細菌第一型DNA聚合酶校對外切酶參與第五型核酸內切酶修復之體內證明

DNA polymerase I proofreading exonuclease activity is required for Endonuclease V pathway in vivo

吳卓遠(Cho-Yuan Wu)

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(2017年)

https://doi.org/10.6342/NTU201700440

摘要

細胞的腺嘌呤 (Adenine; A) 發生自發性的脫胺作用 (deamination) 或受到外源性的氧化壓力等傷害會變成次黃嘌呤 (Hypoxanthine; Hx)。以Hx為鹼基與五碳糖結合，則稱為次黃嘌呤核酸 (Deoxyinosine；dI)。第五型核酸內切酶修復系統 (Endonuclease V alternative excision repair；Endo V AER) 是大腸桿菌主要修復次黃嘌呤核酸的修復途徑之一。
實驗室先前利用噬菌體 M13mp18 衍生的異雙股核酸，以不同基因突變之大腸桿菌萃取液及純化蛋白系統探討試管中次黃嘌呤核酸之修復反應，結果顯示僅需 Endo V、第一型去氧核醣核酸聚合酶 (DNA polymerase I, DNA Pol I) 以及連接酶 (DNA ligase) 即可完成修復。因此推論在 Endo V 產生DNA缺口 (nick) 後，由 DNA Pol I 3’端往 5’端核酸外切酶 (3’-5’ exonuclease, 3’exo-) 活性負責移除 dI，最後再由 DNA ligase 修補缺口完成修復。實驗室也以質體pUC18製作I-T mismatch DNA受質分別轉形到BW25113 (WT)、JW5547 (nfi-)、KA796 (polA+)、KA796 D424A (polA exo-)、四株不同的大腸桿菌中，發現在第五型核酸內切酶缺失的JW5547 (nfi-)細胞株中DNA修復效率和野生株BW25113 (WT)相比大幅降低，而PolI校對活性缺乏的KA796 D424A (polA exo-)大腸桿菌中，其修復效率和對照組KA796 (polA+)無差異，我們懷疑是在第五型核酸內切酶所造成DNA缺口後，第一型DNA聚合酶大次單元無法移除錯誤片段完成修復，進而使DNA複製崩壞所造成的現象，因此無法正確分析DNA受質有無被修復。
本實驗設計一個新的I-T mismatch DNA，其中在I的3’端帶有C-C mismatch，此錯誤配對在大腸桿菌中不會被修復，可做為用來觀察DNA因複製崩壞等因素造成股損失 (strand loss) 的標記，且把此位置設計在endoV pathway對dI的修復範圍外。以此DNA受質分別轉形到BW25113 (WT)、JW5547 (nfi-)、KA796 (polA+)、KA796 D424A (polA exo-)四株不同的大腸桿菌中，隨機挑50個單一菌落，少量放大並抽取DNA，以限制酶進行水解分析，結果可以看到在BW25113 (WT)、KA796 (polA+) 兩株細胞中I-T受質都有70%以上的修復，在JW5547 (nfi-) 細胞中I-T受質只有約3%的背景訊號，在KA796 D424A (polA exo-) 中I-T受質修復約19%，但分析C-C mismatch 發現strand loss現象約有57%。
此實驗確認先前KA796 D424A (polA exo-)大腸桿菌中，其修復效率和對照組KA796 (polA+)無差異是I-T受質無法被修復產生複製崩壞所造成的假性修復現象。並對大腸桿菌中第一型DNA聚合酶3’外切酶活性及第五型核酸內切酶，於第五型核酸內切酶修復系統修復次黃嘌呤核酸的重要性提供有力證據。

關鍵字

脫胺作用 ； 次黃嘌呤核酸 ； 第五型核酸內切酶 ； 第一型去氧核醣核酸聚合酶 ； 第五型核酸內切酶修復系統

並列摘要

The highly mutagenic deoxyinosine (dI) lesion can be produced in DNA spontaneously deamination, and is enhanced by nitrous ion exposure. In Escherichia coli, dI is repaired through endonuclease V (EndoV) pathway. Our previous in vitro assay demonstrated that EndoV, DNA polymerase I (Pol I), and E. coli DNA ligase were sufficient to reconstitute the dI repair. To confirm Pol I proofreading exonuclease is the integral part of Endo V repair pathway, we employed a dI-containing plasmid for in vivo repair assay. The nfi- mutant showed dramatic decrease as expected in dI repair compared to its isogenic wild type. However, the repair efficiency of polAexo- was as high as wild type strains. We suspected this observation might result from mutant Pol I of polAexo- strain inhibited the ligation of the dI-containing strand after EndoV nicking. During plasmid replication the unrepaired nicked strand was lost because of replication fork collapse. Only continuous template strand survived the replication and was scored by our assay. To confirm this hypothesis, we redesigned a substrate containing a C-C mismatch (not repaired in E. coli) away from proposed Endo V repair-patch as the secondary markers for strand loss. For both PolI wild type BW25113 and KA796 (polA+), over 70% of dI were repaired with only dI marker removed and about 20% strain loss by missing both markers on dI-containing strand. For nfi- strain over 85% showed mixed colonies at both markers with less 10% of strand loss and 5% of background repair. As to polAexo- mutant, more than 50% were strand loss, more than 20% were unrepaired, and only less than 20% were repaired. This observation confirmed our hypothesis and provided solid evidence to support Pol I proofreading exonuclease is the major enzyme activity to remove dI lesion in EndoV repair pathway.

並列關鍵字

Deamination ； Deoxyinosine ； Endonuclease V ； DNA polymerase I ； Endo V repair pathway

參考文獻

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