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是均匀的，而是受到细胞核的动态状态如转录和复制的影响。在这项研究中，我们通过结合醛反应探针（ARP）方法、损伤特异性DNA糖基化酶和常规DNA阵列，开发了监测染色体DNA单个基因损伤的基本方法。为了对arp标记的DNA进行高灵敏度检测，使用化学发光检测试剂作为过氧化物酶底物代替传统的显色底物。化学发光法检测ARP的检出限为每10^6个核苷酸1-2个碱基位点。为了用ARP法检测碱基病变，它们需要被DNA糖基酶定量地转化为碱基位点或3'-缺口碱基位点。为此，通过Fenton反应氧化DNA，并用不同量的Endo III或hOGG1处理，分别从DNA中去除氧化的嘧啶和嘌呤病变。通过ARP测定得到的3'-缺口碱基位点。在此基础上，确定定量转化所需的Endo III或hOGG 1的量。在探针杂交过程中，还检测了不同条件下arp标记DNA的稳定性。最后用ARP标记含有碱基位点的c-myc DNA，并在优化的条件下杂交成膜上的DNA阵列。将膜洗净，用亲和素-生物素-辣根过氧化物酶配合物孵育，然后用化学发光检测试剂孵育。c-myc基因有较强的化学发光信号，而其他基因没有。因此，这些数据综合起来表明，通过结合醛反应探针（ARP）方法、损伤特异性DNA糖基化酶和常规DNA阵列，可以检测到染色体DNA单个基因的DNA损伤。

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.