人類細胞亞黃嘌呤修復試管內測試最佳化

Abstract

生物體內細胞中的DNA 進行自發性的反應或是受到外源性的攻擊，導致特定的核酸損傷，例如：亞黃嘌呤(Hypoxanthine;Hx)為生物細胞的腺嘌呤(Adenine;A)發生自發性的脫胺作用、或是遭受到致烷基劑等影響而產生。而以Hx 為含氮鹼基在DNA 構成的核酸稱為deoxyinosine (dI)。因亞黃嘌呤在結構上與鳥嘌呤 (Guanine;G)相似，故在DNA 進行複製時傾向與胞嘧啶(Cytosine;C)配對，一旦出現C-I 的錯誤配對而未被修復，就可能發生A:T→G:C 的transition mutation，進而衍生出疾病。 過往研究已知在人類細胞中的dI 可被alkyladenine DNA glycosylase (AAG)經由鹼基切除修復(base excision repair; BER)去除。而大腸桿菌除了BER 之外，亦能藉由nfi 基因產物endonuclease V (endo V)所主導之alternative excision repair (AER)除去dI。近日文獻在哺乳動物及人類細胞中皆已發現endo V 的homolog，且由試管中實驗證實小鼠細胞之endo V 會如同大腸桿菌於dI 3’端第二個磷酸鍵進行切割，並形成缺刻。但目前在人類細胞中，不論是endo V 起始的後續修復反應之機制、抑或是DNA 中dI 損傷的修復情形皆尚未明瞭。實驗室前人在探討此機制時，設計使用人類細胞萃取液在試管內測試含有G-I, T-I, A-I 受質修復狀況的系統，而本論文將建構新的C-I 異雙股核酸配對受質，讓我們能更完整地了解各種dI 配對受質在人類細胞萃取液中的修復情形，並以不同的因子加入反應中，試著釐清可能參與其中的修復系統及機制；接著對前人所建構在人類細胞萃取液中dI 試管內修復系統進行各種不同條件分析，找尋最適合的反應或是更接近人體生理狀態的條件，最佳化此系統。 實驗結果發現，MMR proficient 的HeLaS3 人類細胞萃取液將C-I 受質修復程度，較MMR deficient (hMLH1 defective)的HCT116 人類細胞萃取液高。而C-I 受質在HeLaS3 及HCT116 人類細胞萃取液之反應需求分析中皆發現，若不加入鎂離子修復反應無法進行；缺乏dNTPs 修復效率會降低；缺乏ATP 或以ATP-γ-S 替代則會使修復效率降低，故推測將C-I 進行辨識及修復的蛋白是需要ATP 結合並且水解。在HeLaS3 細胞萃取液中加入DNA Pol β 抑制劑會使C-I 受質修復效率降低約60%；而加入Pol α、δ、ε 抑制劑則會降低約30%，而額外加入MutLα 至HCT116人類細胞萃取液後，發現G-I 受質被修復效率提升。結果說明除了BER 之外，MMR也可能在人類細胞中參與dI 修復，且不同dI 配對損傷可能會由不同系統修復。 本實驗也利用G-I 受質與HeLaS3 人類細胞萃取液，測試不同修復條件以最佳化實驗室所建立的試管內dI 修復系統。結果發現，G-I 受質修復在30 分鐘時修復效率已達測試最高時間的85%；針對ATP 濃度測試則發現高於2 mM 時會導致核酸修復狀態不穩定，而濃度太低也會導致修復效率不彰；而在鎂離子濃度測試時發現隨濃度上升，修復的效率下降；最後以GTP 代替ATP 則發現修復效率大幅降低。根據以上結果我們未來或能在調整人類細胞萃取液dI 試管內修復系統至更接近生理狀態的條件，讓我們能更有效率地探討人類細胞中dI 修復的機制。

DNA base deamination can occur spontaneously under physiological conditions. Deamination of adenine would generate the highly mutagenic lesion, hypoxanthine(Hx) and the process is enhanced by ROS released upon exposure of DNA to ionizing radiation, UV light, nitrous acid, or heat. Hypoxanthine in DNA can pair with cytosine which results in A:T to G:C transition mutations after DNA replication. In human cells, alkyadenine-DNA glycosylase (AAG) was thought mainly responsible for the recognition and excision of hypoxanthine. In Escherichia coli, deoxyinosine (hypoxanthine deoxyribonucleotide) is removed through an alternative excision repair pathway initiated by endonuclease V. Endo V homolog had been found in mouse and human cells, and biochemical experiment showed that mouse endo V also initiates DNA repair by endonuclease cleavage at the second phosphodieaster bond 3’ to the dI lesion. However, the correction of dI in mammalian cells appeared to be more complex and was not fully understood. To find out what machenisms may involve in dI repair in human cell, we designed dI in vitro repair system using human cell extract. In this study, we applied C-I and G-I substrates to explore the repair pathway and optimize the dI in vitro repair system in human cells. Our results showed that C-I substrate could be repaired more efficiently in HeLaS3 than in HCT116. We also tested the factor requirement for the repair in human cell extracts. Mg2+ is essential in the repair of C-I, and repair levels decreased in cell extracts without dNTPs or ATP or replaced ATP with ATP-γ-S. We concluded that ATP will be utilized and also hydrolyzed for repair process of dI substrates. We assume that DNA Polβ involved not only in BER but also in human endonuclease V-mediated excision repair, so we performed the reaction in the presence of polβ inhibitor, lithicolic acid(LCA). We found that repair levels of C-I substrate decreased about 60%. In the presence of polα、δ、 ε inhibitor aphidicolin (APH), repair levels of C-I decreased about 30% in HeLaS3 extract. HCT116 extract showed lower repair levels of dI than that of HeLaS3 extract, we believed that MMR system played some roles in dI repair system in human cell. We added purified MutLα in repair reaction and found that the deficiency of G-I repair can be partially restored by MutL-α complementation in HCT116 extract. Through these data, we concluded that there were multiple repair systems involved in dI repair, and took charge in different dI pair lesions. According to our previous study, G-I was the best substrate in HeLaS3 extract, threrfore we used G-I as substrate to optimize in vitro repair assay. We found most of substrates could be repair in 30 minutes. The opitimal concentration for ATP is 1 mM. However ATP concentration higher than 2 mM would cause G-I substrates unstable. When ATP was replaced by GTP, the repair level dramatically dropped. With Mg2+ titration test we realized too much Mg2+ would inhibit G-I repair efficiency. The opitimized dI in vitro assay will be very useful for future investigation of this important DNA repair mechanism.