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| DC 欄位 | 值 | 語言 |
|---|---|---|
| dc.contributor.advisor | 冀宏源(Hung-Yuan Chi) | |
| dc.contributor.author | Yi-Hsuan Chang | en |
| dc.contributor.author | 張逸軒 | zh_TW |
| dc.date.accessioned | 2021-06-17T06:24:46Z | - |
| dc.date.available | 2028-08-17 | |
| dc.date.copyright | 2018-08-21 | |
| dc.date.issued | 2018 | |
| dc.date.submitted | 2018-08-17 | |
| dc.identifier.citation | 1. Zeman, M. K., and Cimprich, K. A. (2014) Causes and consequences of replication stress. Nat Cell Biol 16, 2-9
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Beekman, C., Nichane, M., De Clercq, S., Maetens, M., Floss, T., Wurst, W., Bellefroid, E., and Marine, J. C. (2006) Evolutionarily conserved role of nucleostemin: controlling proliferation of stem/progenitor cells during early vertebrate development. Mol Cell Biol 26, 9291-9301 31. Singh, A., and Xu, Y. J. (2016) The Cell Killing Mechanisms of Hydroxyurea. Genes (Basel) 7, 99 32. Sung, P., and Klein, H. (2006) Mechanism of homologous recombination: mediators and helicases take on regulatory functions. Nat Rev Mol Cell Biol 7, 739-750 33. Jasin, M., and Rothstein, R. (2013) Repair of Strand Breaks by Homologous Recombination. Cold Spring Harb Perspect Biol 5, a012740 34. Hashimoto, Y., Puddu, F., and Costanzo, V. (2011) RAD51- and MRE11-dependent reassembly of uncoupled CMG helicase complex at collapsed replication forks. Nat Struct Mol Biol 19, 17-24 35. Petermann, E., Orta, M. L., Issaeva, N., Schultz, N., and Helleday, T. (2010) Hydroxyurea-stalled replication forks become progressively inactivated and require two different RAD51-mediated pathways for restart and repair. Mol Cell 37, 492-502 36. Zellweger, R., Dalcher, D., Mutreja, K., Berti, M., Schmid, J. A., Herrador, R., Vindigni, A., and Lopes, M. (2015) Rad51-mediated replication fork reversal is a global response to genotoxic treatments in human cells. J Cell Biol 208, 563-579 37. Bhat, K. P., and Cortez, D. (2018) RPA and RAD51: fork reversal, fork protection, and genome stability. Nat Struct Mol Biol 25, 446-453 38. Feng, W., and Jasin, M. (2017) Homologous Recombination and Replication Fork Protection: BRCA2 and More! Cold Spring Harb Symp Quant Biol 82, 329-338 39. Schlacher, K., Christ, N., Siaud, N., Egashira, A., Wu, H., and Jasin, M. (2011) Double-strand break repair-independent role for BRCA2 in blocking stalled replication fork degradation by MRE11. Cell 145, 529-542 40. Zadorozhny, K., Sannino, V., Beláň, O., Mlčoušková, J., Špírek, M., Costanzo, V., and Krejčí, L. (2017) Fanconi-Anemia-Associated Mutations Destabilize RAD51 Filaments and Impair Replication Fork Protection. Cell Rep 21, 333-340 41. Chi, P., Van Komen, S., Sehorn, M. G., Sigurdsson, S., and Sung, P. (2006) Roles of ATP binding and ATP hydrolysis in human Rad51 recombinase function. DNA Repair (Amst) 5, 381-391 42. Petermann, E., Maya-Mendoza, A., Zachos, G., Gillespie, D. A., Jackson, D. A., and Caldecott, K. W. (2006) Chk1 requirement for high global rates of replication fork progression during normal vertebrate S phase. Mol Cell Biol 26, 3319-3326 43. Jackson, D. A., and Pombo, A. (1998) Replicon Clusters Are Stable Units of Chromosome Structure: Evidence That Nuclear Organization Contributes to the Efficient Activation and Propagation of S Phase in Human Cells. J Cell Biol 140, 1285-1295 44. Su, W. P., Hsu, S. H., Wu, C. K., Chang, S. B., Lin, Y. J., Yang, W. B., Hung, J. J., Chiu, W. T., Tzeng, S. F., Tseng, Y. L., Chang, J. Y., Su, W. C., and Liaw, H. (2014) Chronic treatment with cisplatin induces replication-dependent sister chromatid recombination to confer cisplatin-resistant