方偉宏臺灣大學:醫學檢驗暨生物技術學研究所尤詠絮Yu, Yung-HsuYung-HsuYu2010-05-112018-07-062010-05-112018-07-062009U0001-1107200914120800http://ntur.lib.ntu.edu.tw//handle/246246/182962Deoxyinosine (hypoxanthine deoxyribonucleoside) in DNA is produced by the hydrolytic or nitrosative deamination of deoxyadenosine. Hypoxanthine pairs with cytosine during replication resulting in A:T to G:C transition mutation if it is not repaired. Deoxyinosine (dI) is excised in a repair pathway that is initiated by endonuclease V (endo V), the nfi gene product, in Escherichia coli. This enzyme cleaves the second phosphodiester bond 3'' to the deoxyinosine. It is suggested that DNA polymerase I and DNA ligase participate in the repair of deoxyinosine but the complete mechanism of deoxyinosine excision after endo V cleavage is not known. We constructed an dI:dG mispair heteroduplex substrate which dI is designed in the XhoI restriction enzyme site. It can be digested by XhoI if dI:dG is repaired to dG:dC. If not, it cannot be recognized by XhoI. We can determine the repair level by evaluating the digested products. Our results showed the repair levels of the dI:dG substrate were not significantly different between mismatch proficient NM522 extracts and mismatch repair-deficient extracts (GM2931, RK1517). We concluded that the involvement of MutHLS pathway in our assay system is insignificant. Furthermore, the repair level of AlkA extracts was similar with that observed in NM522 extracts. Repair of dI heterology was highly dependent on the presence of the nfi gene product in crude extracts. The repair level in endo V deficient extracts decreased to less than 50% of that of endo V-proficient extracts. We also tested the requirement for endo V-mediated repair in E.coli extracts. Mg2+ is indispensable in the repair of dI and addition of exogenous ATP or 4 dNTPs was not required. We also performed the repair reaction with three purified enzymes：endonuclease V, DNA polymerase I, and E. coli DNA ligase. To our surprise, without any additional component from cell extracts, these three enzymes alone were sufficient to reconstitute the repair in vitro. We also replaced DNA polymerase I with klenow fragment 3’→5’ exo- in the reconstitution reaction and found the dI heterology cannot be repaired. This observation clearly demonstrated that 3’→5’ exonuclease activity of DNA polymerase I is essential for the endo V-mediated excision repair assay.總目次 I目次 III文摘要 1文摘要 3寫表 4言 6料與方法 10一、菌株 10二、大腸桿菌細胞萃取物之製備 10三、突變噬菌體M13mp18 mutant之建構 11四、M13mp18系列雙股核酸之製備 12五、M13mp18系列單股核酸之製備 13六、具亞黃嘌呤核酸鹼基之異雙股核酸之製備 13七、異雙股核酸對測定用限制酵素之敏感度分析 15八、試管中之修復反應與結果分析 15九、DNA polymerase I（E.coli）與klenow fragment（3''→ 5''exonuclease-）之gap filling能力測試 16十、利用純化的蛋白質觀察含亞黃嘌呤的環狀異雙股核酸之修復情形 17十一、利用純化的蛋白質觀察直線型異雙股核酸之修復情形 17果 19一、異雙股核酸對分析用限制酵素之敏感度分析 19二、試管中異雙股核酸之修復反應分析 20(1) 反應時間與修復程度之關係 20(2) 含dI受質於MMR蛋白缺失之大腸桿菌細胞萃取液中之修復情形 20(3) 含dI受質於野生型、endonuclease V及AlkA蛋白缺失之大腸桿菌細胞萃取液中之修復情形 20(4) 缺乏Mg2+、ATP、dNTPs及加入ATP-γ-S對修復反應之影響 21三、DNA polymerase I與klenow fragment（3''→ 5'' exonuclease-）之gap filling能力測試 21四、利用純化的蛋白質觀察含亞黃嘌呤的環狀異雙股核酸之修復情形 22五、利用純化的蛋白質觀察直線型異雙股核酸之修復情形 23論 24 26考文獻 41application/pdf1924698 bytesapplication/pdfen-US亞黃嘌呤修復hypoxanthinedeoxyinosineBERAERhttp://ntur.lib.ntu.edu.tw/bitstream/246246/182962/1/ntu-98-R96424027-1.pdf