核酸環修復之反應區段分析

Abstract

核酸聚合酶在DNA複製過程中如發生滑動，將可能產生單股未配對的核酸環，此核酸環若未被修復會提高基因的不穩定性，甚至造成突變。過去的研究已知，核酸環修復由斷股所指引，不需鹼基配對錯誤修復系統蛋白的參與。至今為止大型核酸環修復的詳細機制仍未清楚，本研究目的即在分析大型核酸環修復的反應區段，藉以探討核酸環的修復機制。第一部分以HeLaS3人類細胞核萃取液與大型核酸環的異雙股核酸，在試管內模擬生物體內大型核酸環修復的發生，並分析發生修復的位置及活性。為研究其修復路徑，在修復反應的過程中，藉著不提供dNTPs或使用dNTPαS來限制修復反應的進行，擷取中間產物，並以鹼性瓊脂膠和南方墨點法進行初步分析。結果發現缺乏dNTP將無法進行核酸環修復，而以dNTPαS作用的中間產物訊號只侷限在斷股處。第二部分則以dUTP取代dTTP，於大腸桿菌BD2314 (ung-) 細胞萃取物中進行修復反應，利用其移除uracil功能缺陷的特性，將dUTP嵌入修復路徑中，接著使用uracil glycosylase和AP endonuclease將dUTP移除並打斷該處的鍵結，用同樣方式分析中間產物。初步結果發現大型核酸環修復的中間產物位在斷股和核酸環之間。為了找出詳細的核酸環修復範圍，將反應產物以NheI及SspI做雙重切割，再利用8.3 M urea-5% polyacrylamide gel對信號所在區域做高解析度的分析，結果發現中間產物訊號位在核酸環附近。綜合以上結果，我們推測在大腸桿菌中，大型核酸環的修復可能藉由斷股指引，在核酸環附近進行局部核酸移除和再合成。

DNA loop heterologies are products of DNA polymerase slippage during DNA replication. Such DNA loop heterologies may lead to genomic instability if unrepaired. Previous results have shown that loop repair is nick-directed and independent of the mismatch repair proteins. However, the mechanism of large loop repair is still not clear. To investigate the repair process of large DNA loop, I analyed the repair patch of large loop repair. In the first part, I treated loop heterologies with HeLaS3 human nuclear cell extract to repair large DNA loop in vitro. I trapped repair intermediates by omitting dNTPs. I also did the repair patch mapping by using thiophosphate chemistry by addition of dNTPαS in the reaction. The repair products and intermediates were resolved by alkaline agarose gel electrophoresis and indirect labeling by southern blotting. These results showed that large loop repair is blocked when dNTPs is limited, and the repair reaction is limited to nick when dNTPαS is added. In the second part, I used E. coli cell extracts to study bacterial loop repair process. I replaced dTTP to dUTP in repair reaction, and treated DNA loop substrate with extract of BD2314, a ung- stain of E. coli, then used uracil glyscosylase and AP endonuclease to remove U and broke the phosphate bond. The breakage point was resolved on alkaline agarose gel. These data showed that the repair patch is between loop and nick. I further fine mapped the breakage sites by denaturing polyacylamide gel electrophoresis. I found that the repair synthesis is localized within the loop region. These data indicated that large loop repair in E. coli is nick-directed, and the base excison and resynthesis is localized around the loop.