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研究生:曾筑欣
研究生(外文):Chu-Hsin Tseng
論文名稱:釀酒酵母Hir2之結構與生化特性分析
論文名稱(外文):Structural and Biochemical Characterization of the Saccharomyces cerevisiae Hir2
指導教授:林佳良
指導教授(外文):Chia-Liang Lin
口試委員:李易撰胡念仁
口試委員(外文):Yi-Chuan LiNien-Jen Hu
口試日期:2023-07-20
學位類別:碩士
校院名稱:國立中興大學
系所名稱:生物化學研究所
學門:生命科學學門
學類:生物化學學類
論文種類:學術論文
論文出版年:2023
畢業學年度:111
語文別:中文
論文頁數:66
中文關鍵詞:組蛋白組蛋白伴侶核小體
外文關鍵詞:HistoneHistone ChaperoneHir ComplexNucleosomeHir2WD40
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組蛋白伴侶是一個蛋白質家族,通過保護組蛋白免於非特異性聚集來促進核小體的組裝和分解,組蛋白伴侶結合新合成或回收的組蛋白並將它們沉積到 DNA 上形成核小體,不同種類的組蛋白伴侶負責與 H2A-H2B 或 H3-H4 結合並將其沉積到 DNA 上。HIRA 組蛋白伴侶複合物以不依賴 DNA 合成的方式將組蛋白變體 H3.3 沉積到染色質上,組蛋白 H3.3 的沉積可以促進基因轉錄、修復 DNA 損傷和阻止染色質異常。高度保守的 HIRA 複合物由三個亞基組成,即 HIRA、泛核蛋白 1 (UBN1) 和神經鈣蛋白結合蛋白 1 (CABIN1)。為了探討 HIRA 複合物促進四體組裝的詳細機制,我們重點研究了釀酒酵母 Hir 複合物的結構和生化特性。HIRA 在後生動物中是單條多肽鏈,然而,其在釀酒酵母中的同源物包含兩個亞基:Hir1 和 Hir2,此外,Hpc2 和 Hir3 分別是 UBN1 和 CABIN1 的釀酒酵母同源物。
在本論文中,我們旨在確定 Hir2 的結構與功能活性以及探討 Hir1 和 Hir2 之間的異同。已知 HIRA 與 UBN1 之間有相互作用,故進行 pull-down assay 證實同源蛋白 Hir2 和 Hpc2 之間亦有相互作用。我們成功解析出了 Hir2_WD40 結構域的晶體結構,並同時發現 Hir2_WD40 結構域與其他物種的 WD40 結構域相比存在一個特殊的 Proline 228 (P228),為了探討 P228 的重要性我們建構了 P228A 單突變體,並且發現其失去了與 Hpc2 相互作用的能力,所以 P228 可能是 Hir2_WD40 結構域與Hpc2 相互作用的關鍵胺基酸。此外,我們還純化了 Hir1_WD40 結構域,同樣進行了 pull-down assay,結果顯示Hir1_WD40結構域也能夠和 Hpc2 作用,因此,目前尚未發現 Hir1 與 Hir2 不同之處。
Histone chaperones are a family of proteins that facilitate the assembly and disassembly of nucleosomes by protecting histones from non-specific aggregation. Histone chaperones bind newly synthesized or recycled histones and deposit them onto DNA to form nucleosomes. Different classes of histone chaperones are responsible for binding H2A-H2B or H3-H4 and depositing them on DNA. The HIRA histone chaperone complex deposits histone variant H3.3 onto chromatin in a DNA synthesis-independent manner, and histone H3.3 deposition can promote gene transcription, repair DNA damage and prevent chromatin abnormalities. The highly conserved HIRA complex consists of three subunits, HIRA, ubinuclein 1 (UBN1), and calcineurin 1 (CABIN1). To explore the detailed mechanism by which the HIRA complex promotes tetrasome assembly, we focused on the structural and biochemical properties of the Saccharomyces cerevisiae Hir complex. HIRA is a single polypeptide chain in metazoans. However, its homologue in S. cerevisiae contains two subunits: Hir1 and Hir2. In addition, Hpc2 and Hir3 are the S. cerevisiae homologues of UBN1 and CABIN1, respectively.
In this study, we aimed to determine the structure and functional activity of Hir2 and to explore the similarities and differences between Hir1 and Hir2. It has been known that HIRA interacted with UBN1. A pull-down assay was therefore performed to confirm that there is also an interaction between the homologous protein Hir2 and Hpc2. We successfully resolved the crystal structure of the Hir2_WD40 domain and found that there is a special Proline 228 (P228) in the Hir2_WD40 domain compared with the WD40 domain of other species. To investigate the importance of P228, we constructed a P228A single mutant and found that it lost the ability to interact with Hpc2. Therefore, P228 may be the key amino acid for the interaction between Hir2_WD40 domain and Hpc2. Furthermore, we also purified the Hir1_WD40 domain and performed a pull-down assay. The results showed that the Hir1_WD40 domain can interact with Hpc2 as well. Therefore, no differences between Hir1 and Hir2 have been found so far.
中文摘要 i
英文摘要 ii
目錄 iv
圖表目錄 vii
壹、序論 1
一、組蛋白與核小體 (Histone & Nucleosome) 1
二、組蛋白伴侶 (Histone Chaperone) 2
三、專用 H3-H4 組蛋白伴侶 3
(一)H3.1/2-H4伴侶 3
(二)H3.3-H4伴侶 4
四、研究動機與目的 7
貳、實驗方法與材料 9
一、細菌菌株(Bacterial Strain) 9
二、質體及其建構(Plasmids and Construction) 9
(一) 載體 9
(二) 重組質體 9
三、一般蛋白質之純化 10
(一) 細菌培養 10
(二) 蛋白質的大量表現(over-expression)與純化 11
(三) 蛋白質電泳(Sodium Dodecyl Sulfate Polyacrylamide electrophoresis,SDS-PAGE) 18
(四) 蛋白質染色與退染 19
(五) 蛋白質濃縮 19
(六) 蛋白質濃度測量 19
四、下拉實驗 (Pull-down Assay) 20
五、蛋白質養晶與 X-ray 繞射 20
(一) 結晶方法 20
(二) 繞射數據的收集與分析 20
六、一般分生研究的實驗方法 21
(一) 聚合酶連鎖反應 (Polymerase Chain Reaction, PCR) 21
(二) DNA 片段回收 21
(三) DNA 連接反應 (Infusion) 22
(四) 質體抽取 22
(五) DNA 濃度的測量 23
(六) 大腸桿菌的轉形法(Transformation) 23
參、實驗結果 25
一、蛋白質的表現與純化 25
(一) Hir2_WD40 25
(二) Hir1_WD40 26
(三) Hir2_WD40_P228A 27
(四) GST 28
(五) GST-Hpc2_NHRD+HRD、GST-Hpc2_NHRD+α、GST-Hpc2_NHRD 29
二、證明 Hir2_WD40 與 Hpc2_NHRD之間具有作用力 29
三、證明 Hir1_WD40 與 Hpc2_NHRD 之間具有作用力 30
四、Hir2_WD40 之晶體結構 30
五、證明 Proline 228 影響Hir2_WD40與 Hpc2_NHRD 之間的作用 31
肆、討論 32
一、Hir1 與 Hir2 異同之探討 32
二、Hir2_WD40 結構之探討 32
三、P228 對 Hir2_WD40 與 Hpc2_NHRD 結合能力之影響 33
四、結論 33
伍、圖表 34
陸、參考文獻 64
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