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完整後設資料紀錄
DC 欄位 | 值 | 語言 |
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dc.contributor.advisor | 方偉宏(Woei-horng Fang) | |
dc.contributor.author | Che-Yu Lin | en |
dc.contributor.author | 林哲宇 | zh_TW |
dc.date.accessioned | 2021-06-17T09:08:21Z | - |
dc.date.available | 2021-02-23 | |
dc.date.copyright | 2021-02-23 | |
dc.date.issued | 2021 | |
dc.date.submitted | 2021-02-01 | |
dc.identifier.citation | Baba, T., Ara, T., Hasegawa, M., Takai, Y., Okumura, Y., Baba, M., . . . Mori, H. (2006). Construction of Escherichia coli K-12 in-frame, single-gene knockout mutants: the Keio collection. Mol Syst Biol, 2, 2006 0008. doi:10.1038/msb4100050 Brutlag, D., Kornberg, A. (1972). Enzymatic synthesis of deoxyribonucleic acid. 36. A proofreading function for the 3' leads to 5' exonuclease activity in deoxyribonucleic acid polymerases. J Biol Chem, 247(1), 241-248. Retrieved from https://www.ncbi.nlm.nih.gov/pubmed/4336040 Budke, B., Kuzminov, A. (2006). Hypoxanthine incorporation is nonmutagenic in Escherichia coli. J Bacteriol, 188(18), 6553-6560. doi:10.1128/JB.00447-06 Cowart, M., Gibson, K. J., Allen, D. J., Benkovic, S. J. (1989). DNA substrate structural requirements for the exonuclease and polymerase activities of procaryotic and phage DNA polymerases. Biochemistry, 28(5), 1975-1983. doi:10.1021/bi00431a004 Cronan, J. E., Jr., Narasimhan, M. L., Rawlings, M. (1988). Insertional restoration of beta-galactosidase alpha-complementation (white-to-blue colony screening) facilitates assembly of synthetic genes. Gene, 70(1), 161-170. doi:10.1016/0378-1119(88)90114-x Dalhus, B., Arvai, A. S., Rosnes, I., Olsen, O. E., Backe, P. H., Alseth, I., . . . Bjoras, M. (2009). Structures of endonuclease V with DNA reveal initiation of deaminated adenine repair. Nat Struct Mol Biol, 16(2), 138-143. doi:10.1038/nsmb.1538 Feng, H., Klutz, A. M., Cao, W. (2005). Active site plasticity of endonuclease V from Salmonella typhimurium. Biochemistry, 44(2), 675-683. doi:10.1021/bi048752j Frederico, L. A., Kunkel, T. A., Shaw, B. R. (1990). A sensitive genetic assay for the detection of cytosine deamination: determination of rate constants and the activation energy. Biochemistry, 29(10), 2532-2537. doi:10.1021/bi00462a015 Friedberg, E. C., Walker, G. C., Siede, W., Wolfram, Wolfram, S. (1995). DNA Repair and Mutagenesis: ASM Press. Gates, F. T., 3rd, Linn, S. (1977). Endonuclease V of Escherichia coli. J Biol Chem, 252(5), 1647-1653. Retrieved from https://www.ncbi.nlm.nih.gov/pubmed/14159 He, B., Qing, H., Kow, Y. W. (2000). Deoxyxanthosine in DNA is repaired by Escherichia coli endonuclease V. Mutat Res, 459(2), 109-114. doi:10.1016/s0921-8777(99)00063-4 Hill-Perkins, M., Jones, M. D., Karran, P. (1986). Site-specific mutagenesis in vivo by single methylated or deaminated purine bases. Mutat Res, 162(2), 153-163. doi:10.1016/0027-5107(86)90081-3 Huang, J., Lu, J., Barany, F., Cao, W. (2001). Multiple cleavage activities of endonuclease V from Thermotoga maritima: recognition and strand nicking mechanism. Biochemistry, 40(30), 8738-8748. doi:10.1021/bi010183h Joyce, C. M., Kelley, W. S., Grindley, N. D. (1982). Nucleotide sequence of the Escherichia coli polA gene and primary structure of DNA polymerase I. J Biol Chem, 257(4), 1958-1964. Retrieved from https://www.ncbi.nlm.nih.gov/pubmed/6276402 Karran, P., Lindahl, T. (1980). Hypoxanthine in deoxyribonucleic acid: generation by heat-induced hydrolysis of adenine residues and release in free form by a deoxyribonucleic acid glycosylase from calf thymus. Biochemistry, 19(26), 6005-6011. doi:10.1021/bi00567a010 Klenow, H., Henningsen, I. (1970). Selective elimination of the exonuclease activity of the deoxyribonucleic acid polymerase from Escherichia coli B by limited