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. 2016 Jan;17(1):94-109.
doi: 10.15252/embr.201540964. Epub 2015 Dec 9.

SLFN11 inhibits checkpoint maintenance and homologous recombination repair

Yanhua Mu  1 , Jiangman Lou  1 , Mrinal Srivastava  2 , Bin Zhao  1 , Xin-hua Feng  1 , Ting Liu  3 , Junjie Chen  4 , Jun Huang  5
Affiliations

SLFN11 inhibits checkpoint maintenance and homologous recombination repair

Yanhua Mu et al. EMBO Rep. 2016 Jan.

Abstract

High expression levels of SLFN11 correlate with the sensitivity of human cancer cells to DNA-damaging agents. However, little is known about the underlying mechanism. Here, we show that SLFN11 interacts directly with RPA1 and is recruited to sites of DNA damage in an RPA1-dependent manner. Furthermore, we establish that SLFN11 inhibits checkpoint maintenance and homologous recombination repair by promoting the destabilization of the RPA-ssDNA complex, thereby sensitizing cancer cell lines expressing high endogenous levels of SLFN11 to DNA-damaging agents. Finally, we demonstrate that the RPA1-binding ability of SLFN11 is required for its function in the DNA damage response. Our findings not only provide novel insight into the molecular mechanisms underlying the drug sensitivity of cancer cell lines expressing SLFN11 at high levels, but also suggest that SLFN11 expression can serve as a biomarker to predict responses to DNA-damaging therapeutic agents.

Keywords: DNA damage response; RPA; checkpoint initiation; checkpoint maintenance; homologous recombination repair.

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Figures

Figure 1
Figure 1. SLFN11 is a DNA damage‐responsive protein
  1. A

    Expression analysis of SLFN11 in human cell lines.

  2. B

    Recruitment of endogenous SLFN11 to laser‐induced DNA damage sites. Forty minutes after laser irradiation, cells were stained with antibodies against SLFN11 and RPA2. Scale bar, 10 μm.

  3. C, D

    SLFN11 co‐localizes with RPA2 in DNA damage‐induced nuclear foci. Flag‐tagged SLFN11 was expressed in SF268 (C) or DU145 (D) cells. Foci assembled by this fusion protein and by RPA2 following exposure to CPT (1 μM) for 3 h or IR (10 Gy) for 3 h were detected by immunofluorescence using anti‐Flag and anti‐RPA2 antibodies, respectively. Flag‐SLFN11 foci were detected in green, while RPA2 foci were detected in red. A merged image shows co‐localization. Scale bar, 10 μm.

Source data are available online for this figure.
Figure 2
Figure 2. SLFN11 forms a complex with RPA
  1. A

    HEK293 cells stably expressing SFB‐tagged (S‐tag, Flag epitope tag, and streptavidin‐binding peptide tag) SLFN11 were used for TAP of protein complexes. Tables are summaries of proteins identified by mass spectrometry analysis. Letters in bold indicate the bait proteins.

  2. B

    SLFN11 interacts with RPA, but not with the Morc3 control protein. HEK293 cells were transiently transfected with plasmids encoding SFB‐tagged SLFN11 together with plasmids encoding Myc‐tagged RPA or Morc3. Cell lysates were immunoprecipitated with S beads, and Western blot analysis was performed with anti‐Flag and anti‐Myc antibodies.

  3. C, D

    Association of endogenous SLFN11 with RPA in SF268 (C) or DU145 (D) cells was performed by co‐immunoprecipitation using anti‐SLFN11 or anti‐RPA2 antibody. Nuclease‐treated cell lysates were incubated with protein A agarose beads conjugated with indicated antibodies, and Western blot analysis was performed according to standard procedures.

  4. E

    Direct binding between recombinant GST‐tagged SLFN11 and MBP‐tagged RPA1. Upper panel: SLFN11 was detected by immunoblotting. Lower panel: Purified proteins visualized by Coomassie staining.

  5. F

    Schematic representation of wild‐type and deletion mutants of SLFN11 used in this study.

  6. G

    HEK293 cells were transfected with indicated plasmids. Cell lysates were immunoprecipitated with S beads, and Western blot analysis was performed with anti‐Flag and anti‐Myc antibodies.

Source data are available online for this figure.
Figure 3
Figure 3. RPA recruits SLFN11 to sites of DNA damage
  1. A–C

    SF268 cells stably expressing Flag‐tagged SLFN11 were infected with non‐target or RPA1‐specific lentiviral shRNAs. Forty‐eight hours after infection, cells were treated with CPT (1 μM). Three hours later, cells were subjected to immunostaining using anti‐RPA2 and anti‐Flag antibodies. Representative SLFN11 and RPA2 foci were shown (A). Scale bar, 10 μm. Quantification of RPA2 and SLFN11 foci formation using NIH ImageJ software (B). Error bars represent SD; n = 3. Knockdown efficiency of RPA1 in SF268 cells was confirmed by immunoblotting (C).

  2. D

    RPA1 is required for the accumulation of SLFN11 at laser‐induced DNA damage tracks. A SF268 cell line stably expressing wild‐type RPA1 under the control of a tetracycline‐inducible promoter was generated. The resulting cell line infected with RPA1‐specific lentiviral shRNA targeting the 3′ UTR of RPA1 transcript was induced by doxycycline addition for 48 h and then laser micro‐irradiated. Thirty minutes later, cells were fixed and stained with anti‐RPA2 and anti‐SLFN11 antibodies. Scale bar, 10 μm.

