Rsf-1 의 결합체인 SNF2h 는 Rsf-1 이외에도 다른 여러 단백질들과 결합하여
D. Assembly of γ-H2AX at the DSB-flaking chromatin is regulated by Rsf-1
E. Rsf-1 accumulation at the DNA damage site requires PARP1 activity
그 다음으로 Rsf-1 이 DNA 손상 부위의 어떤 신호를 인지하여 모이는지 확인해보고자 하였다. DNA 손상 시 가장 빠른 신호는 PARP1 에 의한 것으로, 손상 부위에 Poly(ADP-ribosyl) chains (PAR chains) 이 형성되고 이와 결합하여
et al. 2008). Rsf-1 또한 PARP1 과의 연관성이 있을 것이라고 생각되어 제일 먼저
F. PHD domain of Rsf-1 is required for the DNA damage response
Rsf-1 에는 DNA 에 결합한다고 알려진 DTT domain, Glutamic acid 가 많은
다음으로는 Singe double-strand break 을 발생시킬 수 있는 LacO-LacI nuclease
G. Depletion of Rsf-1 diminishes cell survival and DNA damage checkpoint signals
크로마틴 리모델링 인자는 DNA 손상 시 크로마틴 구조를 열어주는 것
활성화 되는 ATR, ATM 의 신호가 30 분부터 4 시간 까지 떨어져 있는 것을 확인하였다. ATR 과 ATM 에 의해 인산화 되어 활성화 되는 체크포인트 단백질인 Chk1, Chk2 또한 4 시간에 현저하게 약화되어져 있는 것을 확인 하였다(Figure 7A). 따라서 Rsf-1 은 DNA 가 손상되면 체크포인트 단백질들의 활성을 조절하여 DNA 손상 복구가 잘 일어날수 있도록 조절 한다는 것을 알 수 있었다. 그 다음으로 Rsf-1 이 없을 때 DNA 손상 복구가 얼마나 잘 일어나는지 확인해보고자 하였다. 세포들에 DNA 를 손상시킬 수 있는 약물들을 처리하고 한 시간 후 배양액을 교체해준 다음 10 일 동안 키운 후 살아남은 세포의 비율을 관찰 하였다. Rsf-1 이 없으면 살아남은 세포가 현저하게 감소되는 것을 확인할 수 있었다(Figure 7B). 외가닥 절단을 유발하는 HU 나 MMS 보다 이중나선절단을 일으키는 Phleomycin 을 처리 하였을 때 손상 복구가 잘 일어나지 않는 것을 확인하였다(Figure 7C). 따라서 Rsf-1 은 DNA 이중나선절단이 발생하였을 때 복구가 잘 일어나도록 작용한다는 것을 알 수 있었다.
Fig. 1. Rsf-1 is recruited to the regions of DNA damage and remained.
Dynamic recruitment of H2AX-GFP, Rsf1-GFP and SNF2h-GFP to DNA damage sites following laser microirradiation. (A) H2AX, Rsf-1 and SNF2h is rapidly recruited to DNA damage sites. Rsf-1 remained after DNA damage upon 10min. (B) Confirm the kinetics of H2AX and Rsf-1 in normal RPE-1 cell line. Scale bars, 10 μm.
Fig. 2. Rsf-1 is recruited to the DNA damage sites.
(A) Endogenous Rsf-1 is recruited to DNA damage sites. DNA damage was generated by laser microirradiation following by immunofluorescence (10 min after damage) with the indicated antibodies. (B) Laser microirradiation induces recruitment of exogenous Rsf-1 to sites of laser-induced DNA damage. (C) Confirm the recruitment of Rsf-1 using FokI system.
(D) Confirm the recruitment of γH2AX, SNF2h and Rsf-1 in normal RPE-1 cell line. Scale bars, 10 μm.
Fig. 3. Rsf-1 contributes to SNF2h recruitment to DNA damage sites.
(A) Recruitment of Rsf-1-GFP in the absence of SNF2h. (B) Cells were assayed as in (A).
The recovery curves were derived from 10 independent cells for each condition. (C) The level of SNF2h in siSNF2h cells compared to sicontrol, was analyzed by western blot. (D) Kinetics of SNF2h-GFP to DSB sites. (E) The curves was monitored in a time course following laser microirradition. (F) The level of Rsf-1. NFU, normalized fluorescence units.
Error bars, SEM. Scale bars, 10 μm.
Fig. 4. Assembly of γ-H2AX at the DSB-flaking chromatin is regulated by Rsf-1.
(A) Kinetics of H2AX and Rsf-1. (B) Cells were assayed as in (A). The recovery curves were derived from 10 independent cells for each condition. (C) The relocation of H2AX and Rsf-1 to DSB(s) in control and knock-down of Rsf-1 cells. (D) Normalized fluorescence of H2AX. NFU, normalized fluorescence units. Error bars, SEM. Scale bars, 10 μm.
Fig. 5. Rsf-1 accumulation at DNA damage sites requires PARP1 activity.
(A) Shown is in vitro IP analysis of Rsf-1-V5 and PARP1 protein. The membrane was denatured and renatured and incubate the membrane with purified bait PAPR1 proteins. (B) Kinetics of PARP1 and Rsf-1 after DNA damage. Cells were treated with (-) and without PARP1 inhibitor (AG-4361) prior to microirradiation. (C, D) Quantification of result in (B).
