DNA Double Strand Break Repair Using SV40-Based Plasmid System
| Autor: | Pei-Fang Chung, 鐘培芳 |
|---|---|
| Rok vydání: | 2000 |
| Druh dokumentu: | 學位論文 ; thesis |
| Popis: | 88 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, pCAT-Promoter plasmid with SV40 Origin was digested with two different restriction enzymes and the linear DNA with mismatched 5’PSS-Blunt, Blunt-Blunt, and 5’PSS-5’PSS ends were then transfected into cultured COS-1 cells. Results from direct Southern analysis demonstrate that these damaged DNA substrate could recircularize and the joining is accompanied by loss a variety of sequence at the junction by deletion and insertion and by the formation of multimers. Among 43 clones analyzed by sequencing, deletions were found at both ends in most cases. The noticeable feature of the junctions in some clones was the presence of insertions of sequence with unknown origin. We also test the effect of repeat sequence flanking the break points on DSBs repair using a plasmid with direct repeat SV40 origin sequences. The result indicates that in contrast to plasmids without repeat sequence repair was achieved through homologous recombination. We also analyzed the influence of CpG methylation on DSBs repair. Methylation is introduced into linear plasmid digested with two restriction enzymes in vitro using the SssI methylase, which specifically methylates the C-5 position of cytosine bases within CpG dinucleotides. DNA substrate were then transfected into cultured COS-1 cells, direct Southern analysis found that methylation decreased DSBs repair efficiency suggesting that methylated sequence may interfere with repair process. 英文摘要……………………………………………………………2 背景…………………………………………………………………3 材料與方法…………………………………………………………9 實驗結果…………………………………………………………..15 討論………………………………………………………………..23 附圖………………………………………………………………..27 附錄………………………………………………………………..46 參考文獻…………………………………………………………..66 |
| Databáze: | Networked Digital Library of Theses & Dissertations |
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