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| DC 欄位 | 值 | 語言 |
|---|---|---|
| dc.contributor.advisor | 王萬波(Won-Bo Wang) | |
| dc.contributor.author | Fang-Yen Chiu | en |
| dc.contributor.author | 邱方彥 | zh_TW |
| dc.date.accessioned | 2021-06-15T11:38:55Z | - |
| dc.date.available | 2021-08-26 | |
| dc.date.copyright | 2016-08-26 | |
| dc.date.issued | 2016 | |
| dc.date.submitted | 2016-08-16 | |
| dc.identifier.citation | Alfieri, C., M. Birkenbach, and E. Kieff (1991). Early events in Epstein-Barr virus infection of human B lymphocytes. Virology 181(2), 595-608.
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| dc.identifier.uri | http://tdr.lib.ntu.edu.tw/jspui/handle/123456789/49634 | - |
| dc.description.abstract | Epstein-Barr virus (EBV) 為一種致癌性的皰疹病毒，與多種人類惡性腫瘤有密切關聯，並能在體外實驗中使B細胞永生化為lymphoblastoid cell lines (LCLs)。Epstein-Barr virus nuclear antigen 2 (EBNA2) 為使B細胞永生化及轉型必要的一個轉活化蛋白 (transactivator)，並能活化許多宿主細胞致癌基因 (oncogene) 的表現，進而造成細胞過度增生 (hyperproliferation) 以及DNA雙股斷裂 (DNA double-strand breaks, DSBs)。先前我們實驗室發現EBNA2能促使U2-OS和Hep-2細胞形成多倍體 (polyploidy) 和微核 (micronuclei)。我們也發現EBNA2能使DSB產生後，mediator of DNA damage checkpoint protein 1 (MDC1) 焦點的形成以及53BP1焦點維持的能力衰減，暗示著EBNA2對於DSB修復有負向的影響。在本研究中，我們發現EBNA2能防止γ-H2AX與MDC1 tBRCT功能域進行交互作用，從而抑制MDC1在DSB處形成焦點。此外，MDC1焦點形成被抑制使得U2-OS細胞對於游離輻射有較高的感受性，以及表現較低的非同源性末端接合 (non-homologous end-joining, NHEJ) 效率。然而，在表現EBNA2的U2-OS細胞中，同源重組 (homologous recombination, HR) 的效率卻被增強，且這個增強可能是由於EBNA2和MDC1交互作用所造成。總結來說，本研究闡述了當細胞產生DSB時，EBNA2調控細胞DSB修復系統的新功能和作用機制，而此功能與機制有可能與EBV潛伏感染時維持病毒基因體穩定和促進宿主細胞腫瘤生成有所關聯。 | zh_TW |
| dc.description.abstract | Epstein-Barr virus (EBV) is an oncogenic herpesvirus closely linked to several human malignancies and can immortalize primary B cells into lymphoblastoid cell lines (LCLs) in vitro. Epstein-Barr virus nuclear antigen 2 (EBNA2), a transactivator which is necessary for B-cell immortalization and transformation, can up-regulate several host oncogenes and cause hyperproliferation and DNA double-strand breaks (DSBs). Previously, our laboratory found that EBNA2 could induce polyploidy and micronuclei formation in U2-OS and Hep-2 cells. We also found that EBNA2 could attenuate the foci formation of mediator of DNA damage checkpoint protein 1 (MDC1) and the foci retention of 53BP1 after DSBs induction, suggesting that EBNA2 might have a negative effect on DSB repair. In this study, we showed that EBNA2 could prevent γ-H2AX from interacting with the tBRCT domain of MDC1, thus inhibiting MDC1 foci formation at DSB sites. Furthermore, the inhibition of MDC1 foci formation led to higher sensitivity of U2-OS cells to irradiation treatment and lower non-homologous end-joining (NHEJ) efficiency in U2-OS cells. However, the HR efficiency in EBNA2 expressed U2-OS cells was enhanced and this enhancement was caused by the interaction between EBNA2 and MDC1. Together, these studies elucidate a novel function and mechanism by which EBNA2 modulates the DSB repair system in cells after DSBs formation, which might be responsible for maintaining viral genomic stability and promoting tumorigenesis of host cells during EBV latent infection. | en |
| dc.description.provenance | Made available in DSpace on 2021-06-15T11:38:55Z (GMT). No. of bitstreams: 1 ntu-105-R03445112-1.pdf: 4725971 bytes, checksum: c3e1caaa4e8db963155ae48272738ce2 (MD5) Previous issue date: 2016 | en |
| dc.description.tableofcontents | 中文摘要 i
