利用飛行時間質譜儀進行基因恆定性相關酵素活性之研究

Abstract

此篇研究主要為利用基質輔助雷射脫附游離飛行時間質譜技術 (MALDI-TOF MS)，搭配未標定的DNA寡核苷酸受質設計，研究三種維護基因體穩定性酵素的活性，提出新的檢測酵素活性的方法，期望未來可應用於生技產業或臨床檢驗。此篇研究分為三個部分，第一部分為第一型DNA聚合酶校正在單一核苷酸插入/缺失錯誤的活性之研究；第二部分為大腸桿菌中DNA修復蛋白Uracil-DNA glycosylase (UDG)活性之研究；第三部分為大腸桿菌第五型內切酶活性檢測方法之建立與研究。 重複序列DNA因為易於滑動，在複製過程中常見插入/缺失錯誤的產生，此錯誤通常可以透過DNA聚合酶校正活性進行校正，但若錯誤位置距離引子3’端較遠，則可能因為末端正確配對使聚合酶直接進行引子延伸。為了研究插入/缺失錯誤之校正活性，我們設計了距離3’端倒數第1至第9個位置的單一核苷酸插入/缺失錯誤，利用MALDI-TOF MS的方法分析 DNA聚合酶的校正活性。使用3種dNTP或4種ddNTP進行校正活性的測定，研究結果發現距離3’端第1至5個核苷酸的插入/缺失錯誤能夠很好的被第一型 DNA 聚合酶進行校正，在距離3’端第6個核苷酸的插入/缺失錯誤僅部分被校正，距離3’端第7至9個核苷酸的插入/缺失錯誤無法被 DNA 聚合酶校正，而會直接進行引子延伸。根據以上結果我們推測此校正活性與傾向，和DNA聚合酶與DNA引子-模板交界的接觸有關。 第二部分為大腸桿菌DNA修復蛋白Uracil-DNA glycosylase (UDG)活性之探討。尿密啶 (Uracil)是DNA中常見的損傷，由胞密啶水解脫氨形成。此DNA損傷若未即時進行修復，則會在DNA複製後形成G:C to A:T的轉換突變。在大腸桿菌中，主要由UDG進行U的修復，此酵素於原核與真核細胞中在演化上高度保留。已知許多測定UDG活性的方法都有其限制性，故我們欲發展高專一性與非標定受質的檢驗方法，期望能應用於臨床上抑制劑篩選的方法。我們利用帶有U的寡核苷酸受質與UDG酵素進行反應，形成帶有apyrimidinic site (AP site) 的產物與U受質分子量相差94，經過MALDI-TOF MS的分析，能夠用質譜圖的信號強度計算酵素的活性。我們利用此方法針對UDG進行酵素活性的研究，結果顯示其Km為50 nM, Vmax 為 0.98 nM/s ，Kcat 為 9.31 s-1，此結果與傳統法所得結果相近。利用uracil DNA glycosylase inhibitor (UGI) 作為抑制物進行抑制效果檢測，結果測得IC50為7.6 pM。另外針對多種的U受質檢測UDG對不同受質的專一性，結果顯示U在正中間且與G配對的受質(T1/U+9)最適合用於檢測UDG的活性，且以質譜儀檢測UDG活性能夠很好的應用於臨床上的檢測。 第三部分則利用MALDI-TOF MS的平台建立用以檢測大腸桿菌內第五型核酸內切酶酵素(Endo V)的活性。DNA 中的腺嘌呤經脫氨作用會形成亞黃嘌呤(dI) 的損傷，若未被及時修復再次複製後會形成A:T to G:C的轉換突變。在大腸桿菌中Endo V為參與dI修復的重要酵素，先前研究發現dI的修復由Endo V、第一型DNA聚合酶(Pol I)與DNA連接酶完成。Endo V在切割完dI受質後會停留在DNA受質上，被認為可能與後續Pol I的作用有關。為了能夠更深入研究Endo V的活性，我們發展以MALDI-TOF MS方法搭配高週轉性的dI受質進行Endo V酵素活性的分析，結果發現高週轉性受質能夠提高Endo V週轉5次的切割活性，並能夠以動力學方法分析發現Endo V對不同DNA受質具有特異性(I-G> I-A≒I-T> I-C)，且利用MS的方法相較於螢光法可提升16倍的靈敏度。我們也利用尿素變性聚丙烯醯胺膠體電泳搭配螢光標定的DNA受質，以研究Endo V與Pol I的交互作用，發現具有3’端至5’端外切酶活性Pol I可以促進Endo V週轉1.5倍的活性，而沒有外切酶活性的Pol I則促進週轉活性能力較差。因此可從這個結果判定Pol I與Endo V之間在修復過程有交互作用。 在此篇論文中我們展現了MALDI-TOF MS此平台檢驗的優勢，快速、高通量、高專一性，搭配非標定的DNA 受質且彈性的受質設計，可以更全面的了解酵素的活性，更重要的是可以做抑制物的檢測，將可應用於生技製藥的研發。

Matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS) has been widely used in the detection of DNA modifications and SNPs. In this study, a non-labeled DNA substrate assay was established using MALDI-TOF MS to study the activities of DNA maintenance enzymes, including DNA polymerase I (Pol I), uracil DNA glycosylase (UDG), and endonuclease V (Endo V). Insertion/deletion errors frequently occur during the replication of repetitive DNA sequences and could be corrected by DNA polymerase proofreading activity. A series of single nucleotide insertion/deletion (indel) error substrates, with the indel errors located 1 to 9 nucleotide (nt) from the 3' terminus, were designed for a proofreading assay and subjected to MALDI-TOF MS. The proofreading activity was performed with three dNTPs or four ddNTPs. The results revealed that indel errors located 1 to 5-nt from the 3' terminus could be effectively proofread by the Pol I, while partially proofread when indel errors at 6-nt from the 3’ terminus. Indel error at 7 to 9-nt from 3’terminus escaped proofreading, leading to primer extension. Based on these results, it suggests that proofreading activity is correlated with the interaction between DNA polymerase and primer-template junction. Uracil (U) is a DNA damage caused by the deamination of cytosine. It would cause G: C to A: T transition mutations if not repaired prior to DNA replication. In E. coli, uracil is mainly repaired by uracil UDG, an enzyme that is highly conserved in both prokaryotic and eukaryotic cells. The UDG detection assay was designed with site-specific uracil and performed by MALDI-TOF MS. UDG activity was determined using a non-labeled double-strand oligonucleotide carrying a uracil on its middle position. Kinetic parameters of UDG with Km at 50 nM, Vmax at 0.98 nM/s, and Kcat of 9.31 s-1 were obtained by mass spectrometric analysis and these parameters were comparable to those revealed by traditional methods. The inhibitor screen assay was performed with a uracil glycosylase inhibitor (UGI), yielding an IC50 at 7.6 pM. Additionally, the UDG substrate specificity for various uracil substrates was tested, concluding that the substrate with uracil located at the center and paired with guanine is the most suitable for detecting UDG activity. Deoxyinosine (dI) is formed by the deamination of adenine and leads to A: T to G: C transition mutations if not repaired before DNA replication. In E. coli, dI repair involves Endo V, Pol I, and DNA ligase. Previous nicking assay showed tightly binding of Endo V to dI-containing DNA suggesting the possible role of dI-bound Endo V as a repair signal for Pol I. To study the interplay of Pol I and Endo V in the repair of dI, the activity of Endo V was determined using a MALDI-TOF MS-based assay and a PAGE assay. Analysis of Endo V activity using MALDI-TOF MS showed a 16-fold higher nicking activity compared to the fluorescence-based method, and substrate specificity in the order of I-G > I-A ≈ I-T > I-C. The interaction between Endo V and Pol I was studied by denaturing urea polyacrylamide gel electrophoresis with fluorescently labeled DNA substrates. The results indicated that Pol I KF with 3' to 5' exonuclease activity enhances Endo V turnover by 1.5 times, while Pol I without exonuclease activity showed much less turnover. These findings suggest a direct interaction between Pol I and Endo V in the repair process. In conclusion, we demonstrate that MALDI-TOF MS is a powerful tool for enzyme activity analysis. It offers rapid, high-throughput, and highly specific results, and allows for flexible substrate design using unlabeled DNA. This approach provides a comprehensive understanding of enzyme activity and shows great potential for inhibitor screening in biomedical settings.