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完整後設資料紀錄
DC 欄位 | 值 | 語言 |
---|---|---|
dc.contributor.advisor | 蔡明道(Ming-Daw Tsai) | |
dc.contributor.author | Mu-Sen Liu | en |
dc.contributor.author | 劉沐森 | zh_TW |
dc.date.accessioned | 2021-06-15T12:43:09Z | - |
dc.date.available | 2019-08-02 | |
dc.date.copyright | 2016-08-02 | |
dc.date.issued | 2016 | |
dc.date.submitted | 2016-07-26 | |
dc.identifier.citation | References
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dc.identifier.uri | http://tdr.lib.ntu.edu.tw/jspui/handle/123456789/50494 | - |
dc.description.abstract | 研究DNA聚合酶之忠實性機制對於化學催化和生物意義上是個基礎且重要的議題。在2000年以前高忠實性複製型DNA聚合酶已被廣泛的研究,然而對於低忠實性修復型DNA聚合酶則較少被研究。本實驗室藉由X-射線結晶學解出12個DNA聚合酶 λ 蛋白質晶體結構並使用酵素動力學測量DNA聚合酶 λ 如何藉由本身結構特性調節忠實性之功能,亦藉由生物物理之測量方法發現DNA聚合酶 λ 具有無需DNA之存在下可以結合dNTP。本實驗室解出第一個apo-型DNA聚合酶 λ,發現apo-型DNA聚合酶 λ 之dNTP結合位已和催化型活化態之三級結構一樣,總和以上發現指出apo-型DNA聚合酶 λ 可以預先結合dNTP,這可以瞭解低忠實性修復型DNA聚合酶 λ 如何保持較低的忠實性。更進一步,DNA聚合酶 λ 之活化區具有一個疏水性核心,由四個胺基酸所組成(Leu431, Ile492, and the Tyr505/Phe506 motif)。本實驗室藉由突變型Leu431Ala發現Leu431扮演調節忠實性之功能。DNA聚合酶具有結合DNA和dNTP之能力,在過往中心教條上DNA聚合酶先結合上DNA,再結合dNTP,此優點可以藉由DNA序列上之鹼基配對來提高複製忠實性。倘若DNA序列上之鹼基受到氧化壓力(ROS)或是太陽光(UV)照射造成鹼基產生突變,會使得高忠實性複製型DNA聚合酶停在DNA複製叉上,若無法通過這些突變鹼基完成複製,進一步造成雙股螺旋DNA斷裂提升基因組不穩定。本論文證實低忠實性修復型DNA聚合酶 λ 具有預先結合dNTP之能力,提供了一條新的路徑,DNA聚合酶可以先結合dNTP越過突變之鹼基優先完成複製,使基因組穩定。 | zh_TW |
dc.description.abstract | The mechanism of DNA polymerase (pol) fidelity is of fundamental importance in chemistry and biology. While high-fidelity pols have been well studied, much less is known about how some pols achieve medium or low fidelity with functional importance. Here we examine how human DNA polymerase λ (Pol λ) achieves medium fidelity by determining 12 crystal structures and performing pre-steady-state kinetic analyses. We showed that apo-Pol λ exists in the closed conformation, unprecedentedly with a preformed MgdNTP binding pocket, and binds MgdNTP readily in the active conformation in the absence of DNA. Since prebinding of MgdNTP could lead to very low fidelity as shown previously, it is attenuated in Pol λ by a hydrophobic core including Leu431, Ile492, and the Tyr505/Phe506 motif. We then predicted and demonstrated that L431A mutation enhances MgdNTP prebinding and lowers the fidelity. We also hypothesized that the MgdNTP-prebinding ability could stabilize a mismatched ternary complex and destabilize a matched ternary complex, and provided evidences with structures in both forms. Our results demonstrate that, while high-fidelity pols follow a common paradigm, Pol λ has developed specific conformations and mechanisms for its medium fidelity. Structural comparison with other pols also suggests that different pols likely utilize different conformational changes and microscopic mechanisms to achieve their catalytic functions with varying fidelities. | en |
dc.description.provenance | Made available in DSpace on 2021-06-15T12:43:09Z (GMT). No. of bitstreams: 1 ntu-105-D00442001-1.pdf: 46201893 bytes, checksum: 1f215978b645470e6328fea5ecd93369 (MD5) Previous issue date: 2016 | en |
dc.description.tableofcontents | Table of contents
論文口試委員審定書 i 致謝 ii 中文摘要 iii Abstract iV Table of contents V List of Figures Vii List of Tables Viii Chapter 1. General introduction 1 1-1. The discovery, function, fidelity of DNA polymerases 1 1-2. Canonical pathway and DNA polymerization mechanism 3 1-3. X-family DNA polymerases 6 1-4. DNA polymerization mechanism in the Pol λ 7 1-5. Some of DNA Polymerases display unique property by prebinding with MgdNTP in the absence of DNA 9 1-6. Deoxynucleotide binding affinity of DNA polymerases uncover the structure-function property 10 Chapter 2. Structural mechanism for the fidelity modulation of DNA polymerase λ 13 2-1. Investigation of fidelity modulation in pol λ 13 2-1-1. Introduction 13 2-1-2. Materials and Methods 16 2-1-2-1. Materials. 16 2-1-2-2. Protein expression and purification. 