본문 바로가기 주메뉴 바로가기
22대 대한민국 국회 국회정보길라잡이 국회의원검색
회원가입 로그인
HOME

정보검색

소장정보 검색

국회도서관 홈으로 정보검색 소장정보 검색

결과 내 검색

동의어 포함

고급검색

인명/단체명

저자정보 상세정보
인명/단체명을 입력하세요.

키워드

  • 대표어

  • 외국어

-

목차보기

Title Page 2

Overall Abstract 5

Contents 8

Nomenclature 13

Chapter 1. Short-range end resection requires PCNA unloading for faithful homologous recombination 16

1.1. Abstract 16

1.2. Introduction 17

1.3. Results 19

1.3.1. PCNA is rapidly loaded on DNA at DSB sites by RFC and later unloaded by ATAD5-RLC 19

1.3.2. PCNA recruitment to DNA damage sites largely depends on MRN-CtIP 24

1.3.3. ATAD5 is important for HR but not for other DSBR pathways 27

1.3.4. ATAD5-depleted cells are sensitive to a drug forming protein-DNA adducts 31

1.3.5. Recruitment of MRN-CtIP and EXO1 to the DSBs and DNA damage checkpoint are intact in ATAD5-depleted cells 34

1.3.6. Long range resection is intact in ATAD5-depleted cells 37

1.3.7. PCNA unloading by ATAD5 is required for MRN-mediated KU removal from DSB ends 40

1.3.8. PCNA loaded on DNA inhibits MRE11/RAD50-mediated resection in vitro 43

1.3.9. DNA repair synthesis is defective in ATAD5-depleted cells 46

1.3.10. Unprocessed DSB ends resulting from ATAD5 depletion are partially repaired by TMEJ 49

1.4. Discussion 52

1.5. Experimental Methods 55

1.5.1. Cell lines and cell culture 55

1.5.2. Small interfering RNAs (siRNAs), and transfection 55

1.5.3. Reagents and antibodies 55

1.5.4. 355-nm UV-laser microirradiation and X-ray irradiation 56

1.5.5. FokI-induced DSB generation 56

1.5.6. Immunostaining 56

1.5.7. Cell cycle specific IR-induced DNA repair synthesis 57

1.5.8. Proximity ligation assay (PLA) 57

1.5.9. I-SceI-induced DSBR assay 58

1.5.10. RPA retention assay using flow cytometry 58

1.5.11. Analysis of metaphase chromosomes 58

1.5.12. Cell cycle analysis 59

1.5.13. Sequence analysis of repair product in DR-GFP construct 59

1.5.14. Protein extraction, immunoprecipitation, and immunoblot analysis 59

1.5.15. Protein purification 60

1.5.16. Preparation of dsDNA substrate for resection assay 60

1.5.17. In vitro resection assay 61

1.5.18. Statistical analysis 62

1.6. Reference 63

Chapter 2. DEK and NUMA1 participate in the UV repair by regulating the nature of XPF and PCNA 69

2.1. Abstract 69

2.2. Introduction 70

2.3. Results 72

2.3.1. DEK and NUMA1 were identified as new NER-related proteins 72

2.3.2. DEK or NUMA1 depletion delays repair of UV lesions 81

2.3.3. DEK or NUMA1 depletion delays UV-induced DNA synthesis 86

2.3.4. DEK or NUMA1 depletion delays XPF recruitment to UV lesion 90

2.3.5. Depletion of DEK or NUMA1 enriched PCNA on UV lesion 93

2.3.6. PCNA enrichment in DEK- or NUMA1- depleted cell was regulated by RFC1, XPA and APE1 depletion in different manner 99

2.3.7. Association of BER pathway in UV repair in DEK, NUMA1 depleted cells 101

2.4. Discussion 105

2.5. Experimental Methods 108

2.5.1. Cell lines and cell culture 108

2.5.2. G1-UV-iPOND-LC-MS 108

2.5.3. SIRF assay 109

2.5.4. Antibodies 110

2.5.5. Transfection & siRNA treatment 110

2.5.6. Western blot, immunoprecipitation 110

2.5.7. Clonogenic cell survival assay 111

2.5.8. Immunofluorescence 111

2.5.9. Recovery of RNA synthesis (RRS) assay, Unscheduled DNA synthesis (UDS) 112

2.5.10. Local UV irradiation assay (6-4PP, CPD) 112

2.5.11. Protein-CPD Co-localization assay 112

2.5.12. Slot-blot assay 113

2.6. Reference 114

Appendix Ⅰ. Curriculum Vitae 120

List of Figures 11

Figure 1.1. PCNA is rapidly recruited to DSBs, and recruitment requires RFC1 but not D... 21

Figure 1.2. PCNA remains on the microirradiated strips for a longer time in ATADS-deple... 23

Figure 1.3. PCNA and ATAD5 are recruited to DSBs, and recruitment requires... 26

Figure 1.4. Diagrams of I-Scel-induced DSBR assay 28

Figure 1.5. ATADS-depleted cells are defective for HR and are hypersensitive to... 30