phenotype in nasopharyngeal carcinoma. Oncotarget 5, 6323-6337 45. Pierce, A. J., Johnson, R. D., Thompson, L. H., and Jasin, M. (1999) XRCC3 promotes homology-directed repair of DNA damage in mammalian cells. Genes Dev 13, 2633-2638 46. Marenchino, M., Armbruster, D. W., and Hennig, M. (2009) Rapid and efficient purification of RNA-binding proteins: application to HIV-1 Rev. Protein Expr Purif 63, 112-119 47. Burgess, R. R. (2009) Chapter 20 Protein Precipitation Techniques. in Guide to Protein Purification, 2nd Edition. pp 331-342 48. Wyatt, H. D., Laister, R. C., Martin, S. R., Arrowsmith, C. H., and West, S. C. (2017) The SMX DNA Repair Tri-nuclease. Mol Cell 65, 848-860 e811 49. Schlacher, K., Wu, H., and Jasin, M. 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(2013) Coordinated Actions of SLX1-SLX4 and MUS81-EME1 for Holliday Junction Resolution in Human Cells. Mol Cell 52, 234-247 61. Lemacon, D., Jackson, J., Quinet, A., Brickner, J. R., Li, S., Yazinski, S., You, Z., Ira, G., Zou, L., Mosammaparast, N., and Vindigni, A. (2017) MRE11 and EXO1 nucleases degrade reversed forks and elicit MUS81-dependent fork rescue in BRCA2-deficient cells. Nat Commun 8, 860 62. Tsai, R. Y., and McKay, R. D. (2005) A multistep, GTP-driven mechanism controlling the dynamic cycling of nucleostemin. J Cell Biol 168, 179-184 63. Bos, J. L., Rehmann, H., and Wittinghofer, A. (2007) GEFs and GAPs: critical elements in the control of small G proteins. Cell 129, 865-877 64. Wittinghofer, A., and Vetter, I. R. (2011) Structure-function relationships of the G domain, a canonical switch motif. Annu Rev Biochem 80, 943-971 | |
| dc.identifier.uri | http://tdr.lib.ntu.edu.tw/jspui/handle/123456789/72128 | - |
| dc.description.abstract | 去氧核糖核酸複製(DNA replication)為細胞分裂(cell division)能順利進行之重要步驟。然而，在去氧核糖核酸複製的過程中，複製叉(replication fork)的行進會因為各種威脅而受到阻礙，導致停滯複製叉(Stalled fork)的形成，甚至進而使崩塌複製叉(Collapsed fork)出現，造成去氧核糖核酸雙股斷裂(DNA double-strand breaks, DSBs)的產生，使細胞遭受複製壓力(replication stress)。增殖細胞(proliferating cells)必須進行大量之去氧核糖核酸複製，因此，其必須利用複製壓力反應(replication stress response)以維持基因體穩定性。Nucleostemin (NS)為一種特別於增殖細胞中高度表現的蛋白質。NS於近期被發現可能為參與複製壓力反應機制中的重要成員之一。NS的缺失會造成去氧核糖核酸雙股斷裂累積於S期(S-phase)的細胞中。另外，NS的缺失會造成去氧核糖核酸雙股斷裂修復蛋白RAD51聚集至去氧核糖核酸斷裂處之能力受到顯著影響。然而，NS於複製壓力反應中行使其功能之詳細機制，目前仍屬未知。
在此研究中，我們分別透過細胞培養以及生物化學實驗，來探討NS於複製壓力反應中的功能性角色。首先，我們透過去氧核糖核酸纖維實驗(DNA fiber assay)，發現NS的缺失會造成停滯複製叉的重啟效率降低。同源重組(homologous recombination, HR)為崩塌複製叉的重要修復機制，透過同源重組報導實驗(HR reporter assay)，我們發現NS的缺失使細胞的同源重組效率顯著降低。另外，我們建立了NS重組蛋白之表達及純化系統以進行生化實驗。藉由電泳遷移實驗(electrophoretic mobility shift assay, EMSA)，我們發現NS對於複製叉之結合能力高於雙股(double-stranded DNA, dsDNA)或是懸伸去氧核糖核酸(overhang DNA)。另一方面，核酸酶保護實驗(nuclease protection assay)的結果顯示，NS可高度保護單股(single-stranded DNA, ssDNA)去氧核糖核酸免於核酸內切酶(endonuclease)之降解。此外，我們發現NS也具有保護單股/雙股去氧核糖核酸接合點(ssDNA/dsDNA junction)之能力。根據親和性沈降實驗(affinity pull-down assay)，我們證實NS與RAD51之間存在直接且具物種專一性之蛋白質交互作用關係。綜合以上結果，我們推測NS參與於停滯及崩塌複製叉之重啟，並可能透過複製叉結合能力以及DNA保護能力，協同RAD51共同於複製壓力反應中行使功能。 | zh_TW |
| dc.description.abstract | DNA replication is a crucial step for proliferating cells to faithfully duplicate the genome during cell cycle. Nevertheless, collapsed replication forks will lead to DNA double-strand breaks (DSBs) and genomic instability. Nucleostemin (NS), a proliferating cell-enriched protein, has been identified as a key regulator for repairing replication-induced DNA damage. Loss of NS accumulates DSBs especially in S-phase cells. More importantly, NS regulates the recruitment of RAD51, an essential repair protein for DSBs and replication stress, to replication-induced DNA damage sites. However, the underlying mechanism for how NS functions in replication stress response remains largely unknown.
