鏈黴菌端點蛋白與染色體末端結構關係之探討

The interactions between Stretomyces lividans terminal protein and its chromosome ends.

陳怡宏(Yi-Hong Chen)

指導教授 ： 楊千金

中原大學/理學院/化學研究所/碩士(2003年)

https://doi.org/10.6840/cycu200300718

摘要

鏈黴菌Streptomyces lividans的染色體是第一個被發現為線形的，它的5’端有一個以共價鍵連接的蛋白稱為端點蛋白（TP），它的末端200bp中富含著迴紋序列，這些迴紋序列使得複製時造成單股的末端形成某些特殊的二級結構。這些特徵也在其他鏈黴菌染色體及線形質體中發現。鏈黴菌的複製是由中間向兩端複製，如此會造成末端無法補足的問題。端點蛋白因此被推測在末端補足的機制中扮演引子的角色。端點蛋白由185個氨基酸組成，且以蘇氨酸與染色體末端共價鍵結。而在共價銜接形成當中，端點蛋白和染色體末端應存在著某種非共價關係。
本論文在討論鏈黴菌端點蛋白與末端序列之間的共價與非共價關係。我們發現在端點蛋白的N端（由pCY02表現）或是C端（由pCY04表現）加上標誌蛋白D-epitope會使端點蛋白喪失和DNA末端形成共價鍵的活性。在共價關係的實驗部分，利用由pCY11（第114氨基酸由蘇氨酸換成絲氨酸）及pCY16（第108氨基酸由蘇氨酸換成絲氨酸）表現的端點蛋白確認形成共價鍵結的蘇氨酸的位置，發現這兩個突變端點蛋白對DNA末端形成共價的能力不及原生的；其中pCY11表現的端點蛋白更是微弱，結果顯示第114個蘇氨酸可能是共價的位置。在非共價鍵結的實驗部分，我們以膠體延遲的方式，發現端點蛋白對富含迴紋序列的末端167bp並不具有特異性，但對單股的親和力明顯高於雙股。

關鍵字

端點蛋白 ； 鏈黴菌

並列摘要

The chromosome of Streptomyces Lividans is the first found linear DNA molecule. Its 5’ ends were proved to be capped covalently with so called terminal proteins (TP). Bidirectional replication of the Streptomycine is happening in the center of the chromosome. It will make the replication uncompleted. It is presumable that the terminal protein works as primer to complete the end replication. The chromosome ends (around 200bp) are rich in palindromes, which may fold into specific secondary structures to be the substrate of TP. These features are common among the chromosomes and linear plasmids of Streptomyces. TP consists of 185 amino acids, one of the eleven threonines will be covalently bound to the chromosome end. It is reasonable to assume that in forming the covalent bond, there must be non-covalent interactions between TP and DNA.

To investigate the covalent bond, we construct and overexpress two mutated TP. One was mutated at the 108th threonine and the other the 114th. In the in vitro nucleotide labeling assay, both of mutated TP showed lower activity than the native one. Especially the 114 mutated TP, which showed very weak signal even after prolonging exposure. This result indicated that the 114th threonine might be the linkage. As to the noncovalent interaction, TP showed higher binding affinity toward single strand end DNA than double strand ones, but both were nonspecific. The binding affinity was determined by means of gel retardation.

並列關鍵字

Stretomyces lividans ； terminal protein

參考文獻

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