第五型核酸內切酶與第一型DNA聚合酶在亞黃嘌呤修復中交互作用之研究

DNA 及核苷酸在生理條件下可自發性發生脫胺作用，而接觸強氧化物或是暴露於輻射﹑紫外光等會促使其反應 ; 其中由腺嘌呤脫胺後與去氧核醣結合形成的亞黃嘌呤，若未經修復，則會在進行 DNA 複製及轉錄時發生錯誤。第五型核酸內切酶修復途徑為大腸桿菌中修復亞黃嘌呤的主要途徑之一，本實驗室先前透過試管中修復試驗，得知在反應中只需第五型核酸內切酶﹑第一型 DNA 聚合酶和DNA 接合酶即可完成亞黃嘌呤之修復反應。過往研究推測第五型核酸內切酶除了與 DNA 上的亞黃嘌呤結合外，還可能促使其他參與修復的酵素進行修復 ; 本實驗室也曾在試管中修復試驗得知在第五型核酸內切酶與第一型 DNA 聚合酶共同存在下，第五型核酸內切酶有出現周轉的現象，然而兩個酵素之間是否有交互作用，目前仍未可知。為證明第五型核酸內切酶與第一型 DNA 聚合酶之間的交互作用，本研究嘗試建立具有第五型核酸內切酶的融合基因，並進行同源置換插入缺乏第五型核酸內切酶之菌株 (JW5547, nfi-)，另一方面以純化之融合蛋白進行試管中修復試驗，觀察第五型核酸內切酶與第一型 DNA 聚合酶的交互作用。我們已嘗試建立出帶有第五型核酸內切酶的融合基因，並以同源置換插入缺乏第五型核酸內切酶之菌株，但透過西方墨點法已確認目前所建立之模型無法表現第五型核酸內切酶。另一方面，透過基質協助雷射去吸附離子化–飛行質譜儀之分析，得知加入克列諾片段確實增加第五型核酸內切酶在修復時的周轉，然而目前以共同免疫沉澱並未觀察到第五型核酸內切酶與缺乏核酸外切酶的克列諾片段有結合。依上述結果得知無法以目前建立之模型進行第五型核酸內切酶的修復試驗，但透過純化蛋白修復反應的結果，推測第五型核酸內切酶和第一型 DNA 聚合酶在修復亞黃嘌呤時存在交互作用，並促使第五型核酸內切酶之周轉。

關鍵字

第五型核酸內切酶；第一型DNA聚合酶；亞黃嘌呤；克列諾片段；基質協助雷射去吸附離子化-飛行質譜儀

並列摘要

Deamination of adenine can occur spontaneously as a result of intracellular stress and can be enhanced by exposure of irradiation, UV light, or nitrosative agent, which generates highly mutagenic deoxyinosine lesion in DNA. Unrepaired deoxyinosine lesion tends to generate A:T to G:C transition mutation during replication. In Escherichia coli (E. coli) , deoxyinosine is primarily removed through the pathway initiated by endonuclese V (Endo V). In previous study, it was suggested that deoxyinosine lesion could be repaired by only endo V, DNA polymearse I (Pol I) and DNA ligase in the in vitro deoxyinosine repair assay. In addition, we found that Endo V turnovered in deoxyinosine lesion repair with Pol I. To further investigate the interaction between Endo V and Pol I, we established maltose-binding protein (MBP)-fused Endo V and knocked into Endo V-deficient E. coli through homologous recombination, to examine that Endo V and Pol I have a contact during dI lesion repair. Unfortunately, we couldn’t get Endo V expression according to the result of Western blot. On the other hand, we discovered that the turnover of Endo V was promoted in the in vitro dI repair assay when reacted together with Klenow fragment. However, we haven’t obsevred interaction between exonuclease-decifient Klenow fragment and Endo V during dI lesion repair yet. In summary, we could not establish functional Endo V to repair dI in vivo. However, we suggest that Endo V could interact with Pol I in repairing dI lesion process and promote the turnover of Endo V.

並列關鍵字

Endonuclease V ； DNA polymerase I ； deoxyinosine ； Klenow fragment ； MALDI-TOF-MS

參考文獻

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第五型核酸內切酶與第一型DNA聚合酶在亞黃嘌呤修復中交互作用之研究

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