精胺對 GGCCTG 重複序列之髮夾型結構的滑動重組動態學的影響

Abstract

很多神經退化性疾病都是由重複序列異常擴張所引起，包含脊髓小腦萎縮症 36 型 (Spinocerebellar ataxia 36, SCA36)。他是由位於 Nop56 基因中的 GGCCTG 六核苷酸重複序列擴張所造成的，會造成一些神經功能障礙，像是肢體不協調及小腦萎縮等症狀。此類重複序列通常會折疊成二級結構，而這些二級結構被認為是在複製、修復或重組過程中造成不正常序列擴張的原因。我們使用單分子螢光共振能量轉移 (single-molecule fluorescence resonance energy transfer, smFRET) 研究重複次數為 3 ~ 8、11 及位於預突變 (premutation) 範圍的 17 的 (GGCCTG)n 重複序列的結構動態學，以及在精胺 (為與 GGCCTG 具有特異性結合的多電荷胺類) 影響下的動力學。我們發現偶數重複的 (GGCCTG)n 會穩定形成 2 個核酸 (2-nt) 突出髮夾結構 (2-nt overhang hairpin)；而奇數重複的 (GGCCTG)n 會在 2-nt 突出髮夾結構與 8-nt 突出髮夾結構 (8-nt overhang hairpin) 之間做轉換，且平衡利於 2-nt 突出髮夾結構。精胺的添加使平衡傾向於 2-nt 突出髮夾結構，使其可能具有更高的重複序列擴張的趨勢。Abnormal repeat expansion is a major cause for many neurodegenerative diseases, including Spinocerebellar Ataxia 36 (SCA36). GGCCTG hexanucleotide repeat expansion in the Nop56 gene is responsible for nervous system dysfunctions, including incoordination and cerebellar atrophy. These tandem repeats DNA usually fold into a secondary structure, which is believed to be crucial in abnormal expansion during replication, repair or recombination process.We used single-molecule fluorescence resonance energy transfer (smFRET) microscopy to study conformational dynamics of (GGCCTG)n sequences with n = 3 ~ 8, 11 and 17, as well as the dynamics under the influence of spermine, a polycharged amine that binds to GGCCTG tandem repeat specifically. We found that even-numbered repeats of (GGCCTG)n form stable 2-nt overhang hairpin structure while odd-numbered repeats form hairpin interconverting between a 2-nt and 8-nt overhang structures, with the equilibrium favors a 2-nt overhang hairpin structure. The addition of spermine makes the equilibrium futher lean toward the 2-nt overhang hairpin structure, which may have higher tendency to undergo repeat expansions.