- 學位論文
發展質譜儀進行DNA修復蛋白- hSMUG1之分析
Development of MALDI-TOF MS based assay for DNA repair enzyme - hSMUG1
摘要
Uracil為細胞中正常存在於RNA上的鹼基，當DNA發生自發性水解脫胺或受到環境中化學物質及UV照射會不正常出現在DNA中，形成U:G錯誤配對，會由Base excision repair (BER)來進行修復，若無法修復，複製後會產生C:G轉為T:A的transition mutation。BER最初由uracil-DNA glycosylases (UDG)來辨認移除damaged base，當細胞中缺乏UDG或其失活會誘發產生許多疾病。在人類細胞中有四種UDG，分別是UNG、TDG、SMUG1及MBD4，其中UNG及SMUG1是最主要的修復蛋白，且研究指出SMUG1針對的受質比UNG更為廣泛。近年來發展許多偵測UDG的方法，包括放射線標定、螢光標記及molecular beacon等方法，但其步驟繁瑣，且有限制性。因此我們想利用基質輔助雷射去吸附離子化-飛行時間質譜儀 (MALDI-TOF MS)以偵測分子量變化來分析人類細胞中hSMUG1修復特性。此外，許多研究指出重金屬會對人體產生危害，和許多癌症相關，其中鎘被歸類為第一類致癌物，會大量累積在體內中，為DNA修復蛋白的抑制劑，影響DNA修復，因此接著會發展以此偵測平台來檢測鎘對hSMUG1修復效率影響的潛在測定方法。 首先測試MALDI-TOF MS能否偵測到hSMUG1的活性及含有dU的DNA受質之穩定性，結果顯示分子量為5541的含U股與hSMUG1反應30分鐘後在分子量5445產生訊號，為AP (apurinic/apyrimidinic)product。接著探討適合用於實驗的hSMUG1濃度，比較50 pmoles的DNA受質和不同濃度hSMUG1的反應效率，發現以1U hSMUG1來反應，在5分鐘內可以觀察到最適合的反應過程，因此接下來實驗使用的酵素濃度為1U。最初發現到hSMUG1可針對單股DNA來進行修復而以此命名，近年文獻指出也可針對雙股DNA來進行反應，於是偵測hSMUG1對於單股及雙股DNA的切除效率，結果雙股DNA的反應效率比單股DNA約高2.5倍，也發現對U:G錯配的修復效率比U:A錯配約高4.5倍，且對dU在序列5’端第三個位置及中間位置的切除效率較快，而根據實驗條件不同發現hSMUG1作用後產生的AP site易受不同化學物質或熱而不穩定斷掉。 接著以MALDI-TOF MS偵測鎘對hSMUG1修復效率影響，首先測試適合的DNA受質及hSMUG1的濃度，以20 pmoles DNA受質和不同濃度hSMUG1反應，發現以1U hSMUG1和DNA受質在30分鐘內最能觀察到反應過程，接著和不同濃度鎘反應，觀察其抑制效果，發現從5μM濃度開始hSMUG1修復效率完全被抑制，且IC50落在約1μM濃度。接下來將此技術應用於細胞層面，結果顯示DNA受質可不被萃取物中外切酶分解，且以MALDI-TOF MS可進行分析。 藉由MALDI-TOF MS透過質量的改變來分析hSMUG1的修復特性，不僅可以觀察到反應的中間產物，且方法也更加快速與簡單，未來可作為screening inhibitor的方法。
並列摘要
Uracil is the base in RNA, and it will abnormally appear in DNA when a spontaneous hydrolytic deamination of cytosine occurs or DNA is exposed to chemicals or UV, and yield U:G mispairs. Uracil is repaired by base excision repair (BER). If it is not repaired, it cause G:C to T:A transition mutation after DNA replication. In BER, uracil-DNA glycosylases (UDG) is the first enzyme to identify and remove damaged bases, and the lack or inactivation of UDG may induce many diseases. There are four types of UDGs in mammalian cells, including UNG, TDG, SMUG1 and MBD4, of which UNG and SMUG1 are the major repair protein and SMUG1 has a broader substrate specificity. Many methods have been developed to detect the activity of UDG, including radioisotope and fluorescent dye labeling. However these methods are time-consuming and with limitation. Therefore, this study attempted to develop MALDI-TOF MS that can efficiently identify the change of molecular weight (MW) to detect uracil cleavage activity and repair characteristics of hSMUG1. In addition, many heavy metals can cause harm to humans, and are related to many cancer. Cadmium was classified as a human carcinogen, which will accumulate in organs in large amounts, and it is an inhibitor of many DNA repair proteins. Therefore, we explore the potential of MALDI-TOF MS assay to detect the effects of cadmium on hSMUG1. At first tested whether the stability of uracil-containing DNA substrates can be detected by MALDI-TOF MS and activity of hSMUG1. After 30 minutes reaction, we found the MW5445 AP product was produced. Comparing the cleavage efficiency of hSMUG1 with different concentration of enzyme in the presence of 50 pmoles DNA substrates, result showed that 1U hSMUG1 was the most suitable reaction for us to observe AP products. hSMUG1 was named because it was initially thought to be more selective for single-strand DNA (ssDNA), but later it was identify to have affinity for double-strand DNA (dsDNA). Thus, we tasted the cleavage efficiency of hSMUG1 toward ssDNA and dsDNA, and result indicated that efficiency of hSMUG1 on dsDNA was about 2.5 times higher than that on ssDNA, and found repair efficiency of hSMUG1 on U:G substrate was approximately 4.5 times higher than that on U:A substrate. In addition, hSMUG1 was more effectively when dU located at the third position of 5'end and middle position of the DNA substrate compared to the 3'end position. And according to different experimental conditions, AP site generated after the action of hSMUG1 are easily affected by different chemicals or heat treatments, and it would be lyased. Furthermore, we detected the effect of cadmium on hSMUG1. Using 20 pmoles DNA substrates and 1U hSMUG1 was favorable to observed the reaction. After that, we added various concentrations of cadmium for reaction, and found that repair efficiency of hSMUG1 was completely inhibited by 5μM of cadmium, and the IC50 was 1μM. In addition, we tried to detect the repair activity in cell extracts by MALDI-TOF MS, result showed that DNA substrates would not be degraded by DNA exonucleases in the EDTA-protected cell extracts, and the cleavage efficiency of UDG could be analyzed. In summary, analyzing the repair characteristics of hSMUG1 through the MW change of oligonucleotide substrate by MALDI-TOF MS provides the possibility to observe the intermediate products of the reaction. The method is also faste and convenient, and it can be used as a method for inhibitor screening in the future.