三核苷酸重複序列之髮夾型結構阻礙 hRPA 蛋白的解旋能力

Abstract

三核苷酸重複序列與許多神經退化性疾病有關，其容易摺疊成二級結構，常會造成DNA滑動，進而導致DNA的異常擴增。人類複製蛋白A (human Replication Protein A, hRPA)為真核生物當中最豐富的單股DNA結合蛋白，在複製、修復、重組期間，可以避免瞬時存在的單股DNA重新黏合回雙股DNA，或是形成二級結構。hRPA亦有能力解開髮夾型結構與G-四聯體結構等二級結構，以維持基因組的穩定性。我們使用CTG重複序列之髮夾型結構作為模型系統，以單分子螢光共振能量轉移(single-molecule fluorescence resonance energy transfer)技術來探討hRPA對三核苷酸髮夾型結構的解開能力。我們的結果顯示hRPA可以接近完全地解開長度為可容納一顆hRPA的CTG重複序列之髮夾型結構。但出乎意料地，當髮夾型結構的長度越來越長，hRPA只能部份地解開髮夾型結構。因此我們推測CTG重複序列之髮夾型結構會快速地發生滑動，並且重新構型，形成阻礙hRPA進一步入侵的對齊髮夾型(blunt-end hairpin)結構。而當我們使用抑制髮夾型結構滑動的突變方法進行實驗後，發現hRPA可以完全地解開CTG重複序列之髮夾型結構。因此證明三核苷酸重複序列的滑動阻礙了hRPA的結構解旋能力，而可能導致此類序列的基因不穩定性。Trinucleotide repeat (TNR) sequences, associated with many neurodegenerative diseases, fold into secondary structures that often cause DNA slippage, ultimately leading to abnormal expansions of DNA. Replication protein A (RPA), the most abundant single-stranded DNA (ssDNA) binding protein in eukaryotes, protects transient ssDNA from reannealing or forming secondary structures during replication, repair, and recombination. RPA is also capable of unwinding secondary structures, such as hairpin and G-quadruplex, to maintain genomic stability. We used CTG repeat hairpins as a model system to examine the unwinding ability of human RPA(hRPA) to the TNR hairpins, utilizing single-molecule fluorescence resonance energy transfer (smFRET) microscopy. Our results revealed that hRPA near-fully unwinds shorter CTG repeat hairpin, which accommodates one hRPA. Surprisingly, hRPA only partially unwinds the CTG repeat hairpin as the length of the hairpin becomes longer. We proposed that the CTG repeat hairpin quickly undergoes hairpin slippage and reorganizes into a blunt-end hairpin that blocks the further invasion of hRPA. This mechanism is further confirmed by finding fully unwound CTG repeat hairpin when we used mutation assays that inhibit the hairpin slippage. We concluded that slippage hairpin reconfiguration of TNR inhibits the hRPA unwinding, hence, potently lead to the genomic instability.