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一成不变的，而是受到转录和复制等核动态的影响。在这项研究中，我们开发了结合醛反应探针(ARP)方法、损伤特异性DNA糖基酶和常规DNA阵列来监测染色体DNA单个基因所产生的DNA损伤的基本方法。为了高灵敏地检测ARP标记的DNA，用化学发光检测试剂代替传统的显色底物作为过氧化物酶底物。当采用化学发光法检测时，ARP的检测下限为每106个核苷酸中有1-2个碱基。用ARP方法检测碱基损伤时，需要用DNA糖基酶将碱基定量转化为碱性位点或3‘-缺口碱性位点。为此，DNA被Fenton反应氧化，并用不同数量的Endo III或hOGG1处理，分别从DNA中去除氧化的嘧啶和嘌呤损伤。通过ARP分析对所得到的3‘-镍碱性位点进行定量。根据数据，确定了定量转化所需的Endo III或Hogg 1的量。还考察了不同条件下ARP标记DNA在探针杂交过程中的稳定性。最后，用ARP标记含有碱性位点的c-myc DNA，并在优化的条件下与其杂交，在膜上构建DNA阵列。将膜洗涤，与抗生物素-生物素-辣根过氧化物酶络合物孵育，再与化学发光检测试剂孵育。观察到c-myc基因有很强的化学发光信号，但阵列中的其他基因没有。因此，这些数据结合在一起表明，结合醛反应探针(ARP)方法、损伤特异性DNA糖基酶和常规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.