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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 | Shi Chen | en |
dc.contributor.author | 陳曦 | zh_TW |
dc.date.accessioned | 2021-06-15T05:19:30Z | - |
dc.date.available | 2012-09-13 | |
dc.date.copyright | 2010-09-13 | |
dc.date.issued | 2010 | |
dc.date.submitted | 2010-07-20 | |
dc.identifier.citation | Adhikari S., Toretsky J.A., Yuan L., Roy R. (2006) Magnesium, essential for base excision repair enzymes, inhibits substrate binding of N-methylpurine-DNA glycosylase. J Biol Chem 281:29525-32.
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(1995) DNA determinants and substrate specificities of Escherichia coli MutY. J Biol Chem 270:23582-8. Memisoglu A., Samson L. (2000) Base excision repair in yeast and mammals. Mutat Res 451:39-51. Moe A., Ringvoll J., Nordstrand L.M., Eide L., Bjoras M., Seeberg E., Rognes T., Klungland A. (2003) Incision at hypoxanthine residues in DNA by a mammalian homologue of the Escherichia coli antimutator enzyme endonuclease V. Nucleic Acids Res 31:3893-900. Ogawa A., Murate T., Suzuki M., Nimura Y., Yoshida S. (1998) Lithocholic acid, a putative tumor promoter, inhibits mammalian DNA polymerase beta. Jpn J Cancer Res 89:1154-9. Patro J.N., Urban M., Kuchta R.D. (2009) Interaction of human DNA polymerase alpha and DNA polymerase I from Bacillus stearothermophilus with hypoxanthine and 8-oxoguanine nucleotides. Biochemistry 48:8271-8. Plotz G., Piiper A., Wormek M., Zeuzem S., Raedle J. (2006) Analysis of the human MutLalpha.MutSalpha complex. Biochem Biophys Res Commun 340:852-9. Saparbaev M., Laval J. (1994) Excision of hypoxanthine from DNA containing dIMP residues by the Escherichia coli, yeast, rat, and human alkylpurine DNA glycosylases. Proc Natl Acad Sci U S A 91:5873-7. Schanz S., Castor D., Fischer F., Jiricny J. (2009) Interference of mismatch and base excision repair during the processing of adjacent U/G mispairs may play a key role in somatic hypermutation. Proc Natl Acad Sci U S A 106:5593-8. Stachelek G.C., Dalal S., Donigan K.A., Campisi Hegan D., Sweasy J.B., Glazer P.M. (2010) Potentiation of temozolomide cytotoxicity by inhibition of DNA polymerase beta is accentuated by BRCA2 mutation. Cancer Res 70:409-17. Tuteja N., Tuteja R. (2004) Prokaryotic and eukaryotic DNA helicases. Essential molecular motor proteins for cellular machinery. Eur J Biochem 271:1835-48. Watkins N.E., Jr., SantaLucia J., Jr. (2005) Nearest-neighbor thermodynamics of deoxyinosine pairs in DNA duplexes. Nucleic Acids Res 33:6258-67. Weiss B. (2008) Removal of deoxyinosine from the Escherichia coli chromosome as studied by oligonucleotide transformation. DNA Repair (Amst) 7:205-12. Wright G.E., Hubscher U., Khan N.N., Focher F., Verri A. (1994) Inhibitor analysis of calf thymus DNA polymerases alpha, delta and epsilon. FEBS Lett 341:128-30. Xia L., Zheng L., Lee H.W., Bates S.E., Federico L., Shen B., O'Connor T.R. (2005) Human 3-methyladenine-DNA glycosylase: effect of sequence context on excision, association with PCNA, and stimulation by AP endonuclease. J Mol Biol 346:1259-74. Yao M., Kow Y.W. (1995) Interaction of deoxyinosine 3'-endonuclease from Escherichia coli with DNA containing deoxyinosine. J Biol Chem 270:28609-16. Yao M., Hatahet Z., Melamede R.J., Kow Y.W. (1994) Purification and characterization of a novel deoxyinosine-specific enzyme, deoxyinosine 3' endonuclease, from Escherichia coli. J Biol Chem 269:16260-8. 尤詠絮, (2009) 亞黃嘌呤核酸鹼基切除修復試管中測定系統之研發,國立台灣大學碩士論文。 王議霆,(2010) 核酸內切酶第五型主導之修復系統於試管中亞黃嘌呤核酸修復區段之分析,國立台灣大學碩士論文。 | |
