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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
佐々木 一美  (株)同仁化学研究所, 研究部長
Project Period (FY) 1997 – 1999
Project Status Completed (Fiscal Year 1999)
Budget Amount *help
¥8,100,000 (Direct Cost: ¥8,100,000)
Fiscal Year 1999: ¥1,700,000 (Direct Cost: ¥1,700,000)
Fiscal Year 1998: ¥2,100,000 (Direct Cost: ¥2,100,000)
Fiscal Year 1997: ¥4,300,000 (Direct Cost: ¥4,300,000)
KeywordsDNA damage / abasic site / ARP / quantitation / DNA repair / MPG / alkylation / アベイシックサイト / デプリネーション / 脆弱部位 / ビオチン / ウラシル
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.

Report

(4 results)
  • 1999 Annual Research Report   Final Research Report Summary
  • 1998 Annual Research Report
  • 1997 Annual Research Report
  • Research Products

    (38 results)

All Other

All Publications (38 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
    • Related Report
      1999 Final Research Report Summary
  • [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
    • Related Report
      1999 Final Research Report Summary
  • [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
    • Related Report
      1999 Final Research Report Summary
  • [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
    • Related Report
      1999 Final Research Report Summary
  • [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
    • Related Report
      1999 Final Research Report Summary
  • [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
    • Related Report
      1999 Final Research Report Summary
  • [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
    • Related Report
      1999 Final Research Report Summary
  • [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
    • Related Report
      1999 Final Research Report Summary
  • [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
    • Related Report
      1999 Final Research Report Summary
  • [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
    • Related Report
      1999 Final Research Report Summary
  • [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
    • Related Report
      1999 Final Research Report Summary
  • [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
    • Related Report
      1999 Final Research Report Summary
  • [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
    • Related Report
      1999 Final Research Report Summary
  • [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
    • Related Report
      1999 Final Research Report Summary
  • [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
    • Related Report
      1999 Final Research Report Summary
  • [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
    • Related Report
      1999 Final Research Report Summary
  • [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
    • Related Report
      1999 Final Research Report Summary
  • [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
    • Related Report
      1999 Final Research Report Summary
  • [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
    • Related Report
      1999 Final Research Report Summary
  • [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
    • Related Report
      1999 Final Research Report Summary
  • [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
    • Related Report
      1999 Final Research Report Summary
  • [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
    • Related Report
      1999 Final Research Report Summary
  • [Publications] Suzuki,T.: "Isolation and characterization of diazoate intermediate upon nitrous acid and nitric oxide treatment of 2'-deoxycytidine."Biochemistry. 38. 7151-7158 (1999)

    • Related Report
      1999 Annual Research Report
  • [Publications] Masaoka,A.: "Enzymatic repair of 5-formyluracil.I.Excision of 5-formyluracil site-specifically incorporated into oligonucleotide substrates by AIKA protein (Escherichia coli 3-methyladenine DNA glycosylase II)."Journal of Biological Chemistry. 274. 25136-25143 (1999)

    • Related Report
      1999 Annual Research Report
  • [Publications] Terato,H.: "Enzymatic repair of 5-formyluracil.II.Mismatch formation between 5-formyluracil and guanine during DNA replication and its recognition by two proteins involved in base excision repair(AIKA) and mismatch repair(MutS)."Journal of Biological Chemistry. 274. 25144-25150 (1999)

    • Related Report
      1999 Annual Research Report
  • [Publications] Oka,S.: "Flow cytometric analysis of thymocyte subpopulations in mice after whole-body X-irradiation."Journal of Veterinary Medical Science. 61. 709-712 (1999)

    • Related Report
      1999 Annual Research Report
  • [Publications] Suzuki,T.: "Formation of 2'-deoxyoxanosine from 2'-deoxyguanosine and nitrous acid:mechanism and intermediates."Nucleic Acids Research. 28. 544-551 (2000)

    • Related Report
      1999 Annual Research Report
  • [Publications] Asaeda,A.: "Substrate specificity of human methylpurine DNA N-glycosylase."Biochemistry. 39(印刷中). (2000)

    • Related Report
      1999 Annual Research Report
  • [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)

    • Related Report
      1998 Annual Research Report
  • [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)

    • Related Report
      1998 Annual Research Report
  • [Publications] Kubo K.: "Induction of fragile sites by flurodeoxyuridine and caffeine accompanieswith misincorpolation of endogenous uridine nucleotide into DNA of feline fibroblasts." Journal of Veterinary Medical Science. 60. 1293-1297 (1998)

    • Related Report
      1998 Annual Research Report
  • [Publications] Suzuki T.: "Misincorporation of 2'-deoxyoxanosine 5'-triphosphate by DNA polymreases and its implication to mutagenesis." Biochemistry. 37. 11592-11598 (1998)

    • Related Report
      1998 Annual Research Report
  • [Publications] Terato,H.: "Recognition of oxidative thymine damage by DNA repair enzymes." Nucleic Acids Symposium Series. 39. 237-238 (1998)

    • Related Report
      1998 Annual Research Report
  • [Publications] Terato,H.: "Formation of highly fluorescent adducts between 2'-deoxyguanosine and amino acids by ionizing radiation." Analytica Chimica Acta. 365. 183-191 (1998)

    • Related Report
      1998 Annual Research Report
  • [Publications] Hiroshi Ide: "Antibodies to oxidative DNA damage : Characterization of antibodies to 8-oxopurines" Cell Biology and Toxicology. 13. 405-417 (1997)

    • Related Report
      1997 Annual Research Report
  • [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)

    • Related Report
      1997 Annual Research Report
  • [Publications] Ayumi Asaeda: "Highly sensitive assay of DNA abasic sites in mammalian cells-Optimization of the aldehyde reactive probe (ARP) method" Analytica Chimica Acta. (印刷中). (1998)

    • Related Report
      1997 Annual Research Report
  • [Publications] Ayumi Asaeda: "Repair kinetics of abasic sites in mammalian cells selectively monitored by the aldehyde reactive prove (ARP)" Nucleosides Nucleotides. (印刷中). (1998)

    • Related Report
      1997 Annual Research Report

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Published: 1997-04-01   Modified: 2016-04-21  

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