研究生: |
匡玉琪 Kuang, Yu-Chi |
---|---|
論文名稱: |
hRPA以不同方向性解開三核苷酸重複序列髮夾結構的機制研究 Polarity-dependent Mechanism of Human Replication Protein A (hRPA) Resolving Trinucleotide Repeat Hairpins |
指導教授: |
李以仁
Lee, I-Ren |
口試委員: |
冀宏源
Chi, Hung-Yuan 李弘文 Li, Hung-Wen 李以仁 Lee, I-Ren |
口試日期: | 2022/07/14 |
學位類別: |
碩士 Master |
系所名稱: |
化學系 Department of Chemistry |
論文出版年: | 2022 |
畢業學年度: | 110 |
語文別: | 中文 |
論文頁數: | 67 |
中文關鍵詞: | 單分子螢光共振能量轉移 、三核苷酸重複序列 、CTG 重複序列 、單股DNA結合蛋白 、人類複製蛋白A 、方向性 |
英文關鍵詞: | single-molecule fluorescence resonance energy transfer, trinucleotide repeat, CTG repeat, single-stranded binding protein, human replication protein A, polarity |
研究方法: | 實驗設計法 |
DOI URL: | http://doi.org/10.6345/NTNU202201093 |
論文種類: | 學術論文 |
相關次數: | 點閱:93 下載:0 |
分享至: |
查詢本校圖書館目錄 查詢臺灣博碩士論文知識加值系統 勘誤回報 |
三核苷酸重複序列的異常擴張容易引發許多神經退化性疾病,這些序列容易折疊成二級結構,導致 DNA 在複製、重組及修復時發生滑動,進而造成不正常擴張。人類複製蛋白 A (human replication protein A, hRPA) 是真核生物中含量最豐富的單股 DNA 結合蛋白,其功能在於幫助 DNA 保持在單股的狀態以維持基因組的穩定性,而其亦具有解開部分二級結構的能力並傾向從 5’ 端往 3’ 端解開。在本論文中,我們以 CTG 重複序列所形成的髮夾型結構作為模型系統,並利用單分子螢光共振能量轉移技術探討 hRPA 在不同方向性上對於 CTG 重複序列之解旋機制,首先我們發現人類複製蛋白 A 難以解開對齊髮夾型結構,因此我們將CTG 重複序列的髮夾結構末端,加上一段 10 個核苷酸的單股 DNA,形成突出型髮夾結構 (overhang hairpin),以利 hRPA 的初始結合,而結果顯示 hRPA 只能部分解開 CTG 重複次數較多的髮夾型結構。我們進一步發現當一顆 hRPA 結合 CTG 重複序列後,髮夾型結構會發生滑動重組,導致形成 hRPA 難以解開的對齊髮夾型結構。然而,在單點突變抑制滑動的實驗中,我們卻發現了與 DNA 方向性有關的結果:當 hRPA 從 3’ 端的突出 DNA 入侵時,單點突變會使其更容易形成完全解開結構;但當 hRPA 從 5’ 端的突出 DNA 入侵時,卻發現其結果有著更低比例的完全解開結構。因此,我們提出了一個模型:在hRPA 從 3’ 端往 5’ 端解開 CTG 髮夾型結構,第二顆 hRPA 便會結合上另一股以 5’ 端往 3’ 端的方向進行更進一步的入侵並解開整個髮夾型解構。然而,在另一個相反的方向性中,第二顆的 hRPA 則不傾向結合上另一股以 3’ 端往 5’ 端的方向解開 CTG 髮夾型結構,進而降低完全解開的效率。因此,我們可以總結在不同的方向性中,hRPA 具有不同的解旋機制。
Abnormal expansions of trinucleotide repeats (TNRs) are responsible for many neurodegenerative disorders. TNRs usually fold into secondary structures that cause DNA slippage during DNA replication, recombination, or repair processes and ultimately lead to abnormal expansions. Human replication protein A (hRPA) is the most abundant single-stranded DNA binding protein in eukaryotes. Its major function is maintaining the single-stranded structure of DNA to keep genomic stability. It is also capable of resolving secondary structures with a polarity preference of 5’ to 3’. In the thesis, we used CTG repeat sequences, which fold into hairpins, as our model system to explore the mechanism of hRPA resolving CTG repeat hairpins in different polarities, utilizing single-molecule fluorescence resonance energy transfer (smFRET) microscopy. We found that hRPA cannot resolve the blunt-end hairpins. We then introduced a short (10-nt) random-coiled overhang to the hairpins for the initial binding of hRPA. The results revealed that hRPA partially resolves long hairpins. We further found that CTG repeat hairpin would undergo hairpin slippage and reorganize into blunt-end hairpin which prohibits the further invasion of hRPA. But when we introduced a single-point mutation to inhibit the slippage reconfiguration, we found polarity-dependent results: The point-mutation boosted the fully resolved hairpin when hRPA invaded from the overhang at 3’ end, while the lowered resolving efficiency was observed when hRPA invaded from the overhang at 5’ end. Hence, we proposed a model: After the hRPA resolves the CTG repeat hairpin from 3’ to 5’, the second hRPA binds to the other strand from 5’ to 3’ and further invades and fully resolves the hairpin. However, with the opposite polarity, the 3’ to 5’ invasion on the other stand is unfavorable and leads to a lowered resolving efficiency. Hence, we can conclude that hRPA has different resolving mechanisms in different polarities.
