請用此 Handle URI 來引用此文件：
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| DC 欄位 | 值 | 語言 |
|---|---|---|
| dc.contributor.advisor | 方偉宏(Woei-Horng Fang) | |
| dc.contributor.author | Ching-I Lin | en |
| dc.contributor.author | 林靜宜 | zh_TW |
| dc.date.accessioned | 2022-11-25T05:36:44Z | - |
| dc.date.available | 2024-10-08 | |
| dc.date.copyright | 2021-11-11 | |
| dc.date.issued | 2021 | |
| dc.date.submitted | 2021-10-08 | |
| dc.identifier.citation | 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. doi:10.1016/s0021-9258(19)45781-5 Cao, W. (2013). Endonuclease V: an unusual enzyme for repair of DNA deamination. Cell Mol Life Sci, 70(17), 3145-3156. doi:10.1007/s00018-012-1222-z Chang, H. L., Su, K. Y., Goodman, S. D., Chou, N. A., Lin, K. C., Cheng, W. C., . . . Fang, W. H. (2020). Proofreading of single nucleotide insertion/deletion replication errors analyzed by MALDI-TOF mass spectrometry assay. DNA Repair (Amst), 88, 102810. doi:10.1016/j.dnarep.2020.102810 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 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 Gates, F. T., III, Linn, S. (1977). Endonuclease V of Escherichia coli. J Biol Chem., 252(5), 1647-1653. doi:10.1016/s0021-9258(17)40598-9 Gut, I. G. (2004). DNA analysis by MALDI-TOF mass spectrometry. Hum Mutat, 23(5), 437-441. doi:10.1002/humu.20023 Jacobsen, H., Klenow, H., Overgaard-Hansen, K. (1974). The N-terminal amino-acid sequences of DNA polymerase I from Escherichia coli and of the large and the small fragments obtained by a limited proteolysis. Eur J Biochem, 45(2), 623-627. doi:10.1111/j.1432-1033.1974.tb03588.x 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. Journal of Biological Chemistry, 257(4), 1958-1964. doi:10.1016/s0021-9258(19)68132-9 Karas, M., Hillenkamp, F. (1988). Laser Desorption Ionization of Proteins with Molecular Masses Exceeding 10000 Daltons. Anal Chem, 60(20), 2299-2301. doi:10.1021/ac00171a028 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 Kaufmann, R. (1995). Matrix-assisted laser desorption ionization (MALDI) mass spectrometry: a novel analytical tool in molecular biology and biotechnology. J Biotechnol., 41(2-3), 155-175. doi:10.1016/0168-1656(95)00009-f Kirpekar, F., Nordhoff, E., Kristiansen, K., Roepstorff, P., Lezius, A., Hahner, S., . . . Hillenkamp, F. (1994). Matrix assisted laser desorption/ionization mass spectrometry of enzymatically synthesized RNA up to 150 kDa. Nucleic Acids Res, 22(19), 3866–3870. doi:10.1093/nar/22.19.3866 Kuraoka, I. (2015). Diversity of Endonuclease V: From DNA Repair to RNA Editing. Biomolecules, 5(4), 2194-2206. doi:10.3390/biom5042194 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. Lindahl, T. (1993). Instability and decay of the primary structure of DNA. Nature, 362(6422), 709-715. doi:10.1038/362709a0 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., Abole, A. K., Cao, W. (2011). Dissecting endonuclease and exonuclease activities in endonuclease V from Thermotoga maritima. Nucleic Acids Res, 39(2), 536-544. doi:10.1093/nar/gkq791 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 Modrich, P. (1987). DNA mismatch