Phase-Separation in response to DNA double-strand breaks
- 주제(키워드) Liquid-Liquid phase separation , CTCF , Homologous recombination , DNA double-strand breaks , Transcription
- 주제(DDC) 570
- 발행기관 아주대학교 일반대학원
- 지도교수 Jong-Soo Lee
- 발행년도 2026
- 학위수여년월 2026. 2
- 학위명 박사
- 학과 및 전공 일반대학원 생명과학과
- 실제URI http://www.dcollection.net/handler/ajou/000000036066
- 본문언어 영어
- 저작권 아주대학교 논문은 저작권에 의해 보호받습니다.
초록/요약
Liquid–liquid phase separation (LLPS) is a phenomenon frequently observed within cell s. Recent studies have reported that LLPS can also be induced by DNA double-strand br eaks (DSBs). Although the physical properties and characteristics of LLPS have been ex tensively investigated, its actual biological functions remain incompletely understood. In this study, I examined the functional role of LLPS induced by DSBs. Condensates form ed through LLPS were generated when DSBs occurred at transcriptionally active region s during the G1 phase. As cells entered the S phase, these condensates recruited key fact ors essential for DNA end resection during homologous recombination (HR), including EXO1 and BLM, as well as RAD51, which is required for nucleoprotein filament forma tion. When condensate formation was impaired, pronounced chromosomal instability wa s observed. In particular, numerous tetrad-like chromosomes were detected, with RAD5 1 distributed along these tetrad-like structures. Moreover, disruption of condensate form ation led to defects in DNA replication forks and a reduction in replication speed. Taken together, this study demonstrates that DSB-induced condensates function to maintain ge nome stability by facilitating efficient DNA damage repair. Graphcal Abstract Key words: Liquid-Liquid phase separation, CTCF, Homologous recombination, DNA double- strand breaks, Transcription
more목차
I. INTRODUCTION 1
II. MATERIALS AND METHODS 7
1. Cells and Reagents 7
2. Immunofluorescence 7
3. Immunoblotting 8
4. RNA Interference 8
5. γ-Irradiation 9
6. FRAP (Fluorescence Recovery After Photobleaching) 9
7. RNA Extraction and Quantitative PCR 9
8. Flow Cytometry 10
9. PLA Combined with Immunofluorescence 10
10. Click Chemistry 11
11. Mitotic Chromosome Spread Analysis 11
12. DNA Fiber Assay 11
13. Antibodies 12
14. Statistical analysis 13
III. RESULTS 14
1. DNA double-strand breaks induce the formation of condensates with LLPS properties 14
2. Condensates enriched in HR factors and formed in a CTCF-dependent manner 25
3. Condensates form during G1 and disassemble upon entry into S/G2 34
4. Execution of HR promotes condensate dissolution upon entry into S phase 41
5. Actively transcribed loci harboring DSBs are sequestered within the condensates 50
6. Transcriptional repression within the condensates is mediated by H3K9me3 59
7. Condensate formation is required for maintaining chromosomal stability 64
8. Condensate formation is required for DNA replication and the maintenance of replication fork i ntegrity 68
IV. DISCUSSION 73
REFERENCES 77
국문요약 84
