Please use this identifier to cite or link to this item: http://hdl.handle.net/11455/60847
標題: 以結構為基礎探討獨特DNA構造及其對神經系統疾病的相關性
Structural basis for unusual DNA conformations:implications for neurological diseases
作者: 羅羽昇
Lo, Yu-Sheng
關鍵字: 剪切式GA配對
sheared G�A pairs
未配對G堆疊
中心粒
DNA重複序列
放線菌素D
結晶結構
unpaired G-stacks
centromere
DNA repeat
actinomycin D
crystal structure
出版社: 基因體暨生物資訊學研究所
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摘要: 脊隨小腦共濟失調症是一群體染色體顯性遺傳的神經退化疾病,其病症主要特徵為小腦共濟失調。脊隨小腦共濟失調症候群31型第二大類脊隨小腦共濟失調症候群,此些病症致病基因在非轉譯區含有五個核苷酸重複序列擴增造成基因變異而導致疾病產生。先前的研究中指出,在染色體16q22.1中,以南方墨點法偵測到有一段長約2.5-3.8 k bp 的(TGGAA)n重複序列插入,此序列的重複插入好發於SCA31的病人,並導致疾病的產生。本研究設計一段TGGAA重複序列,首先以結晶結構探討[d(GTGGAATGGAAC)]2的結晶結構,以sheared G.A配對結合,然而造成鳥嘌呤與鳥嘌呤的堆疊作用,並且在金屬離子Mn2+與DNA主要溝槽的非配對G-G堆疊以配位鍵結合。最後,由結果得知(TGGAA)10以穩定的髮夾結構存在,而此髮夾結構在生理上扮演著重要的角色,可以依據此結果提供SCA 31疾病致病的機制。另一部分在三重覆序列延長會引起神經遺傳性疾病,且這些遺傳性疾病中的DNA容易形成髮夾狀,此DNA序列包含了許多GC鹼基配對,如:(CXG)n重複序列造成不同疾病的產生,其作用機制更是研究的關鍵;Act D為抗癌藥物的一種,此藥物平面結構會嵌入DNA鹼基配對之間,以DNA的GC鹼基有強烈的結合能力,進而造成DNA螺旋結構的翻轉、扭轉與左旋結構變化。本研究主要探討CGG重複序列與Act D之結晶結構,以Act D之phenoxazone ring的嵌入分別造成G6與G15兩個鳥嘌呤的翻轉,而在G15與Act D1 phenoxazone ring是以C-H/π-ring相互作用,以及G6與Act D2 phenoxazone ring以C=O/π-ring交互作用,此結果造成C4與G14之間的鍵結是以Gsyn:Canti結合。進一步以圓二色光譜儀分析與DNA熱穩定性分析不同序列DNA與Act D反應後的變化,因Act D的嵌合作用而改變DNA二級結構並且提高熱穩定性,並且在DNA嵌入藥物中Act D與GC鹼基穩定性最高。進一步以表面薄膜共振分析得知兩個GC結合位之DNA與Act D之間為高結合能力與高解離能力;以Stoichiometry分析得知DNA與Act D是以1:6比例結合,最後以膠體電泳位移實驗得知Act D會抑制CGG延長的作用,進而干擾DNA複製。從以上的結果,可了解Act D與CGG三重複序列的結合機制,並且對於未來治療遺傳性疾病的藥物研發,提供分子結構的基礎。
The repeat expansion diseases are genetic disorders caused by intergenerational expansions of a specific tandem DNA repeat. Spinocerebellar ataxia type 31 (SCA31) is a disease-specific insertion containing penta-nucleotide (TGGAA)n repeats, which occur at the centromeres of human chromosomes. Thus, we have solved the d(GTGGAATGGAAC) DNA structure, and refined it to 2.77 A resolution. d(GTGGAATGGAAC) tandem repeats form an unusual "self-complementary" antiparallel duplex containing (GGA)2 motifs, in which two unpaired guanines from opposite strands intercalate between sheared G.A base pairs. The duplex contains a (GGA)2 motif with intercalative unpaired guanine residues stacking between two sheared G.A base pairs. In addition, through synthetic DNA sequences, (TGGAA)10 was able to fold back into stable hairpin structure. The hairpin form of TGGAA repeats may play important physiological roles in the pathogenic mechanism of SCA31. On the other hands, tandem repeats of the CGG trinucleotide sequences are widely observed in human chromosome and the expansion of this (CGG)n is frequently found in a number of hereditary neurological disorders. Fragile X syndrome (FXS) is present in the 5’-untranslated region (5’-UTR) of CGG repeats, that is correlated with the full mutation (over 200 CGG repeats), which contains many contiguous GpC sites separated. The potent anticancer drug actinomycin D (ActD) acts by intercalating to DNA GpC site, thereby interfering with essential biological processes including replication and transcription. However, earlier studies showed that binding sequence specificity of Act D is the 5’-GpC site, although other sites (e.g., GpG) also have an unusual affinity toward Act D. Thus, we have solved the Act D binding for CGG triplet repeat sequences, and refined it to a 2.6 A resolution with crystal structure of Act D complex to d(ATGCGGCAT). Binding of two ActD molecules to GCGGC causes nucleotide flipping-out, sharp kink and left handed twist in the DNA helix via a two-independent-binding site model. The C-H/π-ring interaction between DNA motif and the chromophore of Act D stabilized the looped-out state guanine bases that resulted from the intercalation of the phenoxazone ring between base pairs of DNA. Interestingly, the C4-G14 step exhibits in common with the unusual G(syn) - C(anti) step in left-handed DNA. The helix DNA which is B-like structure would be structural change arisen from stabilization of the A form type DNA though Act D binding. The duplexes containing two GC step flanking one G:G mispair indeed provide two Act D binding sites contributions to cooperativity upon the results from both melting and binding affinity assay. In addition, through synthetic DNA sequences, 5’-(CGG)16-3’, Act D interfered with DNA expansion via intercalation into the GpC step by gel electrophoretic expansion assay, it might be a potential drug to inhibit CGG trinucleotide repeat expansion during DNA replication, repair, and transcription. Our results may provide the possible biological consequence of ActD bound to CGG triplet repeat sequences toward developing effective therapeutic agents for these hereditary diseases.
URI: http://hdl.handle.net/11455/60847
其他識別: U0005-0811201218572900
文章連結: http://www.airitilibrary.com/Publication/alDetailedMesh1?DocID=U0005-0811201218572900
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