proteolysis. Proc Natl Acad Sci U S A, 65(1), 168-175. doi:10.1073/pnas.65.1.168 Kuraoka, I. (2015). Diversity of Endonuclease V: From DNA Repair to RNA Editing. Biomolecules, 5(4), 2194-2206. doi:10.3390/biom5042194 Lam, W. C., Van der Schans, E. J., Sowers, L. C., Millar, D. P. (1999). Interaction of DNA polymerase I (Klenow fragment) with DNA substrates containing extrahelical bases: implications for proofreading of frameshift errors during DNA synthesis. Biochemistry, 38(9), 2661-2668. doi:10.1021/bi9820762 Lee, C. C., Yang, Y. C., Goodman, S. D., Lin, C. J., Chen, Y. A., Wang, Y. T., . . . Fang, W. H. (2013). The excision of 3' penultimate errors by DNA polymerase I and its role in endonuclease V-mediated DNA repair. DNA Repair (Amst), 12(11), 899-911. doi:10.1016/j.dnarep.2013.08.003 Lee, C. C., Yang, Y. C., Goodman, S. D., Yu, Y. H., Lin, S. B., Kao, J. T., . . . Fang, W. H. (2010). Endonuclease V-mediated deoxyinosine excision repair in vitro. DNA Repair (Amst), 9(10), 1073-1079. doi:10.1016/j.dnarep.2010.07.007 Lehman, I. R., Bessman, M. J., Simms, E. S., Kornberg, A. (1958). Enzymatic synthesis of deoxyribonucleic acid. I. Preparation of substrates and partial purification of an enzyme from Escherichia coli. J Biol Chem, 233(1), 163-170. Retrieved from https://www.ncbi.nlm.nih.gov/pubmed/13563462 Lehman, I. R., Chien, J. R. (1973). Persistence of deoxyribonucleic acid polymerase I and its 5'--3' exonuclease activity in PolA mutants of Escherichia coli K12. J Biol Chem, 248(22), 7717-7723. Retrieved from https://www.ncbi.nlm.nih.gov/pubmed/4584339 Martin, F. H., Castro, M. M., Aboul-ela, F., Tinoco, I., Jr. (1985). Base pairing involving deoxyinosine: implications for probe design. Nucleic Acids Res, 13(24), 8927-8938. doi:10.1093/nar/13.24.8927 Mi, R., Alford-Zappala, M., Kow, Y. W., Cunningham, R. P., Cao, W. (2012). Human endonuclease V as a repair enzyme for DNA deamination. Mutat Res, 735(1-2), 12-18. doi:10.1016/j.mrfmmm.2012.05.003 Moe, A., Ringvoll, J., Nordstrand, L. M., Eide, L., Bjoras, M., Seeberg, E., . . . 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(14), 3893-3900. doi:10.1093/nar/gkg472 Moosmann, P., Rusconi, S. (1996). Alpha complementation of LacZ in mammalian cells. Nucleic Acids Res, 24(6), 1171-1172. doi:10.1093/nar/24.6.1171 Morita, Y., Shibutani, T., Nakanishi, N., Nishikura, K., Iwai, S., Kuraoka, I. (2013). Human endonuclease V is a ribonuclease specific for inosine-containing RNA. Nat Commun, 4, 2273. doi:10.1038/ncomms3273 Myrnes, B., Guddal, P. H., Krokan, H. (1982). Metabolism of dITP in HeLa cell extracts, incorporation into DNA by isolated nuclei and release of hypoxanthine from DNA by a hypoxanthine-DNA glycosylase activity. Nucleic Acids Res, 10(12), 3693-3701. doi:10.1093/nar/10.12.3693 Pang, B., McFaline, J. L., Burgis, N. E., Dong, M., Taghizadeh, K., Sullivan, M. R., . . . Dedon, P. C. (2012). Defects in purine nucleotide metabolism lead to substantial incorporation of xanthine and hypoxanthine into DNA and RNA. Proc Natl Acad Sci U S A, 109(7), 2319-2324. doi:10.1073/pnas.1118455109 Saparbaev, M., Mani, J. C., Laval, J. (2000). Interactions of the human, rat, Saccharomyces cerevisiae and Escherichia coli 3-methyladenine-DNA glycosylases with DNA containing dIMP residues. Nucleic Acids Res, 28(6), 1332-1339. doi:10.1093/nar/28.6.1332 Shapiro, R., Pohl, S. H. (1968). The reaction of ribonucleosides with nitrous acid. Side products and kinetics. Biochemistry, 7(1), 448-455. doi:10.1021/bi00841a057 Su, K. Y., Goodman, S. D., Lai, H. M., Yen, R. S., Hu, W. Y., Cheng, W. C., . . . Fang, W. H. (2018). Proofreading and DNA Repair Assay Using Single Nucleotide Extension and MALDI-TOF Mass Spectrometry Analysis. J Vis Exp(136). doi:10.3791/57862 Su, K. Y., Lai, H. M., Goodman, S. D., Hu, W. Y., Cheng, W. C., Lin, L. I., . . . Fang, W. H. (2018). Application