[1] Margolis, R. L.; McInnis, M. G.; Rosenblatt, A.; Ross, C. A., Trinucleotide Repeat Expansion and Neuropsychiatric Disease. Archives of General Psychiatry 1999, 56 (11), 1019-1031.
[2] Mirkin, S. M., Expandable DNA repeats and human disease. Nature 2007, 447 (7147), 932-940.
[3] McMurray, C. T., Mechanisms of trinucleotide repeat instability during human development. Nat Rev Genet 2010, 11 (11), 786-799.
[4] Walsh, J. M.; Beuning, P. J., Synthetic nucleotides as probes of DNA polymerase specificity. J Nucleic Acids 2012, 2012, 530963.
[5] Kunkel, T. A., Slippery DNA and diseases. Nature 1993, 365 (6443), 207-208.
[6] Greene, A. L.; Snipe, J. R.; Gordenin, D. A.; Resnick, M. A., Functional Analysis of Human FEN1 in Saccharomyces Cerevisiae and Its Role in Genome Stability. Human Molecular Genetics 1999, 8 (12), 2263-2273.
[7] Li, G.-M., Mechanisms and functions of DNA mismatch repair. Cell Research 2008, 18 (1), 85-98.
[8] Ni, C.-W.; Wei, Y.-J.; Shen, Y.-I.; Lee, I. R., Long-Range Hairpin Slippage Reconfiguration Dynamics in Trinucleotide Repeat Sequences. The Journal of Physical Chemistry Letters 2019, 10 (14), 3985-3990.
[9] Waldman, V. M.; Weiland, E.; Kozlov, A. G.; Lohman, T. M., Is a fully wrapped SSB-DNA complex essential for Escherichia coli survival? Nucleic Acids Res 2016, 44 (9), 4317-4329.
[10] Grieb, M. S.; Nivina, A.; Cheeseman, B. L.; Hartmann, A.; Mazel, D.; Schlierf, M., Dynamic stepwise opening of integron attC DNA hairpins by SSB prevents toxicity and ensures functionality. Nucleic Acids Res 2017, 45 (18), 10555-10563.
[11] Fanning, E.; Klimovich, V.; Nager, A. R., A dynamic model for replication protein A (RPA) function in DNA processing pathways. Nucleic Acids Res 2006, 34 (15), 4126-4137.
[12] Brosey, C. A.; Yan, C.; Tsutakawa, S. E.; Heller, W. T.; Rambo, R. P.; Tainer, J. A.; Ivanov, I.; Chazin, W. J., A new structural framework for integrating replication protein A into DNA processing machinery. Nucleic Acids Res 2013, 41 (4), 2313-2327.
[13] Nguyen, B.; Sokoloski, J.; Galletto, R.; Elson, E. L.; Wold, M. S.; Lohman, T. M., Diffusion of human replication protein A along single-stranded DNA. J Mol Biol 2014, 426 (19), 3246-3261.
[14] Safa, L.; Gueddouda, N. M.; Thiébaut, F.; Delagoutte, E.; Petruseva, I.; Lavrik, O.; Mendoza, O.; Bourdoncle, A.; Alberti, P.; Riou, J.-F.; Saintomé, C., 5' to 3' Unfolding Directionality of DNA Secondary Structures by Replication Protein A: G-QUADRUPLEXES AND DUPLEXES. J Biol Chem 2016, 291 (40), 21246-21256.
[15] Ha, T.; Kozlov, A. G.; Lohman, T. M., Single-molecule views of protein movement on single-stranded DNA. Annu Rev Biophys 2012, 41, 295-319.
[16] Juette, M. F.; Terry, D. S.; Wasserman, M. R.; Zhou, Z.; Altman, R. B.; Zheng, Q.; Blanchard, S. C., The bright future of single-molecule fluorescence imaging. Current Opinion in Chemical Biology 2014, 20, 103-111.
[17] Ritort, F., Single-molecule experiments in biological physics: methods and applications. Journal of Physics: Condensed Matter 2006, 18 (32), R531-R583.
[18] Sasmal, D. K.; Pulido, L. E.; Kasal, S.; Huang, J., Single-molecule fluorescence resonance energy transfer in molecular biology. Nanoscale 2016, 8 (48), 19928-19944.
[19] Pietraszewska-Bogiel, A.; Gadella, T. W., FRET microscopy: from principle to routine technology in cell biology. J Microsc 2011, 241 (2), 111-8.
[20] Ishikawa-Ankerhold, H. C.; Ankerhold, R.; Drummen, G. P. C., Advanced fluorescence microscopy techniques--FRAP, FLIP, FLAP, FRET and FLIM. Molecules 2012, 17 (4), 4047-4132.
[21] Fish, K. N., Total internal reflection fluorescence (TIRF) microscopy. Curr Protoc Cytom 2009, Chapter 12, Unit12.18.
[22] Martin-Fernandez, M. L.; Tynan, C. J.; Webb, S. E. D., A ‘pocket guide’ to total internal reflection fluorescence. Journal of Microscopy 2013, 252 (1), 16-22.
[23] Aitken, C. E.; Marshall, R. A.; Puglisi, J. D., An oxygen scavenging system for improvement of dye stability in single-molecule fluorescence experiments. Biophys J 2008, 94 (5), 1826-1835.