育，然后与化学发光检测试剂孵育。观察到c-myc基因的强化学发光信号，但未观察到阵列中的其他基因。因此，这些数据结合在一起表明，染色体DNA的单个基因中的DNA损伤可以通过结合醛反应性探针（阿普）方法、损伤特异性DNA糖基化酶和常规DNA阵列来检测。

The generation and repair of DNA damage are not uniform over the genome, but are affected by the dynamic state of nuclei such as transcription and replication. In this study, we have developed basic methods to monitor DNA damage generated in individual genes of chromosomal DNA by combining the aldehyde reactive probe (ARP) method, damage-specific DNA glycosylase, and conventional DNA arrays. For highly sensitive detection of ARP-labeled DNA, the chemiluminescence detection reagents were used as peroxidase substrates in place of conventional chromogenic substrates. The detection limit of the ARP assay was 1-2 abasic sites per 10^6 nucleotides when chemiluminescence detection was employed. For detection of base lesions by the ARP method, they need to be quantitatively converted to abasic sites or 3'-nicked abasic sites by DNA glycosylases. For this purpose, DNA was oxidized by the Fenton reaction and treated with varying amounts of Endo III or hOGG1 that remove oxidized pyrimidine and purine lesions, respectively, from DNA. The resulting 3'-nicked abasic sites were quantitated by the ARP assay. On the basis of the data, the amount of Endo III or hOGG 1 required for quantitative conversion was determined. The stability of ARP-labeled DNA during probe hybridization was also examined under various conditions. Finally c-myc DNA containing abasic sites were labeled with ARP and hybridized to model DNA arrays on a membrane under optimized conditions. The membrane was washed, incubated with avidin-biotin-horseradish peroxidase complexes, then with the chemiluminescence detection reagents. A strong chemiluminescence signal was observed for the c-myc gene but not for other genes in the arrays. Thus, these data combined together show that DNA damage in individual genes of chromosomal DNA can be detected by combining the aldehyde reactive probe (ARP) method, damage-specific DNA glycosylase, and conventional DNA arrays.