請用此 Handle URI 來引用此文件：
http://tdr.lib.ntu.edu.tw/jspui/handle/123456789/94810完整後設資料紀錄
| DC 欄位 | 值 | 語言 |
|---|---|---|
| dc.contributor.advisor | 詹迺立 | zh_TW |
| dc.contributor.advisor | Nei-Li Chan | en |
| dc.contributor.author | 劉禮玲 | zh_TW |
| dc.contributor.author | Li-Ling Liu | en |
| dc.date.accessioned | 2024-08-19T16:45:06Z | - |
| dc.date.available | 2024-08-20 | - |
| dc.date.copyright | 2024-08-19 | - |
| dc.date.issued | 2024 | - |
| dc.date.submitted | 2024-07-30 | - |
| dc.identifier.citation | 1.Kottemann, M.C. and A. Smogorzewska, Fanconi anaemia and the repair of Watson and Crick DNA crosslinks. Nature, 2013. 493(7432): p. 356-363.
2.Kim, H. and A.D. D'Andrea, Regulation of DNA cross-link repair by the Fanconi anemia/BRCA pathway. Genes & development, 2012. 26(13): p. 1393-1408. 3.Deans, A.J. and S.C. West, DNA interstrand crosslink repair and cancer. Nature reviews cancer, 2011. 11(7): p. 467-480. 4.Kennedy, R.D. and A.D. D'Andrea, The Fanconi Anemia/BRCA pathway: new faces in the crowd. Genes Dev, 2005. 19(24): p. 2925-40. 5.Wang, L.C. and J. Gautier, The Fanconi anemia pathway and ICL repair: implications for cancer therapy. Crit Rev Biochem Mol Biol, 2010. 45(5): p. 424-39. 6.Clauson, C., O.D. Schärer, and L. Niedernhofer, Advances in understanding the complex mechanisms of DNA interstrand cross-link repair. Cold Spring Harb Perspect Biol, 2013. 5(10): p. a012732. 7.Huang, M., et al., The FANCM/FAAP24 complex is required for the DNA interstrand crosslink-induced checkpoint response. Molecular cell, 2010. 39(2): p. 259-268. 8.Walden, H. and A.J. Deans, The Fanconi anemia DNA repair pathway: structural and functional insights into a complex disorder. Annual review of biophysics, 2014. 43: p. 257-278. 9.Meetei, A.R., et al., A novel ubiquitin ligase is deficient in Fanconi anemia. Nat Genet, 2003. 35(2): p. 165-70. 10.Hira, A., et al., Mutations in the gene encoding the E2 conjugating enzyme UBE2T cause Fanconi anemia. The American Journal of Human Genetics, 2015. 96(6): p. 1001-1007. 11.Alpi, A.F., et al., Mechanistic insight into site-restricted monoubiquitination of FANCD2 by Ube2t, FANCL, and FANCI. Molecular cell, 2008. 32(6): p. 767-777. 12.Meier, D. and D. Schindler, Fanconi anemia core complex gene promoters harbor conserved transcription regulatory elements. PLoS One, 2011. 6(8): p. e22911. 13.Al-Hakim, A., et al., The ubiquitous role of ubiquitin in the DNA damage response. DNA Repair (Amst), 2010. 9(12): p. 1229-40. 14.Knipscheer, P., et al., The Fanconi anemia pathway promotes replication-dependent DNA interstrand cross-link repair. Science, 2009. 326(5960): p. 1698-1701. 15.Michl, J., J. Zimmer, and M. Tarsounas, Interplay between Fanconi anemia and homologous recombination pathways in genome integrity. The EMBO Journal, 2016. 35(9): p. 909-923. 16.Niraj, J., A. Färkkilä, and A.D. D'Andrea, The Fanconi Anemia Pathway in Cancer. Annu Rev Cancer Biol, 2019. 3: p. 457-478. 17.Moldovan, G.L. and A.D. D'Andrea, How the fanconi anemia pathway guards the genome. Annu Rev Genet, 2009. 43: p. 223-49. 18.Kim, Y., et al., Mutations of the SLX4 gene in Fanconi anemia. Nat Genet, 2011. 43(2): p. 142-6. 