Here, we report that lack of NS impedes the restart of stalled forks via DNA fiber assay. Moreover, NS depletion causes impaired HR efficiency which is essential for collapsed fork restart. With purified recombinant NS protein, we demonstrate that NS shows DNA binding preference for replication fork substrates over double-stranded or tailed DNA substrates. Using nuclease protection assay, we find that NS strongly protects ssDNA from endonuclease degradation. We further show NS also protects the DNA junction in overhang substrates. By pull-down assay, we demonstrate direct protein-protein interaction between NS and RAD51. Our results implicate that NS have important role in stalled and collapsed fork restart and may functions mechanistically through its fork binding preference and DNA protection activity by coordinated action with RAD51. | en |
| dc.description.provenance | Made available in DSpace on 2021-06-17T06:24:46Z (GMT). No. of bitstreams: 1 ntu-107-R05b46010-1.pdf: 7509310 bytes, checksum: eb9c10943c24f2fcaee752b3a02fe01a (MD5) Previous issue date: 2018 | en |
| dc.description.tableofcontents | 論文口試委員審定書 I
誌謝 II 中文摘要 III ABSTRACT V LIST OF FIGURES IX LIST OF ABBREVIATIONS X CHAPTER 1 INTRODUCTION 1 1-1 DNA replication and genome instability 1 1-2 Molecular pathways to overcome replication stress 1 1-3 The biological significance of Nucleostemin 3 1-4 The role of NS in maintaining genome stability 4 1-5 The roles of RAD51 in the replication stress response 6 1-6 Motivation of my study 8 CHAPTER 2 MATERIALS AND METHODS 9 2-1 Plasmids 9 2-1-1 NS expression plasmids 9 2-1-2 RAD51 expression plasmids 9 2-1-3 I-SceI expression plasmids 9 2-2 Cell culture and transfection 10 2-3 DR-GFP HR reporter assay 10 2-4 Immunoblot analysis 11 2-5 Protein expression and purification 12 2-5-1 MBP-NS-His6 12 2-5-2 RAD51 14 2-6 DNA substrates 15 2-6-1 ΦX174 ssDNA and dsDNA 15 2-6-2 32P-labeled ssDNA 15 2-6-3 32P-labeled 60mer dsDNA and the fork-like substrates 15 2-6-4 The sequence of oligonucleotides 17 2-7 Electrophoretic mobility shift assay (EMSA) 18 2-8 Nuclease protection assay 19 2-9 Affinity pull-down assay 20 CHAPTER 3 RESULTS 21 3-1 NS plays important cellular roles for replication stress 21 3-2 Purification of NS recombinant protein 22 3-3 The DNA-binding characteristics of NS 23 3-4 NS protects DNA against endonuclease digestion 25 3-5 NS has ability to protect ssDNA/dsDNA junction 26 3-6 NS directly interacts with RAD51 27 CHAPTER 4 CONCLUSION AND DISCUSSION 28 4-1 Conclusion 28 4-2 Discussion 29 4-2-1 The role of NS in replication fork dynamics 29 4-2-2 The DNA protection activity of NS 30 4-2-2-1 The unique DNA protection effect of NS against endonuclease 30 4-2-2-2 MUS81-EME2 endonuclease 30 4-2-2-3 MRE11 33 4-2-3 The effect of nucleotide-binding status of NS on its function 34 FIGURES AND FIGURE LEGENDS 35 REFERENCES 41 APPENDICES 47 | |
| dc.language.iso | en | |
| dc.subject | 複製壓力 | zh_TW |
| dc.subject | 去氧核糖核酸雙股斷裂 | zh_TW |
| dc.subject | Nucleostemin | zh_TW |
| dc.subject | RAD51 | zh_TW |
| dc.subject | 同源重組 | zh_TW |
| dc.subject | RAD51 | en |
| dc.subject | DNA double-strand breaks | en |
| dc.subject | Nucleostemin | en |
| dc.subject | Replication stress | en |
| dc.subject | Homologous recombination | en |
| dc.title | 探討Nucleostemin之生化特性及其於DNA複製壓力反應中之功能性角色 | zh_TW |
| dc.title | Investigating the Biochemical Characteristics of Nucleostemin and Its Functional Role in Replication Stress Response | en |
| dc.type | Thesis | |
| dc.date.schoolyear | 106-2 | |
| dc.description.degree | 碩士 | |
| dc.contributor.oralexamcommittee | 廖泓鈞(Hungjiun Liaw),蕭育源(Yu-Yuan Hsiao) | |
| dc.subject.keyword | 複製壓力,去氧核糖核酸雙股斷裂,Nucleostemin,RAD51,同源重組, | zh_TW |
| dc.subject.keyword | Replication stress,DNA double-strand breaks,Nucleostemin,RAD51,Homologous recombination, | en |
| dc.relation.page | 49 | |
| dc.identifier.doi | 10.6342/NTU201803116 | |
| dc.rights.note | 有償授權 | |
| dc.date.accepted | 2018-08-17 | |
| dc.contributor.author-college | 生命科學院 | zh_TW |
| dc.contributor.author-dept | 生化科學研究所 | zh_TW |
| 顯示於系所單位： | 生化科學研究所 | |
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