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完整後設資料紀錄
DC 欄位 | 值 | 語言 |
---|---|---|
dc.contributor.advisor | 方偉宏(Woei-Horng Fang) | |
dc.contributor.author | Yi-An Chen | en |
dc.contributor.author | 陳怡安 | zh_TW |
dc.date.accessioned | 2021-06-13T08:23:13Z | - |
dc.date.available | 2011-10-07 | |
dc.date.copyright | 2011-10-07 | |
dc.date.issued | 2011 | |
dc.date.submitted | 2011-07-19 | |
dc.identifier.citation | Abbotts, J., and Loeb, L.A. (1985). DNA polymerase alpha and models for proofreading. Nucleic Acids Res 13, 261-274.
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DNA polymerase proofreading: active site switching catalyzed by the bacteriophage T4 DNA polymerase. Nucleic Acids Res 35, 5452-5463. Garg, P., Stith, C.M., Sabouri, N., Johansson, E., and Burgers, P.M. (2004). Idling by DNA polymerase delta maintains a ligatable nick during lagging-strand DNA replication. Genes Dev 18, 2764-2773. Goodman, M.F., and Fygenson, K.D. (1998). DNA polymerase fidelity: from genetics toward a biochemical understanding. Genetics 148, 1475-1482. Huang, Y.M., Chen, S.U., Goodman, S.D., Wu, S.H., Kao, J.T., Lee, C.N., Cheng, W.C., Tsai, K.S., and Fang, W.H. (2004). Interaction of nick-directed DNA mismatch repair and loop repair in human cells. J Biol Chem 279, 30228-30235. Johnson, K.A. (2010). The kinetic and chemical mechanism of high-fidelity DNA polymerases. Biochim Biophys Acta 1804, 1041-1048. Karthikeyan, R., Vonarx, E.J., Straffon, A.F., Simon, M., Faye, G., and Kunz, B.A. (2000). Evidence from mutational specificity studies that yeast DNA polymerases delta and epsilon replicate different DNA strands at an intracellular replication fork. J Mol Biol 299, 405-419. Kelman, Z., and O'Donnell, M. (1995). DNA polymerase III holoenzyme: structure and function of a chromosomal replicating machine. Annu Rev Biochem 64, 171-200. Kornberg, A. (1957). Enzymatic synthesis of deoxyribonucleic acid. Harvey Lect 53, 83-112. Lam, W.C., Van der Schans, E.J., Joyce, C.M., and Millar, D.P. (1998). Effects of mutations on the partitioning of DNA substrates between the polymerase and 3'-5' exonuclease sites of DNA polymerase I (Klenow fragment). Biochemistry 37, 1513-1522. Maki, H., and Kornberg, A. (1987). Proofreading by DNA polymerase III of Escherichia coli depends on cooperative interaction of the polymerase and exonuclease subunits. Proc Natl Acad Sci U S A 84, 4389-4392. Maor-Shoshani, A., Reuven, N.B., Tomer, G., and Livneh, Z. (2000). Highly mutagenic replication by DNA polymerase V (UmuC) provides a mechanistic basis for SOS untargeted mutagenesis. Proc Natl Acad Sci U S A 97, 565-570. McCain, M.D., Meyer, A.S., Schultz, S.S., Glekas, A., and Spratt, T.E. (2005). Fidelity of mispair formation and mispair extension is dependent on the interaction between the minor groove of the primer terminus and Arg668 of DNA polymerase I of Escherichia coli. Biochemistry 44, 5647-5659. Morales, J.C., and Kool, E.T. (2000). Importance of terminal base pair hydrogen-bonding in 3'-end proofreading by the Klenow fragment of DNA polymerase I. Biochemistry 39, 2626-2632. Patel, P.H., Suzuki, M., Adman, E., Shinkai, A., and Loeb, L.A. (2001). Prokaryotic DNA polymerase I: evolution, structure, and 'base flipping' mechanism for nucleotide selection. J Mol Biol 308, 823-837. Pursell, Z.F., Isoz, I., Lundstrom, E.B., Johansson, E., and Kunkel, T.A. (2007). Yeast DNA polymerase epsilon participates in leading-strand DNA replication. 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A possible mechanism for the dynamics of transition between polymerase and exonuclease sites in a high-fidelity DNA polymerase. J Theor Biol 259, 434-439. Xie, P. (2009b). A possible mechanism for the dynamics of transition between polymerase and exonuclease sites in a high-fidelity DNA polymerase. Theoretical Biology 434-439. | |
dc.identifier.uri | http://tdr.lib.ntu.edu.tw/jspui/handle/123456789/36928 | - |
dc.description.abstract | 核酸為各個生物體的遺傳物質,因此核酸在複製時的精確性甚為重要,核酸複製的忠誠度主要由三個步驟所決定,一為核酸聚合酶 (DNA polymerase)複製核酸時一連串結構上的改變,以確保將正確的dNTP接上正確的位置;二為核酸聚合酶(DNA polymerase)在遇到末端有錯誤配對時會以3’-5’exonuclease的酵素活性作校正 (proofreading);三為複製完核酸後細胞可以啟動例如 mismatch repair system (MMR)等修復系統修復仍存在的錯誤配對;如此一來可以把核酸複製的錯誤率降至約10-10左右。