19.Hanada, K., et al., The structure-specific endonuclease Mus81-Eme1 promotes conversion of interstrand DNA crosslinks into double-strands breaks. Embo j, 2006. 25(20): p. 4921-32. 20.Kuraoka, I., et al., Repair of an interstrand DNA cross-link initiated by ERCC1-XPF repair/recombination nuclease. J Biol Chem, 2000. 275(34): p. 26632-6. 21.Budzowska, M., et al., Regulation of the Rev1–pol ζ complex during bypass of a DNA interstrand cross‐link. The EMBO Journal, 2015. 34(14): p. 1971-1985. 22.Haynes, B., et al., Crosstalk between translesion synthesis, Fanconi anemia network, and homologous recombination repair pathways in interstrand DNA crosslink repair and development of chemoresistance. Mutat Res Rev Mutat Res, 2015. 763: p. 258-66. 23.Crossan, G.P. and K.J. Patel, The Fanconi anaemia pathway orchestrates incisions at sites of crosslinked DNA. J Pathol, 2012. 226(2): p. 326-37. 24.Baumann, P. and S.C. West, Role of the human RAD51 protein in homologous recombination and double-stranded-break repair. Trends Biochem Sci, 1998. 23(7): p. 247-51. 25.Krejci, L., et al., Homologous recombination and its regulation. Nucleic Acids Res, 2012. 40(13): p. 5795-818. 26.Long, D.T., et al., Mechanism of RAD51-dependent DNA interstrand cross-link repair. Science, 2011. 333(6038): p. 84-7. 27.Alcón, P., et al., FANCD2-FANCI is a clamp stabilized on DNA by monoubiquitination of FANCD2 during DNA repair. Nat Struct Mol Biol, 2020. 27(3): p. 240-248. 28.Yuan, F., et al., FANCI protein binds to DNA and interacts with FANCD2 to recognize branched structures. J Biol Chem, 2009. 284(36): p. 24443-52. 29.Wang, R., et al., DNA clamp function of the monoubiquitinated Fanconi anaemia ID complex. Nature, 2020. 580(7802): p. 278-282. 30.Sijacki, T., et al., The DNA-damage kinase ATR activates the FANCD2-FANCI clamp by priming it for ubiquitination. Nat Struct Mol Biol, 2022. 29(9): p. 881-890. 31.Jones, M.J. and T.T. Huang, The Fanconi anemia pathway in replication stress and DNA crosslink repair. Cell Mol Life Sci, 2012. 69(23): p. 3963-74. 32.Tian, Y., et al., Constitutive role of the Fanconi anemia D2 gene in the replication stress response. Journal of Biological Chemistry, 2017. 292(49): p. 20184-20195. 33.Schwab, R.A., et al., The Fanconi Anemia Pathway Maintains Genome Stability by Coordinating Replication and Transcription. Mol Cell, 2015. 60(3): p. 351-61. 34.Lossaint, G., et al., FANCD2 binds MCM proteins and controls replisome function upon activation of s phase checkpoint signaling. Mol Cell, 2013. 51(5): p. 678-90. 35.Chen, Y.H., et al., ATR-mediated phosphorylation of FANCI regulates dormant origin firing in response to replication stress. Mol Cell, 2015. 58(2): p. 323-38. 36.Schlacher, K., H. Wu, and M. Jasin, A distinct replication fork protection pathway connects Fanconi anemia tumor suppressors to RAD51-BRCA1/2. Cancer Cell, 2012. 22(1): p. 106-16. 37.Sato, K., et al., FANCI-FANCD2 stabilizes the RAD51-DNA complex by binding RAD51 and protects the 5'-DNA end. Nucleic Acids Res, 2016. 44(22): p. 10758-10771. 38.Hashimoto, Y., et al., Rad51 protects nascent DNA from Mre11-dependent degradation and promotes continuous DNA synthesis. Nat Struct Mol Biol, 2010. 17(11): p. 1305-11. 