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研究生:艾蔓達
研究生(外文):Imelda Margaretha Aritonang
論文名稱:藉表現質體體外轉錄強度探討紫外線B與熱休克對斑馬魚胚胎DNA切割修補作用之效應
論文名稱(外文):Differential Effects of Heat Stress and UVB Radiation on Nucleotide Excision Repair in Zebrafish (Danio rerio) Embryos
指導教授:許濤許濤引用關係
指導教授(外文):Todd Hsu
口試委員:許濤招名威易玲輝
口試委員(外文):Todd HsuMing-Wei ChaoLing-Huei Yih
口試日期:2019-01-22
學位類別:碩士
校院名稱:國立臺灣海洋大學
系所名稱:生命科學暨生物科技學系
學門:生命科學學門
學類:生物科技學類
論文種類:學術論文
論文出版年:2019
畢業學年度:107
語文別:英文
論文頁數:33
中文關鍵詞:UVB核酸切割修復作用熱休克體外轉錄
外文關鍵詞:UVBnucleotide excision repairheat stressin vitro transcription
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魚類在早期胚胎階段對環境壓力比其他發育階段較為敏感,因此需要DNA修復系統來維持基因完整性。在快速發育的生物中,若基因修復效率受到干擾,則會增加致命突變的發生機率。本論文主要藉以DNA轉錄為基礎的修復實驗及電泳遷移實驗(EMSA)觀察暴露於+8.5 ᵒC的水中及非致死劑量UV-B下,對斑馬魚胚胎的核酸切割修復(NER)反應之影響。在轉錄修復實驗中,使用UV-C照射表現質體為修復標的,隨著UV-C劑量的上升,愈抑制其轉錄成cDNA的效率。受精後10小時(10 hpf)斑馬魚胚胎的粗蛋白質萃取液與受UV-C照射受損的質體同時培養,能夠提升質體轉錄cDNA的效率,另外,經過急性熱休克(37ᵒC 30分鐘)的10hpf斑馬魚胚胎更能提升轉錄效率,表示熱休克可以提升NER的功能。雖然使用200 J/m2 的UV-B照射胚胎能微量提升NER活性,但是在400 和 800 J/m2 卻有抑制效果。在電泳遷移實驗中,使用6-4光產物(6-4 PP)或環丁烷嘧啶雙體(CPD)的寡核苷酸做為辨識的目標,UV-B照射的胚胎會抑制NER辨識的步驟。因此,UV-B會干擾損傷識別階段來抑制NER的修復效率。
Fish at early embryonic stages are more sensitive to environmental stressors than fish at other developmental stages. DNA repair systems are critical to the maintenance of genetic integrity and lethal mutations may arise in fast-growing organisms if the efficiencies of DNA repair systems are disturbed. The objective of this study was to explore the responses of nucleotide excision repair (NER) in zebrafish (Danio rerio) embryos to a +8.5ᵒC water temperature elevation and sublethal UV-B exposure using a transcription-based DNA repair assay and gel shift assay. Irradiation of an expression plasmid with increasing UV-C dose caused a dose-dependent inhibition of cDNA transcription. Incubation of the plasmid with 10 hpf zebrafish extracts promoted a significant up-regulation of UV-C suppressed cDNA transcription and heat-stressed (37ᵒC for 30 min) 10 hpf zebrafish extracts displayed a better NER capacity than non-stressed zebrafish extracts. UVB at 200 J/m2 induced a slight increase of NER activity, but NER activities were inhibited in embryos exposed to UVB at 400 and 800 J/m2. Gel shift assay using an oligonucleotide carrying a UVC-induced (6-4) photoproducts or cyclobutane pyrimidine dimer (CPD) as the binding target demonstrated that UVB irradiation on 10 hpf embryos generally imposed inhibitory effects on the damage recognition step of NER. Hence, UVB was believed to inhibit NER by disturbing its damage recognition stage.
