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研究生：	陳妍儒 Chen, Yen-Ju
論文名稱：	DNA損傷耐受機制中PARP1自我修飾的功用 The function of PARP1 self-PARylation in the DNA damage tolerance pathway.
指導教授：	廖泓鈞 Liaw, Hung-Jiun
學位類別：	碩士 Master
系所名稱：	生物科學與科技學院 - 生命科學系 Department of Life Sciences
論文出版年：	2017
畢業學年度：	105
語文別：	英文
論文頁數：	40
中文關鍵詞：	PARP1 、DNA損傷耐受機制、同源重組修復
外文關鍵詞：	PARP1, DNA damage tolerance, homologous recombination
相關次數：	點閱：202 下載：20
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外在環境或是體內生理代謝都可能產生對DNA的傷害，而DNA損傷耐受機制DNA damage tolerance (DDT)，又稱為複製後修飾post-replication repair，是細胞發展出的一套機制能確保DNA在遇到損傷或障礙時不間斷的完成完整的複製作用，以降低複製停滯或長時間修復DNA所可能產生的風險。這種從酵母菌到人類都有高度保留的機制可以分為兩條路徑：一是直接越過DNA障礙的translesion synthesis (TLS) 另一個則是利用模板交換的template switching (TS)。在先前的研究中已經發現到同源重組修復機制homologous recombination (HR)會參與在DDT中，但兩者之間詳細的分子機制還有許多的不解之處。而我們實驗室之前的研究中提到，調控TS的其中一個蛋白，HLTF會與PARP1結合並作poly(ADP-ribosyl)ation修飾。在本篇研究中，我們發現到PARP1會與HR相關蛋白—BARD1結合，而且methyl methanesulfonate (MMS) 或是細胞缺乏poly(ADP-ribose) glycohydrolase (PARG)蛋白時，能大幅提升地他們的結合力；但當PARP1上四個自我poly(ADP-ribosyl)ation修飾位突變時，此結合力會明顯減弱。這些結果都說明著PARP1能藉由其自我poly(ADP-ribosyl)ation修飾以調控與BARD1的交互作用。另外若DNA複製時碰到阻礙，細胞缺乏PARP1、HLTF、UBC13和BARD1蛋白會使停滯複製叉的數量大幅提升且顯著地降低DNA複製的速率，並且缺乏這些蛋白會使鼻咽癌細胞對於化療藥物的敏感性大幅地上升。綜合以上實驗結果說明，PARP1/HLTF是藉由PARP1自我修飾去招募BARD1，使受損股插入正常的姊妹染色體產生模板互換的結構，進而促使homologous recombination進行。

To circumvent DNA lesions impeded for the progression of DNA replication, cells have evolved DNA damage tolerance (DDT), also known as post-replication repair to bypass DNA lesions during DNA replication. This mechanism is highly conserved from yeast to human cells and is mediated by two distinct pathways: translesion synthesis (TLS) and template switching (TS). Accumulated evidence revealed that homologous recombination (HR) is participated in DDT but it remains unclear how HR factors are recruited to DNA damage sites to contribute the operation of TS. Our previous studies have demonstrated that HLTF is involved in the TS pathway and interacts with PARP1. In this study, we further extend our understanding of the TS mechanism by showing that PARP1 interacts with BARD1 and this interaction was specifically enhanced by the treatment with methyl methanesulfonate (MMS). Mutations in the poly(ADP-ribose) modification sites of PARP1 decrease the interaction of PARP1 with BARD1. Additionally, the depletion of PARG, a PAR glycohydrolase, enhances the interaction. These results indicate that the interaction of PARP1 with BARD1 is mediated through the PAR modification. Moreover, the depletion of PARP1, HLTF, UBC13 and BARD1 significantly increased the number of stalled replication forks, decreases the progression of replication forks in response to DNA lesions, and sensitizes cells to DNA damaging agents. Taken together, our results suggest that the HLTF/PARP1 complex recruits BARD1, possibly through the PAR modification of PARP1. The recruitment of BARD1 resolves the template-switching structure caused by the invasion of nascent DNA strand into undamaged sister strand through homologous recombination.

Abstract	I
中文摘要	II
致謝	III
Contents	IV
List of figures	VI
Abbreviations	VII
1. Introduction	1
1.1 DNA damage response (DDR)	1
1.2 DNA damage tolerance (DDT)	2
1.3 The mechanism of poly(ADP-ribosyl)ation response to DNA damage.	3
1.4 The aim of this study	4
2. Materials and methods	5
2.1 Cell lines culture and transfection	5
2.2 Plasmid construction	5
2.3 Co-immunoprecipitation (Co-IP)	5
2.4 Western blotting	6
2.5 Lentivirial gene knockdown	6
2.6 Colony formation assay	7
2.7 Cytotoxicity assay	7
2.8 DNA fiber	7
2.9 Table of primers	8
2.10 Table of shRNAs	10
2.11 Table of antibodies	11
3. Results	12
3.1 The ubiquitin E3 ligase HLTF interacts with PARP1	12
3.2 The treatment with ethidium bromide enhanced the interaction between PARP1 and BARD1	12
3.3 The treatment with methyl methanesulfonate (MMS) enhanced the interaction between PARP1 and BARD1	13
3.4 The DBD domain and autoPAR domain of PARP1 interact with BARD1.	14
3.5 Reducing the levels of PARP1 self-PARylation decreases the interaction with BARD1.	14
3.6 The depletion of BARD1 and UBC13 increase the number of stalled replication forks in response to cisplatin, UV, and MMS-caused DNA lesions.	15
3.7 The depletion of BARD1 and UBC13 decreases the progression of replication forks progression in response to UV irradiation.	16
3.8 The depletion of HLTF, UBC13, PARP1, and BARD1 sensitizes HONE6 cells to MMS and cisplatin treatment.	17
4. Discussion	18
5. Reference	21

