{"id":{"repo_id":"ajou","oai_identifier":"oai:repository.ajou.ac.kr:201003/13016"},"canonical_url":"https://search.dev.ndltd.org/etd/ajou/oai:repository.ajou.ac.kr:201003/13016","repository":{"repo_id":"ajou","name":"Ajou University","base_url":"http://repository.ajou.ac.kr/oai/request"},"display":{"title":"Functional characterization of poly(ADP-ribose) dependent ubiquitin E3 ligase in the DNA damage response","abstract":"Ⅰ. INTRODUCTION 1 A. DNA damage response 1 B. DNA damage repair 3 1. Homologous recombination (HR) 4 2. Nonhomologous end joining (NHEJ) 5 C. Ubiquitylation mediated DNA damage response signaling 6 1. Ubiquitylation 6 2. Post-translational modification mediated DNA damage response 7 3. RNF4 functions in the DNA damage response 8 4. Heterochromatin protein 1 functions in the DNA damage response 9 D. Poly(ADP-ribosyl)ation mediated DNA damage response signaling 10 1. DNA damage induced PARP1 activation 10 2. Free pADPr polymer function in DNA damage response 11 E. Aim of this study 12 Ⅱ. MATERIAL AND METHODS 13 1. Cell lines 13 2. Plasmids and antibodies 13 3. Laser micro-irradiation 16 4. HR and NHEJ analysis 17 5. Neutral comet assay 18 6. Chromatin fractionation 18 7. pADPr polymer synthesis 19 8. pADPr and SUMO overlay assay 20 9. Dot blot 20 10. In vitro ubiquitination assay 20 11. In vivo ubiquitination assay 21 12. Western blot 22 13. Mass spectrometry analysis 22 14. Chromatin immunoprecipitation 22 15. BIOID assay 23 16. Immunofluorescence 24 17. Clonogenic assay 25 18. Protein microarray 25 19. Electro-mobility shift assay 26 20. FokI assay 26 21. Biotin pull down assay 27 22. Purification of recombinant protein from insect Sf9 and bacteria cells 27 23. Statistical analysis 28 Ⅲ. RESULTS 29 Part A. Systematic screening of pADPr dependent E3 ligase functions in the DNA damage response 29 1. Identification of pADPr binding protein by using a 17K protein microarray 29 2. Bioinformatic analysis of pADPr binding proteins 35 3. pADPr dependent E3 ligase 38 4. pADPr dependent localization of RNF4 to DNA damage sites 43 5. pADPr enhances the activity and dimerization of RNF4 47 6. pADPr binding activity is critical for RNF4 mediated DSB repair 51 Part B. HP1α, a pADPr dependent substrate of RNF4, regulates homologous recombination 55 1. Systematic screening of pADPr dependent substrate of RNF4 55 2. HP1α is ubiquitinated by RNF4 61 3. HP1α is pADPr binding protein 61 4. RNF4 ubiquitylates HP1α lysine 91 66 5. HP1α K91 ubiquitination by RNF4 is critical for DNA binding 66 6. Lysine 91 ubiquitination is important for HP1α mediated homologous recombination repair 70 Part C. RNF168 is pADPr dependent E3 ligase that regulates PARP1 levels in the DNA damage sites 73 1. RNF168 is a pADPr binding ubiquitin E3 ligase 73 2. RNF168 targets PARP1 for ubiquitination mediated degradation 76 3. RNF168 regulates PARP1 protein levels at DNA damage sites 80 4. Regulation of PARP1 by RNF168 is critical for DNA damage repair 83 Ⅳ. DISCUSSION 85 REFERENCE 94 국문요약 108","abstract_html":"Ⅰ. INTRODUCTION 1 A. DNA damage response 1 B. DNA damage repair 3 1. Homologous recombination (HR) 4 2. Nonhomologous end joining (NHEJ) 5 C. Ubiquitylation mediated DNA damage response signaling 6 1. Ubiquitylation 6 2. Post-translational modification mediated DNA damage response 7 3. RNF4 functions in the DNA damage response 8 4. Heterochromatin protein 1 functions in the DNA damage response 9 D. Poly(ADP-ribosyl)ation mediated DNA damage response signaling 10 1. DNA damage