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研究生:魏立杰
研究生(外文):Li-Chieh Wei
論文名稱:利用含有SV40 origin質體探討DNA雙股斷裂的修復機制
論文名稱(外文):Mechanism of DNA Double Strand Break Repair Using SV-40-Based Plasmid System
指導教授:徐明達徐明達引用關係
指導教授(外文):Ming-Ta Hsu
學位類別:碩士
校院名稱:國立陽明大學
系所名稱:生命科學暨基因體科學研究所
學門:生命科學學門
學類:生物學類
論文種類:學術論文
論文出版年:2009
畢業學年度:97
語文別:英文
論文頁數:66
中文關鍵詞:修補機制
外文關鍵詞:DNA repair
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摘要
DNA雙股斷裂的修復對於維持整個哺乳動物細胞的基因體完整性來說是很重要的,但是到目前為止DNA雙股斷裂的修復機制仍舊是有很多地方需要被探討以及去發現的。為了探討更多有關於DNA雙股斷裂修復的機制, 我利用限制性酵素做切割,在帶有一段SV40 Origin的pUC18質體(PSV)上製造了DNA雙股斷裂 然後再將這些線狀質體轉染到COS-1細胞中,利用細胞的修補機制將斷裂的DNA再修復成為環狀。分析這些修補過後的DNA序列可以發現,有的會在原始的斷裂點處有著DNA序列的刪減, 其刪減長度大多在9~1119 bp左右。除此之外,還有些修補過後的序列伴隨著質體或是細胞本身DNA的插入。綜合全部序列分析過後的結果,序列刪減的情形占絕大多數,特別值得注意的是另外有些序列除了有序列的刪減外還伴隨著序列的插入,插入序列的來源包括來自於刪減區域下游的序列,還有來自於刪減區域內的序列但其位於定序股的互補股序列且插入的方向是以反方向做插入的,這裡我列舉了一些模式來解釋這現象。因為有很多插入的序列是來自於細胞本身的DNA以及質體上的序列,所以我共同轉染了酵素消化過的PSV質體以及lambda phage genomic DNA片段進入COS-1細胞,目的在於探討是否插入的DNA來源僅侷限於細胞內擁有的DNA,結果我發現此時插入的DNA序列來源大多是來自於共同轉染的lambda phage genomic DNA片段,說明了其來源並不只是細胞本身的DNA或是質體DNA而已。另外我也觀察了同源性重組的修補模式在哺乳動物細胞內的重要性,共同轉染兩組PSV DNA到COS-1細胞中,一組是經過限制性酵素消化過後產生有一段DNA缺失的質體,另一組也是經過限制性酵素消化後但產生了較前一組更為大片段的DNA缺失的質體,且此缺失的片段範圍是覆蓋過前一組缺失片段範圍的。此時將損失較小片段的PSV質體和那段被限制性酵素消化後產生的較長且覆蓋過前一組缺失區域的DNA片段共同轉染到細胞中,但序列分析的結果發現到只有極少部分是利用同源性重組來做修補,大部分仍是以非同源性接合為最主要的模式。最後探討是否DNA的甲基化會影響細胞對於斷裂的雙股DNA的修補,利用SssI methylase在CG上進行DNA的甲基化,並且發現這樣的甲基存在不會影響到斷裂DNA的修補,它們仍是以相同於未甲基化的DNA的修復模式被修補起來。
Abstract

DNA double strand breaks(DSBs) repair plays a very important role in the maintenance of genomic integrity in mammalian cells. The mechanism of DSBs repair system has not been fully elucidated. In order to obtain more information about DSBs repair, pUC18 plasmids with SV40 origin of replication were digested with a restriction enzyme to produce the double strand break, and the linearized DNA was then transfected into cultured COS-1 cells. The linear DNA molecules were repaired and re-circularized. Results from DNA sequencing analysis of the re-sealed DB break revealed that the majority of repaired DNA contained deletion from 9 to 1119bp. Furthermore, some of the non-homologous repair was accompanied by the insertion of plasmid or genomic sequences. Among many clones analyzed by sequencing, deletions were found at both ends in most cases. The notable feature of the deletion junctions in some clones was the presence of insertions of sequences which came from the reverse strand in the deleted region or from downstream strand sequence. I postulate a model to explain this phenomenon. Because many of the insertions are of genomic DNA origin, I cotransfected digested plasmids with lambda phage DNA ladders to test whether random sequences could also be inserted at the repair junctions. The result showed that lambda phage DNA could also be inserted. I also tested the efficiency of homologous DNA repair of restoring the DSBs. Co-transfection of a plasmid with a segment of DNA removed with a DNA fragment containing the removed sequences was used to determine the frequency of homologous repair with respect to non-homologous end-joining repair. The result showed that homologous recombination was not the dominant mode in the repair. Finally, I analyzed the influence of CpG methylation on DSBs repair. Methylation was introduced into linear plasmids digested with restriction enzyme using SssI methylase. The result showed methylated DNA sequence could be repaired as efficiently and in the same way as the unmethylated DNA sequence. Therefore, CpG methylation will not interfere with DSBs repair.
Contents

Abstract(中文) ------------------------------------------------1
Abstract(英文) ------------------------------------------------3
Introduction----------------------------------------------------5
Materials&Methods-----------------------------------------15
Results---------------------------------------------------------24
Discussion-----------------------------------------------------30
Figures---------------------------------------------------------35
Appendix------------------------------------------------------53
References----------------------------------------------------57
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