核酸聚合酶 I 對於模版與引子交會處帶有異雙股的核酸受質之校正活性分析

Proofreading Activity of DNA Pol I to Substrate Containing Heterologies within Template-primer Junction

陳怡安(Yi-An Chen)

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(2011年)

https://doi.org/10.6342/NTU.2011.02241

摘要

核酸為各個生物體的遺傳物質，因此核酸在複製時的精確性甚為重要，核酸複製的忠誠度主要由三個步驟所決定，一為核酸聚合酶 (DNA polymerase)複製核酸時一連串結構上的改變，以確保將正確的dNTP接上正確的位置；二為核酸聚合酶(DNA polymerase)在遇到末端有錯誤配對時會以3’-5’exonuclease的酵素活性作校正 (proofreading)；三為複製完核酸後細胞可以啟動例如 mismatch repair system (MMR)等修復系統修復仍存在的錯誤配對；如此一來可以把核酸複製的錯誤率降至約10-10左右。
已知E. coli DNA polymerase I會對於最末端的錯誤配對有校正 (proofreading)的功能，並且過去的研究並沒有發現如果存在足量dNTP的情況之下，primer末端倒數第二個位置有錯誤配對會活化DNA polymerase I的3’-5’ exonuclease的活性。由於核酸聚合酶 (Pol I)在校正 (proofreading)反應完成之後，會繼續往下游複製核酸，合成正確的鹼基配對，於是為了探究DNA polymerase I在核酸校正方面更深入的特性，我們便可以利用限制酵素對於識別序列具有一定程度的專一性，來偵測我們所製備的核酸受質是否有被校正的現象。
我們設計不同的錯誤配對，並且在各種錯誤配對的3’下游一個鹼基後產生一個nick，以測試單純DNA polymerase I作用的情況，並看DNA polymerase I對於倒數第二個位置出現的鹼基錯誤配對，是否仍然可以啟動它校正的活性。由於想觀察酵素反應作用初期的反應狀況，我們將不同的核酸受質，與DNA polymerase I反應，將反應時間縮小為十分鐘之內，每兩分鐘一個間隔觀察核酸聚合酶的反應情況，並比較對於不同的核酸受質，核酸修復效率與反應速率的差異。
實驗結果發現，對於距末端斷股上游第二個位置的鹼基錯誤配對，在T-T，T-G，T-C錯誤配對的受質當中，都發現DNA polymerase I會與受質反應，並且以線狀的受質 (linear DNA substrate)測試結果發現，此反應可以忽略DNA polymerase I在受質上面作nick translation的程度，顯示DNA polymerase I對於倒數第二個位置的錯誤鹼基配對，的確能夠有校正 (proofreading)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 ； 核酸外切活性 ； 錯誤配對 ； 限制酵素 ； 異雙股核酸

並列摘要

DNA is genetic information for all organisms. It is important for maintaining fidelity during replicating. There are three steps that maintain the high fidelity. The first is structure switch for ensuring the right dNTP on the right place. The second is proofreading activity. DNA polymerase can remove the mismatch at the end of the primer then extension continually. The other is repair systems. Based on the above, the error frequency can decrease to about 10-10.
There is no evidence showing that the second last mismatch can activate 3’ to 5’ exonuclease proofreading activity of DNA polymerase I. Besides, we known that after proofreading, Pol I can replicate DNA continually and form correct base pairs. For searching the detail of proofreading, we used the specificity of restriction enzyme to monitor whether the substrate was repaired or not.
We designed different mismatch substrates. Besides, there was a nick downstream from the mismatch. In order to search the preliminary reaction of DNA polymerase I, we designed the reaction time within ten minutes and then monitored every two minutes. In addition, we compared the difference of proofreading efficiency and analysis of enzyme kinetic between different DNA substrates.
Our results showed that DNA polymerase I could react with DNA substrates including T-T, T-G, and T-C. We also used linear substrate to test DNA polymerase I proofreading activity. It showed that this assay can ignore the extent of nick translation. These results suggested that the proofreading activity of DNA polymerase I can also be activated even when the mismatch located not on the end.

並列關鍵字

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

參考文獻

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