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完整後設資料紀錄
DC 欄位 | 值 | 語言 |
---|---|---|
dc.contributor.advisor | 方偉宏 | |
dc.contributor.author | Chun-Chieh Ma | en |
dc.contributor.author | 馬俊傑 | zh_TW |
dc.date.accessioned | 2021-06-15T04:06:16Z | - |
dc.date.available | 2010-03-12 | |
dc.date.copyright | 2010-03-12 | |
dc.date.issued | 2010 | |
dc.date.submitted | 2010-02-08 | |
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Travisano, et al. (2003). 'A role for the mismatch repair system during incipient speciation in Saccharomyces.' J Evol Biol 16(3): 429-37. Grilley, M., J. Griffith, et al. (1993). 'Bidirectional excision in methyl-directed mismatch repair.' J Biol Chem 268(16): 11830-7. Guo, G., Y. Ding, et al. (1997). 'nfi, the gene for endonuclease V in Escherichia coli K-12.' J Bacteriol 179(2): 310-6. Holmes, J., Jr., S. Clark, et al. (1990). 'Strand-specific mismatch correction in nuclear extracts of human and Drosophila melanogaster cell lines.' Proc Natl Acad Sci U S A 87(15): 5837-41. Hou, E. W., R. Prasad, et al. (2007). 'Comparative assessment of plasmid and oligonucleotide DNA substrates in measurement of in vitro base excision repair activity.' Nucleic Acids Res 35(17): e112. Impellizzeri, K. J., B. Anderson, et al. (1991). 'The spectrum of spontaneous mutations in a Saccharomyces cerevisiae uracil-DNA-glycosylase mutant limits the function of this enzyme to cytosine deamination repair.' 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Lutsenko, E. and A. S. Bhagwat (1999). 'The role of the Escherichia coli mug protein in the removal of uracil and 3,N(4)-ethenocytosine from DNA.' J Biol Chem 274(43): 31034-8. Lyamichev, V., M. A. Brow, et al. (1993). 'Structure-specific endonucleolytic cleavage of nucleic acids by eubacterial DNA polymerases.' Science 260(5109): 778-83. Matsumoto, Y. and K. Kim (1995). 'Excision of deoxyribose phosphate residues by DNA polymerase beta during DNA repair.' Science 269(5224): 699-702. Modrich, P. (1997). 'Strand-specific mismatch repair in mammalian cells.' J Biol Chem 272(40): 24727-30. Mourgues, S., M. E. Lomax, et al. (2007). 'Base excision repair processing of abasic site/single-strand break lesions within clustered damage sites associated with XRCC1 deficiency.' Nucleic Acids Res 35(22): 7676-87. Nilsen, H., G. Stamp, et al. (2003). 'Gene-targeted mice lacking the Ung uracil-DNA glycosylase develop B-cell lymphomas.' Oncogene 22(35): 5381-6. Riccio, A., L. A. Aaltonen, et al. 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'The rate of base excision repair of uracil is controlled by the initiating glycosylase.' DNA Repair (Amst) 7(11): 1869-81. Wei, H., C. Therrien, et al. (2008). 'The Fidelity Index provides a systematic quantitation of star activity of DNA restriction endonucleases.' Nucleic Acids Res 36(9): e50. Wyatt, M. D., J. M. Allan, et al. (1999). '3-methyladenine DNA glycosylases: structure, function, and biological importance.' Bioessays 21(8): 668-76. Yao, M. and Y. W. Kow (1994). 'Strand-specific cleavage of mismatch-containing DNA by deoxyinosine 3'-endonuclease from Escherichia coli.' J Biol Chem 269(50): 31390-6. 尤詠絮 (2009) 亞黃嘌呤鹼基切除修復試管中測定系統之研發 國立台灣大學98學年碩士論文 | |
