1999 Fiscal Year Final Research Report Summary
Development of novel probe molecules for specific detection of abasic sites in DNA
| Project/Area Number |
09558070
|
|---|
| Research Category |
Grant-in-Aid for Scientific Research (B)
|
| Allocation Type | Single-year Grants |
|---|
| Section | 展開研究 |
|---|
| Research Field |
環境影響評価(含放射線生物学)
|
|---|
| Research Institution | Hiroshima University |
|---|
Principal Investigator |
IDE Hiroshi Hiroshima Univ., Faculty of Science, Professor, 理学部, 教授 (30223126)
|
|---|
| Co-Investigator(Kenkyū-buntansha) |
KUBO Kihei Osaka Prefect. Univ., College of Agriculture, Professor, 農学部, 教授 (40117619)
TERATO Hiroaki Hiroshima Univ., Faculty of Science, Research Associate, 理学部, 助手 (00243543)
OHYAMA Yoshihiko Hiroshima Univ., Faculty of Science, Associate Professor, 理学部, 助教授 (30169081)
SASAMOTO Kazumi Dojin Co. Ltd., Research Director, 研究部長
|
|---|
| Project Period (FY) |
1997 – 1999
|
| Keywords | DNA damage / abasic site / ARP / quantitation / DNA repair / MPG / alkylation |
|---|
| Research Abstract |
Abasic sites are the most ubiquitous damage found in cellular DNA. Therefore, it is essential to develop a method to detect abasic sites to study cytotoxic and genotoxic effects of DNA damage as well as DNA repair.
In this study, a probe molecule (ARP) specifically reacting with the aldehyde group of abasic sites was synthesized for highly sensitive and convenient detection of abasic sites formed in DNA.
For immobilization of DNA, a protamine sulfate-coated plate was used instead of the conventional UV-irradiated plate to enhance DNA binding. As the result, the time for immobilization was shortened to 1h without any loss of signals. The amount of tritium-labeled DNA bound to the plate was proportional to the DNA concentration employed. When DNA containing AP sites was treated with ARP in solution prior to coating the protamine-plate, the sensitivity of the assay was greatly increased.
HeLa RC355 cells were treated by a very low concentration of an alkylating agent (MMS) that does not affect cell survival. The assay of chromosomal DNA extracted from the cell with ARP revealed that as low as five abasic sites per 10ィイD16ィエD1 nucleotides could be detected, demonstrating a high sensitivity of the ARP method. Furthermore, the ARP assay proved to be very useful for detection of abasic sites resulting from excision of damaged base by DNA N-glycosylases in vitro and in vivo.
In collaboration with Dojindo Co. Ltd., ARP is now commercially available as a reagent or a part of the DNA damage assay kit containing ARP, standard abasic DNA, and plates for immobilization of sample DNA.
|
