(3.235.191.87) 您好!臺灣時間:2021/05/13 03:54
字體大小: 字級放大   字級縮小   預設字形  
回查詢結果

詳目顯示:::

: 
twitterline
研究生:林瑞洋
研究生(外文):Jui-YangLin
論文名稱:Yng2蛋白在DNA雙股斷裂修復中的功能探討
論文名稱(外文):The function of Yng2 in the DNA double-strand break repair
指導教授:廖泓鈞
指導教授(外文):Hung-Jiun Liaw
學位類別:碩士
校院名稱:國立成功大學
系所名稱:生命科學系碩博士班
學門:生命科學學門
學類:生物學類
論文種類:學術論文
論文出版年:2013
畢業學年度:101
語文別:中文
論文頁數:73
中文關鍵詞:DNA雙股斷裂DNA損害反應
外文關鍵詞:NuA4Yng2PHD domainDNA double-strand breakDNA damage responseDSBDDR
相關次數:
  • 被引用被引用:0
  • 點閱點閱:137
  • 評分評分:系統版面圖檔系統版面圖檔系統版面圖檔系統版面圖檔系統版面圖檔
  • 下載下載:6
  • 收藏至我的研究室書目清單書目收藏:0
中文摘要
對細胞內基因組(genome)的整體性來說,DNA雙股斷裂(DNA double-strand break, DSB)是一種最嚴重的損害,若DSB修復途徑失效,則會導致基因組不穩定性(genomic instability)以及形成癌症,從酵母菌(Saccharomyces cerevisiae)至人類的演化過程中,DSB的修復機制具有極高的保留性,在酵母菌中,組蛋白修飾複合體NuA4(在人類中的同功能的同源蛋白為Tip60)在DSB產生後會被招至斷裂點上對組蛋白H2A以及H4進行乙醯化(acetylation),組蛋白被乙醯化的結果被認為是協助染色質結構打開,並讓DNA修復蛋白能進入DSB進行DNA修復,NuA4複合體中有個單元蛋白Yng2,透過體外實驗(in vitro)發現會藉由PHD結構域(Plant HomeoDomain, PHD domain)與組蛋白H3的第四胺基酸賴胺酸(lysine 4)上的兩個甲基(H3K4me2)以及三個甲基修飾(H3K4me3)作用,近年來研究也發現yng2突變株會有DSB修復缺陷,然而Yng2的PHD結構域是否協助NuA4進入DSB依舊未知,在這裡我們發現yng2-PHD酵母突變株沒有生長上以及DNA修復的缺陷,在染色質免疫沉澱(chromatin immunoprecipitation, ChIP)實驗顯示在DNA雙股斷裂(DSB)的初期,組蛋白會從雙股斷裂處脫落,同時組蛋白H2A胺基酸序列129的絲胺酸(serine 129)的磷酸化(phosphorylation)(H2A-S129p)、組蛋白H4胺基酸序列第12的賴胺酸(lysine 12)的乙醯化(H4K12ac)、組蛋白H3的第四胺基酸賴胺酸(lysine 4)上的兩個甲基(H3Kme2)以及三個甲基修飾(H3K4me3)以及組蛋白H3胺基酸序列第36的賴胺酸2甲基化(H3K36me2),這些組蛋白修飾在DSB斷裂點會大量累積,而Yng2也會在DSB上大量累積,這表示NuA4有效率地作用在DSB上,相較於組蛋白H4第四胺基酸賴胺酸(lysine 4)突變(H3K4R)成精胺酸(arginine)的酵母株,模擬H4第四胺基酸無法被甲基化的情況,我們發現Yng2以及H4K12ac在DSB的累積降低、H2A-S129p的累積有延遲的狀況,這是說明H3K4R的菌株在招引NuA4到DSB的能力、DNA修復能力受損,由我們的數據可以說明DSB會誘導H2A-S129p、H3K4me2、H3K4me3、H3K36me2的產生,這些修飾都可以與NuA4的單元蛋白作用並輔助NuA4進入DSB,這些作用包含了H2A-S129p與Arp4、H3K36me2與Eaf3的Chromo結構域以及H3K4me3與Yng2的PHD結構域,這些作用力強而有力的讓NuA4可以專一的作用於DSB上。