dc.identifier.uri | http://tdr.lib.ntu.edu.tw/jspui/handle/123456789/45145 | - |
dc.description.abstract | 在DNA中出現尿嘧啶(Uracil; U)可能來自於DNA進行複製時,在腺嘌呤(Adenine; A)錯配了尿嘧啶,產生不具致突變性的dU:dA錯配。另一種可能則是由胞嘧啶(Cytosine; C)發生自發性或氧化脫胺反應(deamination)而形成尿嘧啶與鳥糞嘌呤(Guanine; G)的配對錯誤。這是一種高致突變性的錯誤,若是未被正確修復,則在複製後會造成C:G轉變成為T:A的突變。
大腸桿菌中主要是利用鹼基切除修復(base excision repair; BER)對DNA中的U進行修復。首先由uracil-DNA glycosylase將DNA中的dU移除形成無鹼基配對處(abasic site; AP site),接著再由AP endonuclease、DNA polymerase、DNA ligase對AP site進行切割修飾後,填入正確的鹼基並進行黏合完成修復反應。 本研究欲利用限制酵素對於識別序列的專一性,發展一個不需要使用放射性同位素即可偵對dU:dG修復的方法。我們製備出雙股環狀DNA在被破壞的XbaI識別序列中包含了dU:dG。dU/dG 經過正確的修復成dC:dG之後則可被XbaI水解,因此可藉限制酵素的水解來評估DNA中dU:dG的修復情形。 我們先用大腸桿菌萃取液進行dU/dG的修復實驗,我們發現無已知修復缺陷的 NM522菌株可有效的修復dU/dG,然而,利用三種不同修復系統缺陷的突變株RK1517 (mutS), JW5547 (nfi), BD2314 (ung)的萃取液進行反應,皆能修復dU:dG且修復程度與 NM522相當,因此我們推測大腸桿菌中有不同的修復系統可對dU:dG進行修復。 利用純化蛋白質UDG、Exo III、Pol I及DNA ligase建立的純化系統可以有效的修復dU:dG,證明我們所設計dU:dG受質為BER的良好受質。我們也利用Endo V、Pol I、DNA ligase建構的純化系統進行dU:dG修復反應,但修復效率比BER比較顯著的降低。 我們也利用具有MMR缺失(HCT 116)以及無MMR缺失(HeLa S3) 的人類細胞核萃取液在對dU:dG進行修復。在HeLa S3實驗中加入uracil glycosylase inhibitor可以抑制對此dU/dG的修復,顯示人類細胞BER參與對於此受質的修復。而在不加入ATP的情況下則修復程度會降低則顯示MMR參與修復的可能。因此推測在人類細胞中有不同系統對dU/dG修復有重疊的現象。 | zh_TW |
dc.description.abstract | Uracil (U) in DNA may come from incorporation of dU instead of dT opposite adenine during replication, outcoming the non-mutagenic dU:dA pair. Uracil also can be introduced by hydrolytic deamination of cytosine which paired with guanine. Highly mutagenic dU:dG mispair may cause a C→T transition mutation upon replication if it was not repaired.
Base excision repair (BER) is the major pathway for repairing the uracil in DNA. It’s initiated by uracil-DNA glycosylase (UDG), which removes the uracil, leaving an abasic site. After AP endonuclease cleave at 5’ to the AP site, DNA polymerase will incorporate the right base and the nick would be ligated by DNA ligase to complete the repair. In this study, we developed a non-radio isotope and convenient method to detect the repair of dU:dG.. The specificity of restriction enzyme was applied to score the repair.. We constructed a circular DNA substrate containing an dU:dG mispair within a disrupted XbaI recognition site. Correction of dU:dG to dC:dG would restore the XbaI site. Thus, the repair level can be evaluated by restriction digestion. We subjected this dU/dG heteroduplex to E. coli cell free extracts for in vitro repair assay. Our results showed that dU:dG substrate can be repaired efficiently by extracts from strain NM522, which has no known defect in DNA repair. We