AGG 插入對於導致 X 染色體脆折症的 CGG 三核苷酸重複序列形成的髮夾型結構與結構動態學的影響

Influences of AGG Insertions on the Structures and Structural Dynamics of CGG Trinucleotide Repeat DNA Hairpins Associated with Fragile X Syndrome

鄭凱駿(Cheng Kai-Chun)

指導教授 ： 李以仁

國立臺灣師範大學/理學院/化學系/碩士(2020年)

https://doi.org/10.6345/NTNU202001316

摘要

染色體脆折症是一種常見的遺傳神經性疾病，其發病原因與人體中 FMR1 中 CGG三核苷酸重複序列不正常擴張有關。在正常個體中，CGG 重複次數低於54次，並會穩定地傳給子代；發病的個體中，CGG 重複次數大於200次，造成 FMR1 基因啟動子甲基化區域過多，使腦部無法正常生成 FMRP (Fragile X Mental Retardation 1 fusion protein)，進而影響智力上的發展。
正常人體中，每8到11個CGG序列存在著1個 AGG 的中斷，且約 65%個體中的 FMR1 5’UTR 的 CGG 重複序列中含有兩個AGG的中斷，但AGG 序列對於 CGG 重複序列的影響仍尚未釐清。因此我們利用單分子螢光共振能量轉移光譜，配合圓二色性光譜，對於 CGG 重複序列與插入 AGG 的 CGG 重複序列做一系列的結構與結構動態學的研究。研究結果顯示富含 CGG 的重複序列在生理相關鹽類濃度下，難以形成鳥嘌呤四聯體，CGG 重複序列的主要構型為接近對齊之露出1個核苷酸的突出的髮夾型結構，透過時間解析發現奇數重複次數的CGG 重複序列存在髮夾型結構的滑動現象，但以後的序列以露出1個核苷酸的突出髮夾型結構為主。當插入一組 AGG 後的序列以露出1個及3個核苷酸的突出髮夾型結構兩種構型為主，與 CGG 重複序列相同都有髮夾型結構的滑動，但平衡向露出3個核苷酸的突出髮夾型結構偏移。插入兩組 AGG 後依中間的 CGG 重複次數分成奇數間隔組與偶數間隔組，奇數間隔組以露出3個核苷酸的突出髮夾型結構為主，偶數間隔組則有露出1個及3個核苷酸的突出髮夾型兩種主要結構，但無論是奇數間隔組或是偶數間隔組皆沒有觀察到構型之間的轉換。
根據本實驗室先前提出的擴張理論，CGG 重複序列傾向形成對齊或接近對齊髮夾型結構，較易進行不正常的擴張而變成更長的序列，而髮夾結構的滑動重組可使序列在擴張後回到對齊或接近對齊髮夾型結構而繼續擴張形成循環。而本篇論文發現，插入一組 AGG 後序列構型由長突出髮夾型結構轉變成接近髮夾型結構的速率降低，減緩了序列的擴張，插入兩組 AGG 後序列的構型則是穩定不易產生滑動的，使序列在複製時更不會被擴張，因此我們認為 AGG 插入對於結構動態滑動至利於擴張的對齊髮夾型結構動態平衡影響，是抑制 CGG 重複序列擴張的可能原因。

關鍵字

單分子螢光共振能量轉移 ； 圓二色性光譜 ； 基因擴張 ； CGG三核苷酸重複序列 ； X染色體脆折症 ； 結構動態學 ； DNA 髮夾型結構

並列摘要

Overexpansion of the CGG trinucleotide repeat (TNR) in the fragile X mental retardation 1 (FMR1) region is responsible for the fragile X syndrome, a common genetic neurodegenerative disease. In healthy individuals, the repeat number of the healthy individuals remain below 54, while in pathological samples, the repeat number goes exceed 200. Abnormal expansion of CGG leads to hypermethylation of the FMR1 gene, which leads to the inhibition of Fragile X Mental Retardation 1 fusion protein (FMRP), a crucial protein for the development of intelligence production. However, the tandem CGG motif is usually interrupted by an AGG triplet in a frequency of one per 8 to 11 repeats in healthy individuals, and ~65% CGG motif in FMR1 5’UTR carries two AGG interruptions. However, the role of AGG insertion in abnormal expansion remains elusive.
Here, we use single-molecule fluorescence resonance energy transfer (smFRET) and circular dichroism (CD) spectroscopy to study the structures and structural dynamics of tandem CGG repeats and those with AGG triplet insertions. Our result shows that G-quadruplexes are unlikely to be formed under physiological conditions in both cases. Instead, CGG repeat folds into a near blunt-end hairpin with 1-nucleotide (nt) overhang. Our time-dependent experiment reveals that the odd-numbered CGG repeat hairpin mainly folds into 3-nt overhang hairpin with transient slippage hairpin structural rearrangement to the 1-nt overhang configuration. With one AGG insertion, interconversion between two major configurations in CGG repeats were still observed, but with a new equilibrium lean to the 3-nt overhang state. With two AGG insertion, parity dependence on the repeat number of CGG spacer units between two AGG was found. The odd-numbered CGG spacer sequences mainly fold into a 3-nt overhang hairpin structure. While the even-numbered CGG spacer unit folds into two major hairpin structures with 1-nt and 3-nt overhang. Neither of them shows interconversion between configurations. With the DNA expansion model proposed by our group, TNR with blunt-end or near blunt-end hairpin configuration has a higher potency of undergoing abnormal expansion. Slippage of TNR hairpins could bring the hairpin with newly synthesized DNA segment back to its (near) blunt-end configuration, and thus, continue another expansion cycle. With one AGG insertion, the conversion from 3-nt overhang hairpin to the 1-nt hairpin gets retarded, which could slow down the abnormal expansion. With two AGG insertions, the slippage hairpin reconfiguration is virtually stopped, possibly inhibits the abnormally expansions. In conclusion, we believe that the impact on the hairpin structures and slippage hairpin reconfiguration by the interruption of AGG insertion to the CGG tandem repeats plays a crucial role in inhibiting the abnormal expansion of CGG repeats.

並列關鍵字

smFRET ； CD spectroscopy ； Gene expansion ； CGG trinucleotide repeat ； Fragile X chromosome ； Structural dynamics ； DNA hairpin

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