of single nucleotide extension and MALDI-TOF mass spectrometry in proofreading and DNA repair assay. DNA Repair (Amst), 61, 63-75. doi:10.1016/j.dnarep.2017.11.011 Su, K. Y., Lin, L. I., Goodman, S. D., Yen, R. S., Wu, C. Y., Chang, W. C., . . . Fang, W. H. (2018). DNA polymerase I proofreading exonuclease activity is required for endonuclease V repair pathway both in vitro and in vivo. DNA Repair (Amst), 64, 59-67. doi:10.1016/j.dnarep.2018.02.005 Weiss, B. (2008). Removal of deoxyinosine from the Escherichia coli chromosome as studied by oligonucleotide transformation. DNA Repair (Amst), 7(2), 205-212. doi:10.1016/j.dnarep.2007.09.010 Yao, M., Hatahet, Z., Melamede, R. J., Kow, Y. W. (1994a). Deoxyinosine 3' endonuclease, a novel deoxyinosine-specific endonuclease from Escherichia coli. Ann N Y Acad Sci, 726, 315-316. doi:10.1111/j.1749-6632.1994.tb52837.x Yao, M., Hatahet, Z., Melamede, R. J., Kow, Y. W. (1994b). Purification and characterization of a novel deoxyinosine-specific enzyme, deoxyinosine 3' endonuclease, from Escherichia coli. J Biol Chem, 269(23), 16260-16268. Retrieved from https://www.ncbi.nlm.nih.gov/pubmed/8206931 Yao, M., Kow, Y. W. (1995). Interaction of deoxyinosine 3'-endonuclease from Escherichia coli with DNA containing deoxyinosine. J Biol Chem, 270(48), 28609-28616. doi:10.1074/jbc.270.48.28609 Yao, M., Kow, Y. W. (1996). Cleavage of insertion/deletion mismatches, flap and pseudo-Y DNA structures by deoxyinosine 3'-endonuclease from Escherichia coli. J Biol Chem, 271(48), 30672-30676. doi:10.1074/jbc.271.48.30672 Yao, M., Kow, Y. W. (1997). Further characterization of Escherichia coli endonuclease V. Mechanism of recognition for deoxyinosine, deoxyuridine, and base mismatches in DNA. J Biol Chem, 272(49), 30774-30779. doi:10.1074/jbc.272.49.30774 Zhang, Z., Jia, Q., Zhou, C., Xie, W. (2015). Crystal structure of E. coli endonuclease V, an essential enzyme for deamination repair. Sci Rep, 5, 12754. doi:10.1038/srep12754 | |
dc.identifier.uri | http://tdr.lib.ntu.edu.tw/jspui/handle/123456789/74827 | - |
dc.description.abstract | DNA 及核苷酸在生理條件下可自發性發生脫胺作用,而接觸強氧化物或是暴露於輻射﹑紫外光等會促使其反應 ; 其中由腺嘌呤脫胺後與去氧核醣結合形成的亞黃嘌呤,若未經修復,則會在進行 DNA 複製及轉錄時發生錯誤。第五型核酸內切酶修復途徑為大腸桿菌中修復亞黃嘌呤的主要途徑之一,本實驗室先前透過試管中修復試驗,得知在反應中只需第五型核酸內切酶﹑第一型 DNA 聚合酶和DNA 接合酶即可完成亞黃嘌呤之修復反應。過往研究推測第五型核酸內切酶除了與 DNA 上的亞黃嘌呤結合外,還可能促使其他參與修復的酵素進行修復 ; 本實驗室也曾在試管中修復試驗得知在第五型核酸內切酶與第一型 DNA 聚合酶共同存在下,第五型核酸內切酶有出現周轉的現象,然而兩個酵素之間是否有交互作用,目前仍未可知。為證明第五型核酸內切酶與第一型 DNA 聚合酶之間的交互作用,本研究嘗試建立具有第五型核酸內切酶的融合基因,並進行同源置換插入缺乏第五型核酸內切酶之菌株 (JW5547, nfi-),另一方面以純化之融合蛋白進行試管中修復試驗,觀察第五型核酸內切酶與第一型 DNA 聚合酶的交互作用。我們已嘗試建立出帶有第五型核酸內切酶的融合基因,並以同源置換插入缺乏第五型核酸內切酶之菌株,但透過西方墨點法已確認目前所建立之模型無法表現第五型核酸內切酶。另一方面,透過基質協助雷射去吸附離子化–飛行質譜儀之分析, 得知加入克列諾片段確實增加第五型核酸內切酶在修復時的周轉,然而目前以共同免疫沉澱並未觀察到第五型核酸內切酶與缺乏核酸外切酶的克列諾片段有結合。依上述結果得知無法以目前建立之模型進行第五型核酸內切酶的修復試驗, 但透過純化蛋白修復反應的結果,推測第五型核酸內切酶和第一型 DNA 聚合酶在修復亞黃嘌呤時存在交互作用,並促使第五型核酸內切酶之周轉。 | zh_TW |
dc.description.abstract | Deamination of adenine can occur spontaneously as a result of intracellular stress and can be enhanced by exposure of irradiation, UV light, or nitrosative agent, which generates highly mutagenic deoxyinosine lesion in DNA. Unrepaired deoxyinosine lesion tends to generate A:T to G:C transition mutation during replication. In Escherichia coli (E. coli) , deoxyinosine is primarily removed through the pathway initiated by endonuclese V (Endo V). In previous study, it was suggested that deoxyinosine lesion could be repaired by only endo V, DNA polymearse I (Pol I) and DNA ligase in the in vitro deoxyinosine repair assay. In addition, we found that Endo V turnovered in deoxyinosine lesion repair with Pol I. To further investigate the interaction between Endo V and Pol I, we established maltose-binding protein (MBP)-fused Endo V and knocked into Endo V-deficient E. coli through