39.Mason, J.M., et al., Non-enzymatic roles of human RAD51 at stalled replication forks. Nature Communications, 2019. 10(1): p. 4410. 40.Bhat, K.P. and D. Cortez, RPA and RAD51: fork reversal, fork protection, and genome stability. Nat Struct Mol Biol, 2018. 25(6): p. 446-453. 41.Liao, H., et al., Mechanisms for stalled replication fork stabilization: new targets for synthetic lethality strategies in cancer treatments. EMBO reports, 2018. 19(9): p. e46263. 42.Liu, W., et al., FANCD2 and RAD51 recombinase directly inhibit DNA2 nuclease at stalled replication forks and FANCD2 acts as a novel RAD51 mediator in strand exchange to promote genome stability. Nucleic Acids Res, 2023. 51(17): p. 9144-9165. 43.Kolinjivadi, A.M., et al., Moonlighting at replication forks - a new life for homologous recombination proteins BRCA1, BRCA2 and RAD51. FEBS Lett, 2017. 591(8): p. 1083-1100. 44.Wang, A.T., et al., A dominant mutation in human RAD51 reveals its function in DNA interstrand crosslink repair independent of homologous recombination. Molecular cell, 2015. 59(3): p. 478-490. 45.Schlacher, K., H. Wu, and M. Jasin, A distinct replication fork protection pathway connects Fanconi anemia tumor suppressors to RAD51-BRCA1/2. Cancer Cell, 2012. 22(1): p. 106-116. 46.Michl, J., et al., FANCD2 limits replication stress and genome instability in cells lacking BRCA2. Nature Structural & Molecular Biology, 2016. 23(8): p. 755-757. 47.Kolinjivadi, A.M., et al., Smarcal1-mediated fork reversal triggers Mre11-dependent degradation of nascent DNA in the absence of Brca2 and stable Rad51 nucleofilaments. Molecular cell, 2017. 67(5): p. 867-881. e7. 48.Halder, S., et al., Strand annealing and motor driven activities of SMARCAL1 and ZRANB3 are stimulated by RAD51 and the paralog complex. Nucleic Acids Res, 2022. 50(14): p. 8008-8022. 49.Reymer, A., et al., Structure of human Rad51 protein filament from molecular modeling and site-specific linear dichroism spectroscopy. Proceedings of the National Academy of Sciences, 2009. 106(32): p. 13248-13253. 50.Yu, D.S., et al., Dynamic control of Rad51 recombinase by self-association and interaction with BRCA2. Mol Cell, 2003. 12(4): p. 1029-41. 51.Shioi, T., et al., Cryo-EM structures of RAD51 assembled on nucleosomes containing a DSB site. Nature, 2024. 628(8006): p. 212-220. 52.Luo, S.-C., et al., Identification of fidelity-governing factors in human recombinases DMC1 and RAD51 from cryo-EM structures. Nature Communications, 2021. 12(1): p. 115. 53.Prasad, T.K., C.C. Yeykal, and E.C. Greene, Visualizing the Assembly of Human Rad51 Filaments on Double-stranded DNA. Journal of Molecular Biology, 2006. 363(3): p. 713-728. 54.Mateus, A., T.A. Määttä, and M.M. Savitski, Thermal proteome profiling: unbiased assessment of protein state through heat-induced stability changes. Proteome Sci, 2016. 15: p. 13. 55.Lu, C.-H., et al., Swi5–Sfr1 stimulates Rad51 recombinase filament assembly by modulating Rad51 dissociation. Proceedings of the National Academy of Sciences, 2018. 115(43): p. E10059-E10068. 56.Lan, W.-H., et al., Rad51 facilitates filament assembly of meiosis-specific Dmc1 recombinase. Proceedings of the National Academy of Sciences, 2020. 