Abstract
DNA double-strand breaks (DSBs) are the most dangerous lesions to the integrity of genome. Failure to repair DSBs properly can lead to genomic instability and cancer. The mechanism of DSB repair is highly conserved from yeast to human. In yeast Saccharomyces cerevisiae, the histone acetyltransferase, NuA4 (functional homolog of Tip60 in human) is recruited to DSBs where it acetylates histone H2A and H4, presumably relaxing the chromatin and allowing the access of repair proteins. A subunit of NuA4, Yng2, can interact with dimethyl K4 of histone H3 (H3K4me2) and trimethyl K4 of histone H3 (H3K4me3) by its plant homeodomain (PHD) in vitro. The yng2 mutant is defective to DSB repair. However, it remains unclear whether the PHD domain of Yng2 directs NuA4 to DSB sites. Here, we demonstrated that mutations in the Yng2 PHD domain (yng2-Δphd) have no significant effect on cell growth or DNA repair. The chromatin immunoprecipitation (ChIP) experiments reveal that high level of phospho-S129 of histone H2A (H2A-S129p), acetyl-K12 of histone H4 (H4K12ac), H3K4me2, H3K4me3 and H3K36me2 are induced at the DSB site. Yng2 is enriched at DSB site, suggesting NuA4 is efficiently recruited to the DSB site. By contrast, the arginine to lysine mutation of K4 of histone H3 (H3K4R) that abrogates the methylation at K4 of histone H3 dramatically reduced the enrichment of Yng2 and H4K12ac at the DSB site. Importantly, activation of H2A-S129p is prolonged, suggesting H3K4R mutation impairs the NuA4 recruitment and repair efficiency. Our results suggest that the DSB can induce specific histone modifications, including H2A-S129p, H4K12ac, H3K4me2, H3K4me3, H3K36me2, which in turns recruits the NuA4 complex to the DSB site by the multiple interactions between these modified histones and subunits of NuA4. These interactions include the interactions between H2A-S129p and Arp4, H3K36me2 and chromodomain and H3K4me3 and PHD domain. These multiple interactions strengthen the recruitment of NuA4 at DSBs.

目次
中文摘要 1
ABSTRACT 3
誌謝 4
目次 5
圖目錄 7
縮寫表 8
壹 緒論 9
第一節 前言: 9
1-1.酵母(Saccharomyces cerevisiae)細胞中的DNA損害反應(DNA DAMAGE RESPONSE, DDR): 9
1-2. 組蛋白修飾(HISTONE MODIFICATION)作為DDR啟動的關鍵之一: 12
1-3. NuA4調控染色質重塑並協助DSB修復路徑相關蛋白進入斷裂點進行修復: 14
1-4. NuA4複合體上的單元蛋白含有一些特殊結構域,可以辨識組蛋白的修飾,協助NUA4專一的作用在修飾點: 16
1-5. 哺乳動物細胞與酵母(Saccharomyces cerevisiae)有非常相似的DNA損害反應(DNA DAMAGE RESPONSE): 18
第二節實驗目的: 22
貳 材料與方法 23
第一節 材料 23
2-1-1.耗材: 23
2-1-2.藥品: 23
2-1-3.培養液相關藥品: 24
2-1-4.抗體: 24
2-1-5.儀器: 24
第二節 實驗方法 25
2-2-1.YNG2與突變質體的製備: 25
2-2-2.yng2剔除酵母的製備: 27
2-2-3.hht1hhf1、hht2hhf2雙基因剔除酵母菌製備: 29
2-2-6.菌株列: 30
2-2-5.酵母菌轉形作用: 32
2-2-7.酵母菌染色體DNA萃取: 33
2-2-8. DNA DAMAGE SENSITIVITY ASSAY: 33
2-2-9.染色質免疫沉澱法(CHROMATIN IMMUNOPRECIPITATION,CHIP): 34
參 結果與討論 40
第一節 結果: 40
3-1. yng2剔除酵母菌株,具有生長緩慢以及對DNA損害藥物有高度敏感性: 40
3-2. yng2菌株對基因毒性(GENOTOXIC)的環境例如:UV、MMS、腐草霉(PHLEOMYCIN),有較高的敏感性(SENSITIVITY): 41
3-3. Yng2的PHD結構域突變對基因毒性的藥物沒有敏感性: 43
3-4.組蛋白H3第四個胺基酸賴胺酸突變(H3K4R),基因毒性的環境壓力下有敏感性: 44
3-5.H2A-S129磷酸化在斷裂點大量累積並且綿延至10Kbp,而Yng2也會輔助NUA4進入DSB位置並協助將DSB斷裂位置的組蛋白H4乙醯化: 45
3-6. H3K4me2、H3K4me3、H3K36me2修飾在DSB斷裂點附近修飾的情形: 47
3-9. H3K4R突變株在DSB產生後無法招引Yng2至斷裂點使得無法對H4K12進行乙醯化的修飾: 49
第二節 討論 51
肆 參考文獻: 56

