應用質譜儀進行DNA修復蛋白的分析

Abstract

Uracil是在細胞中正常存在於RNA上的鹼基，但uracil會在某些情況下不正常的出現在DNA中，並可能會產生由dC變為dT的transition mutation，所以清除掉DNA上的uracil則非常重要。uracil-DNA glycosylase (UDG)為細菌或真核細胞中主要切除DNA中uracil的酵素，許多文獻指出，當細胞中缺乏UDG的功能時會導致很多疾病，因此近年來發表了許多偵測UDG活性方法的研究報告，傳統的偵測方法為放射線及螢光標定，近年來還發展出呈色法、電化學等方法，然而每個方法都有其限制性，為了提供更為精確可靠的測定方法，我們利用基質協助雷射去吸附離子化飛行質譜儀(MALDI-TOF MS)偵測反應後分子量的變化的能力，以測得UDG切除uracil的活性，設計出一種不需要任何同位素或是螢光標記，且步驟簡單的方法測定UDG活性。 首先測試MALDI-TOF MS的操作是否能夠偵測到UDG的活性及含有dU及AP site DNA穩定性，頻譜圖結果顯示原本錯誤鹼基股的訊號在與UDG反應30分鐘後消失並產生分子量減少約96之dU被切除的產物。接下來測試UDG催化反應的酵素動力學，以短時間反應測得UDG反應初速，將不同濃度的DNA受質反應初速繪製成圖表，並計算出UDG的最大反應速率為1.02 sec-1、Km值為51.3 nM，數值與傳統測定結果相合。 接著探討最適合用於實驗的UDG濃度，以50 pmole的DNA受質與不同濃度的UDG作用分析反應效率，發現以0.05 U UDG的反應，在3分鐘內最能夠觀察到其反應過程，因此接下來實驗所使用的酵素濃度為0.05 U。在先前的文獻指出UDG能夠與單股及雙股DNA進行反應且對於單股DNA的反應速率較快，於是我們以質譜儀測試UDG對於單股DNA及雙股DNA的反應效率，結果顯示UDG切除單股DNA上U的效率比起雙股DNA高了約3倍，與先前文獻的結論相同，另外也發現dU在DNA受質的5’端時，UDG的反應速率較快。 前部分的實驗都是以phenol/chloroform終止反應，但phenol/chloroform具有毒性且牽涉相的變化，實驗流程不易自動化，於是我們考慮透過降低pH值終止反應，結果顯示以酸終止法能夠順利使UDG失活。接下來因為希望能夠將此檢測技術應用在人類細胞的層面，我們首先設計能夠用於cell extract試管內實驗的DNA受質，發現UDG能夠與以phosphorothioate bond修飾後的DNA受質作用。並且也測試了人類細胞中UDG的其中一員hSMUG1活性也能由MALDI-TOF MS所偵測到。另外我們以濃度由低至高的UDG的抑制物UGI (UDG inhibitor)與UDG反應，測試出UGI的抑制效果，希望能延伸此偵測平台至藥物的發展上。 經過質譜儀對於UDG反應的探討，此種透過質量改變來分析修復DNA損傷的酵素活性方法，不僅可以觀察到反應的過程及中間產物，並且應用上更加簡單、快速，使得這個方法應用性更加廣泛。

Uracil is a normal base in RNA. However, in DNA it can arise from spontaneous deamination of a deoxycytidine residue. Reactive oxygen species from normal aerobic respiration as well as exposure DNA to nitrate can also enhance the formation of uracil. Uracil in DNA is potentially mutagenic since it prefer to pair with dATP during replication, yielding G-C to A-T transition mutation. Uracil DNA glycosylase (UDG) acts as a key component in base excision repair (BER) pathway to repair hydrolytic deamination of cytosine in DNA, thus is very important in maintaining genome integrity. The abnormal UDG activity in human cells may cause malfunction of uracil excision repair and eventually various diseases. In recent years, new methods have been published to conveniently detect UDG activity. Traditional methods involved in radioactive labeling coupled with gel-electrophoresis or chromatography. Alternative approaches such as fluorescence, electrochemical and G-quadruplex assay are also applicable. However, most of these methods required specific labeling or had limitation of specific sequence design, not suitable for standardization of UDG measurement. Herein, we designed non-labeled and non-radio-isotopic and very specific method to measure UDG activity. An oligodeoxyribonucleotide with a single uracil is annealed to a template DNA forming a defined G-U mismatch and is hydrolyzed by Escherichia coli UDG. Resulting product containing an apurinic/apyrimidinic (AP) site is subjected to Matrix Assisted Laser Desorption/Ionization-time of flight mass spectrometry (MALDI-TOF MS) analysis. The cleavage of uracil is identified by the mass change from uracil substrate to AP product. The high resolution of MS results clearly separate product signal from substrate signal. We evaluated the UDG kinetic assay by MALDI-TOF MS. Assay UDG activity in short time course could obtain the initial rate of the reaction. The value of Vmax and Km were 1.02 sec-1 and 51.3 nM, which were consistent with traditional assay. To evaluate the suitable enzyme concentration for subsequent experiments for substrate specificity, we titrated the UDG cleavage efficiency using 50 pmole of DNA substrate. We concluded that applying 0.05U UDG yielded the best result. We examined UDG activity with single strand and double strand DNAs containing single uracil at various positions of center, near 5’ end, or near 3’end. We found that UDG showed three fold active on single-stranded DNA substrate than that of double-stranded reactions. And we also found that UDG reacted more effectively when dU was at 5’ end of DNA substrate. For a protocol safer and amenable to automation, we tested an alternative quenching protocol with HCl/DEA yielded similar results. We designed and tested endonuclease-resistant DNA substrate with chemical modification intend in vitro human cell assay. We also demonstrated the activity of hSMUG1 which is one of UDG in human cells by MALDI-TOF MS. We also subject uracil substrate to direct comparison of E. coli and mammalian UDG measurements, and we found inconsistency of units defined by different assays from the same commercial source of the enzymes. For the convenient, accurate, precision of this MALDI-TOF analysis should make it potentially a reference method in the future. Uracil DNA Glycosylase inhibitor (UGI) was employed to evaluate inhibition kinetic. The results should demonstrate the high potential for pharmaceutical application of glycosylase inhibitor screening.