NFU, normalized fluorescence units. Error bars, SEM. Scale bars, 10 μm.
Fig. 6. PHD domains of Rsf-1 is required for the damage response.
(A) Schematic presentation of domains for 1. (B) U2OS cells were transfected with Rsf-1-WT, Rsf-1-ΔPHD, Rsf-1-D5 and Rsf-1-D6 and followed by laser microirradiation. (C) Quantitative analysis in (B). (D) Localization of Rsf-1-GFP mutants and FokI wild type(WT) and FokI mutant (D450A). (E) Quantitative analysis of the single foci percentage. NFU, normalized fluorescence units. Error bars, SEM. Scale bars, 10 μm. *P < 0.5, **P < 0.01,
***P < 0.005 by Student’s t-test.
Fig. 7. Depletion of Rsf-1 diminishes cell survival and DNA checkpoint signals.
(A) The number of U2OS cells transfected with siCon and siRsf-1 was seeded and treated with different drugs; HU (100 mM), MMS (0.0002%), and phleomycin (50 μg/ml). (B) The relative percentage of cell survival in control (siCtrl) and Rsf-1 depleted (siRsf-1) cells was calculated. (C) Level of Rsf-1. (D) U2OS cells were transfected with si control (siCon) or siRNA for Rsf-1(siRsf-1) and harvested at the indicated time points after treated IR (10 Gy).
DNA damage checkpoint activation were examined by western blot. *P < 0.5 by Student’s t-test.
Ⅳ. 고 찰
따라서 본 연구에서는 크로마틴 리모델링 인자인 Rsf-1 이 DNA 가 손상
Rsf-1 이 H2AX 보다 먼저 DNA 손상 부위로 모이는 것으로 보아 H2AX 에 의한
리보실화 시키지 못하는것 뿐만 아니라 스스로도 리보실화 시키지 못해서 DNA
단백질들을 조절 한다는 것을 알 수 있었다(Fig. 7). Rsf-1 이 DNA 손상 신호 전달 체계에서 매우 빠른 시기에 관여를 하며 그 다음 단계로 신호를 전달하는 것을 용이하게 해준다는 것을 확인하였고 손상 복구 신호에도 영향이 있다는 것을 확인하였다. DNA 손상 복구 신호에도 여러 가지 경로를 통해 일어나고 복구 되기 때문에 Rsf-1 이 어떤 곳에 연관 되어 있는지에 대한 연구가 계속 진행되면 Rsf-1 의 역할과 기능에 대해서 더 많은 이해를 할 수 있을 것이다.
Ⅴ. 결 론
본 연구에서는 DNA 손상 시 크로마틴 리모델링 인자인 Rsf-1 이 손상 신호 전달 체계에서 손상 부위로 빨리 모여서 작용하고 SNF2h 와 H2AX 가 DNA 손상 부위로 이동하는데 중요하게 요구된다는 결과를 얻었다. 또한 PARP1 활성에 의존적으로 DNA 손상 부위로 모이며, PHD 도메인을 통해 PARP1 과의 결합을 통하여 발생한다는 것을 알 수 있었다. 또한 Rsf-1 이 없을 경우 DNA 손상이 발생하면 세포 주기를 멈추는 단백질들의 활성이 낮아져 있고 세포 생존율이 낮은 것을 통해 DNA 손상 시 복구되는 과정에 있어서도 필요하다는 것을 확인할 수 있었다. 따라서 본 논문은 Rsf-1 가 DNA 손상 시 크로마틴 리모델링 인자로써의 역할과 기전에 대해서 밝힌 논문으로 앞으로 DNA 손상 신호 전달 체계와 복구과정에서 Rsf-1 의 중요성을 제시한 논문이다.
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- ABSTRACT -
A functional role of chromatin remodeling factor, Rsf-1 in
the DNA damage signaling pathway
remodeling factors, RSF complex consists of SNF2h ATPase and Rsf-1. Although it has been reported that SNF2h ATPase is recruited to DNA damage sites (DSBs) in poly(ADP-ribosyl) polymerase 1 (PARP1)-dependent manner in DNA damage response (DDR), the function of Rsf-1 is still elusive. Here we show that Rsf-1 is recruited to DSBs confirmed by various cellular analyses (immunofluorescent microscope, micro-irradiation and stably integrated reporter system at a single DNA double-strand break). Rsf-1 rapidly accumulated at the DSB sites. Signal of γ-H2AX is gradually reduced at 10 minutes after micro-irradiation whereas signals of Rsf-1 and SNF2h are still retained over 30 minutes after DNA damage. Moreover, depletion of Rsf-1 attenuates the recruitment of SNF2h and γ-H2AX. In addition, Rsf-1 accumulation at DNA damage sites requires PARP1 activity throughinteracting with PHD domain. Finally, we demonstrate that depletion of Rsf-1 diminishes DNA damage checkpoint signals and repair. Thus, these results reveal a new function of chromatin remodeler Rsf-1 in coordinating DNA signaling and repair.
key words : DNA damage, chromatin remodeling factor, Rsf-1, PARP1, DNA damage checkpoint