鏈黴菌端粒連結蛋白與末端蛋白參與染色體末端複製

Telomere associate protein (Tap) and terminal protein(Tpg) participate in end patching of Streptomyces

曾淑敏(Shu-Min Tseng)

指導教授 ： 楊千金

中原大學/理學院/化學研究所/碩士(2012年)

https://doi.org/10.6840/cycu201200909

摘要

鏈黴菌線形染色體與線形質體的複製是由中心複製點開始，以一般複製的方式向兩側進行，當複製到末端時會留下3’端突出部分，此再由以蛋白質為引子的補齊機制補齊。以前實驗結果顯示末端蛋白(Tpg)可作為引子；而其共同表現的端粒連結蛋白(Tap)雖具有兩種功能，第一是與末端蛋白有交互作用，第二是與3’端單股形成的二級結構有特定的交互作用[2]，但其結構分析比對結果沒有明顯一般聚合酶的特徵；另外實驗結果顯示兩個基因受損修復聚合酶(DinB, SCO1380、SCO1738)之一可支持鏈黴菌線形複製[10]。然而在體外重建實驗(in vitro)發現，末端蛋白、端粒連結蛋白及3’端單股，不但足以讓末端蛋白與第一個核苷酸dCMP連接並且延伸至第一迴文區端，此結果顯示端粒連結蛋白可能具有DNA聚合酶的活性。

為了證明端粒連結蛋白的DNA聚合酶活性不是來自純化蛋白時受到大腸菌DNA聚合酶(I)的汙染，在使用鎳金屬親和性管柱純化蛋白時，將緩衝液內的NaCl濃度提升至1M並增加沖洗管柱的緩衝液體積以降低大腸菌DNA聚合酶(I)的汙染，另外利用FPLC-superdex 200再次純化，但兩者精化效果不易證明。但原位活性膠體實驗(in situ activity gel)，結果顯示端粒連結蛋白具有DNA聚合酶的活性。

一般蛋白質中螺旋轉螺旋(Helix-turn-Helix)是與DNA連接的區域，端粒連結蛋白刪除此區域(第41~59個胺基酸)而構築迷你質體CY175，可在鏈黴菌中線形複製，體外重建實驗結果顯示具有DNA聚合酶的功能，但其活性下降約5倍。若擴大刪除其N端的區域，而得到迷你質體CY176(刪除第2~81個胺基酸)及CY177(刪除第2~150個胺基酸)，無法在鏈黴菌中線形複製。

我們同時發現延長或刪除末端第一迴文區與第二迴文區的間隙並不影響線形複製體的活性，但刪除間隙與第一迴文區內最後一個核苷酸會消除其活性。

體外重建實驗結果顯示3’端單股序列只需8 mer就足以讓末端蛋白與第一個核苷酸dCMP連接，超過35 mer以上會有明顯延長。刪除第一個及突變第四個核苷酸不影響延長，但反應活性下降；置換第一或第二者G為A時，活性亦下降。

關鍵字

螺旋轉螺旋 ； 末端補齊 ； 端粒連結蛋白 ； 末端蛋白 ； 鏈黴菌

並列摘要

The replication of linear chromosomes and linear plasmids of Streptomyces proceeds from central origin of replication to both sides. It will leave a gap at 3’ end, and the gap is patched by protein-primed DNA synthesis. The previous data suggested that terminal protein (Tpg) worked as the primer; and the co-transcribed telomere associated protein (Tap) could associate with Tpg and specifically interact with the secondary structure of the single strand at 3’ end [2], Tap didn’t have the characteristic of DNA polymerase by alignment. It was found that either one of the two DinB DNA polymerase (SCO1380 and SCO1738) would support the replication of linear chromosomes of Streptomyces[10]. Reconstituted experiments showed that Tpg could link with the first dCMP and extend to the end of palindrome I, in the presence of Tap, Tpg, and single stranded 3’ DNA end , suggesting that Tap has activity of DNA polymerase.
To prove that the DNA polymerase activity of Tap didn’t come from contamination of E.coli DNA polymerase (I), two efforts had been undertaken. First, the volume of washing buffer was increased and the concentration of NaCl was raised up to 1 M and size exclusion chromatography was performed successively to refine the purity, but the results showed no improvements. Second, in situ activity gel was then applied and the result showed that TapC has the activity of DNA polymerase.
Helix-turn-Helix is generally a structure motif of DNA binding. However, when it was deleted from TapC, the resulted plasmid CY175, still exists linear form in MRO4. The matched TapC possesses polymerase activity in vitro, but the activity decreased 5 fold. In contrast, deletion of 2~81a.a or 2~150 a.a of Tap could not support linear plasmids in MRO4.
At the same time we found that addition of GTAC nucleotides after the sequence between palindrome I and II or deletion of the sequence between palindrome I and II did not affect the replication of linear plasmids in MRO4. However, deletion of the sequence between palindrome I and II and the last nucleotide of palindrome I abolished the replication of linear plasmid.
Tpg could bind to the template even when it is only 8-nucleotides long, but it could only extend the DNA synthesis when template is 35-nucleotides long. Deleting or mutating the fourth nucleotide in the template did not affect extension, but both led to reduced activity. Replacement of the first or the second nucleotide G with A not only stopped extension but also resulted in quite low signals.

並列關鍵字

TapC ； TpgC ； helix-turn-turn ； end patching ； Streptomyces

參考文獻

1. Bao, K. and Cohen, S. N. (2001) Terminal proteins essential for the replication of linear plasmids and chromosomes in Streptomyces. Genes Dev. 15: 1518-1527.

11. Huang, C.-H., Lin, Y.-S., Yang, Y.-L., Huang, S.-W. and Chen, C.W. (1998) The1. Bao, K. and Cohen, S. N. (2001) Terminal proteins essential for the replication of linear plasmids and chromosomes in Streptomyces. Genes Dev. 15: 1518-1527.

2. Bao, K. and Cohen, S. N. (2003) 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. 17: 774-785.

4. Birch, A., Häusle, A., R Hütter, R. (1990) Genome rearrangement and genetic instability in Streptomyces spp. Journal of bacteriology. 172, 4138-4142.

5. Chen CW, Lin Y-S, Yang Y-L, Tsou M-F, Chang H-M, Kieser HM, Hopwood DA. (1994) The linear chromosomes of Streptomyces: structure and dynamics. Actinomycetologica 8: 103-112.