ACKNOWLEDGEMENTS i
摘要 ii
ABSTRACT iv
TABLE OF CONTENTS iv
FIGURE INDEX vii
TABLE INDEX vii
CHAPTER I-INTRODUCTION 1
1-1 Thermal Pollution and UVB Exposure Effect 1
1-2 Heat Shock Protein (HSPs) 2
1-3 DNA Damage and Repair 3
1-4 Nucleotide Excision Repair 4
1-5 Research objectives................. 5
CHAPTER II-MATERIAL AND METHOD 6
2-1 Materials................................ 6
2-2 Experimental Equipment 8
2-3 Experimental Method 9
2-3.1 Collection of zebrafish embryos and developing 9
2-3.2 Zebrafish embryo heat treatment 9
2-3.3 Zebrafish embryo UV-B irradiation treatment 10
2-3.4 Preparation of the extracts of zebrafish embryos 10
2-3.5 Protein Quantification use BCA Protein Assay 10
2-3.6 DNA Plasmid Extraction 11
2-3.7 Quantification and charaterization of plasmid 11
2-3.8 Linearization of plasmid 11
2-3.9 DNA Electrophoresis Gel 12
2-3.10 UVC-irradiation on plasmid 12
2-3.11 In vitro transcription based DNA repair assay 12
2-3.12 RNA Electrophoresis Gel of product in vitro Transcription 13
2-3.13 Preparation of 6,4 PP probe 14
2-3.14 Electrophoretic mobility shift assay (EMSA) 14
CHAPTER III RESULTS 16
3.1 UVC-dose dependent of plasmid DNA detected by in vitro transcription assay 16
3.2 Heat stress stimulates NER activities in 10 hpf zebrafish embryo 16
3.3 Low doses of UVB-irradiation in zebrafish embryo induced NER activities 17
3.4 Differential effect of UVB-damaged binding activities on 17
6,4 PP lesion at different UVB doses 17
3.5 Differential effect of UVB-damaged binding activities on 18
6,4 PP lesion at different UVB doses 18
CHAPTER IV DISCUSSION 19
REFERENCES........................ 22
FIGURES SECTION 25
APPENDIX......................... 32
Amin, J. A, Ananthan, J & Voellmy, R. (1988). Key features of heat shock regulatory elements. Molecular and cellular biology, 8(9), 3761-3769.
Altieri, F., Grillo, C., Maceroni, M., & Chichiarelli, S. (2008). DNA damage and repair: from molecular mechanisms to health implications. Antioxidants & redox signaling, 10(5), 891-938.
Balajee, A. S., May, A., & Bohr, V. A. (1999). DNA repair of pyrimidine dimers and 6-4 photoproducts in the ribosomal DNA. Nucleic acids research, 27(12), 2511-2520.
Batty, D. P., & Wood, R. D. (2000). Damage recognition in nucleotide excision repair of DNA. Gene, 241(2), 193-204.
Budden, T., & Bowden, N. A. (2013). The role of altered nucleotide excision repair and UVB-induced DNA damage in melanomagenesis. International Journal of Molecular Sciences, 14(1), 1132-1151.
Calderwood, S. K., Wang, Y., Xie, X., Khaleque, M. A., Chou, S. D., Murshid, A., ... & Zhang, Y. (2010). Signal transduction pathways leading to heat shock transcription. Signal transduction insights, 2, STI-S3994.
Cai, Q., Fu, L., Wang, Z., Gan, N., Dai, X., & Wang, Y. (2014). α-N-methylation of damaged DNA-binding protein 2 (DDB2) and its function in nucleotide excision repair. Journal of Biological Chemistry, jbc-M114.
Chang, Y., Lee, W. Y., Lin, Y. J., & Hsu, T. (2017). Mercury (II) impairs nucleotide excision repair (NER) in zebrafish (Danio rerio) embryos by targeting primarily at the stage of DNA incision. Aquatic Toxicology, 192, 97-104.
Cleaver, J. E. (2005). Cancer in xeroderma pigmentosum and related disorders of DNA repair. Nature Reviews Cancer, 5(7), 564.
Cunniff, N. F., & Morgan, W. D. (1993). Analysis of heat shock element recognition by saturation mutagenesis of the human HSP70. 1 gene promoter. Journal of Biological Chemistry, 268(11), 8317-8324.
de Laat, W. L., Jaspers, N. G., & Hoeijmakers, J. H. (1999). Molecular mechanism of nucleotide excision repair. Genes & development, 13(7), 768-785.
Desgarnier, M. C. D., Fournier, F., Droit, A., & Rochette, P. J. (2017). Influence of a pre-stimulation with chronic low-dose UVB on stress response mechanisms in human skin fibroblasts. PloS one, 12(3), e0173740.