16 2-1-2-3. Crystallization, data collection and structural determination. 16 2-1-2-3-1. Crystallization. 16 2-1-2-3-2. Diffraction data collection and structural determination. 20 2-1-2-4. Figures preparation. 20 2-1-2-5. Isothermal titration calorimetry (ITC) measurements. 20 2-1-2-6. Pre-steady-state kinetics measurements. 20 2-1-3. Results 22 2-1-3-1. Pol λ can bind MgdNTPs selectively in the absence of DNA. 22 2-1-3-2. Apo-Pol λ exists in the closed conformation with preformed dNTP binding pocket. 23 2-1-3-3. Bound dNTP and its binding site in the binary complex adopt conformations very similar to the ternary complex. 28 2-1-3-4. Prediction and demonstration that L431A facilitates MgdNTP binding in the absence of DNA. 30 2-1-3-5. The YF motif favors parallel orientation in the L431A:MgdCTP binary complex. 32 2-1-3-6. High affinity for MgdATP facilitates formation of a mismatched ternary complex of Pol λ with dG:dATP. 36 2-1-3-7. L431A may destabilize the matched ternary complex. 38 2-1-3-8. L431A lowers the fidelity based on pre-steady-state kinetic analysis. 40 2-1-3-9. Y505A mutant changes in the opposite way relative to L431A. 40 2-2. Discussion 43 References 57 Appendix 62 List of Figures Figure 1. Binding curves of MgdNTP to Pol λ measured by ITC. 22, 32, 42 Figure 2. Comparison of apo-Pol λ with other relevant structures. 25 Figure 3. Overlay of apo-Pol λ structures with the 8 kDa lyase subdomain (structure 1a) and without the 8 kDa subdomain (structures 1b and 2a) 26 Figure 4. Structures of Pol λ:dNTP binary complexes. 27 Figure 5. Structural basis for dNTP binding to Pol λ. 29 Figure 6. Conformations of the Tyr505/Phe506 motifs and bound dNTPs. 31 Figure 7. Structures of apo-L431A and L431A:dNTP binary complexes. 34 Figure 8. Conformations of the bound dNTP and the YF motif in the L431A:dNTP binary complexes. 35 Figure 9. Structural information for the dG:CadATP mismatched ternary complex of WT Pol λ (structure 7). 37 Figure 10. The final 2Fo-Fc map (contoured at 1σ) showing the Ca2+ ion, water molecule, templating dG, and bound dATP in the mismatched ternary complex of Pol λ with dG:CadATP (structure 7). 37 Figure 11. Two conformers in the crystal structure of the L431A:dG:CadCTP matched ternary complex (structure 12). 39 Figure 12. The final 2Fo-Fc map (contoured at 1σ) showing the YF motif, templating dG, and bound dCTP in conformer A (red) and conformer B (green) in the dG:dCTP matched ternary complex of L431A (structure 12). 39 Figure 13. Plots of the kpol/Kd,app values of WT versus mutants as a summary of the data in Table 3. 43 Figure 14. Apo-Pol λ preforms MgdNTP binding pocket while apo-Pol μ preforms DNA binding site. 47 List of Tables Table 1. KdMgdNTP values for the binding of dNTP to apo-Pol λ determined by ITC. 22, 32, 41, 51 Table 2. Summary of Pol λ structures and relevant information. 23, 51 Table 3. Summary of pre-steady-state kinetic data. 40, 52 Table 4. Data collection and refinement statistics of crystal structures in Table 2. 53 | |
dc.language.iso | en | |
dc.title | DNA聚合酶λ結構機制探討之忠實度調節 | zh_TW |
dc.title | Structural Mechanism for the Fidelity Modulation of
DNA Polymerase λ | en |
dc.type | Thesis | |
dc.date.schoolyear | 104-2 | |
dc.description.degree | 博士 | |
dc.contributor.oralexamcommittee | 梁博煌(Po-Huang Liang),冀宏源(Hung-Yuan Chi),詹迺立(Nei-Li Chan),袁小琀(Hanna Yuan) | |
dc.subject.keyword | DNA 聚合?λ,忠實性,蛋白質晶體結構, | zh_TW |
dc.subject.keyword | DNA polymerase lambda,crystal structure,dNTP binding,fidelity,kinetics, | en |
dc.relation.page | 111 | |
dc.identifier.doi | 10.6342/NTU201601325 | |
dc.rights.note | 有償授權 | |
dc.date.accepted | 2016-07-27 | |
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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ntu-105-1.pdf 目前未授權公開取用 | 45.12 MB | Adobe PDF |
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