大腸桿菌中大型核酸環修復路徑之分析

Abstract

生物為了維持基因體的穩定，演化出許多不同機制來修復核酸上的錯誤，以避免個體產生病變，甚至死亡。大型核酸環修復系統是近年來才被發現的修復機制，到目前為止已知，在大腸桿菌中此修復機制有下列的特徵：修復過程是由斷股所指引（nick-directed）、需要鎂離子與dNTPs的參與、不需鹼基配對錯誤修復系統的蛋白MutHLS參與、不需要外加ATP來提供能量；此外本實驗室還發現，髮夾結構修復的特徵與大型核酸環相同，因此這兩種錯誤可能由相同機制來修復，但到目前為止，詳細的修復機制與所參與的蛋白卻依然不清楚。因此，為了找出參與修復反應的蛋白，我們取得8種不同核酸修復缺陷的大腸桿菌突變株，利用其細胞萃取液進行測試，結果發現異雙股核酸上的修復反應並沒有被抑制，因此可知UvrA、UvrB、UvrC、UvrD、RecJ、ExoI、ExoVII及SbcCD這8種蛋白，並沒有參與大型核酸環及髮夾結構的修復。在修復路徑 (repair patch)的探討方面，我們藉著不加入dNTPs，或加入ddNTPs，來限制修復反應的進行，並以鹼性瓊酯膠電泳 (alkaline agarose gel)，對反應中所產生的中間產物進行初步分析，再利用變性聚丙醯胺凝膠電泳 (denaturing polyacrylamide gel)，對信號所在區域做細部分析，結果發現大型核酸環與髮夾結構上發生特異性斷股，表示未配對序列可能經由斷股而被移除。了解切除反應 (excision)的範圍，我們利用限制酵素來分析斷股與未配對序列間的核酸是否被移除，結果發現未配對序列兩側的核酸，皆保持雙股狀態。綜合以上結果，我們推測在大腸桿菌中，大型核酸環與髮夾結構的修復，皆由斷股所指引，並直接於未配對序列上進行切割，再進行的修復，但其詳細機制則仍需更進一步的研究。

DNA loop and hairpin are products of normal DNA biosysthetic errors or homeologous recombination and can lead to severe genomic instability if unrepaired. Previous studies showed that a strand break located either 3’ or 5’ to the large loop is sufficient to direct repair to the nicked strand in Escherichia coli cell extracts. This activity is distinct from mismatch repair pathway. Furthermore, our previous results suggested that hairpin and large loop structures would be processed by the same mechanism because of their similar repair characteristics. To investigate what components are involved and understand how heterologies are processed, we used a set of heteroduplexes containing an insertion/deletion large loop or hairpin for our study. Heteroduplexes were tested in the extracts from different E. coli mutant strains for repair efficiency. The results indicated that the correction of large loop and hairpin repair are independent of uvrA, uvrB, uvrC, uvrD, recJ, exoI, exoVII and sbcCD gene products. By limiting the repair synthesis in reactions, the incision or excision intermediates can be trapped and analyzed by denaturing gel electrophoresis. We found that strand- and loop-specific incisions are in the proximity to the heterologies. To further determine the involvement of excision reactions in large loop repair, we employed restriction enzyme digestion assay to the repair intermediates. Our data suggested that large loop and hairpin are processed by specific incision in the heterologies, not processed by the extensive excision from the pre-existing nick toward heterologies.