Functional characterization of LC3B in maintaining genomic stability
- 주제(키워드) DNA damage response , Ubiquitin-like modifiers , DNA double-strand breaks , R-loops , Transcription-associated homologous recombination repair , Autophagy , AU-rich elements , mRNA stability
- 주제(DDC) 570
- 발행기관 아주대학교 일반대학원
- 지도교수 Ho Chul Kang
- 발행년도 2025
- 학위수여년월 2025. 2
- 학위명 박사
- 학과 및 전공 일반대학원 의생명과학과
- 실제URI http://www.dcollection.net/handler/ajou/000000034690
- 본문언어 영어
- 저작권 아주대학교 논문은 저작권에 의해 보호받습니다.
초록/요약
Functional characterization of LC3B maintaining genomic stability Beside ubiquitin, previously, it has been known that various ubiquitin-like modifiers (ULMs), such as NEDD8, SUMOs and FAT10 broadly participate in DNA damage response (DDR). However, it is largely unclear whether other ULMs are involved in DDR. Herein, I identified a critical role of LC3B, an essential factor for the autophagy, in the orchestrating DNA double-strand breaks (DSBs) repair via transcription-associated homologous recombination repair (TA-HRR). Cells that are deficient in LC3B impaired the recruitment of BRCA1 to DNA damage sites and loading of RAD51 on DNA lesions, leading to defect of precise DSB repair by TA-HRR. Notably, the RNA-recognition motif (RRM) of LC3B plays a pivotal role in TA-HRR by regulating translocation of LC3B to DSBs within transcriptionally active regions. In addition, recruitment of LC3B to DSBs is dramatically abolished by inhibition of transcription. Surprisingly, LC3B directly recognizes and interacts with R-loops via its RRM to promoting TA-HRR in autophagy-independent fashion. Disruption of interaction between LC3B and R-loops results in decreasing end-resection, which initial step of HR, and sister chromatid exchanges. Subsequently, interchromosomal fusions (ICFs) are dramatically increased by aberrant non-homologous end joining (NHEJ) in LC3B-depleted cells. In other ways to regulating DSB repair, LC3B enforces efficiency of repair by direct interaction with 3’ UTR of BRCA1 transcripts containing AU-rich elements (AREs) and guarding its stability. These findings indicate that autophagy- independent role of LC3B for maintaining the genomic integrity within transcriptional active areas in the cells and provides insights of TA-HRR and relationship between autophagy and DDR.
more목차
I. INTRODUCTION 1
A. DNA damage response (DDR) 1
B. Double-strand breaks (DSBs) repair pathways 2
C. Post-translational modifications (PTMs) in DSB repair 3
D. Ubiquitin-like modifiers in DSB repair 4
E. Autophagy in the DDR 6
F. Aim of this study 8
II. MATERIALS AND METHODS 9
1. Human cell lines and cell culture 9
2. Primers, siRNAs and oligonucleotides 9
3. Live and fixative cell imaging with laser micro-irradiation (m-IR) 14
4. Monitoring the recruitment of proteins to FokI-induced DSBs 17
5. Fluorescence In Situ Hybridization (FISH) 19
6. HR and NHEJ repair assay 24
7. Clonogenic cell survival assay 25
8. Immunofluorescence 26
9. Immunofluorescence for DNA/RNA hybrids 27
10. Immunofluorescence for newly synthesized RNAs 27
11. Purification of recombinant LC3B WT, RBM, and HBD from sf9 and Escherichia coli 28
12. In vitro R-loop and hybrid binding assay by EMSA 29
13. Comparison of LC3B structure via RCSB Protein Data Bank (RCSB PDB) 31
14. Chromatin immunoprecipitation (ChIP) 31
15. DNA/RNA-immunoprecipitation (DRIP) 32
16. Immunoblotting 33
17. Real-time quantitative PCR (RT-qPCR) 33
18. Dot blot assay for R-loops and biotinylated RNA probes 34
19. Biotin-labeled RNA pull-down assay 35
20. nRIP coupled with high-throughput sequencing (nRIP-seq) 35
21. Bioinformatic analysis of the nRIP-seq DATA set 36
22. Surface plasmon resonance (SPR) 37
23. End resection assay 37
24. Sister chromatid exchange (SCE), interchromatid fusion (ICF), and chromosomal breakage assay 39
25. Image quantification and statistical analysis 40
III. RESULTS 42
LC3B drives transcription-associated homologous recombination via direct interaction with R-loops 42
1. Identification of LC3B as a novel DDR-linked ULM 42
2. LC3B is repair factor for DSBs via HR pathway 50
3. Autophagy is dispensable for LC3B's recruitment to DSBs 57
4. LC3B's translocation to DSBs through R-loops is crucial for R-loop stability 69
5. LC3B interacts directly with DNA/RNA hybrids including R-loops via its RRM 76
6. LC3B acts as a key player in TA-HRR via direct interaction with R-loops 81
7. LC3B regulates BRCA1 mRNA stability through direct interaction with 3' UTR AREs 90
8. LC3B promotes chromosomal stability through TA-HRR guidance 99
IV. DISCUSSION 110
REFERENCES 113
국문요약 123
