Translated Titles

Structural basis for specific binding of echinomycin to methylated DNA with C:C mismatch



Key Words

甲基化胞嘧啶 ; 錯誤配對鹼基 ; 棘黴素 ; 5-methylation ; DNA mismatch ; Echinomycin



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Chinese Abstract

生物能透過DNA聚合酶與錯配修復系統來校正遺傳訊息,但仍然會出現DNA錯配的情況,若DNA修復系統無法修復錯配的DNA,可能會導致遺傳疾病或癌症的發生。在人類細胞的基因組中會透過表觀遺傳學的方式來修飾DNA,甲基化DNA對於生物體發育中的過程扮演著非常重要的角色,而甲基化主要會透過甲基轉移酶(methyltransferase)在CpG位點之胞嘧啶其5號碳上添加甲基基團就會形成5-甲基胞嘧啶。棘黴素(Echinomycin , Echi)是一種歷史悠久的抗癌用藥,其功能可以透過Ala辨識鹼基中的鳥嘌呤,而quinoxaline嵌入CpG位點的DNA,干擾其DNA的複製與轉錄進而達到抑制癌細胞的生成。根據先前的研究結果指出Echinomycin對於富含CpG位點的序列有較高的選擇性結合能力,而CpG island剛好甲基化的熱點區,所以本篇研究探討由Echinomycin專ㄧ性作用於含胞嘧啶錯配之甲基化核酸序列,首先利用生物物理實驗和X光繞射晶體學研究Echinomycin與甲基化DNA(5mC)之結合作用,並設計d(ACGCCGT)2序列中分別含有零個、一個、兩個或三個甲基化胞嘧啶之DNA雙鏈體來完成實驗,藉由 DNA熱穩定性實驗,比較不含甲基化和甲基化之序列間其DNA雙股螺旋解開成單股長鏈之融化溫度(Tm),並加入藥物觀察其變化程度的差異,與非甲基化DNA相比,Echinomycin的添加增加了甲基化DNA的穩定性。接著進一步以結晶結構解析Echinomycin與甲基化與非甲基化DNA的結合構象,結果顯示Echinomycin結合於甲基化DNA後,與非甲基化DNA相比,其晶體堆疊的方式不同;錯配鹼基之氫鍵鍵結數較多,且配對型態較像搖擺(wobble) 配對;其甲基化胞嘧啶之甲基基團與藥物鍵結的數目也較高,使得甲基化DNA-藥物複合物有更高的穩定性。期望我們的發現有助於棘黴素發展成為具潛力之藥物,未來可針對涉及高甲基化之疾病的潛在抗癌藥物奠定了基礎。

English Abstract

Genetic information is ensured by proofreading of DNA polymerase and mismatch repair system. But there are still some mismatches occur. If the DNA repair system is disable to repair the mismatches, it will lead to the occurrence of genetic disease or cancer. DNA methylation plays an important role in normal organismal developmentand methylation mainly occurs by the addition of a methyl group on the carbon at position 5 of cytosine, which leads to the formation of 5-methylcytosine at CpG sites in genomic DNA. Echinomycin (Echinomycin , Echi) is an anticancer drug which functions by intercalating into DNA at CpG sites, it related to their ability to intercalate into the DNA duplex with high affinity, thereby interfering with DNA replication and transcription. In the current study, we employed biophysical experiments and X-ray crystallography to investigate the effects of echinomycin binding to methylated DNA and design the d(ACGCCGT)2 DNA duplex containing one, two or three methylated cytosines were used to carry out melting temperature (Tm) and surface plasmon resonance (SPR) studies with echinomycin. Addition of Echi increased the stability of methylated DNA compared to non-methylated DNA. we compared the Echi-bound conformations of methylated versus non-methylated DNA. The higher stability of the methylated DNA-drug complex is contributed by van der Waals contacts between the quinoxaline ring of Echi and the methylation sites of the cytosines and the mC:mC mismatch adopts a "wobble" confirmation projecting into the major groove and minor groove respectively. Our finding provides the basis to develop potential anticancer drugs against diseases involving hypermethylation.

Topic Category 生命科學院 > 生命科學系所
生物農學 > 生物科學
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