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. 2013 Mar 1;288(9):6342-50.
doi: 10.1074/jbc.M112.411603. Epub 2013 Jan 16.

Alpha thalassemia/mental retardation syndrome X-linked gene product ATRX is required for proper replication restart and cellular resistance to replication stress

Justin Wai-Chung Leung  1 , Gargi Ghosal, Wenqi Wang, Xi Shen, Jiadong Wang, Lei Li, Junjie Chen
Affiliations

"VSports注册入口" Alpha thalassemia/mental retardation syndrome X-linked gene product ATRX is required for proper replication restart and cellular resistance to replication stress

Justin Wai-Chung Leung et al. J Biol Chem. .

Abstract

Alpha thalassemia/mental retardation syndrome X-linked (ATRX) is a member of the SWI/SNF protein family of DNA-dependent ATPases VSports手机版. It functions as a chromatin remodeler and is classified as an SNF2-like helicase. Here, we showed somatic knock-out of ATRX displayed perturbed S-phase progression as well as hypersensitivity to replication stress. ATRX is recruited to sites of DNA damage, required for efficient checkpoint activation and faithful replication restart. In addition, we identified ATRX as a binding partner of MRE11-RAD50-NBS1 (MRN) complex. Together, these results suggest a non-canonical function of ATRX in guarding genomic stability. .

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Figures

FIGURE 1.
FIGURE 1.
Generation of ATRX somatic knock-out cells. A, schematic diagram of the conserved functional domain of ATRX. B, schematic representation of the ATRX genomic DNA and exon distribution. C, targeting and screening strategies for generation of somatic knock-out cells. Homology arms were generated by PCR and cloned into an AAV vector flanked by neo and two loxp sites. Primers for screening were designed outside the homology arms as indicated. D, knock-out cells were verified by PCR as indicated. E and F, ATRX KO cells were confirmed by immunostaining and Western blotting analysis. G, representative micrographs showing immunofluorescence staining of ATRX and PML nuclear bodies in control and ATRX KO cells.
FIGURE 2.
FIGURE 2.
ATRX is involved in replication stress. A, clonogenic survival assay of wild-type and two independent clones of ATRX-deficient HCT116 cells following HU, APH, MMC, and IR treatment as indicated. B, cell-cycle distributions of wild-type and ATRX-deficient HCT116 cells was analyzed by FACS and presented as percentages of cells in G1, S, and G2/M phases. C, cells were released from double thymidine block (1 mm) and pulsed with BrdU 30 min prior harvest. BrdU incorporation was analyzed by staining with anti-BrdU antibody and quantified by flow cytometry. Results were the average of three independent experiments and presented as mean ± S.E. *, p < 0.01. D, FACS analysis of cell cycle and BrdU incorporation after double thymidine block and release. E, cells were fixed 4 h after being released from double thymidine block and stained with antibodies as indicated.
FIGURE 3.
FIGURE 3.
ATRX is recruited to the DNA damage site and promotes checkpoint activation. A, wild-type and ATRX-deficient HCT116 cells were mock-treated or treated with 4 mm HU for 2 h. Cells were fixed and stained with indicated antibodies and counterstained with DAPI. B, cells were treated with indicated doses of HU for 2 h and whole cell extracts were subjected to Western blotting using indicated antibodies. C, HeLa cells were treated with laser induced micro-irradiation and stained with indicated antibodies. D, wild-type and ATRX knock-out HCT116 cells were treated with the indicated concentrations of HU. Cells were harvested, fractionated, and Western blotting was conducted using indicated antibodies. E, cells were treated with indicated doses of HU. Whole cell extracts were prepared and subjected to Western blotting as indicated.
FIGURE 4.
FIGURE 4.
ATRX is required for replication restart. A, mitotic entry after HU release in wild-type and ATRX-deficient HCT116 cells. Cells were pre-treated with 2 mm HU for 24 h before releasing. Data represented averages of three independent experiments and were shown as mean ± S.E. B, replication restart after HU treatment. At least 100 DNA fibers were counted per condition. Error bar represented S.E. C, synchronized cells in early S phase were labeled with IdU for 15 min, treated with HU for 6 h, released and labeled with CIdU for 15–120 min before preparing DNA fibers. The average length of at least 80 replication tracks was plotted for each time point. C, outline of the protocol used to quantify the sites of newly initiated replication fork at indicated time points after the removal of HU. The percentage of newly initiated replication forks was determined by dividing the number of CIdU-containing track (C) by the number of total tracks (A+B+C). *, p < 0.001. CIdU, 5-chloro-2-deoxyuridine; IdU, 5-iodo-2-deoxyuridine; HU, hydroxyurea.
FIGURE 5.
FIGURE 5.
ATRX interacts with MRN complex. A, HEK293T cells stably expressing SFB-ATRX were subjected to tandem affinity purification and mass spectrometry analysis. Red indicates the bait protein, and yellow indicates the MRN complex identified by TAP. The number of peptides recovered from mass spectrometry study was also presented. B, HEK293T cells were transfected with constructs encoding SFB-ATRX together with constructs encoding Myc-NBS1, Myc-MRE11A, or Myc-RAD50. Co-precipitation and immunoblotting were carried out as indicated.
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