DNA聚合酶I於引子末端倒數第二個配對錯誤鹼基校正活性之全面分析

The Proofreading Spectrum of DNA polymerase I to the Different Single Mismatches at 3’-Penultimate Site of the Primer

林千如(Chien-Ju Lin)

指導教授 ： 方偉宏

國立臺灣大學/醫學院/醫學檢驗暨生物技術學研究所/碩士(2012年)

https://doi.org/10.6342/NTU.2012.03297

摘要

DNA為生物體之遺傳物質，其高度複製忠誠度（fidelity）對維持基因穩定性及預防突變發生甚為重要。DNA聚合酶主要透過三種方式降低複製錯誤率：鹼基選擇性配對（base selection）、3端往5端外切酶（3’→5’ exonuclease）之校正能力（proofreading activity）及錯誤配對修復機制（mismatch repair system）。
相關研究指出DNA聚合酶能夠移除引子末端鹼基或末端連續兩個錯誤鹼基，但尚未有研究證實DNA聚合酶能夠校正引子末端倒數第二個配對錯誤鹼基。本實驗室發表文獻指出當DNA聚合酶I與DNA內切酶V（endonuclease V）、DNA連接酶（DNA ligase）及dNTP共同存在下能夠修復G-dI的錯誤配對，應是由DNA內切酶V切斷dI上游第二個磷酸雙酯鍵，活化DNA聚合酶I校正活性導致。為釐清此現象，我們製造十二種斷股上游第二個鹼基為錯誤配對之受質模擬引子與模板交會處，分析DNA聚合酶I對十二種受質之校正活性。
排除DNA聚合酶I行缺口轉譯（nick translation）的可能性後，結果顯示十二種受質皆可以被DNA聚合酶I修復，以purine．purine校正活性最好，purine．pyrimidine校正活性最差（C-A例外）。部分受質在低離子強度（50mM NaCl）下校正活性較好，例如A-C、T-G、G-G、C-T、T-C、A-A；C-C則在高或低離子強度下具有不同的校正活性。我們認為離子可能影響DNA聚合酶I或配對錯誤受質結構上的改變，但其確切造成的影響仍需進一步研究。
文獻顯示purine．purine是出現頻率最高的錯誤配對，兩個大的purine結合造成結構上扭曲，可能造成校正活性較高，得以將發生率高的錯誤配對進行修復。並且，有文獻顯示purine的鹼基堆疊力（base stacking force）較大，並且其N7官能基較易與DNA聚合酶I外切酶活化位之胺基酸結合，也可能提高其校正活性，在我們的研究中發現，當錯誤鹼基為purine且位於引子上時，其校正活性明顯較高，但T-G例外。而purine．pyrimidine則因為其結構與正確配對的Watson-Crick base pairing相似，導致校正活性微弱，但可經由錯誤配對修復機制補償。此外，當DNA聚合酶I往上游切除錯誤鹼基後，往下游繼續行聚合反應時，gap-form受質由於不需要DNA聚合酶I執行5端往3端外切酶活性即可聚合，因此有較高的校正活性。
本篇論文證實DNA聚合酶I之校正活性不僅能移除引子末端錯誤鹼基，其確實能夠校正斷股上游第二個鹼基為錯誤配對之受質，得以解釋本實驗室先前發表論文G-dI之修復現象。並且，DNA聚合酶I不僅能修復正常的鹼基，對於dI此種被修飾過的鹼基也具有校正活性。

關鍵字

校正反應 ； 核酸複製忠誠度 ； 核酸聚合酶核酸聚合酶I ； 核酸外切酶活性 ； 錯誤配對 ； 限制酵素 ； 異雙股核酸

並列摘要

DNA carries genetic information in all organisms. During DNA replication, it is important to maintain genomic fidelity. Three correlating events operate in maintaining the high fidelity of genome：The first is base selection. The second is the proofreading activities of DNA polymerases, which can remove the last mismatched DNA at the primer-template junction. The third is DNA repair systems.
To date, there is no evidence showing that the mismatched DNA at penultimate site of the primer can be edited by DNA polymerase I. Our previous study showed that the proofreading activity of DNA polymerase I could edit deoxyinosine-containing heteroduplex DNA after processing by endonuclease V which created a strand breakage at the second phosphodiester bond 3’ to the deoxyinosine. To figure out how it works, we constructed twelve heteroduplex DNA containing single mismatch at penultimate site of the primer and analysed the proofreading activity.
The involvement of nick translation activity of DNA polymerase I was eliminated. Our results showed that all the twelve heteroduplex DNA can be edited by proofreading activity of DNA polymerase I and there were no general roles for trend of ionic strength in our proofreading assay. We identified purine．purine, the most frequently misinserted mismatches, could be edited well. According to the structure analysis, two large purine bases cause considerable strand strain that may lead to proofreading efficiency elevated. However, purine．pyrimidine mismatches were poorly edited probably due to these structures were similar to the correct Watson-Crick base pairs with minor distortion but the C-A could be edited well.
Furthermore, the mismatch repair system had high efficiency to repair purine．pyrimidine mismatches can compensate to poorly proofreading activity. On the other hand, the large purine bases have increased stacking ability and the common N7 groups may be preferred to bind with the amino acid residue of exonuclease site. We found that the misbase on the primer strand had the more efficiency of proofreading activity but the T-G was not.
Besides, we identified that gap-form substrate had better proofreading activity than nick-form. After removing the wrong base, DNA polymerase I will undergo polymerization. As a result of DNA carrying out polymerization without 5’ to 3’exonuclease activity with the gap-form substrate, it has higher proofreading efficiency.
Conclusively, we identified the proofreading activity of DNA polymerase I can edit DNA mismatches at the penultimate site of the primer. In addition to our previous study, the DNA polymerase I actually could edit deoxyinosine-containing heteroduplex DNA which containing a strand breakage at the second phosphodiester bond 3’ to the deoxyinosine.

並列關鍵字

proofreading ； fidelity ； DNA polymerase ； DNA polymerase I ； exonuclease activity ； mismatch ； restriction enzyme ； heteroduplex DNA

參考文獻

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