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標題: | 尿嘧啶核酸鹼基切除修復試管中測定系統之研發 Development of a New Method for Base Excision Repair of Uracil in vitro |
作者: | Chun-Chieh Ma 馬俊傑 |
指導教授: | 方偉宏 |
關鍵字: | 尿嘧啶,核酸修復, uracil,DNA repair, |
出版年 : | 2010 |
學位: | 碩士 |
摘要: | 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。 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. |
URI: | http://tdr.lib.ntu.edu.tw/jspui/handle/123456789/45145 |
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顯示於系所單位: | 醫學檢驗暨生物技術學系 |
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