induced PARP1 activation 10 2. Free pADPr polymer function in DNA damage response 11 E. Aim of this study 12 Ⅱ. MATERIAL AND METHODS 13 1. Cell lines 13 2. Plasmids and antibodies 13 3. Laser micro-irradiation 16 4. HR and NHEJ analysis 17 5. Neutral comet assay 18 6. Chromatin fractionation 18 7. pADPr polymer synthesis 19 8. pADPr and SUMO overlay assay 20 9. Dot blot 20 10. In vitro ubiquitination assay 20 11. In vivo ubiquitination assay 21 12. Western blot 22 13. Mass spectrometry analysis 22 14. Chromatin immunoprecipitation 22 15. BIOID assay 23 16. Immunofluorescence 24 17. Clonogenic assay 25 18. Protein microarray 25 19. Electro-mobility shift assay 26 20. FokI assay 26 21. Biotin pull down assay 27 22. Purification of recombinant protein from insect Sf9 and bacteria cells 27 23. Statistical analysis 28 Ⅲ. RESULTS 29 Part A. Systematic screening of pADPr dependent E3 ligase functions in the DNA damage response 29 1. Identification of pADPr binding protein by using a 17K protein microarray 29 2. Bioinformatic analysis of pADPr binding proteins 35 3. pADPr dependent E3 ligase 38 4. pADPr dependent localization of RNF4 to DNA damage sites 43 5. pADPr enhances the activity and dimerization of RNF4 47 6. pADPr binding activity is critical for RNF4 mediated DSB repair 51 Part B. HP1α, a pADPr dependent substrate of RNF4, regulates homologous recombination 55 1. Systematic screening of pADPr dependent substrate of RNF4 55 2. HP1α is ubiquitinated by RNF4 61 3. HP1α is pADPr binding protein 61 4. RNF4 ubiquitylates HP1α lysine 91 66 5. HP1α K91 ubiquitination by RNF4 is critical for DNA binding 66 6. Lysine 91 ubiquitination is important for HP1α mediated homologous recombination repair 70 Part C. RNF168 is pADPr dependent E3 ligase that regulates PARP1 levels in the DNA damage sites 73 1. RNF168 is a pADPr binding ubiquitin E3 ligase 73 2. RNF168 targets PARP1 for ubiquitination mediated degradation 76 3. RNF168 regulates PARP1 protein levels at DNA damage sites 80 4. Regulation of PARP1 by RNF168 is critical for DNA damage repair 83 Ⅳ. DISCUSSION 85 REFERENCE 94 국문요약 108","abstract_has_math":false,"creators":["김, 재진"],"institution":null,"degree_name":null,"degree_level":null,"degree_discipline":null,"degree_department":null,"school":null,"contributors":["대학원 의생명과학과","201225182"],"advisors":[],"committee_chairs":[],"committee_members":[],"year":2016,"date_issued":"2016-11-25T10:01:43Z","date_published":"2016-11-25T10:01:43Z","updated_at":"2026-07-24T00:51:53Z","subjects":["Poly(ADP-ribose)","Ubiquitin E3 ligase","Cancer","DNA damage response"],"languages":["en"],"rights":[],"rights_urls":[],"identifier_entries":[{"key":"dc:identifier","label":"Identifier","values":["http://dcoll.ajou.ac.kr:9080/dcollection/jsp/common/DcLoOrgPer.jsp?sItemId=000000023325","000000023325"],"render_values":[{"text":"http://dcoll.ajou.ac.kr:9080/dcollection/jsp/common/DcLoOrgPer.jsp?sItemId=000000023325","href":"http://dcoll.ajou.ac.kr:9080/dcollection/jsp/common/DcLoOrgPer.jsp?sItemId=000000023325","code":true},{"text":"000000023325","href":null,"code":true}]}]},"links":{"outbound_url":"http://repository.ajou.ac.kr/handle/201003/13016","outbound_label":"Repository record","outbound_source":"dc:identifier"},"metadata_groups":[{"id":"people","label":"People","entries":[{"key":"dc:contributor","label":"Contributor","values":["대학원 의생명과학과","201225182","김, 재진"]},{"key":"dc:creator","label":"Author","values":["김, 재진"]}]},{"id":"academic_context","label":"Academic