dc.identifier.uri | http://tdr.lib.ntu.edu.tw/jspui/handle/123456789/46625 | - |
dc.description.abstract | 生物體細胞中的DNA進行自發性的反應或是受到外源性的攻擊,導致特定的核酸損傷,例如腺嘌呤(Adenine; A)產生自發性的水解或是遭受致烷基劑的傷害即會進行脫胺作用而轉變為亞黃嘌呤 (Hypoxanthine;Hx),而以亞黃嘌呤為含氮鹼基在DNA當中構成的base稱為deoxyinosine (dI)。因dI在DNA進行複製時傾向與dC配對,一旦產生這樣的核酸損傷而未被完整的修復,極可能衍生為A:T→G:C的transitoin mutation。
已知人類細胞中的alkyladenine DNA glycosylase (AAG)可將dI移除進行BER (base excision repair)的修復。然而在大腸桿菌中除了BER系統之外,另會藉由nfi基因的產物endonuclease V所主導的 alternative excision repair (AER)途徑修復DNA中的dI。先前文獻在哺乳類動物小鼠以及人類細胞中皆已發現endo V的homolog,且由試管中實驗證實小鼠細胞的endonuclease V會於dI 3’端第二個磷酸鍵進行切除而形成3’端氫氧基與5’端磷酸基,此部分與已知的E.coli endo V-mediated AER作用相同。因此本實驗以人類細胞萃取液將dI受質進行試管中修復來探討有哪些核酸修復系統會參與其中。 實驗結果發現,MMR proficient的HeLaS3以及MMR deficient(hMLH1 defective)的HCT116人類細胞萃取液皆可有效的修復dI-G受質。於HeLaS3細胞萃取液中加入DNA pol α, δ, ε的抑制劑aphidicolin(APH)進行反應,會降低大約50% dI-G受質的修復效率;然而於HCT116細胞萃取液中加入APH對受質修復的效率卻無顯著影響,因此推測MMR系統中的hMLH1會參與部分dI-G受質的修復。 dI-G受質於HeLaS3及HCT116人類細胞萃取液之反應需求進行分析皆發現,若不加入鎂離子修復反應無法進行;缺乏dNTPs修復效率會降低;缺乏ATP或加入ATP-γ-S則會大幅降低修復效率,推測將dI-G受質進行辨識及修復的蛋白是需要與ATP結合並且水解ATP。針對ATP做反應需求之濃度測試,發現ATP濃度太低或太高受質修復效率皆不佳,因此可知ATP對受質的修復扮演了決定性的角色。 Polβ除了參與在BER之中,推測也會涉及人類endonuclease V對dI受質的修復,我們以Polβ抑制劑lithocholic acid(LCA)發現其會抑制HeLaS3以及HCT116細胞萃取液對dI-G受質的修復效率,因此可知polβ對受質的修復具有一定的重要性。 另外,我們也以dI-T受質於相同條件下進行反應並與dI-G之修復效率相互比較後發現,dI-T於HeLaS3細胞萃取液中的修復效率較低;在HCT116細胞萃取液中則幾乎不被修復。這個結果暗示了在人類細胞中可能有不同的活性可以處理dI損傷,而不同的核酸修復系統對於dI-G與dI-T的作用活性也不相同。 | zh_TW |
dc.description.provenance | Made available in DSpace on 2021-06-15T05:19:30Z (GMT). No. of bitstreams: 1 ntu-99-R97424024-1.pdf: 2167536 bytes, checksum: 156061129c2f453c015d0eb9dfb4eb20 (MD5) Previous issue date: 2010 | en |
dc.description.tableofcontents | 總目次
總目次 I 圖目次 IV 中文摘要 1 英文摘要 3 縮寫表 5 前言 7 材料與方法 11 一、菌株 11 二、人類細胞株之繼代培養 11 三、人類細胞核萃取液之製備 12 四、突變噬菌體M13mp18 mutant之建構 13 五、M13mp18系列雙股核酸之製備 15 六、M13mp18系列單股核酸之製備 16 七、具亞黃嘌呤核酸鹼基之異雙股核酸之製備 17 (1) dI-G 異雙股核酸 17 (2) dI-T 異雙股核酸 19 八、異雙股核酸對測定用限制酵素之敏感度分析 20 (1) dI-G 異雙股核酸 20 (2) dI-T 異雙股核酸 21 九、試管中之修復反應與結果分析 22 結果 24 一、異雙股核酸對分析用限制酵素之敏感度分析 24 (1) dI-G 異雙股核酸 24 (2) dI-T 異雙股核酸 25 二、試管中dI-G異雙股核酸之修復反應分析 25 (1) dI-G受質反應時間與修復程度之關係 25 (2) dI-G受質於HeLaS3人類細胞萃取之反應需求以及加入 APH對修復反應之影響 26 (3) dI-G受質於MMR deficient HCT116人類細胞萃取液之反應需求以及加入APH對修復反應之影響 27 (4) 不同濃度ATP對dI-G受質於HeLaS3人類細胞萃取液中修復結果之影響 28 (5) 不同濃度ATP對dI-G受質於MMR deficient HCT116人類細胞萃取液中修復結果之影響 28 (6) Pol β抑制劑對dI-G受質於HeLaS3人類細胞萃取液中修復結果之影響 29 (7) Pol β抑制劑對dI-G受質於MMR deficient HCT116人類細胞萃取液中修復結果之影響 30 三、試管中dI-T異雙股核酸之修復反應分析 30 (1) dI-T受質於HeLaS3人類細胞萃取液之反應需求以及加入APH對修復反應之影響 30 (2) dI-T受質於MMR deficient HCT116人類細胞萃取液之反應需求以及加入APH對修復反應之影響 31 討論 33 圖 39 參考文獻 59 | |
dc.language.iso | zh-TW | |
dc.title | 人類細胞萃取液對亞黃嘌呤之修復機制分析 | zh_TW |
dc.title | Mechanistic Study of Deoxyinosine Excision Repair by Human Cell Extracts | en |
dc.type | Thesis | |
dc.date.schoolyear | 98-2 | |
dc.description.degree | 碩士 | |
dc.contributor.oralexamcommittee | 許濤,蔡芷季,林淑萍 | |
dc.subject.keyword | 亞黃嘌呤,人類細胞萃取液, | zh_TW |
dc.subject.keyword | Deoxyinosine,Human Cell Extracts,Hypoxanthine,alternative excision repair, | en |
dc.relation.page | 61 | |
dc.rights.note | 有償授權 | |
dc.date.accepted | 2010-07-21 | |
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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