肆 參考文獻:
Berndsen, C.E.; Selleck, W.; McBryant, S.J.; Hansen, J.C.; Tan, S.; Denu, J.M.: Nucleosome recognition by the piccolo nua4 histone acetyltransferase complex. Biochemistry 46: 2091-2099 (2007).
Bird, A.W.; Yu, D.Y.; Pray-Grant, M.G.; Qiu, Q.; Harmon, K.E.; Megee, P.C.; Grant, P.A.; Smith, M.M.; Christman, M.F.: Acetylation of histone h4 by esa1 is required for DNA double-strand break repair. Nature 419: 411-415 (2002).
Cahill, D.; Connor, B.; Carney, J.P.: Mechanisms of eukaryotic DNA double strand break repair. Frontiers in bioscience : a journal and virtual library 11: 1958-1976 (2006).
Campos, E.I.; Reinberg, D.: Histones: Annotating chromatin. Annual review of genetics 43: 559-599 (2009).
Choy, J.S.; Kron, S.J.: Nua4 subunit yng2 function in intra-s-phase DNA damage response. Molecular and cellular biology 22: 8215-8225 (2002).
Choy, J.S.; Tobe, B.T.; Huh, J.H.; Kron, S.J.: Yng2p-dependent nua4 histone h4 acetylation activity is required for mitotic and meiotic progression. The Journal of biological chemistry 276: 43653-43662 (2001).
Downs, J.A.; Allard, S.; Jobin-Robitaille, O.; Javaheri, A.; Auger, A.; Bouchard, N.; Kron, S.J.; Jackson, S.P.; Cote, J.: Binding of chromatin-modifying activities to phosphorylated histone h2a at DNA damage sites. Molecular cell 16: 979-990 (2004).
Downs, J.A.; Lowndes, N.F.; Jackson, S.P.: A role for saccharomyces cerevisiae histone h2a in DNA repair. Nature 408: 1001-1004 (2000).
Doyon, Y.; Cote, J.: The highly conserved and multifunctional nua4 hat complex. Current opinion in genetics & development 14: 147-154 (2004).
Elledge, S.J.: Cell cycle checkpoints: Preventing an identity crisis. Science 274: 1664-1672 (1996).
Faucher, D.; Wellinger, R.J.: Methylated h3k4, a transcription-associated histone modification, is involved in the DNA damage response pathway. PLoS genetics 6 (2010).
Harrison, J.C.; Haber, J.E.: Surviving the breakup: The DNA damage checkpoint. Annual review of genetics 40: 209-235 (2006).
Huang, J.; Tan, S.: Piccolo nua4-catalyzed acetylation of nucleosomal histones: Critical roles of an esa1 tudor/chromo barrel loop and an epl1 enhancer of polycomb a (epca) basic region. Molecular and cellular biology 33: 159-169 (2013).
Iijima, K.; Ohara, M.; Seki, R.; Tauchi, H.: Dancing on damaged chromatin: Functions of atm and the rad50/mre11/nbs1 complex in cellular responses to DNA damage. Journal of Radiation Research 49: 451-464 (2008).
Ikura, T.; Ogryzko, V.V.; Grigoriev, M.; Groisman, R.; Wang, J.; Horikoshi, M.; Scully, R.; Qin, J.; Nakatani, Y.: Involvement of the tip60 histone acetylase complex in DNA repair and apoptosis. Cell 102: 463-473 (2000).
Ira, G.; Pellicioli, A.; Balijja, A.; Wang, X.; Fiorani, S.; Carotenuto, W.; Liberi, G.; Bressan, D.; Wan, L.; Hollingsworth, N.M.; Haber, J.E.; Foiani, M.: DNA end resection, homologous recombination and DNA damage checkpoint activation require cdk1. Nature 431: 1011-1017 (2004).
Joshi, A.A.; Struhl, K.: Eaf3 chromodomain interaction with methylated h3-k36 links histone deacetylation to pol ii elongation. Molecular cell 20: 971-978 (2005).
Kast, D.J.; Dominguez, R.: Arp you ready for actin in the nucleus? The EMBO journal 30: 2097-2098 (2011).
Kouzarides, T.: Chromatin modifications and their function. Cell 128: 693-705 (2007).
Kurdistani, S.: In vivo protein–protein and protein–DNA crosslinking for genomewide binding microarray. Methods 31: 90-95 (2003).
