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National Chung Hsing University Institutional Repository - NCHUIR > 生命科學院 > 生物化學研究所 > 依資料類型分類 > 碩博士論文 >  Bacteriophage T2之第二型DNA拓撲異構酶gene 52蛋白次單元的純化與結晶

Please use this identifier to cite or link to this item: http://nchuir.lib.nchu.edu.tw/handle/309270000/97581

標題: Bacteriophage T2之第二型DNA拓撲異構酶gene 52蛋白次單元的純化與結晶
Purification and crystallization of the gene 52 protein of bacteriophage T2 type II topoisomerase
作者: 詹秀倩
Chan, Hsiu-Chien
Contributors: 詹迺立
Nei-Li Chan
國立中興大學
關鍵字: type II topoisomerase;Bacteriophage T2;Purification;crystallization
第二型拓撲異構酶;Bacteriophage T2;純化;結晶
日期: 2003
Issue Date: 2012-08-31 16:54:28 (UTC+8)
Publisher: 生物化學研究所
摘要: 除了某些病毒外,第二型DNA拓撲異構酶是所有生物體中不可或缺的酵素,它主要負責解決細胞中DNA在複製、重組、以及染色體分離時所遭遇到的各種因為DNA拓撲異構形所衍生的問題。此酵素利用ATP的結合及水解所產生的能量,藉由其活性中心tyrosine與DNA的醣磷雙酯骨幹形成共價鍵的方式,先將一雙股DNA(或稱G-segment)的兩條主鏈切斷。接下來,另一段雙股 DNA(或稱T-segment)會在此酵素的幫助下通過這個由DNA與酵素所共同形成的"分子閘門",DNA拓撲構形的變化也在此時完成。反應最後此酵素會將先前在G-segment上所引入的缺口接合,以避免對細胞內的DNA帶來損傷。由於此酵素與細胞內的DNA代謝息息相關,許多的抗癌藥物與抗生素,皆以抑制此一酵素來達到消滅癌細胞和病菌的目的。雖然目前已知yeast topoisomerase II片段以及E. coli. gyrase之A subunit的晶體結構,但此酵素與DNA、以及各種抑制物間結合的分子機制仍待探討。因此,本實驗是研究噬菌體T2的第二型DNA拓撲異構酶中其與DNA結合之區域,進而探討此gene 52蛋白次單元如何辨識 G-segment、此"分子閘門"的結構為何、以及其與抗癌藥物的結合方式為何。先前的研究指出此蛋白的N端為不規則的區域,所以為了解析此酵素的結構,我選用了在此gene 52蛋白的N端去除11個胺基酸之質體pTr-11,利用硫胺沉澱、離子交換層析以及分子篩管柱層析大量純化出Tr-11蛋白,並且在15 % PEG 4K、0.1 M sodium citrate pH 5.6、0.2 M ammonium acetate的條件下得到Tr-11蛋白的晶體。由於蛋白質分子在此晶格的排列並不十分規則,因而只觀察到7Å左右低解析度的繞射訊號。在嘗試尋找其他的結晶條件後,也只得到外觀相似的晶體。由於gene 52蛋白的形狀略為狹長,因此為了提高此蛋白形成高品質晶體的機率,我利用一個可能較易形成晶體之gene 52內部去除突變蛋白進行結晶,並成功的在10 % PEG4K、0.2 M ammonium acetate、0.01 M CaCl2、0.05 M Na cacodylate的結晶條件中得到極可能為此突變蛋白與一 hairpin DNA複合體的晶體。
Type II DNA topoisomerases are essential cellular enzymes which alter the topological state of DNA. They are involved in all aspects of DNA metabolism including DNA replication, transcription, recombination, and chromosome segregation. All type II topoisomerases use an ATP-coupled DNA transport reaction in which the enzyme cleaves and generates a "gate" on one DNA duplex (termed G-segment), transports a second duplex (termed T-segment) through the break, and subsequently religates the cleaved DNA. Many antitumor and antibacterial drugs inhibit type II DNA topoisomerase by trapping covalent enzyme-DNA cleavage complexes. Formation of cleavage complexes is important for the cytotoxicity of these drugs. Although type II enzymes interact extensively with DNA, the protein-DNA interaction is yet to be structurally characterized.
This experiment is aimed to at determing the following crystal structures: 1) the crystal structure of the DNA cleavage/ rejoin domain of bacteriophage T2 topoisomerase (gene 52 protein), 2) the structures of the protein-DNA complex(es), 3) structure(s) of protein-DNA-drug ternary complex(es). Previous studies show that the gene 52 protein has a disorder region in the N- terminus that may interfere with crystallization, therefore I use a truncation mutant of gene 52 protein, (termed Tr-11, in which the 11 N-terminal residues were removed) for structural analysis. Large quantity of Tr-11 protein can be obtained following ammonium sulfate precipitation, hydroxyapatite chromatography, and gel filtration chromatography, and crystals of Tr-11 protein were found in the condition consisted of 15% PEG 4K、0.1 M sodium citrate pH 5.6、0.2 M ammonium acetate. However, only low resolution diffraction was observed from these crystals. Finally I used a internal deletion mutant of gene 52 protein (termed ΔT2-52-5g, in which residues 342 to 418 were replaced by a glycine-linker) for crystallization and crystals were found in 18% PEG 8K、0.1M sodium cacodylate pH 6.5、0.2M calcium acetate .In addition, crystallization of ΔT2-52-5g/DNA complex has also been initated.
Appears in Collections:[依資料類型分類] 碩博士論文

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