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Title: | 去氧核糖核酸聚合酶之動力學研究 Kinetic Studies of DNA Polymerases |
Authors: | Chun-Wei Wang 王竣瑋 |
Advisor: | 蔡明道(Ming-Daw Tsai) |
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, DNA polymerase,kinetics,rate-limiting step,fidelity,G:G mismatch,isothermal titration calorimetry, |
Publication Year : | 2011 |
Degree: | 碩士 |
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。 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. |
URI: | http://tdr.lib.ntu.edu.tw/jspui/handle/123456789/35018 |
Fulltext Rights: | 有償授權 |
Appears in Collections: | 生化科學研究所 |
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