Abstract ii Contents iii Contents of figures vii Contents of supplementary figures viii I. Introduction 1 i. Epstein-Barr virus (EBV) 1 ii. The structure and genome of EBV 2 iii. EBV infection and life cycle 2 A. Attachment and fusion 2 B. Latent infection and latent viral proteins 3 C. Lytic infection and lytic viral proteins 9 iv. DNA damage response (DDR) for DNA double-strand breaks (DSBs) 10 v. Sensing, transducing and amplifying the signals of DNA DSBs 11 vi. DNA DSB repair in DDR 13 A. Cell cycle-dependent pathway choice for DNA DSB repair 13 B. Non-homologous end-joining (NHEJ) 14 C. Homologous recombination (HR) 15 vii. Activation of cell cycle checkpoint and apoptosis in DDR 17 viii. Mediator of DNA damage checkpoint protein 1 (MDC1) 18 II. Specific aim 20 III. Materials and Methods 21 i. Materials 21 A. Chemicals and reagents 21 B. Kits 23 C. Enzymes 24 D. Antibodies 24 E. Others 24 F. Plasmids 25 G. Cell lines 30 ii. Methods 31 A. Transformation 31 B. Transfection 31 C. Whole cell lysate harvest 32 D. Protein quantification 32 E. Western blot 33 F. Glutathione S-transferase (GST) pull-down assay 33 G. Intracellular protein crosslinking 36 H. Co-immunoprecipitation (co-IP) assay 36 I. Immunofluorescence assay (IFA) 37 J. Cell cologenic assay 38 K. In vivo non-homologous end-joining (NHEJ) assay 39 L. Luciferase assay 40 M. In vivo homologous recombination (HR) assay 40 N. Flow cytometry 41 O. γ-irradiation 42 IV. Results 43 i. EBNA2 interacts with the tBRCT domain of MDC1 strongly and with the PST domain of MDC1 weakly 43 ii. EBNA2 interacts with full-length MDC1 in vivo 44 iii. The interaction between EBNA2 and MDC1 prevents the binding of MDC1 to γ-H2AX 44 iv. EBNA2 interacts with the tBRCT domain of MDC1 through its region of amino acid 329-383 45 v. EBNA2 attenuated the foci formation of MDC1 at DNA DSB sites by interacting with the tBRCT domain of MDC1 45 vi. The failed attenuation of MDC1 foci formation in EBNA2Δ329-383 expressed cells is not due to the impaired transactivating activity of EBNA2 46 vii. Nuclear expression of EBNA2 sensitizes the cells to γ-irradiation 47 viii. Expression of EBNA2 in cells inhibits the NHEJ efficiency for DNA DSBs 48 ix. Expression of EBNA2 in cells enhances the HR efficiency for DNA DSBs 49 x. The enhanced HR efficiency in EBNA2-expressing cells may be due to the interaction between EBNA2 and MDC1 49 xi. Increased DNA DSBs does not enhance the HR efficiency as detected by the DR-GFP system of in vivo HR assay in the cells 50 V. Discussion 51 i. Expression of EBNA2 in cells inhibits the NHEJ efficiency for DNA DSBs repair 52 ii. Expression of EBNA2 in cells enhances the HR efficiency for DNA DSBs repair 53 iii. Expression of EBNA2 in cells increases the cellular radiosensitivity 55 iv. The effects of EBNA2-modulated DSB repair system for viral latent replication and cellular pathogenesis 56 VI. Figures 58 VII. Supplementary figures 80 VIII. References 99 | |
| dc.language.iso | en | |
| dc.subject | 同源重組 | zh_TW |
| dc.subject | EB病毒 | zh_TW |
| dc.subject | EBNA2 | zh_TW |
| dc.subject | MDC1 | zh_TW |
| dc.subject | DNA雙股斷裂 | zh_TW |
| dc.subject | 非同源末端接合 | zh_TW |
| dc.subject | DNA double-strand break | en |
| dc.subject | homologous recombination | en |
| dc.subject | non- homologous end-joining | en |
| dc.subject | Epstein-Barr virus | en |
| dc.subject | EBNA2 | en |
| dc.subject | MDC1 | en |
| dc.title | EB病毒核抗原蛋白EBNA2藉由與細胞蛋白MDC1交互作用而調控DNA雙股斷裂修復 | zh_TW |
| dc.title | Epstein-Barr virus EBNA2 protein modulates the repair of DNA double-strand breaks through interacting with MDC1 | en |
| dc.type | Thesis | |
| dc.date.schoolyear | 104-2 | |
| dc.description.degree | 碩士 | |
| dc.contributor.oralexamcommittee | 陳美如(Mei-Ru Chen),鄧述諄(Shu-Chun Teng) | |
| dc.subject.keyword | EB病毒,EBNA2,MDC1,DNA雙股斷裂,非同源末端接合,同源重組, | zh_TW |
| dc.subject.keyword | Epstein-Barr virus,EBNA2,MDC1,DNA double-strand break,non- homologous end-joining,homologous recombination, | en |
| dc.relation.page | 108 | |
| dc.identifier.doi | 10.6342/NTU201602824 | |
| dc.rights.note | 有償授權 | |
| dc.date.accepted | 2016-08-16 | |
| dc.contributor.author-college | 醫學院 | zh_TW |
| dc.contributor.author-dept | 微生物學研究所 | zh_TW |
| 顯示於系所單位： | 微生物學科所 | |
文件中的檔案：
| 檔案 | 大小 | 格式 | |
|---|---|---|---|
| ntu-105-1.pdf 未授權公開取用 | 4.62 MB | Adobe PDF |
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