探討鏈黴菌線形質體SCP1上末端連結蛋白與末端蛋白中影響末端複製的胺基酸

The amino acid residues of Tac and Tpc that affect the end replication of Streptomyces SCP1 linear plasmid

10.6840/cycu201800049

李京儒

鏈黴菌 ； SCP1 ； 端粒連結蛋白 ； 末端蛋白 ； streptomyce ； SCP1 ； terminal associated protein ； terminal protein

中原大學化學系學位論文

2018年

碩士

楊千金

繁體中文

鏈黴菌的染色體與部分質體呈線形。DNA複製時由中央的複製起點開始向兩側進行，當複製到末端時會在3’端留下單股缺口，端粒連結蛋白(Tap)會與缺口結合，牽引末端蛋白(TPs)作為引子，完成末端缺口的補齊。在此，Tap扮演著DNA聚合酶的角色；末端蛋白Tpg則作為引子。 少數鏈黴菌質體如S. coelicolor的SCP1端粒序列及末端蛋白和大多數鏈黴菌有所差異，SCP1末端蛋白(Tpc)基因上游具有端粒連結蛋白(Tac)基因，與Tap及Tpg系統相似，因此也可能以相同機制進行末端的修復。我們將Tac、Tap、用蛋白質做引子的Φ29 DNA聚合酶以及具有DNA聚合酶同源性的E. coli Pol II的胺基酸序列做比對，發現有三處位置的天門冬胺酸是四種蛋白共有，其中Asp398、Asp670對應到Φ29 DNA聚合酶和E. coli Pol II與鎂離子結合的位置；在Φ29 DNA聚合酶上的YCDTDS基序(motif)與其活性有關，其中的Thr具有高度保守性。本次研究目標之一是測試Tac與金屬離子結合的天門冬胺酸與YCDTDS基序中的蘇胺酸，將Asp398與Asp670突變成無法與二價金屬離子結合的天門冬醯胺或丙胺酸；Thr669突變成丙胺酸，再以鏈黴菌體內實驗測試，由於鏈黴菌要維持線形質體或染色體複製必須依靠Tac，若Tac失去活性則無法得到線形質體，pLUS894是一個能在鏈黴菌中維持線形複製的迷你質體，我們將三個突變後分別換入pLUS894中的tac，用限制酶AseI線形化後送到鏈黴菌內，抽取單株的質體，結果無法得到質體。 Tpc對鏈黴菌維持線形質體或染色體也極為重要，其主要的功能為引子，可以鍵結在DNA上讓Tac順利進行複製。先前實驗顯示，在Tap與Tpg系統中，Tpg是利用親水性高的第114個蘇胺酸和DNA鍵結。我們利用放射性磷的dG*作為標記，Tpc為引子，Tac為DNA聚合酶進行體外補齊實驗，再用二維TLC片推測Tpc可能利用絲胺酸與DNA鍵結，Tpc總共有13個絲胺酸，將胺基酸序列做親水性分析，結果Ser206為Tpc上較為親水的絲胺酸。本研究第二個目標為定位出Tpc與DNA鍵結的絲胺酸，先將Ser206突變成丙胺酸，再送入鏈黴菌做體內實驗，結果無法得到線形質體；之後將突變成蘇胺酸、酪胺酸以及具有咪唑的組胺酸，測試絲胺酸可否被以上胺基酸取代；另外我們也測試Ser178，將其突變成丙胺酸測試是否會有影響，若Tpc無法鍵結到DNA上則無法維持線形複製，將突變送入鏈黴菌的結果為Ser178換成丙胺酸無法得到線形質體，而Ser206換成上述三種胺基酸仍可以得到線形質體，但換成丙胺酸則無法得到線形質體，初步結果顯示Tpc的Ser206可能不是與DNA鍵結的位置，而丙胺酸的突變可能因為其含有His-tag而失去活性；Ser178還需要做其他突變進行更明確的判斷是否是Tpc與DNA結合的位置。

Some plasmids and chromosomes of Streptomyces are linear. The replication of streptomyces proceeds bidirectionally from central origin to the end of DNA, and leaves a 3’ overhang. Next, terminal asscoiated protein (Tap) links to the 3’ overhang and recruit terminal protein (Tpg) to repair the 3' gap. In this case, Tap serves as a DNA polymerase, and Tpg serves as a primer. Some plasmids, like SCP1, have different telomeric DNA sequence that can’t align with others. The terminal asscoiated protein of SCP1 named Tac, and terminal protein named Tpc. The tac (gene of Tac) is on the upstream of tpc (gene of Tpc). This characteristic is similar to Tap-Tpg system, and therefore they may have the same mechanism of end-patching. We align the amino acid sequence of Tac, Tap, Φ29 DNA polymerase, and E.coli DNA Pol II. The alignmet showed that there are three conservative aspartic acid residues. Two of them have been identified that are the Mg2+ binding site of Φ29 DNA polymerase and E.coli DNA pol II[1,29], and the corresponding positions in Tac are Asp398 and Asp670.The essentialty of these two residues was examined in vivo by mutating the corresponding positions in tac gene in plasmid pLUS892L, a mini linear SCP1 plasmid. The mutants of them named TacD398Q and TacD670A.The results showed that there had no linear plasmids formed. Additionally, Φ29 DNA polymerase has a motif (YCDTDS) to do with protein-primed and polymeration. The threonine is important for its activity. The essentialty of Thr669 of Tac was also tested. The in vivo results suggested that it might be critical. The essential aminio acid residues on Tpc were also examined. We use phosphoamino acid analysis (PAA) to determine that Tpc used serine to bond to the end of DNA. There are thirteen serines in Tpc, we first check Ser206 because of its hydrophilicity. It was mutated to alanine, and named TpcS206A. The in vivo result suggested that Tpc might use Ser206 to bond to DNA end. Further, Ser206 was mutated to histidine, threonine and tyrosine. The in vivo results showed that Ser206 can be replace by those amino acid. The result of TpcS206A might be influence by his-tag, hence there had no linear plasmid formed. We also picked Ser178 to mutate to alanine then had in vivo testing. The result identicated that Ser178 can’t get any linear plasmid. There was an unecpected mutation caused by polymerase chain reaction (PCR), the His135 of Tpc mutated to tyrosine. The in vivo result that His135 might also be critical.

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