Li, H.; Ilin, S.; Wang, W.; Duncan, E.M.; Wysocka, J.; Allis, C.D.; Patel, D.J.: Molecular basis for site-specific read-out of histone h3k4me3 by the bptf phd finger of nurf. Nature 442: 91-95 (2006).
Li, X.; Heyer, W.D.: Homologous recombination in DNA repair and DNA damage tolerance. Cell research 18: 99-113 (2008).
Liang, D.; Burkhart, S.L.; Singh, R.K.; Kabbaj, M.H.; Gunjan, A.: Histone dosage regulates DNA damage sensitivity in a checkpoint-independent manner by the homologous recombination pathway. Nucleic acids research 40: 9604-9620 (2012).
Lieber, M.R.: The mechanism of double-strand DNA break repair by the nonhomologous DNA end-joining pathway. Annual review of biochemistry 79: 181-211 (2010).
Martin, D.G.; Baetz, K.; Shi, X.; Walter, K.L.; MacDonald, V.E.; Wlodarski, M.J.; Gozani, O.; Hieter, P.; Howe, L.: The yng1p plant homeodomain finger is a methyl-histone binding module that recognizes lysine 4-methylated histone h3. Molecular and cellular biology 26: 7871-7879 (2006).
Mitchell, L.; Lambert, J.P.; Gerdes, M.; Al-Madhoun, A.S.; Skerjanc, I.S.; Figeys, D.; Baetz, K.: Functional dissection of the nua4 histone acetyltransferase reveals its role as a genetic hub and that eaf1 is essential for complex integrity. Molecular and cellular biology 28: 2244-2256 (2008).
Moyal, L.; Lerenthal, Y.; Gana-Weisz, M.; Mass, G.; So, S.; Wang, S.Y.; Eppink, B.; Chung, Y.M.; Shalev, G.; Shema, E.; Shkedy, D.; Smorodinsky, N.I.; van Vliet, N.; Kuster, B.; Mann, M.; Ciechanover, A.; Dahm-Daphi, J.; Kanaar, R.; Hu, M.C.; Chen, D.J.; Oren, M.; Shiloh, Y.: Requirement of atm-dependent monoubiquitylation of histone h2b for timely repair of DNA double-strand breaks. Molecular cell 41: 529-542 (2011).
Nakada, D.; Matsumoto, K.; Sugimoto, K.: Atm-related tel1 associates with double-strand breaks through an xrs2-dependent mechanism. Genes & development 17: 1957-1962 (2003).
Nakanishi, S.; Lee, J.S.; Gardner, K.E.; Gardner, J.M.; Takahashi, Y.H.; Chandrasekharan, M.B.; Sun, Z.W.; Osley, M.A.; Strahl, B.D.; Jaspersen, S.L.; Shilatifard, A.: Histone h2bk123 monoubiquitination is the critical determinant for h3k4 and h3k79 trimethylation by compass and dot1. The Journal of cell biology 186: 371-377 (2009).
Nyberg, K.A.; Michelson, R.J.; Putnam, C.W.; Weinert, T.A.: Toward maintaining the genome: DNA damage and replication checkpoints. Annual review of genetics 36: 617-656 (2002).
Rogakou, E.P.; Pilch, D.R.; Orr, A.H.; Ivanova, V.S.; Bonner, W.M.: DNA double-stranded breaks induce histone h2ax phosphorylation on serine 139. The Journal of biological chemistry 273: 5858-5868 (1998).
Schulze, J.M.; Wang, A.Y.; Kobor, M.S.: Reading chromatin: Insights from yeast into yeats domain structure and function. Epigenetics 5: 573-577 (2010).
Schwartz, M.F.; Duong, J.K.; Sun, Z.; Morrow, J.S.; Pradhan, D.; Stern, D.F.: Rad9 phosphorylation sites couple rad53 to the saccharomyces cerevisiae DNA damage checkpoint. Molecular cell 9: 1055-1065 (2002).
Seiler, D.M.; Rouquette, J.; Schmid, V.J.; Strickfaden, H.; Ottmann, C.; Drexler, G.A.; Mazurek, B.; Greubel, C.; Hable, V.; Dollinger, G.; Cremer, T.; Friedl, A.A.: Double-strand break-induced transcriptional silencing is associated with loss of tri-methylation at h3k4. Chromosome research : an international journal on the molecular, supramolecular and evolutionary aspects of chromosome biology 19: 883-899 (2011).
