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1999 Fiscal Year Final Research Report Summary

Development of novel probe molecules for specific detection of abasic sites in DNA

Research Project

Project/Area Number 09558070
Research Category

Grant-in-Aid for Scientific Research (B)

Allocation TypeSingle-year Grants
Section展開研究
Research Field 環境影響評価(含放射線生物学)
Research InstitutionHiroshima 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
KeywordsDNA 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.

  • Research Products

    (22 results)

All Other

All Publications (22 results)

  • [Publications] Ide,H.: "Antibodies to oxidative DNA damage:Characterization of antibodies to 8-oxopurines"Cell Biology and Toxicology.

    • Description
      「研究成果報告書概要(和文)」より
  • [Publications] 井出 博: "酸化的損傷を特異的に含むDNA基質と生化学研究への応用"放射線生物研究. 32. 307-325 (1997)

    • Description
      「研究成果報告書概要(和文)」より
  • [Publications] Asaeda A.: "Repair kinetics of abasic sites in mammalian cells selectively monitored by the aldehyde reactive prove(ARP)"Nucleosides Nucleotides. 17. 503-513 (1998)

    • Description
      「研究成果報告書概要(和文)」より
  • [Publications] Asaeda A.: "Highly sensitive assay of abasic sites in mammalian cells.Optimization of aldehyde reactive prove(ARP)method"Analytica Chimica Acta. 365. 35-41 (1998)

    • Description
      「研究成果報告書概要(和文)」より
  • [Publications] Kubo K.: "Induction of fragile sites by fluorodeoxyuridine and caffeine accompanieswith misincorpolation of endogenous uridine nucleotide into DNA of feline fibroblasts"Journal of Veterinary Medical Science. 60. 1293-1297 (1998)

    • Description
      「研究成果報告書概要(和文)」より
  • [Publications] Suzuki T.: "Misincorporation of 2'-deoxyoxanosine 5'-triphosphate by DNA polymreases and its implication to mutagenesis"Biochemistry. 37. 11592-11598 (1998)

    • Description
      「研究成果報告書概要(和文)」より
  • [Publications] Terato,H.: "Recognition of oxidative thymine damage by DNA repair enzymes"Nucleic Acids Symposium Series. 39. 237-238 (1998)

    • Description
      「研究成果報告書概要(和文)」より
  • [Publications] Terato,H.: "Formation of highly fluorescent adducts between 2'-deoxyguanosine and amino acids by ionizing radiation"Analytica Chimica Acta. 365. 183-191 (1998)

    • Description
      「研究成果報告書概要(和文)」より
  • [Publications] Oka,S.: "Flow cytometric analysis of thymocyte subpopulations in mice after whole-body Xirradiation"Journal of Veterinary Medical Science. 61. 709-712 (1999)

    • Description
      「研究成果報告書概要(和文)」より
  • [Publications] Suzuki,T.: "Formation of 2'-deoxyoxanosine from 2'-deoxyguanosine and nitrous acid:mechanism and intermediates"Nucleic Acids Research. 28. 544-551 (2000)

    • Description
      「研究成果報告書概要(和文)」より
  • [Publications] Asaeda,A.: "Substrate specificity of human methylpurine DNA N-glycosylase"Biochemistry. 39. 1959-1965 (2000)

    • Description
      「研究成果報告書概要(和文)」より
  • [Publications] Ide. H. et al.: "Antibodies to oxidative DNA damage : Characterization of antibodies to 8-oxopurines"Cell Biology and Toxicology. 13. 405-417 (1997)

    • Description
      「研究成果報告書概要(欧文)」より
  • [Publications] Ide. H.: "Preparation of DNA substrates containing specific oxidative damage and their application for biochemical research."Radiation Biology Research Communications. 32. 307-325 (1997)

    • Description
      「研究成果報告書概要(欧文)」より
  • [Publications] Asaeda A. et al.: "Repair kinetics of abasic sites in mammalian cells selectively monitored by the aldehyde reactive prove (ARP)."Nucleosides Nucleotides. 17. 503-513 (1998)

    • Description
      「研究成果報告書概要(欧文)」より
  • [Publications] Asaeda A. et al.: "Highly sensitive assay of abasic sites in mammalian cells. Optimization of aldehyde reactive prove (ARP) method."Analytica Chimica Acta. 365. 35-41 (1998)

    • Description
      「研究成果報告書概要(欧文)」より
  • [Publications] Kubo K. et al.: "Induction of fragile sites by fluorodeoxyuridine and caffeine accompanieswith misincorpolation of endogenous uridine nucleotide, into DNA of feline fibroblasts."Journal of Veterinary Medical Science. 60. 1293-1297 (1998)

    • Description
      「研究成果報告書概要(欧文)」より
  • [Publications] Suzuki T. et al.: "Misincorporation of 2'-deoxyoxanosine 5'-triphosphate by DNA polymreases and its implication to mutagenesis."Biochemistry. 37. 11592-11598 (1998)

    • Description
      「研究成果報告書概要(欧文)」より
  • [Publications] Terato, H. et al.: "Recognition of oxidative thymine damage by DNA repair enzymes."Nucleic Acids Symposium Series. 237-238 (1998)

    • Description
      「研究成果報告書概要(欧文)」より
  • [Publications] Terato, H. et al.: "Formation of highly fluorescent adducts between 2'-deoxyguanosine and amino acids by ionizing radiation."Analytica Chimica Acta. 365. 183-191 (1998)

    • Description
      「研究成果報告書概要(欧文)」より
  • [Publications] Oka, S. et al.: "Flow cytometric analysis of thymocyte subpopulations in mice after whole-body X-irradiation."Journal of Veterinary Medical Science. 61. 709-712 (1999)

    • Description
      「研究成果報告書概要(欧文)」より
  • [Publications] Suzuki, T. et a1.: "Formation of 2'-deoxyoxanosine from 2'-deoxyguanosine and nitrous acid : mechanism and intermediates."Nucleic Acids Research. 28. 544-551 (2000)

    • Description
      「研究成果報告書概要(欧文)」より
  • [Publications] Asada A. et al.: "Substrate specificity of human methylpurine DNA N-glycosylase."Biochemistry. 39. 1959-1965 (2000)

    • Description
      「研究成果報告書概要(欧文)」より

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Published: 2001-10-23  

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