以單分子光譜觀測 CTG 重複序列的滑動現象

Abstract

三核苷酸重複序列會導致許多種神經退化性遺傳疾病，而其中的 CTG 重複序列是導致神經退化性脊髓小腦失調症第8型、肌強直性型肌肉萎縮症後群第一型、類亨丁頓舞蹈症第二型的主要因素，這些疾病的病患的染色體上，能夠發現其所擁有的 CTG 重複序列會有過表達的現象 (至少 35 組以上)。造成 DNA 重複序列擴張的原因，是由於 DNA 本身會形成非典型的二級結構，因此誘導 DNA 在複製、修復與重組的過程中發生滑動現象。在本篇研究中，我們使用單分子螢光共振能量轉移光譜研究 CTG 重複序列的結構動態學，其結構會與 CTG 的重複次數有關，偶數重複的 (CTG)n 會穩定折疊成兩端對齊髮夾結構；而奇數重複的 (CTG)n 則會在兩端對齊髮夾結構與單組 CTG 突出的髮夾兩種結構間，進行互相轉換，且無論重複次數多寡，構型皆傾向於兩端對齊髮夾結構。我們提出一個構型間轉換過程的局部不穩定傳遞模型，其是先由髮夾結構中的環處改變構型，並形成氣泡狀突出，之後以氣泡移動的方式，連續往末端移動，直到構型改變。而重複序列增長時，雖然總體穩定性增加，但因為構型轉換是由局部不穩定所驅動，因此此一滑動機制可以延伸到更長的序列，可能為導致DNA擴張而致病的原因之一。

Tandem nucleotide repeats are responsible for many genetic neurodegenerative disorders. Among them, the trinucleotide, CTG, repeat is the primary cause of Spinocerebellar Ataxia Type 8, Myotonic Dystrophy Type I, and Huntington disease like 2. Overexpression (>35 bases) of CTG repeat can be found in the chromosome of every patient. The possible cause of error-prone expansion is the DNA slippage induced by the folded atypia structures during DNA replication, repair or recombination process. Here, we present our single-molecule fluorescence resonance energy transfer study on the size-dependent structure and structural dynamics. A parity (even or odd) dependence was found: The even-numbered sequence folds into a stable blunt-end hairpin structure, while the odd-numbered repeat mainly folds into two hairpin structures including single-repeat overhang structure and blunt-end hairpin structure. Interestingly, dynamic interconversions between the blunt-end and the overhang configurations were observed in the longer odd-numbered-repeat sequences and the equilibrium lean toward to the blunt-end configuration. We propose a multi-step bulge transfer model: Local conformational transition at the looping region induces a bulge formation followed by stepwise bulge transferring toward the end of the hairpin and ultimately achieves the conformational change. This mechanism allows the DNA slippage extends to longer repeats that carry greater global stability and can possibly be the cause of the disease-prone expansion.