Dreze, M., Calkins, A. S., Galicza, J., Echelman, D. J., Schnorenberg, M. R., Fell, G. L., ... & Lazaro, J. B. (2014). Monitoring repair of UV-induced 6-4-photoproducts with a purified DDB2 protein complex. PloS one, 9(1), e85896.
Fernandes, M., Xiao, H., & Lis, J. T. (1994). Fine structure analyses of the Drosophila and Saccharomyces heat shock factor-heat shock element interactions. Nucleic acids research, 22(2), 167-173.
Ghosh, R., Tummala, R., & Mitchell, D. L. (2003). Ultraviolet radiation‐induced DNA damage in promoter elements inhibits gene expression. FEBS letters, 554(3), 427-432.
Guo CX, Tang TS, Friedberg EC (2010). SnapShot : Nucleotide Excision Repair. Cell. 140(5):754-U169.
Huang, J. C., Svoboda, D. L., Reardon, J. T., & Sancar, A. (1992). Human nucleotide excision nuclease removes thymine dimers from DNA by incising the 22nd phosphodiester bond 5'and the 6th phosphodiester bond 3'to the photodimer. Proceedings of the National Academy of Sciences, 89(8), 3664-3668.
Iyama, T., & Wilson, D. M. (2013). DNA repair mechanisms in dividing and non-dividing cells. DNA repair, 12(8), 620-636.
Jezierska, B., Ługowska, K., & Witeska, M. (2009). The effects of heavy metals on embryonic development of fish (a review). Fish physiology and biochemistry, 35(4), 625-640.
Jonak, C., Klosner, G., & Trautinger, F. (2009). Significance of heat shock proteins in the skin upon UV exposure. Front Biosci, 14, 4758-4768.
Kemp, M. G., Reardon, J. T., Lindsey-Boltz, L. A., & Sancar, A. (2012). Mechanism of release and fate of excised oligonucleotides during nucleotide excision repair. Journal of Biological Chemistry, 287(27), 22889-22899.
Kimmel, C. B., Ballard, W. W., Kimmel, S. R., Ullmann, B., & Schilling, T. F. (1995). Stages of embryonic development of the zebrafish. Developmental dynamics, 203(3), 253-310.
Khobta, A., & Epe, B. (2012). Interactions between DNA damage, repair, and transcription. Mutation Research/Fundamental and Molecular Mechanisms of Mutagenesis, 736(1-2), 5-14.
Ling, L. B., Chang, Y., Liu, C. W., Lai, P. L., & Hsu, T. (2017). Oxidative stress intensity-related effects of cadmium (Cd) and paraquat (PQ) on UV-damaged-DNA binding and excision repair activities in zebrafish (Danio rerio) embryos. Chemosphere, 167, 10-18.
Li, J., Wang, Q. E., Zhu, Q., El-Mahdy, M. A., Wani, G., Prætorius-Ibba, M., & Wani, A. A. (2006). DNA damage binding protein component DDB1 participates in nucleotide excision repair through DDB2 DNA-binding and cullin 4A ubiquitin ligase activity. Cancer research, 66(17), 8590-8597.
Lo, H. L., Nakajima, S., Ma, L., Walter, B., Yasui, A., Ethell, D. W., & Owen, L. B. (2005). Differential biologic effects of CPD and 6-4PP UV-induced DNA damage on the induction of apoptosis and cell-cycle arrest. BMC cancer, 5(1), 135.
Marteijn, J. A., Lans, H., Vermeulen, W., & Hoeijmakers, J. H. (2014). Understanding nucleotide excision repair and its roles in cancer and ageing. Nature reviews Molecular cell biology, 15(7), 465.
Mellon, I., Spivak, G., & Hanawalt, P. C. (1987). Selective removal of transcription-blocking DNA damage from the transcribed strand of the mammalian DHFR gene. Cell, 51(2), 241-249.
Moser, J., Volker, M., Kool, H., Alekseev, S., Vrieling, H., Yasui, A., ... & Mullenders, L. H. (2005). The UV-damaged DNA binding protein mediates efficient targeting of the nucleotide excision repair complex to UV-induced photo lesions. DNA repair, 4(5), 571-582.
