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| DC 欄位 | 值 | 語言 |
|---|---|---|
| dc.contributor.advisor | 朱雪萍 | zh_TW |
| dc.contributor.advisor | Hsueh-Ping Chu | en |
| dc.contributor.author | 張庭嘉 | zh_TW |
| dc.contributor.author | Ting-Chia Chang | en |
| dc.date.accessioned | 2023-09-22T16:27:33Z | - |
| dc.date.available | 2023-11-09 | - |
| dc.date.copyright | 2023-09-22 | - |
| dc.date.issued | 2023 | - |
| dc.date.submitted | 2023-08-09 | - |
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| dc.identifier.uri | http://tdr.lib.ntu.edu.tw/jspui/handle/123456789/89866 | - |
| dc.description.abstract | 當DNA結構受到外部或內在產生的有毒因素時（例如：紫外線作為外源毒性或R-loop的形成視為內源因素），會對genomic DNA中造成DNA損傷。如果未能即時修復這些DNA損傷，最終可能會導致細胞的衰亡，也因此細胞發展了各種機制來維護DNA結構的完整性。R-loop是由單股DNA和DNA-RNA hybrid所組成，而R-loop的生成會使DNA結構的不穩定，也因此R-loop的解開是被需要的。
DHX8 是RNA helicase，本身作為splicing factor可以從spliceosome上釋出mRNA而聞名。先前研究中已經證實DHX8在體外可以解開DNA-RNA hybrid，且我們發現DHX8 會影響R-loop的累積。此外，我們先前的研究亦有發現DHX8參與了ATR-Chk2 repair pathway，並且在缺乏DHX8的細胞中，造成RPA無法累積在 DNA 斷裂處。 然而，我們的最近結果顯示出：當加入DNA 損傷藥劑CPT 時，DHX8對於消除double-strand break（DSB）上的DNA-RNA hybrid是沒有效用的。此外，我們意外的發現：加入DNA 損傷藥劑CPT，在缺乏DHX8的情況下，會無法累積在DNA斷裂處上的DNA-RNA hybrid。 因前人的研究指出RNA polymerase（RNAPII）可以轉錄DNA-induced long non-coding RNA（dilncRNA），且該RNA可以參與DNA損傷（DDR）的啟動。基於上述的研究，我們推測DHX8與召集RNAPII到DSB上具有潛在作用，我們的結果顯示出：在缺乏DHX8的細胞中，RNAPII被召集至DSB的數量明顯減少，這說明DHX8在促進RNAPII被召集到DSB中發揮了關鍵作用。 | zh_TW |
| dc.description.abstract | Once the DNA structure is challenged by the toxic external or internal factors, such as ultraviolet light as exogenous toxicity or the formation of R-loops as an endogenous factor, it can result in the generation of DNA lesions within the genomic DNA. Failure to repair these DNA lesions can ultimately lead to cell death. Therefore, cells have developed various mechanisms to preserve genomic integrity.
R-loop is a structure composed of single-strand DNA and DNA-RNA hybrid. The formation of R-loops destabilizes the DNA structure, and therefore is required to be unwound. DHX8, an RNA helicase known for mRNA releasing from spliceosome, has been demonstrated to unwind DNA-RNA hybrid in vitro. Our previous study showed that DHX8 depletion leads to accumulation of genomic R-loop, and inhibits the activation of the ATR-Chk1 repair pathway. Depletion of DHX8 displays a defect of replication protein A (RPA) recruitment to DNA damage sites, thereby impeding the further steps of the DNA repair pathway. Surprisingly, our results demonstrate that DHX8 depletion fails to accumulate DNA-RNA hybrids at double-strand break (DSB) sites upon camptothecin (CPT) treatment. We show that DHX8 is associated with RNA polymerase II (RNAPII), R-loops and DNA breaks induced by CPT. Recent studies indicate that RNAPII can transcribe DNA damage-induced long non-coding RNAs (dilncRNAs), which are involved in initiating DNA damage response (DDR). To test the potential involvement of DHX8 in the recruitment of RNAPII to double-strand breaks (DSBs), proximity ligation assay (PLA) for RNAPII and 𝛾H2AX was performed to detect RNAPII at DSBs. The result shows that depletion of DHX8 leads to a decrease of RNAPII at DSB sites, suggesting that DHX8 plays a critical role in regulating the recruitment of RNAPII to DSBs, and this recruitment is important for the initiation of DNA repair. | en |
| dc.description.provenance | Submitted by admin ntu (admin@lib.ntu.edu.tw) on 2023-09-22T16:27:33Z No. of bitstreams: 0 | en |
| dc.description.provenance | Made available in DSpace on 2023-09-22T16:27:33Z (GMT). No. of bitstreams: 0 | en |