Context","entries":[{"key":"dc:date","label":"Dc Date","values":["2016-11-25T10:01:43Z","2016"]},{"key":"dc:type","label":"Dc Type","values":["Thesis","Theses"]}]},{"id":"subjects_keywords","label":"Subjects and Keywords","entries":[{"key":"dc:subject","label":"Dc Subject","values":["Poly(ADP-ribose)","Ubiquitin E3 ligase","Cancer","DNA damage response"]}]},{"id":"language_rights","label":"Language and Rights","entries":[{"key":"dc:language","label":"Dc Language","values":["en"]}]},{"id":"identifiers","label":"Identifiers","entries":[{"key":"dc:identifier","label":"Identifier","values":["http://repository.ajou.ac.kr/handle/201003/13016","http://dcoll.ajou.ac.kr:9080/dcollection/jsp/common/DcLoOrgPer.jsp?sItemId=000000023325","000000023325"]}]},{"id":"additional","label":"Additional Metadata","entries":[{"key":"dc:description","label":"Description","values":["Ⅰ. INTRODUCTION 1 A. DNA damage response 1 B. DNA damage repair 3 1. Homologous recombination (HR) 4 2. Nonhomologous end joining (NHEJ) 5 C. Ubiquitylation mediated DNA damage response signaling 6 1. Ubiquitylation 6 2. Post-translational modification mediated DNA damage response 7 3. RNF4 functions in the DNA damage response 8 4. Heterochromatin protein 1 functions in the DNA damage response 9 D. Poly(ADP-ribosyl)ation mediated DNA damage response signaling 10 1. DNA damage induced PARP1 activation 10 2. Free pADPr polymer function in DNA damage response 11 E. Aim of this study 12 Ⅱ. MATERIAL AND METHODS 13 1. Cell lines 13 2. Plasmids and antibodies 13 3. Laser micro-irradiation 16 4. HR and NHEJ analysis 17 5. Neutral comet assay 18 6. Chromatin fractionation 18 7. pADPr polymer synthesis 19 8. pADPr and SUMO overlay assay 20 9. Dot blot 20 10. In vitro ubiquitination assay 20 11. In vivo ubiquitination assay 21 12. Western blot 22 13. Mass spectrometry analysis 22 14. Chromatin immunoprecipitation 22 15. BIOID assay 23 16. Immunofluorescence 24 17. Clonogenic assay 25 18. Protein microarray 25 19. Electro-mobility shift assay 26 20. FokI assay 26 21. Biotin pull down assay 27 22. Purification of recombinant protein from insect Sf9 and bacteria cells 27 23. Statistical analysis 28 Ⅲ. RESULTS 29 Part A. Systematic screening of pADPr dependent E3 ligase functions in the DNA damage response 29 1. Identification of pADPr binding protein by using a 17K protein microarray 29 2. Bioinformatic analysis of pADPr binding proteins 35 3. pADPr dependent E3 ligase 38 4. pADPr dependent localization of RNF4 to DNA damage sites 43 5. pADPr enhances the activity and dimerization of RNF4 47 6. pADPr binding activity is critical for RNF4 mediated DSB repair 51 Part B. HP1α, a pADPr dependent substrate of RNF4, regulates homologous recombination 55 1. Systematic screening of pADPr dependent substrate of RNF4 55 2. HP1α is ubiquitinated by RNF4 61 3. HP1α is pADPr binding protein 61 4. RNF4 ubiquitylates HP1α lysine 91 66 5. HP1α K91 ubiquitination by RNF4 is critical for DNA binding 66 6. Lysine 91 ubiquitination is important for HP1α mediated homologous recombination repair 70 Part C. RNF168 is pADPr dependent E3 ligase that regulates PARP1 levels in the DNA damage sites 73 1. RNF168 is a pADPr binding ubiquitin E3 ligase 73 2. RNF168 targets PARP1 for ubiquitination mediated degradation 76 3. RNF168 regulates PARP1 protein levels at DNA damage sites 80 4. Regulation of PARP1 by RNF168 is critical for DNA damage repair 83 Ⅳ. DISCUSSION 85 REFERENCE 94 국문요약 108","Doctor"]},{"key":"dc:format","label":"Dc