117(21): p. 11257-11264. 57.Hsu, H.-F., et al., Investigating Deinococcus radiodurans RecA protein filament formation on double-stranded DNA by a real-time single-molecule approach. Biochemistry, 2011. 50(39): p. 8270-8280. 58.Chan, K.L., et al., Replication stress induces sister-chromatid bridging at fragile site loci in mitosis. Nat Cell Biol, 2009. 11(6): p. 753-60. 59.Naim, V. and F. Rosselli, The FANC pathway and BLM collaborate during mitosis to prevent micro-nucleation and chromosome abnormalities. Nat Cell Biol, 2009. 11(6): p. 761-8. | - |
| dc.identifier.uri | http://tdr.lib.ntu.edu.tw/jspui/handle/123456789/94810 | - |
| dc.description.abstract | DNA 股間交聯（DNA interstrand crosslinks, ICL）會阻礙 DNA 鏈分離，從而抑制 DNA 複製，導致細胞損傷。修復 ICL 的主要機制是范可尼貧血（Fanconi anemia, FA）途徑，是維持基因組穩定性至關重要的途徑。在 FA 途徑中FANCI 和 FANCD2 會形成蛋白複合物（ID complex）並扮演活化途徑的重要角色，當ID complex ICL存在時會誘發泛素化同時會鎖定在受損的 DNA 上，並招募核酸內切酶，將ICL 移除，再透過同源重組 (homologous recombination, HR) 進行DNA修復。先前的研究顯示，FANCI、FANCD2和RAD51 會在停滯的複製叉處大量聚集，以防止 DNA 被外切酶降解。最近的發現進一步指出，ID complex可以穩定 RAD51-DNA核蛋白絲，並協同保護停滯的複製叉。然而，ID complex與 RAD51 之間相互作用的結構基礎以及這種相互作用如何穩定停滯的複製叉仍然未知。我們的目標是了解 ID-RAD51 複合物的結構和功能以及ID complex如何穩定 RAD51蛋白絲。利用ID complex與RAD51在脊椎動物間的高度同源性，我們首先通過下拉實驗(pull down assay)確認了Gallus gallus ID complex（GgID complex）和 Homo sapiens RAD51（HsRAD51）在生理溫度下能較好的相互作用。並藉由電泳遷移率實驗（Electrophoretic Mobility Shift Assays, EMSA），確認了 HsRAD51 和 GgID 複合物可同時存在於 5'-flap DNA 上。我們還透過單分子 TPM 實驗 (single-molecule TPM experiment)，證明了 GgID complex可藉由促進 HsRAD51核蛋白絲形成和降低核蛋白絲解離的速率，來穩定 HsRAD51核蛋白絲。最後為了進一步了解 ID complex與 RAD51 之間的相互作用，我們重組了 ID-RAD51 complex，並以負染電鏡(negative staining EM) 和冷凍電鏡（cryo-electron microscopy, cryo-EM）進行分析。結果顯示GgID complex存在時會延長HsRAD51 形成的蛋白絲比，未來也將繼續優化組成複合物的條件，以提高 ID-RAD51 complex在cryo-EM分析中的顆粒的品質，並透果結構解析，更深入ID complex和RAD51交互作用機制。 | zh_TW |
| dc.description.abstract | DNA interstrand crosslinks (ICLs) are cytotoxic lesions that block DNA strand separation, thereby inhibiting DNA replication and transcription. The primary mechanism for repairing ICLs is the Fanconi anemia (FA) pathway, which is crucial for maintaining genome stability. The protein complex formed by FANCI and FANCD2 (ID complex) plays an essential role in the FA pathway; when monoubiquitinated, the ID complex locks onto damaged DNA and recruits endonucleases, to introduce DNA double-strand breaks (DSBs). After ICL removal, the DNA lesion is repaired by homologous recombination. Recent findings have revealed that the ID complex can stabilize the RAD51-DNA filament and cooperatively protect the stalled replication fork. However, the structural basis underlying the interaction between the ID complex and RAD51 and how this interaction stabilizes stalled replication forks remains unknown. Our aim is to understand the structure and function of the ID-RAD51 complex and how the ID complex stabilizes the RAD51 filament. Since the ID complex and RAD51 are well conserved in vertebrates, we first confirmed that the Gallus gallus ID complex (GgID complex) and Homo sapiens RAD51 (HsRAD51) interact more effectively at physiological temperatures using a pull-down assay. Electrophoretic mobility shift assay (EMSA) was conducted to confirm the simultaneous presence of