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http://tdr.lib.ntu.edu.tw/jspui/handle/123456789/6043完整後設資料紀錄
| DC 欄位 | 值 | 語言 |
|---|---|---|
| dc.contributor.advisor | 張茂山(Mau-Sun Chang) | |
| dc.contributor.author | Yen-Yu Gong | en |
| dc.contributor.author | 龔彥宇 | zh_TW |
| dc.date.accessioned | 2021-05-16T16:19:58Z | - |
| dc.date.available | 2013-08-17 | |
| dc.date.available | 2021-05-16T16:19:58Z | - |
| dc.date.copyright | 2013-08-17 | |
| dc.date.issued | 2013 | |
| dc.date.submitted | 2013-08-07 | |
| dc.identifier.citation | Deng, L., Wang, C., Spencer, E., Yang, L., Braun, A., You, J., . . . Chen, Z. J. (2000). Activation of the IkappaB kinase complex by TRAF6 requires a dimeric ubiquitin-conjugating enzyme complex and a unique polyubiquitin chain. Cell, 103(2), 351-361.
Fnu, S., Williamson, E. A., De Haro, L. P., Brenneman, M., Wray, J., Shaheen, M., . . . Hromas, R. (2011). Methylation of histone H3 lysine 36 enhances DNA repair by nonhomologous end-joining. Proc Natl Acad Sci U S A, 108(2), 540-545. doi: 10.1073/pnas.1013571108 Hershko, A., & Ciechanover, A. (1986). The ubiquitin pathway for the degradation of intracellular proteins. Prog Nucleic Acid Res Mol Biol, 33, 19-56, 301. Hickson, I., Zhao, Y., Richardson, C. J., Green, S. J., Martin, N. M., Orr, A. I., . . . Smith, G. C. (2004). Identification and characterization of a novel and specific inhibitor of the ataxia-telangiectasia mutated kinase ATM. Cancer Res, 64(24), 9152-9159. doi: 10.1158/0008-5472.CAN-04-2727 Jazayeri, A., Balestrini, A., Garner, E., Haber, J. E., & Costanzo, V. (2008). Mre11-Rad50-Nbs1-dependent processing of DNA breaks generates oligonucleotides that stimulate ATM activity. EMBO J, 27(14), 1953-1962. doi: 10.1038/emboj.2008.128 Jeggo, P. A., & Lobrich, M. (2005). Artemis links ATM to double strand break rejoining. Cell Cycle, 4(3), 359-362. Kanaar, R., & Wyman, C. (2008). DNA repair by the MRN complex: break it to make it. Cell, 135(1), 14-16. doi: 10.1016/j.cell.2008.09.027 Lee, J. H., & Paull, T. T. (2005). ATM activation by DNA double-strand breaks through the Mre11-Rad50-Nbs1 complex. Science, 308(5721), 551-554. doi: 10.1126/science.1108297 Li, F., Mao, G., Tong, D., Huang, J., Gu, L., Yang, W., & Li, G. M. (2013). The Histone Mark H3K36me3 Regulates Human DNA Mismatch Repair through Its Interaction with MutSalpha. Cell, 153(3), 590-600. doi: 10.1016/j.cell.2013.03.025 Lloyd, J., Chapman, J. R., Clapperton, J. A., Haire, L. F., Hartsuiker, E., Li, J., . . . Smerdon, S. J. (2009). A supramodular FHA/BRCT-repeat architecture mediates Nbs1 adaptor function in response to DNA damage. Cell, 139(1), 100-111. doi: 10.1016/j.cell.2009.07.043 Mahaney, B. L., Meek, K., & Lees-Miller, S. P. (2009). Repair of ionizing radiation-induced DNA double-strand breaks by non-homologous end-joining. Biochem J, 417(3), 639-650. doi: 10.1042/BJ20080413 Matsuoka, S., Ballif, B. A., Smogorzewska, A., McDonald, E. R., 