方偉宏臺灣大學：醫事技術學研究所陳盈坤Chen, Ying-KunYing-KunChen2007-11-292018-07-062007-11-292018-07-062004http://ntur.lib.ntu.edu.tw//handle/246246/62830演化過程中所保留下來的MMR可以藉由修復DNA複製錯誤，抑制發生在部分解離DNA序列之間的非同質性重組，與不同的DNA損害作用以及一些其他的功能來維持真核與原核生物基因體的穩定性。在核酸配對錯誤復上已有相當透澈的瞭解。但是大型核酸環的修復，一個迥異於配對錯誤的修復機制，卻是個未知的系統。因此研究這兩種修復系統間的差異，將是個令人感興趣的議題。 人類特異股配對修復及大型核酸環修復均需外加dNTP，同時修復反應也受到aphidicolin的抑制。之前關於配對錯誤修復片段的研究多半是在HeLa細胞核萃取物修復異雙股核酸反應中，不外加dNTP或加入aphidicolin的條件下，截取修復反應的中間產物。於試管中停止再合成反應的情況下，觀察移除反應後斷裂的單股核酸產物，以進行移除反應範圍的分析。因此觀察到移除反應區域橫跨在錯誤至斷端這範圍。然而這類型的研究方法沒有使修復反應作用完全，因此不能排除所得結果是來自中止反應所衍生副反應的訊號。 為了避免此項疑慮，因此改用含硫磷酸的化學方法來探討可能修復路徑。利用dNTPαS和iodoethanol處理下，核酸配對錯誤、核酸環、或兩者同時存在下的3’含斷股之異雙股核酸呈現出個別的修復路徑。在核酸配對錯誤修復方面，最遠的取代訊號出現在距離錯誤配對5’端100~150 鹼基長度的位置。這與一般移除反應路徑分析終止端位置結果一致。在核酸環修復方面，修復的訊號則僅位於斷端與核酸環兩點。因此推論：核酸配對錯誤與核酸環修復是經由不同的機制修復，然而當核酸配對錯誤與核酸環共同存在時，則會影響彼此的修復反應。Evolutionarily conserved mismatch repair (MMR) systems promote genetic stability in most eukaryotic and prokaryotic organisms by correcting DNA replication errors, antagonizing homeologous recombination between partially diverged DNA sequences, interacting with variety of DNA lesions, and performing other functions. The study about mismatch repair has been analyzed thoroughly. But the large loop repair, a distinct repair from mismatch repair, is an unknown repair system. Studying the differences between these two repair systems seems to be an interesting project. Both human strand-specific mismatch repair and large loop repair depend on presence of exogenous dNTPs and are inhibited by aphidicolin. Most of previous studies about mismatch repair patches were to trap the intermediates produced from the course of heteroduplex correction in HeLa nuclear extracts either in the absent of exogenous dNTP or in the supplement of aphidicolin. In vitro excision tracts have been mapped as single-strand gaps produced when repair is provoked under condition of restricted DNA synthesis. Analysis of gaps produced on these heteroduplex resulted in excision tracts spanning the path between lesion and the nick. However, according to this methodology, this repair reaction is incomplete. It is not able to eliminate the possibility of the result generated from side reaction caused by the restricted to show that condition. To avoid the argument, we employed thiophosphate chemistry in the repair patch mapping. Under the condition of dNTPΠ substitution and iodoethanol treatment, mismatch, large loop, or mismatch and large loop co-exist heteroduplexses with 3’ nick showed distinct repair patches. In mismatch repair, the most extreme ladders shift to 5’ direction approximately 100 to 150 bases beyond the mismatch, which is consistent with the excision tract endpoint mapped and agrees with the results of others. In loop repair, the repair signal locates on nick and loop site. These results further ascertain that the repair of mismatch and large loop are processed through different mechanisms. When mismatch and loop co-exist, there is interference between these two repair patches.總目次 I 表目次 III 圖目次 IV 附錄目次 V 中文摘要 VI 英文摘要 VII 縮寫表 IX 前言 1 材料與方法 8 一、菌株 8 二、人類細胞株之繼代培養 8 三、桿菌細胞萃取物之製備 9 四、人類細胞核萃取液之製備 10 五、突變噬菌體M13mp18 mutant、f1PM mutant之建構 11 六､M13mp18與f1PM系列複製型雙股核酸及單股核酸之製備 12 七、異雙股核酸之建構 14 八､異雙股核酸對測定用限制酵素之敏感度分析 15 九、試管中反應條件之選定與修復反應 16 十､試管中修復情形之分析 17 十一､γ-P32[ATP]標記探針 17 十二､電泳分析核酸產物 18 十三､南方墨點分析 19 十四､核酸產物分析 19 結果 21 一、具斷股異雙股核酸之建構 21 二、異雙股核酸對分析用限制酵素之敏感度分析 21 三、試管中修復反應條件之選定 22 四、試管中異雙股核酸之修復反應定量用限制酵素之 敏感度分析 23 五、在大腸桿菌萃取液以及人類細胞核萃取液中添加 dCTPαS對修復反應之影響 24 六、鹼性瓊脂凝膠電泳分析 25 七、8.3 M尿素-5%聚丙醯胺凝膠電泳分析 26 甲、 核酸環修復與核酸配對錯誤修復 26 乙、 核酸配對錯誤與核酸環共同修復 27 討論 30 附表 33 附圖 36 附錄 47 參考文獻 522409688 bytesapplication/pdfen-US修復路徑移除反應配對錯誤核酸環修復DNA修復核酸修復mismatch repairrepair patchDNA repairthiophosphate chemistryLoop repairexcision mappingotherhttp://ntur.lib.ntu.edu.tw/bitstream/246246/62830/1/ntu-93-R91424011-1.pdf