Figure 1. PARP1 can interact with BARD1.	25
Figure 2. The interaction of PARP1 with HLTF and BARD1 is enhanced by treatment with EtBr.	26
Figure 3. The interaction between PARP1 and BARD1 is enhanced by the MMS treatment.	27
Figure 4. The interaction between BARD1 and PARP1 is dependent on the DBD and autoPAR domains of PARP1.	28
Figure 5. Mutations in PARylation sites of PARP1 reduces the interaction of PARP1 with BARD1.	29
Figure 6. The depletion of PARG enhances the interaction of PARP1 with BARD1.	30
Figure 7. The BARD1 - and UBC13 - deficient HONE6 cells show elevated numbers of stalled forks in the presence of cisplatin treatment.	32
Figure 8. The depletion of BARD1 and UBC13 increases the number of stalled forks and decreases the progression of DNA replication in the presence of UV treatment.	34
Figure 9. The depletion of BARD1, PARP1 and UBC13 increases the number of stalled forks and decreases the progression of DNA replication in the presence of MMS treatment.	36
Figure 10. HLTF-, PARP1-, BARD1- and UBC13- deficient HONE6 cells are sensitive to MMS.	38
Figure 11. HLTF-, PARP1- and BARD1-deficient HONE6 cells are sensitive to cisplatin.	40


                                    

