아주대학교

목차

CHAPTER I : Introduction 1
A. DNA damage response (DDR) 1
B. Mechanisms of DNA double-strand break repair 2
C. P53-mediated transcriptional regulation in the DNA damage response 6
D. The chromatin remodeling function of RSF1 10
E. 53BP1 function in DNA repair and transcription 12
F. Phase separation in the DNA damage response 16
G. Aims of this study 20
CHAPER II : RSF1 coordinates p300 and FACT to regulate p53-dependent transcription 22
A. Chapter Introduction 23
B. Materials and Methods 25
1. Cell culture 25
2. Plasmid and RNA interference 25
3. Western blot analysis 26
4. Antibodies and reagents 26
5. Flow cytometric (FACS) analysis 28
6. Whole-cell extraction 28
7. Immunofluorescence microscopy 28
8. Pull-down assay 29
9. Immunoprecipitation assay 29
10. Real-time PCR analysis 30
11. Reporter gene activity measurement using luciferase assay 30
12. Chromatin immunoprecipitation (ChIP) assay 30
13. Statistical analysis 32
C. Results 33
1. RSF1 promotes p53-dependent transcription and cell cycle arrest in response to DNA damage 33
2. RSF1 controls p53 transcriptional activity by regulating its acetylation at lysine 382 37
3. RSF1 facilitates p53-dependent transcription by coordinating p300 activity and pre-initiation complex assembly 41
4. RSF1 enhances p21 transcription by bridging the FACT complex and RNA polymerase II to promoter regions 45
CHAPER III : RSF1 supports 53BP1 repair condensate formation through chromatin remodeling 49
A. Chapter Introduction 50
B. Materials and Methods 52
1. Cell culture 52
2. Cloning and plasmids 52
3. siRNA sequences, antibodies, and chemicals 53
4. OptoDroplet assay 55
5. Fluorescence recovery after photobleaching (FRAP) 56
6. Laser microirradiation and immunofluorescence 56
7. Immunoblotting 56
8. Recombinant Protein Expression and Purification from Sf9 Insect Cells and E. coli 57
9. Poly-ADP-ribosylation in vitro assay 57
10. Quantitative real-time PCR 58
11. Reporter gene activity measurement using luciferase assay 58
12. Chromatin immunoprecipitation (ChIP) assay 59
13. Image quantification 59
14. Statistical analysis 60
C. Results 61
1. RSF1 predominantly comprises intrinsically disordered regions (IDRs) 61
2. RSF1 alone does not form liquid condensates 65
3. RSF1 is required for the 53BP1-mediated liquid condensate formation 69
4. The C-terminal IDR3 of RSF1 is required for 53BP1 condensate formation 73
5. RSF1 modulates 53BP1 condensate formation through PARylation dynamics 77
6. RSF1 is required for 53BP1 condensate formation but does not affect FUS-driven condensates 81
7. RSF1 facilitates timely PARG recruitment to DNA damage sites, independent of PARP1 accumulation 84
8. Delayed PARG recruitment underlies 53BP1 condensate defects in RSF1 C1-expressing cells 87
9. Disruption of phase separation attenuates p53 target gene transcription following DNA damage 90
10. RSF1-mediated condensates promote p53 recruitment to chromatin and transcriptional activation 93
CHAPER IV : Discussion 96
REFERENCES 106
국문요약 123