correction. Annu Rev Biochem., 56, 435-466. doi:10.1146/annurev.bi.56.070187.002251 Nordhoff, E., Cramer, R., Karas, M., Hillenkamp, F., Kirpekar, F., Kristiansen, K., Roepstorff, P. (1993). Ion stability of nucleic acids in infrared matrix-assisted laser desorption:ionization mass spectrometry. Nucleic Acids Res. doi:10.1093/nar/21.15.3347 Pieles, U., Zürcher, W., Schär, M., Moser, H. E. (1993). Matrix-assisted laser desorption ionization time-of-flight mass spectrometry: a powerful tool for the mass and sequence analysis of natural and modified oligonucleotides. Nucleic Acids Res, 21(14), 3191–3196. doi:10.1093/nar/21.14.3191 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 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 Wu, K. J., Steding, A., Becker, C. H. (1993). Matrix-assisted laser desorption time-of-flight mass spectrometry of oligonucleotides using 3-hydroxypicolinic acid as ultraviolet-sensitive matrix. Rapid Commun Mass Spectrom, 7(2), 142-146. doi:10.1002/rcm.1290070206 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(23), 16260-16268. doi:10.1016/s0021-9258(17)34002-4 Yao, M., Kow, Y. W. (1994). Strand-specific cleavage of mismatch-containing DNA by deoxyinosine 3'-endonuclease from Escherichia coli. Journal of Biological Chemistry, 269(50), 31390-31396. doi:10.1016/s0021-9258(18)31706-x 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, W., Evans, D. H. (1995). DNA strand transfer catalyzed by the 5'-3' exonuclease domain of Escherichia coli DNA polymerase I. Nucleic Acids Res, 23(22), 4620-4627. doi:10.1093/nar/23.22.4620 | |
| dc.identifier.uri | http://tdr.lib.ntu.edu.tw/jspui/handle/123456789/82140 | - |
| dc.description.abstract | 在一般生理環境下，DNA的腺嘌呤 (adenine; A)會自發性水解脫胺(deamination)產生脫氧肌苷 (deoxyinosine ; dI)。Deoxyinosine如果未修復，在DNA複製時，因為dI偏好與dCTP結合，會造成A:T變G:C的transition mutations。當DNA暴露由輻射、UV、亞硝酸鹽、熱等引起的ROS環境時，會促進adenine的deamination。在Escherichia coli (E. coli)中，dI的修復反應由endonuclease V (Endo V; nfi gene product) nicking作為起始。先前本實驗室進行 in vitro minimum component reconstitution assay時，發現Endo V、DNA polymerase I (Pol I)和E. coli DNA ligase共同反應後成功修復dI，而且發現Pol I的3’-5’ exonuclease對於修復十分重要。先前研究發現Endo V在辨識dI後，會在dI 3’端第二個phosphodiester bond產生一個nick，之後Endo V會結合在DNA上，不會turnover。然而，先前本實驗室進行 in vitro assay時，加入有proofreading能力的Pol I或Klenow fragment (KF)的情況下，Endo V與Pol I turnover增加至少4倍。這結果顯示Endo V與Pol I在修復反應過程中可能有交互作用。 為了證明Endo V和Pol I之間在修復dI時可能有交互作用，我們使用MALDI-TOF MS進行分析，當Endo V修復有dI的DNA時會產生nicking product，並且有分子量上的改變，藉由分子量的差異可以辨識出nicking product。使用dI ssDNA substrate、I-T dsDNA substrate和dI與不同正常鹼基配對的DNA substrate分析Endo V nicking活性，發現針對不同substrate具有不同的修復效率。其中，Endo V對含dI ssDNA有較高的活性。 使用40 bp I-T substrate進行實驗時，發現Endo V在反應一段時間後幾乎沒有turnover，之後追加等量的Endo V進行反應，發現增加等比例的nicking products。而當在反應中加入KF時，Endo V total product有明顯增加，顯示KF可能是藉由proofreading exonuclease活性移除dI來促進Endo V turnover。為了驗證這個假設，我們使用proofreading exonuclease deficiency KF (KF exo-)和24 bp I-T substrate進行實驗。我們意外發現KF exo-也可以促進Endo V turnover。之後我們使用40 bp I-T和I-C substrate並在反應中加入KF或KF exo-進行實驗，我們推論Endo V和KF之間的interaction是透過Endo V nicking後使dI露出，讓KF辨識後進行修復，並且值得進一步深入研究。為了進一步研究in vitro和in vivo中，Pol I和Endo V在修復過程中的交互作用，我們設計帶有CC marker 的I-T異雙股質體進行實驗。我們已經構築出有selected marker的質體來進行substrate preparation。 | zh_TW |
