유전자 손상 복구 반응을 조절하는 PARP 의존적 유비퀴틴 중합 효소와 탈유비퀴틴 효소의 기능 분석
Functional Characterization of PARP Dependent Ubls and Dubs Regulating DNA Damage Response
- 주제(키워드) DNA damage response (DDR) , Ubiquitin E3 ligase (Ubl) , Deubiquitylating enzymes (Dub) , Poly(ADP-ribose) (PAR) polymerase (PARP)
- 발행기관 아주대학교
- 지도교수 강호철
- 발행년도 2017
- 학위수여년월 2017. 2
- 학위명 박사
- 학과 및 전공 일반대학원 의생명과학과
- 실제URI http://www.dcollection.net/handler/ajou/000000024452
- 본문언어 영어
- 저작권 아주대학교 논문은 저작권에 의해 보호받습니다.
초록/요약
DNA damages induced by either endogenous or exogenous factors lead to alteration of DNA structure causing mutation of DNA. Thus, DNA mutations cause a wide range of human disease such as cancers and neuronal disorders. Hence, all organisms have internal surveillance system for monitoring of DNA lesions, thereby control DNA damage response (DDR) for the error-free repair of genetic code. In the DDR, post-translational modifications play the central roles for signal transmission for DNA restoration. Particularly, Poly(ADP-ribose) (PAR)-substrates-ubiquitin axis is a pivotal regulatory network for the recruiting of DNA repair machineries to the DNA lesions that regulates DNA repair processes such as homologous recombination (HR) and non-homologous end joining (NHEJ). Although it has been little known that repair process of double strand breaks (DSBs) can be controlled by ubiquitination, but the direct connection between PAR and ubiquitin signal in DDR remains unclear. Herein, using a systematic screening, we identified the PAR coupled ubiquitin E3 ligase (Ubls) and deubiquitylating enzymes (Dubs) in DDR. We identified 15 Ubls and 6 Dubs linked to PAR signaling pathway and further investigated the functional roles of DIZELs and USP39 on DDR. These proteins were rapidly recruited to DNA lesions via direct interaction with PAR moiety. The DIZELs contain bipartite C2H2-type zinc finger Drought-induced 19 (Di19) motif. By systematic experimental approaches, it has been revealed that C2H2(I), the first motif of the two conserved C2H2 Di-19 region plays an important role for generation of unanchored K11 chains (UnK11Ubcs) but later C2H2(II) motif involves in the direct interaction with PAR moieties. Besides, we also found that UnK11Ubcs synthesized by DIZELs is a crucial factor for recruitment of NPL4/VCP complex and 53BP1 at the DNA lesions. In parallel, we revealed that USP39 also strongly binds to PAR polymer through its N-terminal RG repeated motif and regulates HR or NHEJ repair process, in a splicing-dependent or -independent manner, respectively. Furthermore, we found that a spliceosome independent USP39 shows distinct role for the recruitment of XRCC4/LIG4 complex to regulate NHEJ repair process. Taken together, we have identified that PAR-substrates-ubiquitin axis is a master regulatory network for the regulation of Ubls or Dubs activity, thereby allows monitoring and maintaining of genomic stability.
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ABSTRACT i
TABLE OF CONTENTS iii
LIST OF FIGURES vii
LIST OF TABLE ix
ABBREVIATION x
Ⅰ. INTRODUCTION 1
A. DNA damage response 1
B. DNA repair pathway 2
C. DNA repair signaling 4
1. Post-translation modification in DDR pathway 4
2. Ubiquitination and deubiquitination 5
3. PARylation and PAR binding 7
D. Aim of this study 7
Ⅱ. MATERIAL AND METHODS 9
1. cDNA Library Cloning 9
2. Cell Lines and Culture 9
3. Plasmids and Antibodies 10
4. Homologues Recombination (HR) and Non-Homologous End-Joining (NHEJ) Repair Analysis 18
5. G2/M Cell Cycle Checkpoint Analysis 18
6. Clonogenic Survival Assay 18
7. Live Cell Imaging with Laser Micro-irradiation 19
8. Immunofluorescence 19
9. FokI Assay 20
10. Neutral Comet Assay 20
11. Purification of Recombinant Protein from Sf9 Cells 21
12. In Vitro Ubiquitination Assay 22
13. In Vivo Ubiquitination Assay 23
14. Immunoprecipitation 23
15. Immunoblot Assay 24
16. Mass Spectrometric Analysis 24
17. Synthesis of Biotin-Labeled PAR Polymer 24
18. PAR Overlay Assay 25
19. Quantitative PCR 25
20. Image Quantification 28
21. Statistical Analyses 28
Ⅲ. RESULTS 29
Part A. DIZELs generate unanchored K11 ubiquitin chains (UnK11Ubc), driving PARP1-coupled DNA damage signaling 29
1. Screening of novel Ubls at DNA damage sites 29
2. Classification of ATM- or PARP1- dependent Ubls on DDR 33
3. Systematic functional characterization of DDR-linked Ubls 36
4. DIZELs are key player regulating DDR 40
5. DIZELs are novel PAR binding Ubls coupled to DDR 43
6. RNF114 and RNF166 generate unanchored ubiquitin chains (UnUbcs) with UbcH5 family 47
7. DIZELs generate UnUbc and accelerate their pool in a PAR-dependent manner 50
8. DEZELs utilize only Lys11 of ubiquitin to generate homogenous UnK11Ubcs 54
9. C2H2(I) motif of DIZELs is an essential region for generation of UnK11Ubcs 57
10. C2H2(II) motif of DIZELs is required for interaction with PAR and its recruitment at DNA lesions 60
11. RNF114 generates UnK11Ubcs via its tyrosine residue on C2H2(I) in mammalian cells 63
12. UnK11Ubcs are crucial for the 53BP1 foci formation 66
13. NPL4/VCP complex recognizes the UnK11Ubc at DNA lesions 69
14. UnK11Ubc is required for 53BP1 recruitment at DNA lesions 72
Part B. USP39 promotes NHEJ repair process in a PARP1 activity dependent manner 75
1. Systematic screening of PARP coupled Dubs in DDR 75
2. USP39 is recruited to DNA damage sites in PARP1 activity dependent manner 78
3. USP39 play a key role for the DNA repair process 81
4. N-terminal region of USP39 is crucial for its recruitment to DNA lesions 86
5. Arginine-Glycine rich motif of USP39 play a key role in its recruitment
to DSBs by interplaying with PAR polymer 89
6. USP39 is an indispensable factor for recruitment of NHEJ regulatory
proteins to DNA lesions 92
7. XRCC4/LIG4 complex translocate to DNA damage sites in USP39 dependent manner 95
8. USP39 controls XRCC4 translocation to DNA lesions in a spliceosome
activity independent manner 98
9. N-terminal and ZF domain of USP39 are essential for NHEJ repair process 101
Ⅳ. DISCUSSION 105
REFERENCE 110
국문요약 120
