利用突變分析的方法探討Streptomyces lividans染色體端粒的複製機制

Mutational analysis of terminal patching of Streptomyces lividans chromosome telomeres

林澤庠(Tse-Hsiang Lin)

指導教授 ： 黃志宏

國立臺北科技大學/工程學院/生物科技研究所/碩士(2007年)

https://doi.org/10.6841/NTUT.2007.00106

摘要

鏈黴菌，是存在於土壤中的格蘭式陽性菌且含量非常豐富，具有線型的染色體以及質體。先前的研究發現當鏈黴菌進行線型DNA複製時 ，由於在lagging strand的非連續性複製導致在3’端留下一缺口。而3’端突出的單股可能會往內自我摺疊形成二級結構後會被一個末端相關蛋白(terminal associate protein，Tap)所辨認。Tap會與末端蛋白(terminal protein，TP)彼此交互作用後將TP 帶往端粒，接著利用TP當作引子來進行末端缺口的修復工作。
在比較了其他鏈黴菌物種的末端序列後發現它們具有高度的保守性。第一，含有緊密相連的迴文結構。第二，迴文結構比序列還要具有保守性。第三，最末端13 bp(palindrome I)的DNA序列是完全相同的。為了探討末端端粒序列在DNA複製時所扮演的功能，我們主要針對Streptomyces lividans染色體的端粒來進行研究。我利用定位突變(site direct mutagenesis)來產生許多不同的突變端粒並且在活體中測試這些線型質體在DNA複製的功能。假設突變端粒可以支持線型DNA複製，則突變的區域對於末端修復則是不必要的；若是突變區域對於末端修復的必要區域，鏈黴菌無法進行線型DNA複製就會改成環型DNA來進行複製。利用這策略我得到了以下結果：(1)末端端粒僅需90 bp就足夠支持線型DNA複製。(2) palindrome II對於線型DNA是必須的，而非palindrome III。(3)最末端第一個鹼基C是重要的，而非第十三個鹼基G。這些結果指出了末端端粒對於末端缺口修補的必要區域。

關鍵字

鏈黴菌 ； 端粒 ； 線型染色體端粒

並列摘要

The Gram-positive Streptomyces, which are abundant in soil, carried linear chromosomes and plasmids. In previous studies, during replication of the Streptomyces linear replicons the discontinuous synthesis of the lagging strands will leave a gap on the 3’ end of the DNA. The expose 3’ overhang may fold back and form a secondary structure which is recognized by the terminal associated protein (Tap). Tap will recruit terminal protein (TP) to the telomere, where the TP acts a primer for patching synthesis that fill up the single-strand gaps.
Comparison of terminal sequence of Streptomyces shows a high degree of conservation. Firstly, they are composed by tightly spaced palindromes. Secondly, the palindromic structure is more conserved than primary sequence. Thirdly, the terminal 13 bp(palindrome I)DNA are conserved in all. To study the function of the telomere sequences during replication, we focused on the telomere of Streptomyces lividans. By different site direct mutagenesis methods, we generated a series of mutant telomeres and examined their function on linear plasmids in vivo. If the mutated telomeres can support the replication of the linear plasmids in Streptomyces, the mutated nucleotide(s) is not essential for end patching. Using this strategy, we found that (1) only the first 90 bp of the telomere was sufficient; (2) the palindrome II region was required for linear DNA replication, but not palindrome III; and (3) the first nucleotide (C) was essential but not the thirteenth nucleotide (G). These results define the essential telomere structure for end patching.

並列關鍵字

Streptomyces ； telomere ； linear chromosome telomere

參考文獻

[1] Bao, K. and S. N. Cohen. "Terminal proteins essential for the replication of linear plasmids and chromosomes in Streptomyces." Genes Dev, vol 15, no. 12, 2001, pp 1518-27.

[2] Bao, K. and S. N. Cohen. "Recruitment of terminal protein to the ends of Streptomyces linear plasmids and chromosomes by a novel telomere-binding protein essential for linear DNA replication." Genes Dev, vol 17, no. 6, 2003, pp 774-85.

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