Format","values":["application/pdf"]},{"key":"dc:title","label":"Title","values":["Functional characterization of poly(ADP-ribose) dependent ubiquitin E3 ligase in the DNA damage response","DNA 손상복구 반응에서 poly(ADP-ribose) 의존적 유비퀴틴 접합효소의 기능 분석"]}]}],"canonical_facts":{"dc:contributor":["대학원 의생명과학과","201225182","김, 재진"],"dc:creator":["김, 재진"],"dc:date":["2016-11-25T10:01:43Z","2016"],"dc:description":["Ⅰ. INTRODUCTION 1 A. DNA damage response 1 B. DNA damage repair 3 1. Homologous recombination (HR) 4 2. Nonhomologous end joining (NHEJ) 5 C. Ubiquitylation mediated DNA damage response signaling 6 1. Ubiquitylation 6 2. Post-translational modification mediated DNA damage response 7 3. RNF4 functions in the DNA damage response 8 4. Heterochromatin protein 1 functions in the DNA damage response 9 D. Poly(ADP-ribosyl)ation mediated DNA damage response signaling 10 1. DNA damage induced PARP1 activation 10 2. Free pADPr polymer function in DNA damage response 11 E. Aim of this study 12 Ⅱ. MATERIAL AND METHODS 13 1. Cell lines 13 2. Plasmids and antibodies 13 3. Laser micro-irradiation 16 4. HR and NHEJ analysis 17 5. Neutral comet assay 18 6. Chromatin fractionation 18 7. pADPr polymer synthesis 19 8. pADPr and SUMO overlay assay 20 9. Dot blot 20 10. In vitro ubiquitination assay 20 11. In vivo ubiquitination assay 21 12. Western blot 22 13. Mass spectrometry analysis 22 14. Chromatin immunoprecipitation 22 15. BIOID assay 23 16. Immunofluorescence 24 17. Clonogenic assay 25 18. Protein microarray 25 19. Electro-mobility shift assay 26 20. FokI assay 26 21. Biotin pull down assay 27 22. Purification of recombinant protein from insect Sf9 and bacteria cells 27 23. Statistical analysis 28 Ⅲ. RESULTS 29 Part A. Systematic screening of pADPr dependent E3 ligase functions in the DNA damage response 29 1. Identification of pADPr binding protein by using a 17K protein microarray 29 2. Bioinformatic analysis of pADPr binding proteins 35 3. pADPr dependent E3 ligase 38 4. pADPr dependent localization of RNF4 to DNA damage sites 43 5. pADPr enhances the activity and dimerization of RNF4 47 6. pADPr binding activity is critical for RNF4 mediated DSB repair 51 Part B. HP1α, a pADPr dependent substrate of RNF4, regulates homologous recombination 55 1. Systematic screening of pADPr dependent substrate of RNF4 55 2. HP1α is ubiquitinated by RNF4 61 3. HP1α is pADPr binding protein 61 4. RNF4 ubiquitylates HP1α lysine 91 66 5. HP1α K91 ubiquitination by RNF4 is critical for DNA binding 66 6. Lysine 91 ubiquitination is important for HP1α mediated homologous recombination repair 70 Part C. RNF168 is pADPr dependent E3 ligase that regulates PARP1 levels in the DNA damage sites 73 1. RNF168 is a pADPr binding ubiquitin E3 ligase 73 2. RNF168 targets PARP1 for ubiquitination mediated degradation 76 3. RNF168 regulates PARP1 protein levels at DNA damage sites 80 4. Regulation of PARP1 by RNF168 is critical for DNA damage repair 83 Ⅳ. DISCUSSION 85 REFERENCE 94 국문요약 108","Doctor"],"dc:format":["application/pdf"],"dc:identifier":["http://repository.ajou.ac.kr/handle/201003/13016","http://dcoll.ajou.ac.kr:9080/dcollection/jsp/common/DcLoOrgPer.jsp?sItemId=000000023325","000000023325"],"dc:language":["en"],"dc:subject":["Poly(ADP-ribose)","Ubiquitin E3 ligase","Cancer","DNA damage response"],"dc:title":["Functional characterization of poly(ADP-ribose) dependent ubiquitin E3 ligase in the DNA damage response","DNA 손상복구 반응에서 poly(ADP-ribose) 의존적 유비퀴틴 접합효소의 기능 분석"],"dc:type":["Thesis","Theses"]},"updated_at":"2026-07-24T00:51:53Z"}