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研究生:吳秉憲
研究生(外文):Ping-Hsien Wu
論文名稱:核酸聚合酶第一型核酸內切酶於亞黃嘌呤修復中之切除長度分析
論文名稱(外文):Mapping the DNA polymerase I proofreading exonuclease excision track in deoxyinosine repair
指導教授:方偉宏方偉宏引用關係
指導教授(外文):Woei-Horng Fang
口試委員:蔡芷季楊雅倩許濤
口試委員(外文):Zhi-Ji CaiYa-Chien YangTodd Hsu
口試日期:2015-07-29
學位類別:碩士
校院名稱:國立臺灣大學
系所名稱:醫學檢驗暨生物技術學研究所
學門:醫藥衛生學門
學類:醫學技術及檢驗學類
論文種類:學術論文
論文出版年:2015
畢業學年度:103
語文別:中文
論文頁數:53
中文關鍵詞:亞黃嘌呤單核酸擴展焦磷酸定序DNA核酸聚合酶核酸內切酶第五型
外文關鍵詞:dIDNA polymerase Iproofreadingendonuclease V3''-5'' exonuclease
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亞黃嘌呤 (hypoxanthine, Hx)是腺嘌呤(adenine)經由外源性或自發性刺激,進行去胺化作用(deamination)的產物,與五碳糖結合時,稱之為亞黃嘌呤核酸(deoxyinosine, dI)。亞黃嘌呤核酸(dI)在DNA複製時傾向於與dC配對,如未修復則將會形成A to G的transition mutation。因此,DNA修復系統對於維持基因複製的忠誠性,扮演著相當重要的角色
目前已知大腸桿菌會利用endonuclease V-mediated alternative excision repair (AER)的途徑修復DNA中的亞黃嘌呤核酸 (dI),機制為: 由endonuclease V (endo V)辨認出dI錯誤,並於dI 3''端第二個磷酸二酯鍵切出缺口,DNA polymerase I (Pol I)再將錯誤部分去除,並合成正確的核酸,最後再由DNA ligase補起缺口完成修復作用。由先前本實驗室建構出純化蛋白系統,證明了AER是修復dI的主要路徑,且發現僅endo V、Pol I,以及DNA ligase的參與即可對dI進行修復,也顯示Pol I的3''-5'' exonuclease活性對於dI修復有非常重要的影響。為了釐清dI修復的細節,我們於不具3''-5'' exonuclease活性的聚合酶中,外加入Pol I,進行焦磷酸定序;我們也利用單核酸擴展的概念,設計多種配對錯誤組合,進行修復反應後以質譜儀偵測,發現Pol I可以連同dI與配對錯誤的部分一起去除,同時在僅有dI錯誤存在時,則僅切除至dI的位點,但以缺乏3''-5'' exonuclease活性的聚合酶進行反應時,則無法產生切除現象。這個結果能夠幫助我們釐清Pol I的3''-5'' exonuclease 活性於AER中去除錯誤的詳細狀況。


DNA is subjected to deamination at a physiologically significant rate. Deoxyinosine in DNA can arise from deamination of deoxyadenosine, which can be spontaneous or promoted by exposure of DNA to ionizing radiation, UV light, or nitrous acid. Deoxyinosine in DNA can pair with cytosine during replication resulting in A:T to G:C transitions. Specific mechanisms for removal of deaminated bases have been evolved. Several bacterial, archaeal and eukaryotic organisms contain an evolutionary conserved enzyme, endonuclease V (endoV) that recognizes deaminated adenine in DNA. In E. coli, endoV incises DNA at the second phosphodiester bond 3'' to the dI lesion, leaving a 3'' OH and a 5'' P termini. Previously, we developed an in vitro assay to score the dI repair with cell extracts and purified proteins. We found that dI lesions were repaired by endoV mediated repair with high efficiency. In a purified system, endoV, DNA polymerase I (Pol I) and ligase are sufficient for the repair. We also demonstrated that 3'' to 5'' exonuclease (exo) activity of Pol I was very important for processing deoxyinosine lesions. To get better understanding how the dI lesion is processed by Pol I 3'' exo, in this study we designed dI-containing oligo-duplex and utilized proofreading in trans concept supplementing Pol I to proofreading deficient polymerase based reaction mixture in pyro-sequencing dI-containing procedure. We also utilized single nucleotide extension approach to map excision path during dI processing by mass spectrometry. We found Pol I could remove mismatched terminal base and activate sequencing reaction for the reaction otherwise to be inactive when containing proofreading deficient polymerase alone. The correction patch was mapped to the last mismatched base-pair, and no extra paired nucleotide was removed. This study might provide valuable information regarding the excision role of Pol I in endoV DNA repair pathway.

總目次
總目次 I
圖目次 III
表目次 IV

中文摘要 1
英文摘要 2
縮寫表 3
前言 5
材料與方法 11
一、寡鏈核苷酸 11
二、酵素 12
三、核苷酸 12
四、Pyrosequencing kit 12
五、偵測機台 12
六、焦磷酸定序測試 12
七、外加具有校對外切酶之DNA聚合酶進行焦磷酸定序 13
八、dNTPαS取代dNTP進行焦磷酸定序 14
九、焦磷酸定序加入endonuclease V模擬dI修復狀況 14
十、利用質譜儀系統偵測primer和template並偵測分析修復區 段 14

結果 16
一、焦磷酸定序測試 16
二、外加具有校對外切酶之DNA聚合酶進行焦磷酸定序 17
三、dNTPαS取代dNTP進行焦磷酸定序 18
四、焦磷酸定序加入endonuclease V模擬dI修復狀況 19
五、利用質譜儀系統偵測primer和template 19
六、以質譜儀系統偵測分析修復區段 21

討論 23
參考文獻 51

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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。


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