Morimoto, R. I., & Tissières, A. (1994). The biology of heat shock proteins and molecular chaperones (No. 577.112 BIO).
Marteijn, J. A., Lans, H., Vermeulen, W., & Hoeijmakers, J. H. (2014). Understanding nucleotide excision repair and its roles in cancer and ageing. Nature reviews Molecular cell biology, 15(7), 465.
Nakajima, S., Lan, L., Kanno, S. I., Takao, M., Yamamoto, K., Eker, A. P., & Yasui, A. (2004). UV light-induced DNA damage and tolerance for the survival of nucleotide excision repair-deficient human cells. Journal of Biological Chemistry, 279(45), 46674-46677.
Pasheva, E. A., Pashev, I. G., & Favre, A. (1998). Preferential binding of high mobility group 1 protein to UV-damaged DNA Role of the COOH-terminal domain. Journal of Biological Chemistry, 273(38), 24730-24736.
Rastogi, R. P., Kumar, A., Tyagi, M. B., & Sinha, R. P. (2010). Molecular mechanisms of ultraviolet radiation-induced DNA damage and repair. Journal of nucleic acids, 2010.
Rosen, M. A., Bulucea, C. A., Mastorakis, N. E., Bulucea, C. A., Jeles, A. C., & Brindusa, C. C. (2015). Evaluating the thermal pollution caused by wastewaters discharged from a chain of coal-fired power plants along a river. Sustainability, 7(5), 5920-5943.
Rylander, M. N., Feng, Y., Bass, J. O. N., & Diller, K. R. (2006). Thermally induced injury and heat‐shock protein expression in cells and tissues. Annals of the New York Academy of Sciences, 1066(1), 222-242.
Saxowsky, T. T., & Doetsch, P. W. (2006). RNA polymerase encounters with DNA damage: transcription-coupled repair or transcriptional mutagenesis?. Chemical reviews, 106(2), 474-488.
Scrima, A., Koníčková, R., Czyzewski, B. K., Kawasaki, Y., Jeffrey, P. D., Groisman, R., ... & Thomä, N. H. (2008). Structural basis of UV DNA-damage recognition by the DDB1–DDB2 complex. Cell, 135(7), 1213-1223.
Shen, Y. C., Hsu, T., Ling, L. B., You, W. C., & Liu, C. W. (2017). Identification of low-molecular-weight vitellogenin 1 (Vg1)-like proteins as nucleotide excision repair (NER) factors in developing zebrafish (Danio rerio) using a transcription-based DNA repair assay. Fish physiology and biochemistry, 43(2), 663-676.
Stephanou, A., & Latchman, D. S. (2011). Transcriptional modulation of heat-shock protein gene expression. Biochemistry research international, 2011
Tang, J. Y., Hwang, B. J., Ford, J. M., Hanawalt, P. C., & Chu, G. (2000). Xeroderma pigmentosum p48 gene enhances global genomic repair and suppresses UV-induced mutagenesis. Molecular cell, 5(4), 737-744.
Xiao, H., & Lis, J. T. (1988). Germline transformation used to define key features of heat-shock response elements. Science, 239(4844), 1139-1142.
van Gool, A. J., van der Horst, G. T., Citterio, E., & Hoeijmakers, J. H. (1997). Cockayne syndrome: defective repair of transcription?. The EMBO journal, 16(14), 4155-4162.
Weindling, E., & Bar-Nun, S. (2015). Sir2 links the unfolded protein response and the heat shock response in a stress response network. Biochemical and biophysical research communications, 457(3), 473-478.
Wittschieben, B. Ø., Iwai, S., & Wood, R. D. (2005). DDB1-DDB2 (xeroderma pigmentosum group E) protein complex recognizes a cyclobutane pyrimidine dimer, mismatches, apurinic/apyrimidinic sites, and compound lesions in DNA. Journal of Biological Chemistry, 280(48), 39982-39989.
Wu, C. (1995). Heat shock transcription factors: structure and regulation. Annual review of cell and developmental biology, 11(1), 441-469.
Åkerfelt, M., Morimoto, R. I., & Sistonen, L. (2010). Heat shock factors: integrators of cell stress, development and lifespan. Nature reviews Molecular cell biology, 11(8), 545.
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