homologous recombination, to examine that Endo V and Pol I have a contact during dI lesion repair. Unfortunately, we couldn’t get Endo V expression according to the result of Western blot. On the other hand, we discovered that the turnover of Endo V was promoted in the in vitro dI repair assay when reacted together with Klenow fragment. However, we haven’t obsevred interaction between exonuclease-decifient Klenow fragment and Endo V during dI lesion repair yet. In summary, we could not establish functional Endo V to repair dI in vivo. However, we suggest that Endo V could interact with Pol I in repairing dI lesion process and promote the turnover of Endo V. | en |
dc.description.provenance | Made available in DSpace on 2021-06-17T09:08:21Z (GMT). No. of bitstreams: 1 U0001-0102202110103800.pdf: 3149869 bytes, checksum: 026fa440be527a42bd7eb37fd4893005 (MD5) Previous issue date: 2021 | en |
dc.description.tableofcontents | 總目次 致謝 .............................................................................................................. I 中文摘要 .................................................................................................... II Abstract ..................................................................................................... III 圖目次 ....................................................................................................... VI 縮寫表 .................................................................................................... VIII 一﹑背景介紹 ............................................................................................. 1 1.1 亞黃嘌呤的產生 .................................................................................................... 1 1.2 第五型核酸內切酶 ............................................................................................... 2 1.3 第一型 DNA 聚合酶 ............................................................................................ 3 1.4 亞黃嘌呤之修復機制 ............................................................................................ 4 1.5 研究動機及目的 ................................................................................................... 5 二﹑材料與方法 ......................................................................................... 6 2.1 材料 ....................................................................................................................... 6 2.1.1 菌株 ............................................................................................................... 6 2.1.2 質體 ............................................................................................................... 6 2.1.3 核酸序列 ....................................................................................................... 7 2.2 構築具有nfi 上﹑下游序列,以及MBP-Endo V 之質體 ................................ 8 2.2.1 構築nfi 上﹑下游序列 ................................................................................. 8 2.2.2 構築MBP-Endo V ....................................................................................... 10 2.2.3 嵌入四環黴素抗藥基因(Tetracycline resistance gene, TcR) ..................... 11 2.3 抽取細胞萃取液 ................................................................................................... 12 2.4 西方墨點法 ........................................................................................................... 13 2.5 同源置換 ............................................................................................................. 13 2.6 以MALDI-TOF MS 分析Endo V 與Pol I 共同作用 ...................................... 14 2.7 共同免疫沉澱法 ................................................................................................... 15 2.7.1 試管中dI 修復試驗 ...................................................................................... 