Figure 1.6. ATADS-depleted cells are hypersensitive to camptothecin 33

Figure 1.7. Recruitment of MRN-CtIP and EXO1 to the DSBs and DNA damage... 36

Figure 1.8. Long-range resection is intact in ATADS-depleted cells 39

Figure 1.9. DNA end processing is defective in ATADS-depleted cells 42

Figure 1.10. PCNA loaded on DNA blocks MR-mediated resection in vitro 45

Figure 1.11. DNA repair synthesis is defective in ATAD5-depleted cells 48

Figure 1.12. RAD51 foci intensity is higher in ATADS-depleted cells 51

Figure 2.1. DEK and NUMA1 were identified and confirmed as new UV repair... 76

Figure 2.2. Basic confirmation about Gl-UV-iPOND and involvement of DEK and... 78

Figure 2.3. Cell cycle profile was not altered in DEK- or NUMA1-depleted cells 80

Figure 2.4. DEK- or NUMAl-depleted cells showed defects in repair of UV lesions 83

Figure 2.5. Depletion of DEK or NUMA1 resulted in slight defects in UV lesion repair in sl... 85

Figure 2.6. Figure 3. DEK- or NUMA1-depleted cells showed mild delay in repair DNA syn... 88

Figure 2.7. Depletion of DEK or NUMA1 did not affect the expression of XP proteins 89

Figure 2.8. DEK- or NUMAl-depleted cells showed abnormal XPF recruitment and... 92

Figure 2.9. DEK- or NUMAl-depleted cells showed abnormal PCNA accumulation 96

Figure 2.10. RFC1 association at chromatin was increased in DEK- or NUMA1- depleted c... 98

Figure 2.11. enrichment of PCNA showed different dependency on XPA and APE1 at diffe... 103

Figure 2.12. Model of the study 104

초록보기

 Mutations driven by exogenous mutagens, failure in DNA replication and deficient DNA repair mechanisms directly fuel the momentum of evolution. However, the accumulation of mutation within the somatic cells can lead to catastrophic consequences - senescence and onset of various diseases - for certain organisms. Guided by humanistic values, we prioritize the health and longevity of each organism more than ever before, highlighting the importance of maintenance of genomic integrity. DNA repair pathways serve as the guardians of our genome. While their fidelity is commendable, our extended lifespan prompts us to strive for perfection in these processes, to mitigate common causes of death like senescence and cancer. Extensive studies on the DNA repair processes have elucidated the major players of each repair pathway. However, intricate molecular nature within the cellular systems and distinct applications of repair pathways in cancerous contexts present formidable challenges for us to overcome. During my Ph.D., I tried to uncover novel contributors to DNA repair processes, aiming to broaden our comprehension in DNA repair pathways while hoping our findings will contribute to the big goal of mankind.

Homologous recombination (HR) repair restores DNA double-strand breaks (DSB) occurring during the S/G2 cell phase. Unlike other homology-directed repairs (HDR) and non-homologous end-joining (NHEJ), HR is error-free, by using homologous chromosomes as a template. HR is comprised of end resection to digest bulky DSB lesions and to remain single-strand DNA (ssDNA) strand, strand invasion into the homologous chromosome, and repair DNA synthesis to ensure the integrity of genomic information. While numerous DNA repair proteins contribute to HR, the significance of accessory factors is becoming increasingly evident. Here, I elucidated the importance of proper regulation of PCNA by ATAD5 in short-range end resection. PCNA is localized at DSB sites dependent on RFC1 and MRN-CtIP mediated endonucleolytic cleavage. Uncontrolled PCNA localization at DSB sites impedes short-range end resection, hindering the removal of the KU70/80 complex and impeding DNA repair synthesis, thereby compromising HR. Depletion of ATAD5 leads to HR defects, heightened sensitivity to camptothecin (CPT), and increased reliance on end-joining pathways, emphasizing the necessity of ATAD5-mediated PCNA unloading for faithful completion of HR.

Nucleotide excision repair (NER) is a versatile pathway that detects and removes various DNA lesions accompanying structural distortion in the DNA helix, largely induced by UV irradiation and chemotherapeutic reagents. Hereditary loss of core NER factors results in severe genetic diseases such as Xeroderma pigmentosum (XP), Cockayne syndrome (CS), and trichothiodystrophy (TTD).

Despite the established importance of the NER pre-incision complex, the later DNA repair synthesis process remains poorly understood, necessitating the identification of novel regulators in this process.

Employing a mass spectrometry-based approach, I identified DEK oncoprotein (DEK) and nuclear mitotic apparatus protein 1 (NUMA1) as novel contributors to repair DNA synthesis during NER. Depletion of DEK or NUMA1 resulted in increased sensitivity to UV irradiation, moderate deficiency in UV lesion removal, and delayed DNA synthesis. Mechanistic studies revealed that loss of DEK or NUMA1 delayed the recruitment and dissociation of XPF, leading to elevated PCNA accumulation at UV lesions mediated by RFC1. Furthermore, I uncovered NER-independent PCNA accumulation at later timepoints post-UV irradiation, which was dependent on APE1-mediated long-patch base excision repair (Lp-BER), underscoring the roles of DEK and NUMA1 in regulating PCNA accumulation in both NER and BER pathways. Based on this, we propose the contribution of DEK and NUMA1 in UV repair, while suggesting further questions to be answered.

추천서가 (다양한 추천 자료를 만나보세요)

권호기사보기

권호기사 목록 테이블로 기사명, 저자명, 페이지, 원문, 기사목차 순으로 되어있습니다.
기사명 저자명 페이지 원문 기사목차

권호

기사명 원문보기 기사목차
닫기