Wilson, D.M., 3rd, V.A. Bohr, and P.J. McKinnon. DNA damage, DNA repair, ageing and age-related disease. Mech Ageing Dev (2008); 129(7-8): p. 349-52.
2. Martin, G.M. Modalities of gene action predicted by the classical evolutionary biological theory of aging. Ann N Y Acad Sci (2007); 1100: p. 14-20.
3. Hoeijmakers, J.H. DNA damage, aging, and cancer. N Engl J Med (2009); 361(15): p. 1475-85.
4. Ciccia, A. and S.J. Elledge. The DNA damage response: making it safe to play with knives. Mol Cell (2010); 40(2): p. 179-204.
5. Lee, J.H. and T.T. Paull. ATM activation by DNA double-strand breaks through the Mre11-Rad50-Nbs1 complex. Science (2005); 308(5721): p. 551-4.
6. Bakkenist, C.J. and M.B. Kastan. DNA damage activates ATM through intermolecular autophosphorylation and dimer dissociation. Nature (2003); 421(6922): p. 499-506.
7. Zou, L. and S.J. Elledge. Sensing DNA damage through ATRIP recognition of RPA-ssDNA complexes. Science (2003); 300(5625): p. 1542-8.
8. Byun, T.S., et al. Functional uncoupling of MCM helicase and DNA polymerase activities activates the ATR-dependent checkpoint. Genes Dev (2005); 19(9): p. 1040-52.
9. Cimprich, K.A. and D. Cortez. ATR: an essential regulator of genome integrity. Nat Rev Mol Cell Biol (2008); 9(8): p. 616-27.
10. Majka, J., A. Niedziela-Majka, and P.M. Burgers. The checkpoint clamp activates Mec1 kinase during initiation of the DNA damage checkpoint. Mol Cell (2006); 24(6): p. 891-901.
11. Schreiber, V., et al. Poly(ADP-ribose): novel functions for an old molecule. Nat Rev Mol Cell Biol (2006); 7(7): p. 517-28.
12. Harper, J.W. and S.J. Elledge. The DNA damage response: ten years after. Mol Cell (2007); 28(5): p. 739-45.
13. Misteli, T. and E. Soutoglou. The emerging role of nuclear architecture in DNA repair and genome maintenance. Nat Rev Mol Cell Biol (2009); 10(4): p. 243-54.
14. Bergink, S. and S. Jentsch. Principles of ubiquitin and SUMO modifications in DNA repair. Nature (2009); 458(7237): p. 461-7.
15. Friedberg, E.C. Suffering in silence: the tolerance of DNA damage. Nat Rev Mol Cell Biol (2005); 6(12): p. 943-53.
16. Howardfl.P and R.P. Boyce. DNA Repair and Genetic Recombination - Studies on Mutants of Escherichia Coli Defective in These Processes. Radiation Research (1966); S: p. 156-&.
17. Rupp, W.D. and P. Howard-Flanders. Discontinuities in the DNA synthesized in an excision-defective strain of Escherichia coli following ultraviolet irradiation. J Mol Biol (1968); 31(2): p. 291-304.
18. Ouyang, K.J., L.L. Woo, and N.A. Ellis. Homologous recombination and maintenance of genome integrity: Cancer and aging through the prism of human RecQ helicases. Mechanisms of Ageing and Development (2008); 129(7-8): p. 425-440.
19. Huang, D.Q., B.D. Piening, and A.G. Paulovich. The Preference for Error-Free or Error-Prone Postreplication Repair in Saccharomyces cerevisiae Exposed to Low-Dose Methyl Methanesulfonate Is Cell Cycle Dependent. Molecular and Cellular Biology (2013); 33(8): p. 1515-1527.
20. Hishida, T., et al. RAD6-RAD18-RAD5-pathway-dependent tolerance to chronic low-dose ultraviolet light. Nature (2009); 457(7229): p. 612-U124.
21. Sale, J.E. Translesion DNA synthesis and mutagenesis in eukaryotes. Cold Spring Harb Perspect Biol (2013); 5(3): p. a012708.
22. Sale, J.E., A.R. Lehmann, and R. Woodgate. Y-family DNA polymerases and their role in tolerance of cellular DNA damage. Nat Rev Mol Cell Biol (2012); 13(3): p. 141-52.
23. Motegi, A., et al. Polyubiquitination of proliferating cell nuclear antigen by HLTF and SHPRH prevents genomic instability from stalled replication forks. Proc Natl Acad Sci U S A (2008); 105(34): p. 12411-6.
24. Blastyak, A., et al. Yeast Rad5 protein required for postreplication repair has a DNA helicase activity specific for replication fork regression. Mol Cell (2007); 28(1): p. 167-75.
25. Burkovics, P., et al. Strand invasion by HLTF as a mechanism for template switch in fork rescue. Nucleic Acids Res (2014); 42(3): p. 1711-20.
26. Luo, X. and W.L. Kraus. On PAR with PARP: cellular stress signaling through poly(ADP-ribose) and PARP-1. Genes Dev (2012); 26(5): p. 417-32.
27. Hassa, P.O., et al. Nuclear ADP-ribosylation reactions in mammalian cells: where are we today and where are we going? Microbiol Mol Biol Rev (2006); 70(3): p. 789-829.
28. Sakura, H., et al. Natural occurence of a biopolymer, poly (adenosine diphosphate ribose). Nucleic Acids Res (1977); 4(8): p. 2903-15.
29. Min, W. and Z.Q. Wang. Poly (ADP-ribose) glycohydrolase (PARG) and its therapeutic potential. Front Biosci (Landmark Ed) (2009); 14: p. 1619-26.
30. Li, M., et al. The FHA and BRCT domains recognize ADP-ribosylation during DNA damage response. Genes Dev (2013); 27(16): p. 1752-68.
31. Li, M. and X.C. Yu. Function of BRCA1 in the DNA Damage Response Is Mediated by ADP-Ribosylation. Cancer Cell (2013); 23(5): p. 693-704.
32. Robu, M., et al. Role of poly(ADP-ribose) polymerase-1 in the removal of UV-induced DNA lesions by nucleotide excision repair. Proc Natl Acad Sci U S A (2013); 110(5): p. 1658-63.
33. Pines, A., et al. PARP1 promotes nucleotide excision repair through DDB2 stabilization and recruitment of ALC1. J Cell Biol (2012); 199(2): p. 235-49.
34. Kim, M.Y., T. Zhang, and W.L. Kraus. Poly(ADP-ribosyl)ation by PARP-1: 'PAR-laying' NAD+ into a nuclear signal. Genes Dev (2005); 19(17): p. 1951-67.
35. Ali, A.A., et al. The zinc-finger domains of PARP1 cooperate to recognize DNA strand breaks. Nat Struct Mol Biol (2012); 19(7): p. 685-92.
36. Jackson, S.P. and J. Bartek. The DNA-damage response in human biology and disease. Nature (2009); 461(7267): p. 1071-8.
37. Li, M. and X. Yu. Function of BRCA1 in the DNA damage response is mediated by ADP-ribosylation. Cancer Cell (2013); 23(5): p. 693-704.
38. Westermark, U.K., et al. BARD1 participates with BRCA1 in homology-directed repair of chromosome breaks. Mol Cell Biol (2003); 23(21): p. 7926-36.
39. Su, W.P., et al. Chronic treatment with cisplatin induces replication-dependent sister chromatid recombination to confer cisplatin-resistant phenotype in nasopharyngeal carcinoma. Oncotarget (2014); 5(15): p. 6323-37.
40. Shiu, J.L. The HLTF-PARP1 interaction mediates homologous recombination to bypass DNA damage. Thesis of Master Degree (2015).
41. Hu, Y., et al. PARP1-driven poly-ADP-ribosylation regulates BRCA1 function in homologous recombination-mediated DNA repair. Cancer Discov (2014); 4(12): p. 1430-47.
42. Gagne, J.P., et al. Quantitative site-specific ADP-ribosylation profiling of DNA-dependent PARPs. DNA Repair (Amst) (2015); 30: p. 68-79.

校內：2022-08-01公開
校外：2022-08-01公開

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