阿拉伯芥多功能核酸酶的單鏈脫氧核醣核酸結合位的結構研究

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研究生：	尤宗富
論文名稱：	阿拉伯芥多功能核酸酶的單鏈脫氧核醣核酸結合位的結構研究 Structural insights of the ssDNA binding site in the multifunctional endonuclease AtBFN2 from Arabidopsis thaliana
指導教授：	孫玉珠王惠鈞
口試委員:	蕭傳鐙林彩雲馬徹
學位類別：	博士 Doctor
系所名稱：	生命科學暨醫學院 - 生物資訊與結構生物研究所 Institute of Bioinformatics and Structural Biology
論文出版年：	2014
畢業學年度：	103
語文別：	英文
論文頁數：	54
中文關鍵詞：	X光繞射、核酸酶、磷酸二酯酶、n-糖基化、三核金屬酵素
相關次數：	點閱：3 下載：0
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多功能S1/P1核酸酶AtBFN2在酵素分類上被歸類為EC3.1.30.1，它是由阿拉伯芥At1g68290基因編碼的糖蛋白，它的功能為可分解消化RNA，單鏈DNA和雙鏈DNA。AtBFN2需要三個鋅離子的幫助裂解DNA和RNA的3'-OH而得到5'-核苷酸。此外，AtBFN2的酵素活性與聚醣有很強的依賴性。植物的鋅離子依賴性核酸內切酶呈現出獨特的結構折疊，屬於Phospholipase C (PLC)/ P1核酸酶家族。在這個研究工作中，我們提出第一個完整的AtBFN2蛋白質結構，以及硫酸鹽複合物結構，磷酸鹽複合物結構和單鏈DNA的複合物結構。根據這些實驗結果，我們能夠提供更好的聚醣結構組成和可能的酵素機制。與其它核酸酶結構比較，AtBFN2/ligand-free和AtBFN2/PO4模型顯示與之前提出相似的催化機制。我們的數據還證實磷酸鹽和釩酸鹽可以透過佔據活性位點抑制核酸酶的活性。更重要的是，AtBFN2/A5T結構揭示了一個新穎的且保守的第二個單鏈DNA結合位點，其似乎對植物的鋅離子依賴性核酸內切酶是很重要的。基於以上這些發現，我們提出了一個合理的單鏈DNA與AtBFN2結合的模式，其中單鏈DNA包裹自己周圍的蛋白質和連接表面的聚醣，進而強化穩定此複合物結構。

List of Contents
Abstract (in Chinese)……………………………………………I
Abstract………………………………………………………………………………Ⅱ
List of Contents…………………………………………………………Ⅲ
List of Tables………………………………………………………………Ⅴ
List of Figures……………………………………………………………Ⅵ
Abbreviations…………………………………………………………………Ⅷ
List of Publication…………………………………………………Ⅹ

1. Introduction……………………………………..…………………1
2. Materials and Methods……………………………………4
2.1 Cloning, overexpression and mutagenesis of AtENDO2……………………….4
2.2 Modified purification of AtENDO2………………………5
2.3 In-gel ssDNase activities assays………………………5
2.4 Western blotting…………………………………………………………………6
2.5 ssDNA activity inhibition and kinetic assay……………………………………7
2.6 Screening and modified crystallization…………………………8
2.7 Data collection and structure determinati…………………8
2.8 PC-PLC activity assay for AtENDO2………………………………………9
2.9 Phosphomonoesterase activity assay……………………………………9
3. Results……………………………………………………………………………10
3.1 Improved AtBFN2 protein and crystal quality………………………………...10
3.2 Overview of AtENDO2 structures……………………………………………10
3.3 Zinc cluster active-site configuration………………………11
3.4 N-linked polysaccharide conformation……………………………12
3.5 Mutagenesis study of zinc cluster active-site……13
3.6 Discovery of the nuclease inhibition and inhibitors………13
3.7 Multifunctional roles of AtBFN2…………………………………………15
4. Discussions………………………………………………………………………16
5. Tables and Figures……………………………………………………22

References…………………………………………………………………………51

Appedix
                                

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