| dc.description.provenance | Made available in DSpace on 2022-11-25T05:36:44Z (GMT). No. of bitstreams: 1 U0001-0410202111280400.pdf: 3579667 bytes, checksum: 5dea93876881b1deb8de1af3547583a7 (MD5) Previous issue date: 2021 | en |
| dc.description.tableofcontents | 致謝 I 摘要 II Abstract IV 總目次 VI 圖目次 IX 附錄目次 X 縮寫表 XI 第一章、 背景介紹及研究動機 1 1.1 DNA出現亞黃嘌呤 (Hypoxanthine) 1 1.2 亞黃嘌呤的修復機制 1 1.3 第五型核酸內切酶 2 1.4 第一型DNA聚合酶 3 1.5 基質輔助雷射去吸附離子化飛行時間質譜儀 (Matrix-Assisted Laser Desorption Ionization-Time of Flight Mass Spectrometry ; MALDI-TOF MS) 4 1.6 研究動機 5 第二章、 實驗材料與方法 7 2.1 核酸序列 7 2.2 菌株 8 2.3 質體 8 2.4 噬菌體 8 2.5 酵素與試劑 9 2.5.1 酵素 9 2.5.2 試劑 9 2.6 MALDI-TOF MS測定Endo V修復dI活性 9 2.7 MALDI-TOF MS測定額外Endo V促進修復dI活性 10 2.8 MALDI-TOF MS測定Endo V與KF修復dI活性 11 2.9 MALDI-TOF MS測定Endo V對於不同dI配對的修復活性 12 2.10 MALDI-TOF MS測定Endo V與KF的交互作用 12 2.11 MALDI-TOF MS 13 2.12反應產物濃度計算 14 2.12.1 訊號標準化 14 2.12.2 Endo V nicking product 濃度計算 14 2.12.3 Endo V與KF repair product 濃度計算 14 2.12.4 4S28TI+24I nicking product濃度計算 15 2.13製備帶有CC marker的I-T異雙股質體 15 2.13.1構築CC marker 15 2.13.2 菌落分離 16 2.13.3 XhoI (+)質體構築第二段oligonucleotide 17 2.13.4製備XhoI oligo2單股DNA 19 2.13.5 製備AflII (+)質體雙股DNA 19 2.13.6製備缺口 (gap)異雙股質體 21 2.13.7製備超螺旋 (super coil) I-T異雙股質體 21 第三章、 實驗結果 23 3.1 MALDI-TOF MS測定Endo V修復反應 23 3.2 MALDI-TOF MS測定endoV對於dI與不同鹼基配對的修復反應 23 3.3 MALDI-TOF MS測定KF促進Endo V turn over 24 3.4 MALDI-TOF MS測定Endo V和KF的交互作用 25 3.5 製備帶有CC marker的I-T異雙股質體 26 3.5.1構築CC marker及菌落分離 26 3.5.2 XhoI (+)質體構築第二段oligonucleotide並製備單股DNA 27 3.5.3製備超螺旋 (Super coil)異雙股質體 27 第四章、 總結與討論 29 4.1 使用MALDI-TOF MS探討Endo V與KF對dI修復反應 29 4.2 Endo V對於dI與不同鹼基配對的修復反應 29 4.3 KF促進Endo V修復dI 30 4.4 CC marker的I-T異雙股質體製備問題 31 4.5 結論 31 第五章、 圖 33 附錄 53 參考文獻 59 | |
| dc.language.iso | zh-TW | |
| dc.subject | 第五型核酸內切酶 | zh_TW |
| dc.subject | 第一型DNA聚合酶 | zh_TW |
| dc.subject | 脫氧肌苷 | zh_TW |
| dc.subject | 克列諾片段 | zh_TW |
| dc.subject | 基質輔助雷射去吸附離子化飛行時間質譜儀 | zh_TW |
| dc.subject | matrix-Assisted Laser Desorption Ionization-Time of Flight Mass Spectrometry | en |
| dc.subject | deoxyinosine | en |
| dc.subject | endonuclease V | en |
| dc.subject | DNA polymerase I | en |
| dc.subject | Klenow fragment | en |
| dc.title | 應用質譜儀進行第五型核酸內切酶與第一型DNA聚合酶修復亞黃嘌呤之研究 | zh_TW |
| dc.title | Application of MALDI-TOF Mass Spectrometry for studying endonuclease V and DNA polymerase I repair of deoxyinosine | en |
| dc.date.schoolyear | 109-2 | |
| dc.description.degree | 碩士 | |
| dc.contributor.oralexamcommittee | 蘇剛毅(Hsin-Tsai Liu),郭靜穎(Chih-Yang Tseng),蔡芷季,許濤 | |
| dc.subject.keyword | 脫氧肌苷,第五型核酸內切酶,第一型DNA聚合酶,克列諾片段,基質輔助雷射去吸附離子化飛行時間質譜儀, | zh_TW |
| dc.subject.keyword | deoxyinosine,endonuclease V,DNA polymerase I,Klenow fragment,matrix-Assisted Laser Desorption Ionization-Time of Flight Mass Spectrometry, | en |
| dc.relation.page | 62 | |
| dc.identifier.doi | 10.6342/NTU202103527 | |
| dc.rights.note | 同意授權(限校園內公開) | |
| dc.date.accepted | 2021-10-08 | |
| 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 |
| dc.date.embargo-lift | 2024-10-08 | - |
| 顯示於系所單位： | 醫學檢驗暨生物技術學系 | |
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