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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 | 蔡明道(Ming-Daw Tsai) | |
dc.contributor.author | Chun-Wei Wang | en |
dc.contributor.author | 王竣瑋 | zh_TW |
dc.date.accessioned | 2021-06-13T06:38:55Z | - |
dc.date.available | 2016-08-01 | |
dc.date.copyright | 2011-08-01 | |
dc.date.issued | 2011 | |
dc.date.submitted | 2011-07-25 | |
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(2005) Kinetic studies of yeast polyA polymerase indicate an induced fit mechanism for nucleotide specificity, Biochemistry-Us 44, 7777-7786. 53. Luft, J. R., Wolfley, J. R., and Snell, E. H. (2011) What's in a drop? Correlating observations and outcomes to guide macromolecular crystallization experiments, Cryst Growth Des 11, 651-663. | |
dc.identifier.uri | http://tdr.lib.ntu.edu.tw/jspui/handle/123456789/35018 | - |
dc.description.abstract | 去氧核糖核酸聚合酶在核酸的複製與受到損壞時的修補上扮演非常重要的角色。在此我們描述兩種在結構上很相似,並且參與對於維持基因完整性(genetic integrity)上非常重要的鹼基切除修復(base excision repair)的聚合酶。(1) Pol β是一種具有高保真度(high fidelity)的聚合酶,並且是做為動力學研究催化核酸轉移機制的理想模型。從動力學截流儀(stopped flow)探討鎂離子濃度以及R258A突變型研究中,我們提出了鎂離子抑制現象的機制之假說。藉由酸鹼指示劑測量質子生成的實驗中,我們直接證實在高pH值的時候其速率決定步驟(rate limiting step)是在於其子域(subdomain)重新開啟釋出產物的步驟。(2) ASFV Pol X因為其低保真度、並且參與獨特的dGTP對帶有dG的核酸模板(DNA template)之專一催化而顯得非常特別,因此有假設說它可能對於病毒基因的策略性突變(strategic mutagenesis)相當重要。之前從我們的核磁共振(nuclear magnetic resonance)研究已經發現His115對於Pol X能夠形成這種G:G錯誤配對(mismatch)很重要,因此我們進一步利用等溫滴定微量熱儀(isothermal titration calorimetry)來測量Pol X-dNTP的解離常數(Kd)。結果顯示Pol X對於dGTP有獨特的專一性,但是當帶有dG的核酸模板存在條件下,其比較傾向dCTP三元複合體(ternary complex)的生成。H115A突變顯著地降低了dNTP的結合,並且在動力學上對於G:G和G:C的嵌入模板效率(incorporation rate)也有顯著下降的影響。這些結果更進一步證實先前假設Pol X可能利用低保真度的特性來促進ASFV策略性突變。 | zh_TW |
dc.description.abstract | DNA polymerases play a central role in DNA replication and DNA repair upon damage. Here we describe two structurally similar DNA polymerases involved in DNA base excision repair (BER) pathway, which is crucially important in maintaining genetic integrity. (1) DNA polymerase β (Pol β) is a high fidelity DNA polymerase, and serves as an ideal kinetic model for studying the mechanism of nucleotidyl transfer catalysis. By stopped flow, we proposed a hypothesis for the Mg2+ inhibition mechanism from [Mg2+] dependence and R258A mutant studies. From measuring proton formation by pH indicator, we provide direct evidence that the rate-limiting step during catalysis at high pH is the subdomain re-opening after chemistry. (2) African swine fever virus (ASFV) DNA polymerase X (Pol X) is distinct in that it is considered to be low fidelity that involves specific catalysis of dGTP insertion toward DNA template dG, which may be important in the strategic mutagenesis of the viral genome. Our NMR study has implicated the importance of His115 for Pol X nucleotide specificity in G:G mismatch, thus the binding constants for Pol X-dNTP complexes were determined by isothermal titration calorimetry (ITC). Free Pol X shows distinct nucleotide specificity on dGTP, but in DNA with template dG, the formation of the dCTP ternary complex is favored instead. The H115A mutation significantly weakens nucleotide binding, and impairs catalysis of both G:G and G:C insertion. These results provide another example to support previous hypothesis of the error prone Pol X on facilitating strategic mutagenesis in ASFV. | en |
dc.description.provenance | Made available in DSpace on 2021-06-13T06:38:55Z (GMT). No. of bitstreams: 1 ntu-100-R98B46008-1.pdf: 1894232 bytes, checksum: 28fc4b7c5aab3955fba7645914d186ac (MD5) Previous issue date: 2011 | en |
dc.description.tableofcontents | 謝誌 I