Sharma, S.; Raghavan, S.C.: Nonhomologous DNA end joining in cell-free extracts. Journal of nucleic acids 2010 (2010).
Shi, X.; Hong, T.; Walter, K.L.; Ewalt, M.; Michishita, E.; Hung, T.; Carney, D.; Pena, P.; Lan, F.; Kaadige, M.R.; Lacoste, N.; Cayrou, C.; Davrazou, F.; Saha, A.; Cairns, B.R.; Ayer, D.E.; Kutateladze, T.G.; Shi, Y.; Cote, J.; Chua, K.F.; Gozani, O.: Ing2 phd domain links histone h3 lysine 4 methylation to active gene repression. Nature 442: 96-99 (2006).
Shi, X.; Kachirskaia, I.; Walter, K.L.; Kuo, J.H.; Lake, A.; Davrazou, F.; Chan, S.M.; Martin, D.G.; Fingerman, I.M.; Briggs, S.D.; Howe, L.; Utz, P.J.; Kutateladze, T.G.; Lugovskoy, A.A.; Bedford, M.T.; Gozani, O.: Proteome-wide analysis in saccharomyces cerevisiae identifies several phd fingers as novel direct and selective binding modules of histone h3 methylated at either lysine 4 or lysine 36. The Journal of biological chemistry 282: 2450-2455 (2007).
Shim, E.Y.; Chung, W.H.; Nicolette, M.L.; Zhang, Y.; Davis, M.; Zhu, Z.; Paull, T.T.; Ira, G.; Lee, S.E.: Saccharomyces cerevisiae mre11/rad50/xrs2 and ku proteins regulate association of exo1 and dna2 with DNA breaks. The EMBO journal 29: 3370-3380 (2010).
Shroff, R.; Arbel-Eden, A.; Pilch, D.; Ira, G.; Bonner, W.M.; Petrini, J.H.; Haber, J.E.; Lichten, M.: Distribution and dynamics of chromatin modification induced by a defined DNA double-strand break. Current biology : CB 14: 1703-1711 (2004).
Symington, L.S.; Gautier, J.: Double-strand break end resection and repair pathway choice. Annual review of genetics 45: 247-271 (2011).
Tao, R.; Chen, H.; Gao, C.; Xue, P.; Yang, F.; Han, J.D.; Zhou, B.; Chen, Y.G.: Xbp1-mediated histone h4 deacetylation contributes to DNA double-strand break repair in yeast. Cell research 21: 1619-1633 (2011).
van Attikum, H.; Fritsch, O.; Gasser, S.M.: Distinct roles for swr1 and ino80 chromatin remodeling complexes at chromosomal double-strand breaks. The EMBO journal 26: 4113-4125 (2007).
van Attikum, H.; Gasser, S.M.: The histone code at DNA breaks: A guide to repair? Nature reviews. Molecular cell biology 6: 757-765 (2005).
Van Dyck, E.; Stasiak, A.Z.; Stasiak, A.; West, S.C.: Binding of double-strand breaks in DNA by human rad52 protein. Nature 398: 728-731 (1999).
van Gent, D.C.; Hoeijmakers, J.H.; Kanaar, R.: Chromosomal stability and the DNA double-stranded break connection. Nature reviews. Genetics 2: 196-206 (2001).
Xu, C.; Cui, G.; Botuyan, M.V.; Mer, G.: Structural basis for the recognition of methylated histone h3k36 by the eaf3 subunit of histone deacetylase complex rpd3s. Structure 16: 1740-1750 (2008).
Zhou, B.B.; Elledge, S.J.: The DNA damage response: Putting checkpoints in perspective. Nature 408: 433-439 (2000).
Zou, L.; Elledge, S.J.: Sensing DNA damage through atrip recognition of rpa-ssdna complexes. Science 300: 1542-1548 (2003).

連結至畢業學校之論文網頁點我開啟連結
註: 此連結為研究生畢業學校所提供,不一定有電子全文可供下載,若連結有誤,請點選上方之〝勘誤回報〞功能,我們會盡快修正,謝謝!
QRCODE
 
 
 
 
 
                                                                                                                                                                                                                                                                                                                                                                                                               
第一頁 上一頁 下一頁 最後一頁 top
系統版面圖檔 系統版面圖檔