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標題: 以結構為基礎探討鎳離子與色霉素A3形成的複合體與神經系統疾病相關之CCG三核苷酸重複序列的特異性結合
Structural basis for the specific binding of the Ni(II) complex of dimeric chromomycin A3 to CCG trinucleotide repeats associated with neurological disease
作者: Wen-Hsuan Tseng
關鍵字: Chromomycin A3
trinucleotide repeats
neurological disease
X-ray crystallography
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摘要: With technologies nowadays, scientists have found out that some neurological diseases are correlated with expansion of trinucleotide repeats, such as fragile X syndrome (FXS) , which is caused by the expansion of (CCG)n trinucleotide repeats. The massive (CCG)n trinucleotide repeats expansion may be attributed to the strands slippage along with the transient formation of hairpin structures during DNA replication, and the hairpin structures consist of many contiguous GpC sites flanked by mismatched C:C base pair. Chromomycin A3 (Chro) is an anti-cancer metalloantibiotic, Chro can form a stable dimeric complex via chelation with a single divalent metal ion. Chro binds selectively to GC-rich DNA sequences, and the antitumor properties of Chro may be attributed to its inhibitory effects on replication and transcription during cell proliferation. Our previous studies found a new GC specific DNA-targeting metalloantibiotics, NiII(Chro)2, which shows much greater potential than those of other dimeric Chro complexes on DNA-acting efficacy, including its DNA-binding affinity, DNA stabilization capacity and the inhibition of transcription both in vitro and within cells, and CoII(Chro)2 comes second. When (CCG)n trinucleotide repeats forms intrastrand hairpin structures, it offers many GpC sites flanking by a C:C mispair for NiII(Chro)2 binding. This study focus on the binding of NiII(Chro)2 to DNA with CCG trinucleotide repeats. We solved the crystal structure of NiII(Chro)2 bound to d(TTCCGCCGCCGAA). The binding of NiII(Chro)2 to CCG trinucleotide repeats causes many unexpected conformational changes including nucleotide flipping out and a distortion in the DNA helix, and these binding characteristics have also been observed in the crystal stucture of CoII(Chro)2 -d(TTCCGCCGCCGAA) complex. We also solved the crystal structure of the NiII(Chro)2 complex in the absence of DNA duplex at 0.89 A resolution, and than compared these two Chro dimer conformations with/without DNA duplex. It revealed that the Chro dimer of the the X-ray structure without DNA exhibits shows a larger dihedral angle than the Chro dimer bound to DNA. Furthermore, we conducted a biophysical study to determine the effects of these MetalII(Chro)2 complexes on the CNG trinucleotide repeats. Heat denaturation and surface plasmon resonance analyses shows that NiII(Chro)2 exhibits higher DNA-stabilizing effects towards CCG trinucleotide repeats than do the other trinucleotide repeats. In addition, (CCG)n repeats at least three times provide a favorable binding s ite for NiII(Chro)2. Our result firmly establishes the detailed structural information regarding the DNA binding model of the metal-mediated dimer of the drug, it also points out a useful direction for future new drugs design in the treatment of neurological disease.
迄今已發現許多神經退化疾病與三核苷酸重複序列相關,像是CCG重複序列的異常擴增會導致X染色體脆裂綜合症(Fragile X syndrome, FXS)。CCG重複序列的大量擴增歸因於DNA複製過程中因滑動形成不穩定的髮夾結構,此富含G/C鹼基的髮夾結構中,包含許多連續的G:C配對鹼基彼此間隔C:C錯配鹼基。色霉素A3 (Chromomycin A3, Chro)為作用在DNA之抗腫瘤藥物,Chro可與二價金屬離子螯合形成二聚體結構,選擇性的結合於富含G/C鹼基之DNA序列上,進而干擾細胞增殖時DNA之複製以及轉錄作用。本實驗室先前研究結果發現,一種新型可結合於富含G/C鹼基DNA之metalloantibiotics類藥物-NiII(Chro)2,不論其形成二聚體結構的穩定性、與DNA的結合親和力,以及在體外與細胞內抑制轉錄作用的能力等,都優於其他金屬離子和Chro所形成之二聚體結構,而CoII(Chro)2則表現次之。當CCG重複序列形成髮夾結構時,結構中連續的G:C配對鹼基彼此間隔C:C錯配鹼基提供了NiII(Chro)2許多結合位置,因此本篇研究著重於探討NiII(Chro)2與CCG重複序列的結合機制,我們利用結晶結構解析NiII(Chro)2與d(TTCCGCCGCCGAA)的結合機制,結果顯示NiII(Chro)2結合於CCG重複序列導致非預期之結構,像是造成錯配胞嘧啶(Cytosine, C)翻轉以及DNA的扭轉,此結構特徵也同時出現在CoII(Chro)2- d(TTCCGCCGCCGAA)複合體晶體結構。我們同時也解析了解析度達0.89 A 之NiII(Chro)2二聚體結構,在比較NiII(Chro)2與CCG重複序列結合前後構型變化的結果後,顯示出NiII(Chro)2二聚體的單體間夾角及扭轉角度在與DNA結合後明顯改變。我們也進一步以生物物理方法探討MetalII(Chro)2對於不同三核苷酸重複序列的結合機制,由DNA熱穩定性分析結果發現不同三核苷酸重複序列中,NiII(Chro)2能明顯提高CCG重複序列之熱穩定性,而表面電漿共振技術進行動力學分析結果也顯示,NiII(Chro)2對於CCG重複序列的結合親和力隨著序列中CCG重複次數增多而上升。研究結果皆指出相較於其他三核苷酸重複序列,NiII(Chro)2能特異性的結合於CCG重複序列,並顯著提升其DNA結構穩定性,且CCG重複次數達三次以上可以提供NiII(Chro)2良好的結合位置。了解NiII(Chro)2與CCG重複序列的結合機制後,期望此研究有助於NiII(Chro)2發展成具潛力之藥物並有效應用於CCG重複序列相關之疾病。
其他識別: U0005-1312201306070400
文章公開時間: 10000-01-01
Appears in Collections:基因體暨生物資訊學研究所



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