- 學位論文
第一型 DNA 聚合酶校正機制之研究分析
Mechanistic analysis of DNA Polymerase I proofreading activity
摘要
互補的核苷酸互相配對時，便被稱為 DNA 誤配(DNAmismatch)。生物體中，DNA 誤配主要 DNA 聚合酶的校正(proofread)以及誤配修補系統(mismatch repair, MMR)進行修復，其中，DNA 聚合酶的校正主要針對正在進行複製的 DNA 進行。 本實驗室過去曾研究 Klenow fragment(KF)對於誤配核苷酸與插入/缺失錯誤(insertion/ deletion error, indel error)的校正活性，發現 KF 對於自引子股 3’端數來 1至 4 個位置的的異常核苷酸有著較高的校正活性。實驗室據此提出了假說，認為KF 能夠與距離 DNA 引子股 3’端 4 個核苷酸直接接觸，因此在這個範圍內的異常核苷酸能夠受到較強的校正作用。 在本篇研究中，我們首先使用 KF 與在引子 3’端數來 1 至 7 個位置帶有單一誤配核苷酸的 DNA 進行反應並以 MALDI-TOF MS 分析，發現 KF 對於自引子股3’端的 1 至 3 個位置的誤配核苷酸有著較高的校正活性，4 號位置的校正活性較低，5 至 7 號位置則無校正活性而進行引子延伸。接著，我們測試帶有兩個誤配核苷酸的 DNA 的校正活性，發現當兩個誤配核苷酸包含彼此在內相距 4 個核苷酸以內時若有一個誤配核苷酸位於可被校正的範圍內，另一個誤配核苷酸會一併進行校正。另外也發現誤配序列的種類會影響誤配核苷酸共同受到校正的活性。 為了能夠進一步了解 KF 進行校正反應的過程，我們利用含有兩個誤配核苷酸的 DNA 與 KF 反應並進行 time course 實驗，在實驗中，我們觀察到了 KF 中3’→5 外切酶(3’→5’ exonuclease)的非連續性(non-processive)作用。最後，結合本篇研究的實驗結果，我們對 KF 的校正反應機制提出了以下模型: 當 KF 對 DNA進行校正時，將引子股 3’端送往 3’→5’外切酶活性位 (3’→5’ exonuclease site) 並且水解一個核苷酸後送回聚合酶活性位 (polymerase site)，若 DNA 上仍有異常序列，便會重複以上步驟，直到異常序列受到水解為止。
並列摘要
DNA mismatch is defined as the misincorporation of nucleotide. In livingorganism, DNA mismatch could be corrected by either DNA proofreading or mismatchrepair (MMR). In previous research devoted by our laboratory, the proofreading activity of Klenow fragment(KF) for mismatch error and insertion/deletion error(indel error) was studied. The results showed relatively strong proofreading activity for errors within 4 nucleotides from primer 3’ end in both experiments. Thus, our laboratory proposed a hypothesis that KF may physically contact with 4 nucleotides from primer 3’ end, therefore preform strong proofreading ability within such range. In this study, proofreading activity of KF for single mismatched nucleotide was examined. The data revealed that polymerase showed strong proofreading activity for mismatched nucleotide within 3 nucleotides distant from primer 3’ end. Less proofreading activity to the fourth nucleotide from primer 3’ end. As for those whose distance from primer 3’ end is over 5 nucleotides, KF failed to correct the mismatched nucleotide. After that, proofreading efficiency of KF for DNA which contains two mismatches was tested. The data suggests that if the distance between two mismatches is beyond 3 nucleotides, KF would tend to correct both mismatched nucleotides as long as one of them could be recognized by the polymerase. Also, the proofreading activity for the two mismatched base pairs would also be influenced by the type of mismatches. To investigate the process of proofreading activity carried out by KF, time course experiment of a double-mismatched DNA proofread by KF were practiced. According to the result, we found the non-processive activity of KF 3’→5 exonuclease. Finally, based on the experimental result obtained in this study, we proposed a model of KF proofreading process. Upon recognizing errors on the DNA, the single stranded primer would be transferred to the 3’→5 exonuclease site, after that, a nucleotide would be hydrolyzed. The primer would then be transferred back into polymerization site, there, KF would determine whether the error has been removed. If not, the primer would be transferred to the 3’→5 exonuclease site once again, and the process would be repeated once and again until the error sequence was corrected.