已知E. coli DNA polymerase I會對於最末端的錯誤配對有校正 (proofreading)的功能,並且過去的研究並沒有發現如果存在足量dNTP的情況之下,primer末端倒數第二個位置有錯誤配對會活化DNA polymerase I的3’-5’ exonuclease的活性。由於核酸聚合酶 (Pol I)在校正 (proofreading)反應完成之後,會繼續往下游複製核酸,合成正確的鹼基配對,於是為了探究DNA polymerase I在核酸校正方面更深入的特性,我們便可以利用限制酵素對於識別序列具有一定程度的專一性,來偵測我們所製備的核酸受質是否有被校正的現象。 我們設計不同的錯誤配對,並且在各種錯誤配對的3’下游一個鹼基後產生一個nick,以測試單純DNA polymerase I作用的情況,並看DNA polymerase I對於倒數第二個位置出現的鹼基錯誤配對,是否仍然可以啟動它校正的活性。由於想觀察酵素反應作用初期的反應狀況,我們將不同的核酸受質,與DNA polymerase I反應,將反應時間縮小為十分鐘之內,每兩分鐘一個間隔觀察核酸聚合酶的反應情況,並比較對於不同的核酸受質,核酸修復效率與反應速率的差異。 實驗結果發現,對於距末端斷股上游第二個位置的鹼基錯誤配對,在T-T,T-G,T-C錯誤配對的受質當中,都發現DNA polymerase I會與受質反應,並且以線狀的受質 (linear DNA substrate)測試結果發現,此反應可以忽略DNA polymerase I在受質上面作nick translation的程度,顯示DNA polymerase I對於倒數第二個位置的錯誤鹼基配對,的確能夠有校正 (proofreading)活性。 | zh_TW |
dc.description.abstract | DNA is genetic information for all organisms. It is important for maintaining fidelity during replicating. There are three steps that maintain the high fidelity. The first is structure switch for ensuring the right dNTP on the right place. The second is proofreading activity. DNA polymerase can remove the mismatch at the end of the primer then extension continually. The other is repair systems. Based on the above, the error frequency can decrease to about 10-10.
There is no evidence showing that the second last mismatch can activate 3’ to 5’ exonuclease proofreading activity of DNA polymerase I. Besides, we known that after proofreading, Pol I can replicate DNA continually and form correct base pairs. For searching the detail of proofreading, we used the specificity of restriction enzyme to monitor whether the substrate was repaired or not. We designed different mismatch substrates. Besides, there was a nick downstream from the mismatch. In order to search the preliminary reaction of DNA polymerase I, we designed the reaction time within ten minutes and then monitored every two minutes. In addition, we compared the difference of proofreading efficiency and analysis of enzyme kinetic between different DNA substrates. Our results showed that DNA polymerase I could react with DNA substrates including T-T, T-G, and T-C. We also used linear substrate to test DNA polymerase I proofreading activity. It showed that this assay can ignore the extent of nick translation. These results suggested that the proofreading activity of DNA polymerase I can also be activated even when the mismatch located not on the end. | en |
dc.description.provenance | Made available in DSpace on 2021-06-13T08:23:13Z (GMT). No. of bitstreams: 1 ntu-100-R98424018-1.pdf: 1078196 bytes, checksum: 92619cd9d8e2aedb116601a86985b7c3 (MD5) Previous issue date: 2011 | en |
dc.description.tableofcontents | 總目次 I
圖目次 III 表目次 IV 中文摘要 1 英文摘要 3 縮寫表 4 前言 6 材料與方法 11 一、菌株 11 二、酵素 11 三、突變噬菌體f1PM mutant之建構 11 四、f1PM系列雙股核酸之製備 13 五、f1PM系列單股核酸之製備 14 六、異雙股核酸之製備 15 七、異雙股核酸對測定用限制酵素之敏感度分析 16 八、DNA Pol I校正活性測定 16 結果 18 一、核酸受質之構建與限制酵素敏感度分析 18 二、核酸聚合酶 I 對核酸受質作用之活性分析 19 三、核酸聚合酶 I 對線狀異雙股核酸受質之作用分析 20 四、以KF和3’ exo- KF取代Pol I測試其修復反應 20 五、異雙股核酸受質經Pol I 作用後之修復股確認 21 六、核酸聚合酶 I 對核酸受質作用之酵素反應初期分析 22 (1) T-T 異雙股核酸 22 (2) T-G 異雙股核酸 22 (3) T-C 異雙股核酸 22 七、核酸聚合酶 I 對不同核酸受質的反應速率與效率比較 23 討論 25 圖 30 表 47 參考文獻 50 | |
dc.language.iso | zh-TW | |
dc.title | 核酸聚合酶 I 對於模版與引子交會處帶有異雙股的核酸受質之校正活性分析 | zh_TW |
dc.title | Proofreading Activity of DNA Pol I to Substrate Containing Heterologies within Template-primer Junction | en |
dc.type | Thesis | |
dc.date.schoolyear | 99-2 | |
dc.description.degree | 碩士 | |
dc.contributor.oralexamcommittee | 高照村,許濤,蔡芷季 | |
dc.subject.keyword | 校正反應,核酸複製忠誠度,核酸聚合酶,核酸聚合酶,I,核酸外切活性,錯誤配對,限制酵素,異雙股核酸, | zh_TW |
dc.subject.keyword | proofreading,fidelity,polymerase,polymerase I,exonuclease activity,mismatch,restriction enzyme,heteroduplex DNA, | en |
dc.relation.page | 53 | |
dc.rights.note | 有償授權 | |
dc.date.accepted | 2011-07-20 | |
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 |
顯示於系所單位: | 醫學檢驗暨生物技術學系 |
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