15 2.7.2 免疫沉澱 ........................................................................................................ 15 2.7.3 西方墨點法 .................................................................................................... 16 三﹑結果 ................................................................................................... 17 3.1 構築nfi 上﹑下游序列以及MBP-Endo V ....................................................... 17 3.1.1 構築nfi 上﹑下游序列 ................................................................................ 17 3.1.2 構築MBP-Endo V 及TcR .......................................................................... 17 3.2 以同源置換將MBP-Endo V 送入缺乏Endo V 之大腸桿菌 ........................... 18 3.3 MBP-Endo V 蛋白表現及活性之試驗 .............................................................. 18 3.4 以MALDI-TOF-MS 觀察KF 協同Endo V 進行修復反應 ............................. 19 3.5 Endonuclease V 與DNA polymerase I 之交互作用 .......................................... 19 四﹑討論及結論 ....................................................................................... 20 4.1 構築pBSK-Endo V-TcR 之過程 ....................................................................... 20 4.2 MBP-Endo V 於E.coli 中不表現 ....................................................................... 20 4.3 KF 協同Endo V 進行dI 修復反應 .................................................................... 21 4.4 Endo V 與Pol I 之交互作用 ............................................................................... 22 4.5 結論及未來展望 ................................................................................................. 22 參考文獻 ................................................................................................... 45 附錄 ........................................................................................................... 49 圖目次 圖一﹑含MBP-Endo V 之質體設計圖 ................................................. 24 圖二﹑於pBlueScript II SK(+)上正確嵌入nfi 上游序列 (H1) ........... 25 圖三﹑於pBlueScript II SK(+)上正確嵌入nfi 下游序列 (H2) ........... 26 圖四﹑於pBlueScript II SK(+)上同時嵌入nfi 上﹑下游序列 ............. 27 圖五﹑於pET His6 MBP TEV LIC (2M-T)上嵌入nfi 序列 ................. 29 圖六﹑於pBSK-H12 上嵌入MBP-Endo V .......................................... 31 圖七﹑於pBSK-Endo V 上嵌入TcR ...................................................... 33 圖八﹑於JW5547 中成功轉形pKD46 ................................................. 35 圖九﹑使MBP-Endo V 置換插入JW5547 genomic DNA .................... 36 圖十﹑轉形pBSK-Endo V 的細胞萃取液未發現MBP-Endo V 表現 . 37 圖十一﹑KF 促使Endo V 於修復dI 反應時進行周轉 ........................ 39 圖十二﹑以Anti-Pol I 偵測KF 3’exo- .................................................. 42 圖十三﹑Endo V 與KF 3’exo-於修復dI 反應時無交互作用 .............. 43 附錄 附錄一﹑亞黃嘌呤之形成途徑 .............................................................. 49 附錄二﹑亞黃嘌呤之修復途徑 ............................................................ 50 附錄三﹑Pol I 對於不同正常鹼基與dI 配對時的校正比例 ................ 51 附錄四﹑基質協助雷射去吸附離子化–飛行時間質譜儀 .................... 52 附錄五﹑Pol I 或Klenow fragment 可協同Endo V 進行dI 修復反應 53 附錄六﹑pol I exo-表現阻礙pol I 與Endo V 進行修復dI 反應 ......... 54 | |
dc.language.iso | zh-TW | |
dc.title | 第五型核酸內切酶與第一型DNA聚合酶在亞黃嘌呤修復中交互作用之研究 | zh_TW |
dc.title | Study of interaction of endonuclease V and DNA polymerase I in deoxyinosine repair | en |
dc.type | Thesis | |
dc.date.schoolyear | 109-1 | |
dc.description.degree | 碩士 | |
dc.contributor.oralexamcommittee | 蘇剛毅(Kang-Yi Su),郭靜穎(Ching-Ying Kuo),蔡芷季(Jyy-Jih Tsai) | |
dc.subject.keyword | 第五型核酸內切酶,第一型DNA聚合酶,亞黃嘌呤,克列諾片段,基質協助雷射去吸附離子化-飛行質譜儀, | zh_TW |
dc.subject.keyword | Endonuclease V,DNA polymerase I,deoxyinosine,Klenow fragment,MALDI-TOF-MS, | en |
dc.relation.page | 54 | |
dc.identifier.doi | 10.6342/NTU202100313 | |
dc.rights.note | 有償授權 | |
dc.date.accepted | 2021-02-02 | |
dc.contributor.author-college | 醫學院 | zh_TW |
dc.contributor.author-dept | 醫事技術學研究所 | zh_TW |
顯示於系所單位: | 醫學檢驗暨生物技術學系 |
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