Identification and characdterization of DNA glycosylases that recognize and remove oxidative base damage in DNA
| Project/Area Number |
16510035
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| Research Category |
Grant-in-Aid for Scientific Research (C)
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| Allocation Type | Single-year Grants |
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| Section | 一般 |
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| Research Field |
Risk sciences of radiation/Chemicals
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| Research Institution | Kyoto University |
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Principal Investigator |
ZUANG Qiu-Mei Kyoto University, Graduate School of Science, Associate Professor, 大学院・理学研究科, 助教授 (00260604)
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| Co-Investigator(Kenkyū-buntansha) |
YONEI Shuji Kyoto University, Graduate School of Science, Professor, 大学院・理学研究科, 教授 (60093340)
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| Project Period (FY) |
2004 – 2005
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| Project Status |
Completed (Fiscal Year 2005)
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| Budget Amount *help |
¥3,900,000 (Direct Cost: ¥3,900,000)
Fiscal Year 2005: ¥1,000,000 (Direct Cost: ¥1,000,000)
Fiscal Year 2004: ¥2,900,000 (Direct Cost: ¥2,900,000)
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| Keywords | Ionizing radiation / Reactive oxygen species / DNA repair / Base excision repair / DNA glycosylas / Mutation / Oxidative base damage / 8-oxo-7,8-dihydroguanine / APサイト / 忠実性 / DNAポリメラーゼ / APエンドヌクレアーゼ / 塩基相補性 / DNAポリメラーゼβ / フレームシフト |
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| Research Abstract |
(1) Reactive oxygen species cause a wide variety of oxidative modifications to purines and pyrimidines in DNA. Bacteria and eukaryotes have evolved base excision repair mechanisms for oxidative base damage in DNA. E.coli has three kinds of DNA glycosylase, MutM, Nth and Nei, that recognize and remove oxidatively damaged bases from DNA. These DNA glycosylases are able to recognize and remove 5-formyluracil and 5-hydroxymethyluracil in DNA. However, several evidence showed that there are residual activities in crude extract from E.coli mutM nth nei triple mutant. In this study, we identified the abilities of novel enzyme(s) to recognize double-stranded oligonucleotides containing 5-foU. (2) 7,8-dihydro-8-oxoguanine (8-oxoG) is the most important product of oxidative base damage in DNA, and causes G:C to T:A transversions in bacteria and mammalian cells. 8-oxoG is repaired by MutM in E.coli and 8-oxoG-DNA glycosylase (Ogg1) in yeast and mammalian cells. In the present study, we identified
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and characterized an ascidian homolog of the human hOgg1 in Ciona intestinalis (CiOgg1). Introduction of the CiOgg1 gene significantly reduced the frequency of spontaneous G:C to T:A transversions in E.coli mutM mutY strain. Purified GST-CiOgg1 fusion protein had 8-oxoG DNA glycosylase/AP lyase activity. It formed Schiff base intermediates with 8-oxoG-containing duplex oligonucleotides and removed 8-oxoG preferentially from 8-oxoG/C. The CiOgg1 cleaved 8-oxoG-containing duplex DNA via β-elimination reaction. Furthermore, the expression level of CiOgg1 was compared in various tissues in Ciona intestinalis. The highest expression level was observed in testis. (3) In this study, AP endonuclease in Ciona inteslinalis (CiAPE) was purified and characterized. The properties of CiAPE were compared with those of human APE1. The CiAPE protein was about 34 kDa. It efficiently cleaved tetrahydrofuran (THF)-containing duplex oligonucleotides. In addition, the transformation of E.coli, BW9093 Δxth with CiAPE complemented the H2O2 sensitivity. These results demonstrated that CiAPE has AP endonuclease activity to prevent against oxidative stress in Ciona intestinalis. Less
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Report
(3 results)
Research Products
(26 results)
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[Book] 電磁場生命科学(共著書)2005
Author(s)
張秋梅, 米井脩治
Total Pages
366
Publisher
京都大学学術出版会
Description
「研究成果報告書概要(和文)」より
Related Report
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