3rd, Hurov, K. E., Luo, J., . . . Elledge, S. J. (2007). ATM and ATR substrate analysis reveals extensive protein networks responsive to DNA damage. Science, 316(5828), 1160-1166. doi: 10.1126/science.1140321 Meek, K., Dang, V., & Lees-Miller, S. P. (2008). DNA-PK: the means to justify the ends? Adv Immunol, 99, 33-58. doi: 10.1016/S0065-2776(08)00602-0 Mellor, J. (2006). It takes a PHD to read the histone code. Cell, 126(1), 22-24. doi: 10.1016/j.cell.2006.06.028 Mendez, J., & Stillman, B. (2000). Chromatin association of human origin recognition complex, cdc6, and minichromosome maintenance proteins during the cell cycle: assembly of prereplication complexes in late mitosis. Mol Cell Biol, 20(22), 8602-8612. Roberts, S. A., Strande, N., Burkhalter, M. D., Strom, C., Havener, J. M., Hasty, P., & Ramsden, D. A. (2010). Ku is a 5'-dRP/AP lyase that excises nucleotide damage near broken ends. Nature, 464(7292), 1214-1217. doi: 10.1038/nature08926 Stender, J. D., Pascual, G., Liu, W., Kaikkonen, M. U., Do, K., Spann, N. J., . . . Glass, C. K. (2012). Control of proinflammatory gene programs by regulated trimethylation and demethylation of histone H4K20. Mol Cell, 48(1), 28-38. doi: 10.1016/j.molcel.2012.07.020 Williams, R. S., Williams, J. S., & Tainer, J. A. (2007). Mre11-Rad50-Nbs1 is a keystone complex connecting DNA repair machinery, double-strand break signaling, and the chromatin template. Biochem Cell Biol, 85(4), 509-520. doi: 10.1139/O07-069 Wu, L., Luo, K., Lou, Z., & Chen, J. (2008). MDC1 regulates intra-S-phase checkpoint by targeting NBS1 to DNA double-strand breaks. Proc Natl Acad Sci U S A, 105(32), 11200-11205. doi: 10.1073/pnas.0802885105 Zhou, B. B., & Elledge, S. J. (2000). The DNA damage response: putting checkpoints in perspective. Nature, 408(6811), 433-439. doi: 10.1038/35044005 | |
| dc.identifier.uri | http://tdr.lib.ntu.edu.tw/jspui/handle/123456789/6043 | - |
| dc.description.abstract | 當細胞受到外在環境的刺激進而影響到基因體的完整性時，細胞內會有許多的反應，例如細胞週期的調控，轉錄作用的調控，DNA修補作用，或是細胞凋亡，其目的是避免基因體的完整性遭到破壞，稱之為DNA損害反應。DNA 損害反應中，ATM和ATR扮演重要的角色，Matsuoka等人利用可辨識ATM和ATR受質的抗體進行免疫沉澱後將所獲得的樣品進行質譜儀分析，發現有七百多種蛋白質會被ATM和ATR磷酸化，PHRF1被發現是ATM和ATR磷酸化的受質，但功能未知。
我們的研究以PHRF1為核心，根據胺基酸分析， PHRF1上有許多重要的蛋白質功能區域，包含PHD domain、Ring finger、SQ/TQ cluster domain。以這些資訊為出發點，我們想了解PHRF1在DNA損害反應中所扮演的角色。免疫螢光染色結果顯示PHRF1是一個核蛋白；當細胞遭受基因毒性壓力時， PHRF1會從細胞核質移動到染色質，而將內生性缺乏ATM表現的細胞株進行細胞分層萃取，發現PHRF1受到基因毒性壓力後由核質到染色質的移動能力消失，因此 ATM對PHRF1磷酸化會調控PHRF1移動到染色質的能力。 在2011年，Fnu等人發現H3K36me2會促進非同源末端黏合（non-homologous end-joining，NHEJ）。藉由免疫沉澱， PHRF1分別可以跟H3K36me2、Nbs1及Ku70交互作用，而末端黏合實驗更進一步證實PHRF1可以影響NHEJ，因此我提出假說認為PHRF1是連接H3K36me2和Nbs1及Ku70調控細胞在DNA斷裂時負責非同源末端黏合修補。 | zh_TW |
| dc.description.abstract | The role of histone methylation in double-strand break repair by non-homologous end-joining (NHEJ) is not well defined. Previous studies indicate that H3K36me2 links Nbs1 and Ku70 to promote NHEJ, but which protein is responsible for this connection is unknown.
