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FUNCTION OF NOVEL MAMMALIAN GLYCOSYLASE FOR OXYDATIVE PYRIMIDINE

Research Project

Project/Area Number 15510059
Research Category

Grant-in-Aid for Scientific Research (C)

Allocation TypeSingle-year Grants
Section一般
Research Field Risk sciences of radiation/Chemicals
Research InstitutionOSAKA PREFECTURE UNIVERSITY

Principal Investigator

KUBO Kihei  OSAKA PREFECTURE UNIVERSITY, AGRICULTURE AND BIOLOGICAL SCIENCES, PROFESSOR, 農学生命科学研究科, 教授 (40117619)

Co-Investigator(Kenkyū-buntansha) MATSUYAMA Satoshi  OSAKA PREFECTURE UNIVERSITY, AGRICULTURE AND BIOLOGICAL SCIENCES, ASSOCIATE PROFESSOR, 農学生命科学研究科, 助教授 (10254442)
UI Masahiro  OSAKA PREFECTURE UNIVERSITY, AGRICULTURE AND BIOLOGICAL SCIENCES, RESEARCH ASSOCIATE, 農学生命科学研究科, 助手 (10336810)
Project Period (FY) 2003 – 2004
Project Status Completed (Fiscal Year 2004)
Budget Amount *help
¥3,100,000 (Direct Cost: ¥3,100,000)
Fiscal Year 2004: ¥1,200,000 (Direct Cost: ¥1,200,000)
Fiscal Year 2003: ¥1,900,000 (Direct Cost: ¥1,900,000)
KeywordsDNA REPAIR / REACTIVE OXYGEN / GLYCOSYLASE / BASE DAMAGE / AP SITES / THYMINE GLYCOL / BASE EXCISION REPAIR / MAMMALIAN / マウス / グルコシラーゼ
Research Abstract

Reactive oxygen species induced by ionizing radiation and intracellular metabolic process attack DNA bases to create various oxidative damages, such as thymine glycol(TG). These damaged bases are mainly repaired by base excision repair(BER) pathway. The novel monofunctional thymine glycol(TG) activity was extracted from isolated nuclei of mouse stomach and spleen after removing mNTH1 and major AP endonuclease by the high-salt treatment. The western blot analysis revealed that the extract prepared did not include a detectable amount of mNTH1 protein. The nuclear extract was further purified either by the combination of UNO-S1 and UNO-Q1 column or by hydrophobic, hydroxyapatite and UNO-S1 column chromatography. The optimum pH and salt condition of the target activity were 100 mM KCl and pH 8.0, respectively. The activity was resistant to 0.58 mM EDTA. After SDS-PAGE and 2D-electrophesis analyses, we found several candidate protein spots or bands ; 2 major (61,56 kDa) with 6 minor candidates (70,53,37,33,27,25 kDa). We analyzed them with MALDI-FTICR Mass Spectrometry. No known TG-DNA glycosylases (mNth1 and mNei1) were included in the candidate proteins analyzed by Mascot Search. XP_140547, BAB_32018, XP_359164 (NCBI protein data base) were identified in the analysis. These proteins were PCR-cloned and the pET-expression systems were constructed.
To analyze the action of the glycosylase, a novel AP site detection method was developed using fluorescein-conjugated ARP (FARP-1). The intracellular AP sites were successfully and quantitatively detected by the method. By the aid of this new method, intranuclear BER reaction was successfully monitored after MMS treatment of HeLa cells.

Report

(3 results)
  • 2004 Annual Research Report   Final Research Report Summary
  • 2003 Annual Research Report
  • Research Products

    (8 results)

All 2004 2003 Other

All Journal Article (6 results) Book (1 results) Publications (1 results)

  • [Journal Article] Detection of endonuclease III- and 8-oxoguanine glycosylase-sensitive base modifications in γ-irradiate DNA and cells by the aldehyde reactive probe (ARP) assay.2004

    • Author(s)
      Ali, M.M., Kurisu, S., Yoshioka, Y., Terato, H., Oyama, Y., Kubo, K., Ide, H
    • Journal Title

      J.Radiat.Res. 45・2

      Pages: 229-237

    • NAID

      110004041021

    • Description
      「研究成果報告書概要(和文)」より
    • Related Report
      2004 Final Research Report Summary
  • [Journal Article] 塩基除去修復とその相補機構2004

    • Author(s)
      正岡綾, 久保喜平
    • Journal Title

      放射線生物研究 39・4

      Pages: 396-407

    • NAID

      40006576838

    • Description
      「研究成果報告書概要(和文)」より
    • Related Report
      2004 Annual Research Report 2004 Final Research Report Summary
  • [Journal Article] Detection of endonuclease III- and 8-oxoguanine glycosylase-sensitive base modifications in g-irradiated DNA and cells by aldehyde reactive probe(ARP) assay2004

    • Author(s)
      Ali, M.M., Kurisu, S., Yoshioka, Y., Terato, H., Ohyama, Y, Kubo, K., Ide, H.
    • Journal Title

      J.Radiat.Res. 45(2)

      Pages: 229-237

    • Description
      「研究成果報告書概要(欧文)」より
    • Related Report
      2004 Final Research Report Summary
  • [Journal Article] Detection of endonuclease III- and 8-oxoguanine glycosylase-sensitive base modifications in γ-irradiated DNA and cells by the aldehyde reactive probe (ARP) assay.2004

    • Author(s)
      Ali, M.M., Kurisu, S., Yoshioka, Y., Terato, H., Oyama, Y., Kubo, K., Ide, H
    • Journal Title

      J.Radiat.res. 45・2

      Pages: 229-237

    • NAID

      110004041021

    • Related Report
      2004 Annual Research Report
  • [Journal Article] Detection of DNA Damage by the Aldehyde Reactive Probe (ARP) Assay : Principles and its Application2003

    • Author(s)
      Ide, H., Kurisu, S., Tanaka R., Asaeda, A., Kubo, K
    • Journal Title

      Recent Res.Devel.Biochem.Transworld research Network, Kelala, India 1

      Pages: 33-47

    • Description
      「研究成果報告書概要(和文)」より
    • Related Report
      2004 Final Research Report Summary
  • [Journal Article] Detection of DNA Damage by the Aldehyde Reactive Probe(ARP) Assay : Principles and its Application2003

    • Author(s)
      Ide, H., Kurisu, S., Tanaka R., Asaeda, A., Kubo, K.
    • Journal Title

      recent Res.Devel.Biochem., Transworld research Network, Kelala, India

    • Description
      「研究成果報告書概要(欧文)」より
    • Related Report
      2004 Final Research Report Summary
  • [Book] Recent Res.Devel.Nucleosides & Nucleotides2003

    • Author(s)
      Ide, H., Kurisu, H., Tanaka, R., Asaeda, A., Kubo, K.
    • Total Pages
      15
    • Related Report
      2004 Annual Research Report
  • [Publications] M.M.Ali et al.: "Detection of Endonuclease III- and 8-Oxoguanine Glycosylase-Sensitive Base Modifications in γ-Irradiated DNA and Cells by the Aldehyde Reactive Probe"Journal of Radiation Research. (印刷中).

    • Related Report
      2003 Annual Research Report

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Published: 2003-04-01   Modified: 2016-04-21  

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