| dc.description.tableofcontents | Contents
誌謝 i 中文摘要 ii Abstract iii Contents v Content of figures and supplementary figures x Content of tables xi Abbreviations xii Chapter 1 Introduction 1 1-1 Splicing and spliceosomes 1 1-2 DExD/H-box proteins within spliceosomes 2 1-3 DEAH-box proteins and DEAH-box helicase 8 (DHX8) 4 1-4 DNA damage response and DNA damage repair pathway 5 1-5 Telomeric repeat-containing RNA (TERRA) and Alternative lengthening of telomere (ALT) cancer cells 8 1-6 Camptothecin (CPT) 10 1-7 DNA-RNA hybrid formation after DNA damage 11 1-8 Recruitment and regulation of RNA polymerases after DNA damage 12 1-9 DHX8 depletion results in TERRA R-loop accumulation at telomeres 13 Chapter 2 Materials and Methods 16 2-1 Cell culture 16 2-2 siRNA knockdown and CPT treatment 16 2-3 Protein-protein Immunostaining 17 2-4 Western blotting 18 2-5 Proximity ligation assay (PLA) 19 2-6 Quantitative and statistical analysis 22 Chapter 3 Results 24 3-1 DHX8 is recruited to CPT-induced DNA damage sites. 24 3-2 DHX8 is recruited to DNA-RNA hybrids. 26 3-3 DHX8 depletion reduces RNA-DNA hybrids at DNA damage sites. 27 3-4 DHX8 interacts with RNA polymerase II in response to CPT-induced DNA damage. 29 3-5 DHX8 is required for RNA polymerase II recruitment at DNA damage sites. 30 Chapter 4 Discussion 32 Chapter 5 Figures and supplementary figures 39 Chapter 6 Tables 60 Chapter 7 References 67 Content of figures and supplementary figures Figure 1. DHX8 is recruited to DNA damage sites. 41 Figure 2. DHX8 is recruited to DNA-RNA hybrids after CPT treatment. 44 Figure 3. DHX8 is required for the formation of DNA-RNA hybrids at DNA double-stranded break sites. 48 Figure 4. DHX8 is associated with RNA polymerase (RNAPII) upon CPT treatment. 50 Figure 5. DHX8 is required for RNA polymerase II recruitment to the DNA double-stranded break sites. 52 Figure 6. Analysis of PLA signals in U2OS cells treated with CPT. 53 Figure 7. Models. 55 Supplementary Figure 1. DHX8 depletion leads to accumulation of telomeric R-loops in ALT cancer cells. 58 Content of tables Table 1. Seeding density and CSKT/ TrixtonX-100 treatment time of each cell lines in cell staining experiment 60 Table 2. Antibodies used in IF and PLA 61 Table 3. Antibodies used in WB 62 Table 4. siRNA used in transfection 63 Table 5. Reagents 63 | - |
| dc.language.iso | en | - |
| dc.subject | DNA損害反應 | zh_TW |
| dc.subject | DHX8 | zh_TW |
| dc.subject | 剪接因子 | zh_TW |
| dc.subject | DNA-RNA hybrid | zh_TW |
| dc.subject | RNA聚合酶II | zh_TW |
| dc.subject | splicing factor | en |
| dc.subject | DHX8 | en |
| dc.subject | DNA-RNA hybrid | en |
| dc.subject | DDR | en |
| dc.subject | RNAPII | en |
| dc.title | DHX8對DNA雙股斷裂點招募RNA聚合酶II的影響 | zh_TW |
| dc.title | DHX8 impacts the recruitment of RNA polymerase II to DNA double-strand break sites | en |
| dc.type | Thesis | - |
| dc.date.schoolyear | 111-2 | - |
| dc.description.degree | 碩士 | - |
| dc.contributor.oralexamcommittee | 吳青錫;譚婉玉 | zh_TW |
| dc.contributor.oralexamcommittee | CHING-SHYI WU;Woan-Yuh Tarn | en |
| dc.subject.keyword | DNA損害反應,剪接因子,DHX8,DNA-RNA hybrid,RNA聚合酶II, | zh_TW |
| dc.subject.keyword | DDR,splicing factor,DHX8,DNA-RNA hybrid,RNAPII, | en |
| dc.relation.page | 72 | - |
| dc.identifier.doi | 10.6342/NTU202303302 | - |
| dc.rights.note | 同意授權(限校園內公開) | - |
| dc.date.accepted | 2023-08-10 | - |
| dc.contributor.author-college | 生命科學院 | - |
| dc.contributor.author-dept | 分子與細胞生物學研究所 | - |
| dc.date.embargo-lift | 2028-08-07 | - |
| 顯示於系所單位： | 分子與細胞生物學研究所 | |
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