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研究生: 王義翔
Wang, Yi-Hsiang
論文名稱: 增生細胞核抗原藉DNA複製增進過氧化氫引起之DNA損傷修復
Proliferating Cell Nuclear Antigen Facilitates Repair of Hydrogen Peroxide-Induced DNA Damage by Coupling to DNA Replication
指導教授: 劉銀樟
Liu, Yin-Chang
口試委員: 汪宏達
李財坤
學位類別: 碩士
Master
系所名稱: 生命科學暨醫學院 - 分子醫學研究所
Institute of Molecular Medicine
論文出版年: 2012
畢業學年度: 100
語文別: 英文
論文頁數: 45
中文關鍵詞: 增生細胞核抗原去氧核糖核酸複製鹼基切除修復
外文關鍵詞: Proliferating Cell Nuclear Antigen, DNA Replication, Base Excision Repair
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  • 細胞增生核蛋白已知為去氧核糖核酸合成過程中的重要因子,同時也被證實參與在核苷酸切除修復和鹼基切除修復之去氧核糖核酸修復機制中。我們先前研究發現核苷酸切除修復和鹼基切除修復同時發生時,鹼基切除修復強勢過於核苷酸切除修復,並造成核苷酸切除修復延遲。進一步,我們分析增生細胞核蛋白多寡如何影響鹼基切除修復。由結果指出,血清缺乏狀況的細胞於過氧化氫引起的去氧核糖核酸氧化損傷中,將使延鹼基切除修復過程延長,而核苷酸切除修復卻不受其影響。相同地,移除細胞增生核蛋白的細胞也具有延遲鹼基切除修復作用的效果,反之,若過量表現細胞增生核蛋白有助於弭除血清缺乏狀況的鹼基切除修復延遲。此外,同時過量表現細胞增生核蛋白及抑制去氧核糖核酸複製,將癱瘓鹼基切除修復作用而無法消除氧化損傷。此研究證實,細胞增生核蛋白具有在去氧核糖核酸複製機制中,共同參與鹼基切除氧化傷害修復的重要角色。


    Proliferating cell nuclear antigen (PCNA), known as processive protein factor during DNA synthesis, has been shown to be involved in all types of DNA repair mechanisms including nucleotide excision repair (NER) and base excision repair (BER). Our previous study has found that when NER is encountered with BER, BER appears to be dominant over NER, but NER is delayed to allow BER. As an effort to characterize the BER dominancy, we assessed how the levels of PCNA might affect BER. The results were showed that the repair of hydrogen peroxide induced DNA lesions was greatly delayed in serum-deprived cells. Knockdown of PCNA produced similar inhibitory effect, while the overexpression of PCNA abolished the serum deprivation effect. However, the overexpression of PCNA were unfavorable BER in DNA replication inhibition. The results demostrated that PCNA appears to be tightly coupled with DNA replication as the presence of oxidative damages.

    Abstract ......................................................................................................................... 3 中文摘要........................................................................................................................ 4 I. Introduction .............................................................................................................. 5 II. Materials and methods ........................................................................................... 8 Cell cultures ........................................................................................................... 8 Ultraviolet (UV) radiation ..................................................................................... 8 Comet assay ........................................................................................................... 8 Whole cell extract ................................................................................................ 10 Determination of protein concentration .............................................................. 10 Western blotting ................................................................................................... 10 Expression plasmids ............................................................................................. 11 Transient transfection .......................................................................................... 12 ELISA of 8-OHdG ................................................................................................ 12 Immunostaining of 8-OHdG ................................................................................ 14 Cell Proliferation by BrdU incorporation ........................................................... 14 III. Results and Discussion ........................................................................................ 16 i. Repair of oxidative DNA damage is slower in serum starved cells where PCNA levels are low. ................................................................................................... 16 ii. Repair of oxidative DNA damage can be tuned up or down by manipulation of PCNA protein levels. ....................................................................................... 16 iii. Effect of PCNA on repair of oxidative DNA damage is coupled with DNA replication. ....................................................................................................... 18 iv. Concluding remarks and Model ...................................................................... 19 IV. Figures and Figure Legends ................................................................................ 21 V. Reference ................................................................................................................ 38 VI. Appendix ............................................................................................................... 43

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