第一型DNA聚合酶校正特異性之研究分析

Abstract

DNA 複製的高保真度對於維持細胞的遺傳穩定性與防止有害突變的累積至關重要。DNA 聚合酶在 DNA 合成過程中，透過其 3’→5’外切酶活性進行校對，能有效移除錯誤嵌入的核苷酸，降低 DNA 合成過程中的錯誤率。然而，雖然先前研究已確認大腸桿菌 DNA 聚合酶 I（Pol I）可校正位於引子股 3’端倒數 1 至 4 個核苷酸範圍內的錯配，但針對不同類型錯誤配對在各位置的校對特異性仍未釐清。因此，本研究旨在針對引子股 3’端倒數 1 至 4 個核苷酸位置，系統性探討 Pol I 對各類型錯誤配對的校對特異性，並嘗試建立體外與體內的校對分析模型。 本研究利用設計錯配於特定位點的引子與模板 DNA，建構體外延伸反應平台，搭配 MALDI-TOF 質譜技術分析產物組成與校對比例，並控制反應條件以模擬生理環境，避免非特異性延伸與序列偏差干擾。結果顯示，Pol I 的校對效率高度依賴錯配位置，特別是在引子股倒數第一個位置，即使為結構不穩定的 purine-purine 錯配亦能完全校正；相對地，當錯配位於倒數第三或第四個核苷酸時，即便為常見的 G:T 錯配亦無法觸發有效校對反應。此結果突顯 Pol I 校對活性的空間限制，顯示其 proofreading 僅侷限於 3’端末端數個鹼基內。 為進一步評估細胞內的修正機制，本研究亦嘗試建立體內校對分析模型，將含有特定位錯配的 DNA 序列轉型至錯配修復功能缺失的菌株，初步驗證錯配可否逃脫 Pol I 校對並交由其他修復系統處理。實驗結果顯示，Pol I 無法修正的 G:T 錯配若未即時修復，將可能累積為突變，顯示校對與錯配修復系統在細胞內具互補分工。 此外，結合結構功能的初步觀察，本研究亦關注一可能參與錯配辨識的高度保守結構: J-helix。此結構被推測與引子末端錯配識別與修正行為相關，並可能參與催化活性位點與錯配間的構型變化。 結合以上觀點來看，本研究揭示 Pol I 對錯配的校對特性受錯配位置顯著影響，並與細胞內錯配修復系統形成互補協作。所建立之 MALDI-TOF 平台提供一套可量化、具可擴充性的 proofreading 分析工具，未來可應用於探討其他 DNA 聚合酶之錯配辨識特性，亦為後續聚合酶結構功能與基因體穩定性研究奠定基礎。

High-fidelity DNA replication is essential for maintaining genomic stability and preventing the accumulation of harmful mutations. DNA polymerases achieve this fidelity in part through their 3’→5’ exonuclease activity, which proofreads and removes in-correctly incorporated nucleotides during DNA synthesis. Although previous studies have demonstrated that Escherichia coli DNA polymerase I (Pol I) is capable of proof-reading mismatches located within the last one to four nucleotides at the 3’ end of the primer strand, the mismatch-type and position-specific proofreading specificity of Pol I remains poorly understood. Therefore, this study aims to systematically investigate the proofreading specificity of Pol I against various mismatch types located at positions 1 to 4 from the 3’ terminus of the primer and to establish both in vitro and in vivo platforms for proofreading analysis. In vitro assays were conducted using synthetic primer-template duplexes contain-ing site-specific mismatches, analyzed by MALDI-TOF mass spectrometry under con-trolled reaction conditions to mimic physiological environments. Results revealed a strong positional dependency of Pol I proofreading activity. Near-complete correction was observed for purine-purine mismatches at the terminal position, while mismatches located at the third or fourth nucleotide from the 3’ end—such as G:T—were not effi ciently repaired. This spatial limitation suggests that Pol I proofreading is confined to a narrow range near the primer terminus. Preliminary in vivo experiments using mismatch-containing plasmids transformed into mismatch repair-deficient strains further indicate a cooperative relationship be tween Pol I proofreading and the mismatch repair (MMR) system. Additionally, struc tural analysis highlights the potential role of the conserved J-helix motif in mismatch recognition and catalysis. Overall, this study elucidates the position-dependent proofreading mechanism of Pol I and provides a quantitative platform applicable to the study of proofreading speci ficity in other DNA polymerases.