also subjected dU/dG substrate to extracts from several DNA repair deficient strain such as RK1517 (mutS)), JW5547 (nfi), BD2314 (ung) and we found the repair levels were comparable to that from NM522. These results suggested that there were overlapping of repair pathways in processing dU:dG in E. coli extracts. We used the purified proteins to reconstitute the BER (UDG, Exo III Pol I, DNA ligase) and Alternative excision repair (Endo V, Pol I, DNA ligase ) pathways. We found that dU:dG was the good substrate for BER, however the repair efficiency of dU/dG was much reduced in AER system. The dU/dG heteroduplex was also tested in human cell nuclear extracts from MMR deficient HCT 116 and MMR proficient HeLa S3. We also found there were an overlapping of repair pathways in processing dU/dG. Additon of uracil glycosylase inhititor reduced the repair level of HeLa S3 indicating the involvement of BER system. Omission of exogenous ATP reduced repair indicating the possible involvement of MMR in processing dU/dG. | en |
dc.description.provenance | Made available in DSpace on 2021-06-15T04:06:16Z (GMT). No. of bitstreams: 1 ntu-99-R96424020-1.pdf: 1089909 bytes, checksum: 57124f9244380f1d86f07cfaa1fb4747 (MD5) Previous issue date: 2010 | en |
dc.description.tableofcontents | 總目次 I
圖目次 III 表目次 V 中文摘要 1 Abstract 2 縮寫表 3 前言 5 材料與方法 10 一、菌株 10 二、人類細胞株之繼代培養 10 三、限制酵素對含尿密定序列的專一性測試 11 四、突變噬菌體M13mp18 mutant之建構 12 五、M13mp18系列雙股核酸之製備 13 六、M13mp18系列單股核酸之製備 14 七、具尿嘧啶核酸鹼機織異雙股核酸之製備 15 巴、異雙股核酸對測定用限制酵素之敏感度分析 16 九、大腸桿菌細胞萃取物之製備 17 十、人類細胞核萃取物之至倍 17 十一、試管中之修復反應與結果分析 18 十二、利用純化的蛋白質觀察鹼基切除修復系統對含尿嘧啶環狀異雙股核酸之修復情形 20 十三、利用純化的蛋白質觀察AER系統對含尿嘧啶環狀異雙股核酸之修復情形 21 十四、利用純化的蛋白質觀察AER系統對含尿嘧啶直線型異雙股核酸之修復情形 21 結果 23 一、限制酵素對含尿嘧啶序列的專一性測試 23 二、雙股核酸對測定用限制酵素之敏感度分析 23 三、試管中之修復反應與結果分析 23 (1) 含dU受質於MMR蛋白缺失之大腸桿菌細胞萃取液中之修復情形 23 (2) 反應時間與修復程度的關係 24 (3) 含dU受質於不同mutant大腸桿菌細胞萃取液中之修復情形 24 (4) 大腸桿菌修復尿嘧啶之反應需求 25 (5) UDG對於大腸桿菌修復尿嘧啶之影響 26 四、利用純化的蛋白質觀察E. coli鹼基切除修復系統對含尿嘧啶環狀異雙股核酸之修復情形 26 五、利用純化的蛋白質觀察AER系統對含尿嘧啶環狀異雙股核酸之修復情形 27 六、利用純化的蛋白質觀察AER系統對含尿嘧啶直線型異雙股核酸之修復情形 27 七、試管中人類細胞對異雙股核酸之修復反應分析 28 (1)受質M13x22U於不同量之人類細胞核萃取液中之修復情形 28 (2) M13x22U受質於MMR缺陷知人類細胞核萃取液中之修復情形 28 (3)加入uracil glycosylase inhibitor對人類細胞修復反應之影響 29 (4)人類細胞修復尿嘧啶修復尿嘧啶之反應需求 29 討論 31 圖 36 表 61 參考文獻 62 | |
dc.language.iso | zh-TW | |
dc.title | 尿嘧啶核酸鹼基切除修復試管中測定系統之研發 | zh_TW |
dc.title | Development of a New Method for Base Excision Repair of Uracil in vitro | en |
dc.type | Thesis | |
dc.date.schoolyear | 98-1 | |
dc.description.degree | 碩士 | |
dc.contributor.oralexamcommittee | 許濤,高照村,蔡芷季 | |
dc.subject.keyword | 尿嘧啶,核酸修復, | zh_TW |
dc.subject.keyword | uracil,DNA repair, | en |
dc.relation.page | 65 | |
dc.rights.note | 有償授權 | |
dc.date.accepted | 2010-02-08 | |
dc.contributor.author-college | 醫學院 | zh_TW |
dc.contributor.author-dept | 醫學檢驗暨生物技術學研究所 | zh_TW |
顯示於系所單位: | 醫學檢驗暨生物技術學系 |
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