| Co-Investigator(Kenkyū-buntansha) |
KUBO Kihei Osaka Prefecture Univ., Graduate School of Agriculture and Biological Science, Professor, 大学院・農学生命科学研究科, 教授 (40117619)
TERATO Hiroaki Hiroshima Univ., Graduate School of Science, Research Associate, 大学院・理学研究科, 助手 (00243543)
OHYAMA Yoshihiko Hiroshima Univ,, Graduate School of Science, Associate Professor, 大学院・理学研究科, 助教授 (30169081)
SASAMOTO Kazumi Dojin Co. Ltd., Research Director, 研究所, 研究本部長
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| Research Abstract |
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.
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