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| DC 欄位 | 值 | 語言 |
|---|---|---|
| dc.contributor.advisor | 蔡明道 | |
| dc.contributor.author | "Liang Hin, Lim" | en |
| dc.contributor.author | 林良興 | zh_TW |
| dc.date.accessioned | 2021-06-17T02:14:28Z | - |
| dc.date.available | 2021-01-04 | |
| dc.date.copyright | 2018-01-04 | |
| dc.date.issued | 2017 | |
| dc.date.submitted | 2017-11-13 | |
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| dc.identifier.uri | http://tdr.lib.ntu.edu.tw/jspui/handle/123456789/68192 | - |
| dc.description.abstract | DNA polymerase beta (Pol beta) is a key enzyme involved in base excision repair and other DNA metabolism pathway involving gap-filling DNA synthesis. Recently, numerous of biochemical studies illustrated that Pol beta interacts with various proteins and the properties of the enzyme changed during the interaction event. However, there is no any structural and mechanism study about Pol beta with interacting partners has been solved and published until now. In the first part of the present study, we aim to understand the binding mechanism of Pol beta with proliferating cell nuclear antigen (PCNA) by determining the crystal structures of Pol beta/PCNA complex and the Pol beta/PCNA/DNA ternary complex. We have succeeded to obtain the of Pol beta/PCNA complex crystals and the Pol beta/PCNA/DNA ternary complex crystals and both of the complexes only diffracted to 4.5 Å resolution. The diffraction analysis indicated that Pol beta/PCNA complex belonged to space group P222, with unit-cell parameters a = 63, b = 155, c = 185 Å and the Pol beta/PCNA/DNA ternary complex belonged to space group P1, with unit-cell parameters a = 74, b = 111, c = 113 Å. We will further to optimize the crystallization conditions as well as the cryo-protection conditions to facilitate structural determination.
The second part of this study, we focus on the binding mechanism of Pol beta with protein arginine methyltransferase 6 (PRMT6) by determining the crystal structure. Our result showed that Pol beta binds to PRMT6 tightly with a Kd value of 16.8 ± 2.7 μM. However, we failed to crystallize the Pol beta/PRMT6 complex and we only crystallized PRMT6 alone at the crystallization experiments. We will also continue to screen for the optimized conditions and also the additive that can stabilize and crystalize the Pol beta/ PRMT6 complex for the structure determination. | en |
| dc.description.provenance | Made available in DSpace on 2021-06-17T02:14:28Z (GMT). No. of bitstreams: 1 ntu-106-R01b46012-1.pdf: 4280957 bytes, checksum: cff33f444e7d15d1cf815ee8877829bb (MD5) Previous issue date: 2017 | en |
| dc.description.tableofcontents | 論文口試委員審定書 i
謝誌 ii 中文摘要 iii Abstract iv Table of contents vi List of Figures viii List of Tables x Chapter 1 Introduction 1 1-1 DNA Damage and Repair 1 1-2 Base Excision Repair (BER) 2 1-3 DNA Polymerase Beta ( Pol beta) 4 1-4 Proliferating Cell Nuclear Antigen (PCNA) 6 1-5 The Interaction of Pol beta and PCNA 8 1-6 Protein Arginine Methyltransferases（PRMTs） 9 1-7 Protein Arginine Methyltransferase 6（PRMT6） 11 1-8 The Interaction of Pol beta and PRMT6 13 1-9 The Scope of This Thesis 14 Chapter 2 Materials and Methods 15 2-1 The Structural Study of Pol Beta/PCNA/DNA Complex 15 2-1-1 Overexpression and Purification of Pol beta 15 2-1-2 Overexpression and Purification of PCNA 17 2-1-3 DNA Annealing 18 2-1-4 The Preparation of Pol beta/PCNA Complex 20 2-1-5 The Preparation of Pol Beta/PCNA/DNA Complex 20 2-1-6 The Crystallization of Pol beta/PCNA Complex 22 2-1-7 The Crystallization of Pol Beta/PCNA/DNA Complex 26 2-1-8 Data Collection and Processing 31 2-2 The Structural Study of Pol Beta/PRMT6 Complex 33 2-2-1 Overexpression and Purification of Human PRMT6 33 2-2-2 Overexpression and Purification of Mouse PRMT6 35 2-2-3 The Preparation of Pol Beta/Mouse PRMT6 Complex 36 2-2-4 Characterizing Pol Beta/Mouse PRMT6 by Analytical Ultracentrifugation (AUC) 2-2-5 The Biolayer Interferometry Study of Pol Beta and Mouse PRMT6 38 2-2-6 The Crystallization of Pol Beta/Mouse PRMT6 Complex 38 2-2-7 Data Collection and Processing 41 Chapter 3 Results and Discussions 42 3-1 The Structural Study of Pol Beta/PCNA/DNA Complex 42 3-1-1 The Characteristic of Pol Beta/PCNA Complex in Solution 42 3-1-2 The Crystallization of Pol Beta/PCNA Complex 44 3-1-3 The Characteristic of Pol Beta/PCNA/DNA Complex in Solution 48 3-1-4 The Crystallization of Pol Beta/PCNA/DNA Complex 50 3-2 The Structural Study of Pol Beta/PRMT6 Complex 52 3-2-1 The Characteristic of Human PRMT6 and Mouse PRMT6 52 3-2-2 The Characteristic of Pol beta/mouse PRMT6 Complex in Solution. 52 3-2-3 The Crystallization of Pol Beta/ Mouse PRMT6 Complex 55 References 56 Appendixes 61 | |
| dc.language.iso | en | |
| dc.subject | PRMT6 | zh_TW |
| dc.subject | 甲基移?? | zh_TW |
| dc.subject | 鹼基切除修復 | zh_TW |
| dc.subject | PCNA | zh_TW |
| dc.subject | DNA聚合?β | zh_TW |
| dc.subject | X-ray繞射 | zh_TW |
| dc.subject | X-ray | en |
| dc.subject | PCNA | en |
| dc.subject | BER | en |
| dc.subject | Methyltransferase | en |
| dc.subject | PRMT6 | en |
| dc.subject | DNA polymerase beta | en |
| dc.title | DNA 聚合酶 β與其交互作用蛋白之結構機制探討 | zh_TW |
| dc.title | Structural Studies of DNA Polymerase Beta with Interacting Partners | en |
| dc.type | Thesis | |
| dc.date.schoolyear | 106-1 | |
| dc.description.degree | 碩士 | |
| dc.contributor.oralexamcommittee | 張震東,張茂山 | |
| dc.subject.keyword | DNA聚合?β,PCNA,鹼基切除修復,甲基移??,PRMT6,X-ray繞射, | zh_TW |
| dc.subject.keyword | DNA polymerase beta,PCNA,BER,Methyltransferase,PRMT6,X-ray, | en |
| dc.relation.page | 90 | |
| dc.identifier.doi | 10.6342/NTU201704361 | |
| dc.rights.note | 有償授權 | |
| dc.date.accepted | 2017-11-13 | |
| dc.contributor.author-college | 生命科學院 | zh_TW |
| dc.contributor.author-dept | 生化科學研究所 | zh_TW |
| 顯示於系所單位： | 生化科學研究所 | |
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