複製中鏈黴菌DNA端粒的引子酶辨認序列之研究

鏈黴菌是帶有線型染色體及質體的革蘭氏陽性菌，其線型DNA的複製是由中間開始向兩端同時進行雙股複製，複製到末端時其延遲股(lagging strand)無法複製到最末段，在不同的端粒(telomere)序列會留下不同長度的單股間隙，是為端粒複製中間產物，然後此中間產物的單股DNA部分預期會依其豐富的廻紋序列自動形成穩定的二級結構供Tap蛋白所辨識結合，再藉由Tap與TP(末端蛋白)及DNA聚合酶的互相作用，將後兩者帶至這中間產物上，然後DNA聚合酶以TP作為複製起始所需的引子、此中間產物為模板，將其單股DNA片段複製完成。所以這端粒複製中間產物的出現，應該就是端粒複製的起始訊號。雖這中間產物在不同端粒序列上所出現的單股DNA長度的不同，推測除了決定岡崎片段位置的引子酶(primase)的辨識序列在不同的端粒序列上的位置不同外，其豐富的廻紋序列的位置可能也有關。為了研究這個問題，藉由找尋最後一個岡崎片段出現的位置來得知鏈黴菌的引子酶辨識序列是首需解決的問題，並可進一步探討其位置對端粒複製的影響。本論文以帶有S. lividans染色體端粒片段的線型質體pLUS970L做為研究對象，藉由在端粒序列上的各種突變來觀察其延遲股上最後一個岡崎片段位置是否受影響，來找尋其引子酶辨識序列的位置。本實驗室的先前研究，已將其位置縮小到10個核苷酸之間，而我繼續縮小範圍到五個核苷酸之間。此外我也藉由在線型質體上構築相同長度的不同端粒序列，比較其岡崎片段位置的不同位置來找是否有保守序列來幫助尋引子酶辨識序列，結果發現有CGGSCS的保守序列，值得作為後續研究的起始目標。而比較這些不同端粒上最後岡崎片段的位置與廻紋序列區域的關係，發現其位置都是落在廻紋序列區域的外面，可能暗示廻紋序列可能與最後一個岡崎片段出現的位置也有關係。

關鍵字

鏈黴菌；端粒複製；引子酶辨認序列

並列摘要

Streptomyces is gram-positive bacteria with linear chromosome/plasmid. Replication of linear DNA in Streptomyces is initiated from an internal origin and proceeds bidirectionally towards the ends. In this mechanism, lagging strand leaves a single-strand gap at 3’ end. This gap, known as a telomeric replication intermediate, is rich in palindromes and is expected to fold into stable secondary structure for Tap (telomere-associated protein) binding. TP (terminal protein) and DNA polymerase are recruited to this intermediate by interaction with Tap, then the DNA polymerase employees TP as primer and the intermediate as template to complete the single-strand gap. Thus, the appearance of replication intermediate might be considered as an initiating signal of telomere replication. The length of replication intermediates vary in different telomeres. We hypothesized that the gap is not only decided by primase recognition site responsible for the last Okazaki fragment, the pattern and locations of palindromes might also take part in it. Localization of the last primase recognition sequence will be the first step to further reveal the mechanism of Streptomyce telomeric replication and how the position of primase recognition sequence can affect telomere replication. Our lab’s previous studies used linear plasmid pLUS970L with S. lividans telomeres as research material, several telomere mutations were generated to examine the position of last Okazaki fragment in telomere. By this strategy, the last primase recognition sequence had narrowed down in 10 base pairs. In this study, I continued to narrow it to 5 base pairs. I also tried to find primase recognition sequence by comparing the conserved sequences in the upstream of last Okazaki fragment in pLUS970L derivatives with the same length of different telomeres. I found that the conserve region contenting CGGSCS can be a start point of further studies. Comparing the position of the last Okazaki fragment and the distribution of palindromes, I found that the last Okazaki fragment always located outside the clustered palindromes. It suggest that the palindromes might also affect the position of last Okazaki fragment.

並列關鍵字

Streptomyces ； telomere replication ； primase recognition sequence

參考文獻

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複製中鏈黴菌DNA端粒的引子酶辨認序列之研究

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