透過您的圖書館登入
IP:44.220.131.93
  • 學位論文

鏈黴菌端粒連結蛋白活性中心兩個天門冬胺酸的定位

Identification of the two aspartic acid residues involved in the activity of Tap of Streptomyces

指導教授 : 楊千金

摘要


鏈黴菌的染色體與部分質體是線形的,進行複製時是以中心的複製起點開始,用一般複製的方式向兩側前進,在兩側的3'末端會留下單股缺口,這段單股缺口具有特殊的迴文序列會形成穩定的二級結構,端粒連結蛋白(TapC)會與第二、第三迴文序列區域結合,牽引末端蛋白(TpgC)作為引子,完成末端缺口的補齊。TapC在此扮演著DNA聚合酶的角色,合成3'末端前13個核酸。 雖然TapC並沒有已知DNA聚合酶的基序(motif),不過我們將其胺基酸序列與S.coelicolor線型質體SCP1的端粒連結蛋白Tac、屬於Family B的E.coli DNA Pol II、以及同為Family B而且與TapC一樣能用蛋白質做為引子的 29 DNA聚合酶做比對,發現有三個位置的天門冬胺酸是四個序列共有的,其中兩個位置TapC Asp433與Asp662都分別對應到E.coli DNA pol II和 29 DNA聚合酶與Mg2+結合必需胺基酸的位置。本論文研究目的之一是找出維持TapC活性的必要天門冬胺酸,我們分別做了Asp177、Asp433、Asp628與Asp662四個位置的突變,將這些位置的天門冬胺酸替換成無法與二價金屬離子配位的丙胺酸或是天門冬醯胺,用N端His-tag把蛋白純化出來,以鏈黴菌體外末端單股序列補齊實驗檢測活性,因鏈黴菌染色體3'末端前三個鹼基都是G(GGG),我們只加入含放射性同位素的dCTP做為DNA合成的材料,有活性的TapC會在TpgC上補齊前三個鹼基對(CCC),最後證實Asp433和Asp662為必要的胺基酸。之後我們以體內測試再做一次驗證,TapC是維持鏈黴菌線形染色體或質體複製的關鍵,如果TapC沒有活性,我們將無法得到線形的染色體,而pLUS981是一個能在鏈黴菌中維持線形複製的迷你染色體,我們將Asp433、Asp662兩個位置的突變分別換到裡面的tapC,用限制酶AseI線型化後送進鏈黴菌體內,抽取單株的質體並用限制酶SpeI或SpeI+ClaI檢驗,結果我們並沒有得到任何線形質體。 為了維持DNA合成的正確性,DAN聚合酶隨著演化得到分辨dNTP與NTP的能力,根據胺基酸序列比對的結果,TapC Phe438對應到控制 29 DNA 聚合酶核苷酸選擇性的Tyr254,Tyr上的芳香環是辨識dNTP與NTP的關鍵,而Phe也是具有芳香環的胺基酸,本論文第二個研究目的是找出與TapC dNTP與NTP辨識能力有關的胺基酸。我們把苯丙胺酸(Phe)改為沒有芳香環的丙胺酸(Ala),在末端單股序列補齊實驗同時加入含放射性同位素的dCTP與CTP做為合成材料,結果TapCF438A在TpgC上合成的CCC數量明顯下降,表示這個位置的突變確實會影響TapC的dNTP與NTP辨識性,使TapCF438A增加了CTP的親和性,稀釋了選取dCTP的數量。

並列摘要


The chromosomes and some plasmids of Streptomyces are linear. The replication of them starts from a central origin with bidirectional process and leaves a 3' end single strand overhang which subsequently forms stable secondary structures through its specific palindrome sequence.On this secondary structure, terminal asscoiated protein (TapC) binds specifically to the palindrome II and III area and recruits terminal protein (TpgC) as a primer to patch the 3' gap. The DNA polymerase activity of Tap limited to synthesize the first 13 nucleotides is also novel. Although the amino acid sequence of TapC does not show any of the characterized DNA polymerase motifs, the aligment with  29 DNA polymerase and E.coli DNA Pol II, members of Family B, and Tac, the telomere associated protein of SCP1( the linear plasimd of S.coelicolor) showed that there are three conserved aspartic acid residues. Two of these three have been identified to be essential for Mg2+-binding in the activity of  29 DNA polymerase and E.coli DNA pol II ; and the corresponding positions in TapC are Asp433 and Asp662. Each of them was mutated to asparagine (N) and purified as TapCD433N and TapCD662N through the technique of His-tag. Both of them were not able to add dCTP to TpgC by means of in vitro end patching system. We also test the essentialty of these two residues in vivo by mutating the corresponding positions in tapc gene in plasmid pLUS981, a mini linear chromosome.The results showed that there were no linear plasmid formed. We were also interested in Phe438, since the corresponding position Try254 of  29 DNA polymerase is involved in the discrimination of deoxynucleotide (dCTP) and nucleotide (CTP). The alanine substitution, TapC, reduced the discrimination as  29.

參考文獻


2. Lin, Y. S., Kieser, H. M., Hopwood, D. A., and Chen, C. W. (1994) The chromosomal DNA of Streptomyces lividans 66 is linear. Molecular microbiology 14, 1103
4. Huang, C. H., Lin, Y. S., Yang, Y. L., Huang, S. W., and Chen, C. W. (1998) The telomeres of Streptomyces chromosomes contain conserved palindromic sequences with potential to form complex secondary structures. Molecular microbiology 28, 905-916
5. Bentley, S. D., Brown, S., Murphy, L. D., Harris, D. E., Quail, M. A., Parkhill, J., Barrell, B. G., McCormick, J. R., Santamaria, R. I., Losick, R., Yamasaki, M., Kinashi, H., Chen, C. W., Chandra, G., Jakimowicz, D., Kieser, H. M., Kieser, T., and Chater, K. F. (2004) SCP1, a 356,023 bp linear plasmid adapted to the ecology and developmental biology of its host, Streptomyces coelicolor A3(2). Molecular microbiology 51, 1615-1628
6. Huang, C. H., Tsai, H. H., Tsay, Y. G., Chien, Y. N., Wang, S. L., Cheng, M. Y., Ke, C. H., and Chen, C. W. (2007) The telomere system of the Streptomyces linear plasmid SCP1 represents a novel class. Molecular microbiology 63, 1710-1718
7. Zuker, M. (2003) Mfold web server for nucleic acid folding and hybridization prediction. Nucleic acids research 31, 3406-3415

延伸閱讀