Coupled Functions of CTCF in Transcription & Repair in Response to DNA Double-Strand Breaks
- 주제(키워드) NELF-E , DNA Double-Strand Breaks , CTCF , DNA damage repair
- 주제(DDC) 570
- 발행기관 아주대학교
- 지도교수 이종수
- 발행년도 2023
- 학위수여년월 2023. 8
- 학위명 석사
- 학과 및 전공 일반대학원 생명과학과
- 실제URI http://www.dcollection.net/handler/ajou/000000033009
- 본문언어 한국어
- 저작권 아주대학교 논문은 저작권에 의해 보호받습니다.
초록/요약
A variety of factors are commonly or differentially involved in the repair of DNA damage depending on types of DNA insults. Among them, DNA double-strand breaks (DSBs) are the most severe lesions that, if left unrepaired, can lead to serious genome aberrations, potentially affecting cell survival. Two major DSB repair pathways include non-homologous end joining (NHEJ) and homologous recombination (HR). At the vicinity of DSBs occurring within transcriptional active regions of the genome, several HR proteins are preferentially recruited and DNA-RNA hybrid R loop structures are formed via the negative elongation factor NELF-dependent transient transcription suppression. Little is known about the mechanisms that ensure coupled transcription suppression and HR-mediated repair in response to DSBs. In this study, I identified the multifunctional transcriptional factor CCCTC-binding factor (CTCF), which engages in the initial step in HR by recruiting the resection endonuclease CtIP, as a crucial factor that facilitates the rapid recruitment of NELF-E and its resultant R-loop formation via transcription suppression at DSBs. CTCF interacts with NELF-E. Depletion of CTCF attenuates DSB recruitment of NELF-E and resultantly impairs the DSB-induced transcriptional suppression and R-loop generation at DSBs. Either CTCF or NELF-E depletion impairs recruitment of several HR factors including BARD1, BRCA1, and RAD51, and heterochromatin protein1γ (HP1γ) at DSBs, thereby resulting in compromised DSB R-loop formation and HR. However, NELF-E depletion has little effects on the CTCF recruitment at DSB, suggesting that CTCF acts upstream of NELF-E and R-loop in the HR pathway. Overall, my findings demonstrate that CTCF directs rapid and transient transcription suppression and HR-mediated repair of DSBs at transcriptionally active regions through establishing CTCF-NELF-HR factor axis.
more목차
Ⅰ. Introduction 1
Ⅱ. Material and Methods 4
1. Cell culture 4
2. Plasmid construction and transfection 4
3. Live Laser micro-irradiation 4
4. Immunofluorescence 5
5. Immunoprecipitation 5
6. HR, canonical and alternative NHEJ, and single-strand annealing (SSA) DNA repair assays 5
Ⅲ. Results 9
1. CTCF regulates NELF-E recruitment at DSB site 9
2. CTCF Promotes R-loop formation at DSB site via NELF-E recruitment 13
3. NELF-E regulates the recruitment of HR factors 19
4. NELF-E regulates HR in a CTCF dependent manner 27
Ⅳ. Discussion 34
Ⅴ. References 36
