ASTRIN mediates RIF1-related DNA damage response
- Issued Date
- 2018
- Keyword
- DNA damage response
- Abstract
- DNA double strand breaks (DSBs) is repaired by two major pathway non-homologous end joining (NHEJ) and homologous recombination (HR). RIF1 pro-motes non-homologous end joining repair and NBS1 is essential for homologous recombination repair. DSB repair choice is decided by the DNA end resection. However, the molecular mechanism of DNA end resection competition remains elusive. Here, we have identified an ASTRIN as a novel RIF1 and NBS1-binding protein by yeast-two hybrid assay. We suggest that ASTRIN is a new player that determines DNA double strand breaks repair pathway.|DNA 이중나선 절단에 의한 DNA 손상은 주로 비상동말단결합과 상동재조합에 의해 복구된다. RIF1은 비상동말단결합을 촉진하며, NBS1은 상동재조합에 있어서 필수적이다. 이러한 DNA 이중나선 절단 복구의 선택은 DNA 말단절제 유무에 의해 결정된다. 그러나 이런 DNA 말단절제 조절에 대한 자세한 기전은 아직 밝혀지지 않았다. 본 연구에서는 Yeast two-hybrid screening을 통해 DNA 말단절제를 조절하는 중요한 단백질인 RIF1, NBS1과 결합하는 ASTRIN이라는 단백질을 동정하였다. 본 연구는 ASTRIN이 RIF1/NBS1과 결합하여, DNA 말단절제를 조절함으로써 DNA 이중나선 절단에 의한 비상동말단결함/상동재결합을 선택하는데 중요한 조절자임을 제안한다.
- Alternative Title
- ASTRIN mediates RIF1-related DNA damage response
- Alternative Author(s)
- Yang, Eun-Bi
- Affiliation
- 조선대학교 일반대학원
- Department
- 일반대학원 의과학과
- Advisor
- 이정희
- Awarded Date
- 2019-02
- Table Of Contents
- KOREAN ABSTRACT ………………………………………………………… ⅳ
INTRODUCTION …………………………………………………………………… 1
MATERIALS AND METHODS
1. Cell culture and treatment of ionizing radiation ……………………………… 6
2. siRNA transfection …………………………………………………………………… 7
3. co-Immunoprecipitation assay …………………………………………………… 8
4. Western blot analysis ……………………………………………………………… 9
5. Antibodies …………………………………………………………………………… 10
6. Proximity Ligation Assay (PLA) ………………………………………………… 11
7. Plasmid …………………………………………………………………………… 13
8. Colony survival assay ……………………………………………………………… 13
9. Immunofluorescence microscopy ……………………………………………… 14
10. Non-homologous end joining assay …………………………………………… 15
11. Homologous recombination assay ……………………………………………… 16
12. 5’end resection assay …………………………………………………………… 17
13. Metaphase chromosome spreads assay ……………………………………… 18
14. Statistical analysis ………………………………………………………………… 19
RESULTS
1. ASTRIN interacts with RIF1 and NBS1 ……………………………………… 20
2. The N-term domain of ASTRIN is critical for interaction of RIF1 and NBS1 ………………………………………………………………………………………………… 24
3. Knockdown of ASTRIN is hypersensitive to ionizing radiation …………… 27
4. ASTRIN upregulates RIF1 foci formation after ionizing radiation ……… 32
5. ASTRIN promotes Non-homologous end joining …………………………… 35
6. ASTRIN downregulates NBS1 foci formation at DSBs …………………… 39
7. ASTRIN impairs Homologous recombination ………………………………… 44
8. ASTRIN deficiency increases resection of DSBs …………………………… 47
9. ASTRIN is involved in maintaining genome stability …………………… 51
DISCUSSION
Discussion ……………………………………………………………………………… 54
ABSTRACT
Abstract …………………………………………………………………………………… 57
REFERENCES
References ……………………………………………………………………………… 59
- Degree
- Master
- Publisher
- 조선대학교 일반대학원
- Citation
- 양은비. (2018). ASTRIN mediates RIF1-related DNA damage response.
- Type
- Dissertation
- URI
- https://oak.chosun.ac.kr/handle/2020.oak/13700
http://chosun.dcollection.net/common/orgView/200000267046
- Appears in Collections:
- General Graduate School > 3. Theses(Master)
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