Human PHRF1/KIAA1542 contains a plant homeodomain (PHD), a putative methylated histone binding domain, and is identified as a phosphorylation substrate of ATM/ATR kinase. However, very little is known about its function in DNA damage response. Immunofluorescence and subcellular fractionation results revealed that PHRF1 mainly localized in the nucleus prior to genotoxic stress, but PHRF1 increased onto chromatin upon DNA damage in an ATM-dependent manner. Immunoprecipitation suggested that PHRF1, Nbs1, Ku70, and dimethylated H3K36 (H3K36me2) were in the same immunocomplex. In vivo end-joining assay using a linearized luciferase reporter suggested that end-joining efficiency was decreased in PHRF1 knockdown HEK293 cells but significantly increased in PHRF1 overexpressing U2OS cells, suggesting that the presence of PHRF1 may link H3K36 methylation to non-homologous end joining by interaction with Nbs1 and Ku70. | en |
| dc.description.provenance | Made available in DSpace on 2021-05-16T16:19:58Z (GMT). No. of bitstreams: 1 ntu-102-R00b46007-1.pdf: 1309918 bytes, checksum: 5ab8b8a1b76801d956e9b0a92e05c799 (MD5) Previous issue date: 2013 | en |
| dc.description.tableofcontents | 中文摘要………………………………………………………………………….........i
英文摘要……………………………………………...……………………………….ii 縮寫表………………………………………………………………………………...iii 目錄………………………………………………………………………………….. .v 第一章 前言…………………………………………………………………………..1 1.1 DNA損害反應（DNA damage response）…………………………………1 1.2細胞週期的調控…………………………………………………………….1 1.3 DNA修補反應………………………………………………………………1 1.3.1同源重組（Homologous recombination，HR）……………………….…2 1.3.2非同源末端黏合（Non-homologous end-joining，NHEJ）……….…….2 1.4 PHRF1/KIAA1542（PHD and ring finger domains 1）……………............3 1.5 研究動機……………………………………………………………............3 第二章 材料與方法………………………………………………..………..………..4 2.1.1 細胞株培養……………………………………………..………..……….4 2.1.2 細胞計數…………………………………………………..……….……..4 2.1.3 加藥處理…………………………………………………..…..………….4 2.2.1 細胞蛋白質萃取 (Whole cell extract)……………………..…..……...…5 2.2.2 細胞分層萃取 (Fractionation)………………….…………..…..………..5 2.3 免疫沉澱…………………………………………………………..……..6 2.4 免疫螢光染色……………………………………………………..……..6 2.5.1 基因轉染………………………………………………………………...7 2.5.2 大量表現細胞株建立…………………………………………………...7 2.5.3 少量表現細胞株建立…………………………………………………...8 2.5.4 In vivo ubiquitination assay……………………………………………...8 2.6.1 SDS-PAGE膠體電泳……………………………………………………9 2.6.2 西方墨點法……………………………………..…………………...…10 2.7 末端黏合檢驗(End joining assay)……………………….……………..10 第三章 實驗結果…………………………………………..………………………11 3.1 PHRF1蛋白質的表現大小………………………………………………11 3.2 PHRF1在細胞內的分布…………………………………………………11 3.3 PHRF1受到genotoxic stress會結合到染色質..…………….………….11 3.4 PHRF1可和組蛋白交互作用…………………...........................………12 3.5 PHRF1的SCD domain使其具備移動能力…………………………….12 3.6 ATM可以促進PHRF1的移動…………………………………………..13 3.7 PHRF1可以和Nbs1交互作用…………………………………………..13 3.8 PHRF1可以影響末端黏合的效率………………………………………13 第四章 總結與討論………………………………………………………..………14 第五章 實驗結果圖表……………………………………………………..………17 附錄………………………………………………………………………..………..26 參考文獻…………………………………………………………………..………..30 | |
| dc.language.iso | zh-TW | |
| dc.title | PHRF1參與DNA損害反應 | zh_TW |
| dc.title | PHRF1 in DNA damage response | en |
| dc.type | Thesis | |
| dc.date.schoolyear | 101-2 | |
| dc.description.degree | 碩士 | |
| dc.contributor.oralexamcommittee | 余榮熾(Lung-Chih Yu),陳宏文(Hungwen Chen),張震東(Geen-Dong Chang),冀宏源(Hung-Yuan Chi) | |
| dc.subject.keyword | PHRF1,ATM,H3K36me2,Nbs1,NHEJ, | zh_TW |
| dc.relation.page | 32 | |
| dc.rights.note | 同意授權(全球公開) | |
| dc.date.accepted | 2013-08-07 | |
| 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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