摘要 II Abstract III Chapter 1 Introduction 1 1.1 The importance of DNA polymerase 1 1.2 The conserved structure of DNA polymerase 2 1.3 The catalytic mechanism of DNA polymerase 3 1.4 Pol β in base excision repair 3 1.5 Pol β in X-ray crystal structure studies 5 1.6 Previous kinetic studies on Pol β 6 1.7 African swine fever virus 7 1.8 Characterization of Pol X: the error prone polymerase 8 1.9 The unique G:G mismatch of Pol X in NMR study 9 1.10 Goal of this Thesis research 11 Chapter 2 Materials and Methods 12 2.1 DNA substrates preparation 12 2.2 Tritium-labeled nucleotides 13 2.3 Protein expression system of Pol β and Pol X 13 2.4 Protein purification of Pol β and Pol X 14 2.5 Protein expression of Pol β and Pol X for NMR study 16 2.6 Site-directed mutagenesis 17 2.7 Stopped flow fluorescence study 18 2.8 Stopped flow absorbance study 19 2.9 Rapid chemical quench flow study 20 2.10 Isothermal titration calorimetry 21 2.11 Pol X single nucleotide incorporation assay 22 2.12 Pol X additive screening 23 Chapter 3 Results and Discussions 25 3.1 The advantages of using short hairpin DNA substrates 25 3.2 Interpretation of Pol β stopped flow fluorescence kinetic results 25 3.3 The Mg2+ effect on Pol β in catalysis 26 3.4 The mechanism of Mg2+ inhibition by mutagenesis study 27 3.5 Pol β stopped flow absorbance kinetic assay on proton release 28 3.6 Confirm the nucleotidyl transfer by rapid quench 30 3.7 Pol β shows no preference to nucleotide in NMR 30 3.8 Pol X shows distinct nucleotide specificity in ITC 31 3.9 Nucleotide titration to Pol X in the presence of DNA template 33 3.10 Pol X H115A loses the nucleotide specificity 34 3.11 DNA compensates for the loss of binding energy from His115 35 3.12 Determination of initial rate in nucleotide incorporation for Pol X 37 3.13 Pol X solubility test for additive screening 38 3.14 Pol X nucleotide incorporation under single turnover condition 39 3.15 The kinetic scheme for nucleotide incorporation by Pol X 40 Chapter 4 Conclusion and Perspective 42 List of Figures 44 Figure 1. The predicted hairpin structure of DNA substrates. 45 Figure 2. The structural comparison of Pol X and Pol β. 46 Figure 3. The protein purification of Pol β and Pol X. 47 Figure 4. Pol β Stopped flow fluorescence on [Mg2+] dependent study. 48 Figure 5. Comparison of Mg2+ inhibition effect on kpol at pH 7.4 and 7.7. 49 Figure 6. The mechanism of Mg2+ inhibition by mutagenesis study. 50 Figure 7. Comparison of Pol β WT and R258A on Mg2+ inhibition 51 Figure 8. Pol β stopped flow absorbance and Trp fluorescence kinetics. 52 Figure 9. Stopped flow absorbance kinetics of enzymatic proton formation. 53 Figure 10. Confirm the nucleotidyl transfer by rapid quench. 54 Figure 11. Titration of four different dNTPs to Pol X WT by ITC. 55 Figure 12. Titration of dGTP or dCTP to Pol X WT with dhDNA. 57 Figure 13. Titration of dGTP or dCTP to Pol X H115A. 58 Figure 14. Titration of dGTP or dCTP to Pol X H115A with dhDNA. 59 Figure 15. Qualitative kinetic results of dGTP and dCTP insertion by Pol X WT and H115A. 60 Figure 16. The kinetic scheme for nucleotide incorporation by Pol X. 61 List of Tables 62 Table 1. List of sequences of DNA substrates in this study. 62 Table 2. List of designed primers for site-directed mutagenesis. 63 Table 3. Thermodynamic parameters for titration of dNTP to Pol X (WT or H115A) and Pol X (WT or H115A)-DNA complex by ITC. 64 References 65 Appendixes 70 Appendix 1. Schematic of nucleotidyl transfer mechanism of Pol β. 70 Appendix 2. Common base excision repair (BER) pathway. 71 Appendix 3. Scheme of common mechanism for DNA polymerases. 72 Appendix 4. Structural-based sequence alignment of Pol X with C-terminal of rat Pol β and human Pol β. 73 Appendix 5. Catalytic efficiencies of Pol X. 74 Appendix 6. His115 is critical for Pol X in G:G mismatch formation. 75 Appendix 7. Structural study of Pol X-DNA(dG)-dGTP complex by NMR. 76 Appendix 8. 2D 1H,15N-TROSY-HSQC of Pol β-dNTP binary complex. 77 Appendix 9. Catalytic efficiency and fidelity of WT Pol X and H115A. 78 | |
dc.language.iso | en | |
dc.title | 去氧核糖核酸聚合酶之動力學研究 | zh_TW |
dc.title | Kinetic Studies of DNA Polymerases | en |
dc.type | Thesis | |
dc.date.schoolyear | 99-2 | |
dc.description.degree | 碩士 | |
dc.contributor.oralexamcommittee | 梁博煌(Po-Huang Liang),楊維元(Wei-Yuan Yang) | |
dc.subject.keyword | 去氧核糖核酸聚合酶,動力學,速率決定步驟,保真度,G:G錯誤配對,等溫滴定微量熱儀, | zh_TW |
dc.subject.keyword | DNA polymerase,kinetics,rate-limiting step,fidelity,G:G mismatch,isothermal titration calorimetry, | en |
dc.relation.page | 78 | |
dc.rights.note | 有償授權 | |
dc.date.accepted | 2011-07-25 | |
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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