The p150 large subunit of chromatin assembly factor 1 regulate ATM pathway in double strand breaks repair.

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DNA DSB repair|CAF1 p150|ATM pathway
Chromatin assembly factor 1 (CAF-1)은 게놈 보전과 DNA 수복에 관여하는 중요한 유전자다. 그렇지만, CAF-l이 DNA 수복에 어떻게 관여하는지는 많이 연구되어지지 않았다. 본 연구에서는 DNA double strand breaks (DSBs) 수복에서 CAF-1 p150 의 생물학적인 역할을 연구했다. CAF-1 p150은 DSBs 수복 반응에서 우선적으로 활성화 되어지는 ATM (ataxia telangiectasia mutated) 유전자와 직접 상호작용을 한다. 또한, CAF-1 p150은 DSBs 수복이 일어나는 곳에 보충되고, 동시에 ATM과 함께 집중된다. RNA interference를 통한 CAF-1 p150의 silencing은 ATM과 H2AX의 인산화를 감소시켰고, foci 형성 또한 약간 감소시켰다. 그러나, 53BP1, MDC1, BRCA1, Chk2 and MRN complex같은 DSBs 수복에 관여하는 DNA 수복 단백질의 보충에는 영향을 미치지 않았다. 또한, CAF-1 p150이 knockdown된 세포에서 Chk2의 인산화는 지연되었고 감소되었다. 그렇지만, BRCA1과 p53의 인산화는 증가되었다. DSBs를 일으키는 물질인 Neocasinostatin (NCS)를 처치한 후에 DNA가 손상된 세포는 G2/M기에서 세포주기가 정지하였다. 이러한 결과는 DSBs 수복에서 CAF-1 p150이 ATM 신호전달 경로를 조절함으로써 세포주기 정지에 관여한다는 것을 나타낸다. 결론적으로, DNA double strand breaks 수복에서 CAF-1 p150은 G2/M기 DNA checkpoint를 조절하고 ATM 신호전달 경로를 통해서 DNA 수복에 관여할 것으로 사료된다.|Chromatin assembly factor 1 (CAF-1) is involved in genome integrity and DNA repair. Here we investigated the biological role of CAF-1 p150 to DNA double strand breaks (DSBs) in HeLa cell. CAF-1 p150 directly interacted with ATM ( ataxia telangiectasia mutated), which is primarily activated in response to DSBs. CAF-1 p150 is also recruited to foci with kinetics to, and colocalizes with ATM in response to DSBs. Silencing of CAF-1 p150 via RNA interference decreased phosphorylation levels of ATM and H2AX and foci formation a little, but didn’t affect to recruit another DNA repair protein including 53BP1, MDC1, BRCA1, Chk2 and MRN complex in response to DSBs, although BRCA1 foci slightly induced. Chk2 phosphorylation were delayed and decreased in CAF-1 p150 depleted cells. However, BRCA1 and p53 phosphorylation surprisingly increased in CAF-1 p150 depleted cells. DNA damaged cells after necocasinostatin (NCS) treatment causing DSBs arrested G2/M phase in cell cycle. These results indicate that CAF1 p150 involved in cell cycle arrest by regulating ATM signaling pathway in DSBs. Collectively, our results suggest that CAF-1 p150 regulates G2/M phase DNA checkpoint and involved in DNA repair through ATM signaling pathway in DNA double strand breaks.
Alternative Title
DNA DSB repair에서 CAF1 p150에 의한 ATM pathway 조절 연구
Alternative Author(s)
Cho, Byoung Ok
조선대학교 대학원
일반대학원 의학과
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Table Of Contents
국문초록 = 1
1. Plasmids construction = 10
2. Yeast Two Hybrid screening using mating method = 10
3. Maintenance of Cell Lines = 11
4. Reagents and DNA damage = 11
5. RNA interference = 11
6. Co-immunoprecipitation = 12
7. Immunofluorescence microscopy = 13
8. Western blotting = 14
9. Flow cytometry by PI staining = 15
10. Cytotoxicity assay = 15
1. p150 interacts with ATM = 16
2. Colocalization of p150 and pATM on S1981 following NCS treatment = 16
3. CAF1 p150 was required for the phosphorylation of ATM in DNA DSBs repair following NCS treatment = 16
4. Colocalization of pATM on S1981 and γ-H2AX in 200ng/ml NCS treated HeLa cells after siRNA transfection = 17
5. The involvement of p150 of recruitment of DNA damage response proteins at the double strand breaks site = 17
6. p150 affect checkpoint signal in response to DNA damage = 18
7. Silencing of p150 by RNAi induced G2/M arrest and decreased cell viability after DNA damage = 18
8. Recruitment of p150 and γ-H2AX in DNA double strand breaks site = 19
조선대학교 대학원
조병옥. (2008). The p150 large subunit of chromatin assembly factor 1 regulate ATM pathway in double strand breaks repair.
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General Graduate School > 4. Theses(Ph.D)
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