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系統識別號 U0026-1508201419041200
論文名稱(中文) 組蛋白修飾和HLTF蛋白在DNA修復機制中的功能
論文名稱(英文) The function of histone modification and HLTF in DNA repair
校院名稱 成功大學
系所名稱(中) 生命科學系
系所名稱(英) Department of Life Sciences
學年度 102
學期 2
出版年 103
研究生(中文) 林怡如
研究生(英文) Yi-Ju Lin
學號 L56011106
學位類別 碩士
語文別 中文
論文頁數 60頁
口試委員 指導教授-廖泓鈞
口試委員-曾淑芬
口試委員-張松彬
口試委員-邱文泰
中文關鍵字 抗藥性  後複製修復路徑  DNA雙股斷裂  組蛋白修飾  染色體重塑 
英文關鍵字 post-replication repair  HLTF  template-switch  DSB  NuA4 
學科別分類
中文摘要 順鉑 (cisplatin) 是現今廣泛使用於治療癌症的藥物,其藥理機制是透過將DNA變為交連 (crosslink) 結構而造成DNA複製受阻,最後使細胞走向凋亡。但我們發現在具有抗藥性的HONE6鼻咽癌細胞株中,其後複製修復路徑 (post-replication repair, PRR) 有強化的現象,因此,我們對後複製修復路徑做更進一步的探討。後複製修復路徑可以不必移除DNA損傷部位對DNA進行修復,先前研究指出HLTF和SHPRH蛋白參與在後複製修復中的模板置換路徑,並擔任E3泛素連接酶 (E3 ligase) 的角色,對增殖細胞核抗原 (proliferating cell nuclear antigen, PCNA)進行多泛素化 (polyubiquitination)。我們的實驗發現HLTF也會與DNA損傷修復相關的蛋白DNA-PK及PARP1結合,因為DNA-PK是phosphatidylinositol-3 kinase-like kinase (PIKK)家族的一員且參與在DNA雙股斷裂修復路徑中,所以我們發現HLTF蛋白可能被DNA-PK磷酸化且參與DNA雙股斷裂修復機制。PARP1使用NAD當作基質將ADP-ribose催化轉移至核蛋白上,並在原本的ADP-ribose上連接60-80個ADP-ribose單元,在核蛋白上形成poly(ADP-ribose),且PARP1會參與在DNA單股及雙股斷裂的修復機制中,我們實驗結果顯示HLTF可能利用DNA-PK及PARP1參與在後複製修復路徑中。
DNA雙股斷裂是所有DNA損傷中最嚴重的一種,現今所採用的癌症治療方式也大多是讓癌細胞產生DNA雙股斷裂而導致細胞凋亡。因此,了解細胞的DNA雙股斷裂修復機制有助於癌症治療的發展。從酵母研究中,我們知道當DNA雙股斷裂發生時,H2A的第129 serine (S129) 位置會被進行磷酸化修飾,接著召集Ino80、Swr1及NuA4聚集到斷裂處進行染色體重塑 (Chromatin remodeling)。NuA4是乙醯轉移酶 (Histone acetyltransferase, HAT),能對組蛋白H4進行乙醯化修飾使染色體結構鬆開,我們實驗發現SET1有對組蛋白進行甲基化修飾的能力,而NuA4會藉由次蛋白Yng2的PHD domain去辨識組蛋白H3第4 lysine (K4) 位置上的甲基化修飾,證明DNA雙股斷裂處附近的組蛋白甲基化修飾有助於NuA4進入DNA斷裂處。
英文摘要 Post-replication repair (PRR) is the DNA damage tolerance pathway that bypasses DNA lesions without removing the lesions during DNA replication. Here, we discovered that several genes in the PRR pathway is highly expressed in cisplatin-resistant nasopharyngeal carcinoma (NPC) cells, HONE6, indicating the importance of the PRR pathway in the drug resistant phenotype. We further demonstrate that HLTF can associate with DNA-PK and PARP1. DNA-PK belongs to the phosphatidylinositol-3 kinase-like kinase (PIKK) superfamily and participates in DNA double-strand break (DSB) repair pathway, indicating that HLTF may be phosphorylated and related to the DSB repair pathway. PARP1 uses NAD as substrate to catalyze both the covalent transfer of ADP-ribose to a variety of nuclear protein acceptors and subsequently the transfer of an additional 60 to 80 ADP-ribose units to the initial moiety. PARP1 has been known to be involved in repairing DNA single-strand breaks and also double-strand breaks. Our results suggest that HLTF may utilize DNA-PK and PARP1 to participate in the PRR pathway. In addition, we study the function of histone modifications at DSBs in yeast Saccharomyces cerevisiae. We demonstrate that Set1 is recruited to the DSB site, where Set1 methylates lysine 4 of histone H3 at the DSB site. This process facilitates the enzyme activity of NuA4 at DSB sites through the interaction between the plant homeodomain (PHD) of Yng2 and methylated K4 of histone H3, thus facilitating DSB repair.
論文目次 中文摘要 I
Abstract III
致謝 VI
目錄 VII
圖目錄 VIII
第一章 前言 1
第二章 實驗目的 12
第三章 實驗方法 14
第四章 結果 32
第五章 討論 40
參考文獻 45
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