HsRAD51 and the GgID complex on a 5’-flap DNA. We also employed single-molecule TPM experiments to demonstrate that the GgID complex stimulates HsRAD51 filament formation and reduces filament disassembly, thereby stabilizing the HsRAD51 filament. To gain further insight into the interaction between the ID complex and RAD51, we reconstituted the ID-RAD51 complex for structural characterization using negative staining EM and cryo-electron microscopy (cryo-EM). Preliminary analysis with negative staining EM revealed that HsRAD51 forms longer filaments in the presence of the GgID complex compared to when it is alone. We will continue to optimize incubation conditions to improve the homogeneity of the ID-RAD51 complex to facilitate structural analysis using in cryo-EM analysis. | en |
| dc.description.provenance | Submitted by admin ntu (admin@lib.ntu.edu.tw) on 2024-08-19T16:45:06Z No. of bitstreams: 0 | en |
| dc.description.provenance | Made available in DSpace on 2024-08-19T16:45:06Z (GMT). No. of bitstreams: 0 | en |
| dc.description.tableofcontents | 口試委員審定書 i
誌謝 ii 摘要 iii Abstract v Abbreviations vii Contents viii List of Figures x List of Tables xii 1. Introduction 1 1.1 The Fanconi anemia pathway in DNA interstrand crosslink repair 2 1.2 The function of FA protein in replication stress 4 1.3 Role of RAD51 at stalled replication forks 5 1.4 Specific aim 6 2.Material and Methods 8 2.1 Plasmids and expression 9 2.2 Protein purification 10 2.3 DNA substrates 14 2.4 Protein assay 14 2.5 Negative stain EM and Cryo-EM sample preparation and data collection 18 2.6 Single-Molecule TPM experiments 19 3.Results 21 3.1 Purification ID complex and RAD51 22 3.2 Evaluate the interaction between the ID complex and RAD51 24 3.3 RAD51-induced stabilization of ID complex 25 3.4 Identification of the ID complex-RAD51 interacting with 5’ flap DNA 25 3.5 ID complex stimulates RAD51 nucleoprotein filament assembly 27 3.6 ID complex prevent RAD51 nucleoprotein filament disassembly 28 3.7 Structural analysis of ID complex-RAD51 and ID complex-RAD51 with DNA 29 4.Discussion 31 5. Figures 35 6. Tables 60 7. Supplemental Figure 69 8. References 83 | - |
| dc.language.iso | en | - |
| dc.title | 探討 ID complex和RAD51之間的交互作用與對RAD51蛋白絲的影響 | zh_TW |
| dc.title | Investigation of the interaction between ID complex and RAD51 and its impact on the formation of RAD51 filament | en |
| dc.type | Thesis | - |
| dc.date.schoolyear | 112-2 | - |
| dc.description.degree | 碩士 | - |
| dc.contributor.oralexamcommittee | 林敬哲;李弘文;冀宏源 | zh_TW |
| dc.contributor.oralexamcommittee | Jing-Jer Lin;Hung-Wen Li;Hung-Yuan Chi | en |
| dc.subject.keyword | 范可尼貧血,FANCI-FANCD2複合物,同源重組蛋白RAD51,冷凍電子顯微鏡, | zh_TW |
| dc.subject.keyword | Fanconi anemia (FA),FANCI-FANCD2 complex,RAD51,cryo-EM, | en |
| dc.relation.page | 88 | - |
| dc.identifier.doi | 10.6342/NTU202401951 | - |
| dc.rights.note | 同意授權(全球公開) | - |
| dc.date.accepted | 2024-07-30 | - |
| dc.contributor.author-college | 醫學院 | - |
| dc.contributor.author-dept | 生物化學暨分子生物學研究所 | - |
| 顯示於系所單位： | 生物化學暨分子生物學科研究所 | |
文件中的檔案：
| 檔案 | 大小 | 格式 | |
|---|---|---|---|
| ntu-112-2.pdf | 3.88 MB | Adobe PDF | 檢視/開啟 |
系統中的文件，除了特別指名其著作權條款之外，均受到著作權保護，並且保留所有的權利。