GGCCTG 六核苷酸重複髮夾結構的構型多樣性與精胺的特異性結合研究

Abstract

串聯重複DNA序列的異常擴張是神經退行性疾病的主要原因之一。脊髓小腦性共濟失調症第36 型 (SCA36) 是一種遺傳性神經退行性疾病，由 Nop56 基因中 GGCCTG 六核苷酸重複序列的擴張引起，具有肢體不協調和小腦萎縮等症狀。由串聯重複 DNA 形成的二級結構被認為在複製、修復和重組過程中的不正常擴張有關連。多胺為許多細胞過程中，包括 DNA 複製和轉錄，不可缺少的多價陽離子，先前的結晶學研究顯示自然界中豐富的精胺與GGCCTG具有特異性的結合。因此，我們使用單分子螢光共振能量轉移光譜研究 GGCCTG 重複序列的構型動態學以及精胺的影響。我們發現奇數重複的 (GGCCTG)n 折疊成兩個可相互轉換的髮夾結構，分別為具有2-nt與8-nt的垂懸的髮夾結構，而偶數重複的 (GGCCTG)n 折疊成一個穩定的 2-nt 懸垂髮夾。利用滯留時間分析得到在8-nt垂懸髮夾狀態具有兩種FRET無法區分的構型。在精胺的存在下，其中一種滯留時間較長的構型，會形成了緊湊的結構。而在其互補序列 (CAGGCC)5 髮夾構型中未觀察到類似行為，表明精胺與 (GGCCTG)5 髮夾構型具有獨特的結合。Abnormal expansion of tandem repeat DNA sequences is one of the major causes of neurodegenerative diseases. Spinocerebellar ataxia type 36 (SCA36), a hereditary neurodegenerative disorder, is caused by the expansion of a GGCCTG hexanucleotide repeat in the Nop56 gene with symptoms of limb incoordination and cerebellar atrophy. Secondary structures formed by tandem repeat DNA are believed to facilitate expansion during the replication, repair, and recombination processes. Polyamines are essential in many cellular processes, including DNA replication and transcription. Previous crystallographic study demonstrated a specific binding of spermine, a naturally abundant polyamine, to GGCCTG sequences. Thus, we used single-molecule fluorescence resonance energy transfer (smFRET) to investigate the dynamics and stability of GGCCTG repeats and the influence of spermine. Odd-numbered GGCCTG repeats fold into two interconvertible hairpin structures with 2-nt and 8-nt overhangs, while even-number repeats fold into a stable 2-nt overhang hairpin. Kinetics analysis shows two structural sub-states with different dwell time (long-lived and short-lived) distributions and virtually identical FRET distributions. In the presence of spermine, the structure of the long-lived species becomes more compact. Similar behavior was not observed in the antisense (CAGGCC)5 hairpins, indicating a specific and unique binding of spermine to the (GGCCTG)5 hairpins.