  3. E

    The C‐terminus of SLFN11 is required for its foci formation. SF268 cells were transfected with plasmids encoding Flag‐tagged wild‐type or mutant SLFN11. Immunostaining experiments were performed 3 h after CPT (1 μM) treatment using the indicated antibodies. Scale bar, 10 μm.

  4. F–H

    SF268 cells stably expressing Flag‐tagged SLFN11 were transfected twice with the indicated siRNAs. Forty‐eight hours after second transfection, cells were treated with CPT (1 μM). Three hours later, cells were subjected to immunostaining using anti‐RPA2 and anti‐Flag antibodies. Representative SLFN11 and RPA2 foci were shown (F). Scale bar, 10 μm. Quantification of RPA2 and SLFN11 foci formation using NIH ImageJ software (G). Error bars represent SD; n = 3. Knockdown efficiency of Mre11 and CtIP in SF268 cells was confirmed by immunoblotting (H).

Source data are available online for this figure.
Figure 4
Figure 4. SLFN11 inhibits maintenance but not initiation of DNA damage checkpoint
  1. A

    Analysis of the kinetics of CHK1 and CHK2 phosphorylation in cell lines expressing SLFN11 at high (SF268 and DU145) and very low or undetectable levels (HeLa and U2OS), respectively. The indicated cells were treated with CPT (1 μM) for 1 h. Cells were then washed, shifted to fresh medium (time 0), and harvested at the indicated time points for immunoblotting with the indicated antibodies.

  2. B, C

    HeLa (B) or U2OS (C) cells overexpressing exogenous SLFN11 display a specific defect in checkpoint maintenance. A HeLa or U2OS cell line to express Flag‐tagged SLFN11 under the control of a tetracycline‐inducible promoter was generated. The resulting cells were treated with CPT (1 μM) for 1 h. Cells were then washed, shifted to fresh medium (time 0), and harvested at the indicated time points for immunoblotting with the indicated antibodies.

  3. D

    Overexpression of exogenous SLFN11 rendered both HeLa and U2OS cells more sensitive to CPT treatment. A HeLa or U2OS cell line stably expressing Flag‐tagged SLFN11 was generated. The resulting cells were treated with various doses of CPT for 24 h, then shifted to fresh medium and permitted to grow for 14 days before staining. Error bars represent SD; n = 3.

  4. E

    Downregulation of SLFN11 delays the decline of phosphorylated CHK1 and CHK2 in SF268 cells. SF268 cells were transfected twice with control siRNA or siRNAs specific for SLFN11. Forty‐eight hours after transfection, cells were treated with CPT (1 μM) for 1 h. Cells were then washed, shifted to fresh medium (time 0), and harvested at the indicated time points for immunoblotting with the indicated antibodies.

  5. F

    SLFN11 knockout SF268 cells were treated with CPT (1 μM) for 1 h. Cells were then washed, shifted to fresh medium (time 0), and harvested at the indicated time points for immunoblotting with the indicated antibodies.

  6. G

    A SLFN11‐deficient SF268 cell line stably expressing Flag‐tagged SLFN11 was generated. The resulting cell line was treated with CPT (1 μM) for 1 h. Cells were then washed and shifted to fresh medium. Twenty‐four hours later, cells were harvested for immunoblotting with the indicated antibodies.

Source data are available online for this figure.
Figure EV1
Figure EV1. SLFN11 inhibits maintenance but not initiation of DNA damage checkpoint

  1. Characterization of SLFN11 antibody. HeLa cells stably expressing Flag‐tagged SLFN11 were lysed and immunoblotting was performed using anti‐Flag or anti‐SLFN11 antibody.

  2. Downregulation of SLFN11 delays the decline of phosphorylated CHK1 and CHK2 in DU145 cells. DU145 cells were transfected twice with control siRNA or siRNAs specific for SLFN11. Forty‐eight hours after transfection, cells were treated with CPT (1 μM) for 1 h. Cells were then washed, shifted to fresh medium (time 0), and harvested at the indicated time points for immunoblotting with the indicated antibodies (the asterisk indicates a non‐specific band).

  3. Knockout of SLFN11 delays the decline of phosphorylated CHK1 and CHK2 in SF268 cells. SLFN11 knockout SF268 cells were treated with IR (10 Gy) for 1 h. Cells were then washed, shifted to fresh medium (time 0), and harvested at the indicated time points for immunoblotting with the indicated antibodies.

Figure EV2
Figure EV2. The effects of SLFN11 deficiency on DNA replication and cell cycle progression

  1. SLFN11 deficiency has no effect on DNA replication under normal conditions. BrdU incorporation assays were carried out as described in the Materials and Methods section.

  2. SLFN11 deficiency caused an increased percentage of cells in the G2 phase of the cell cycle after CPT withdrawal, accompanied by a decrease in the percentage of S phase cells. Cell cycle analysis was carried out as described in the Materials and Methods section.

Figure 5
Figure 5. SLFN11 inhibits homologous recombination repair
  1. A, B

    SLFN11 suppresses SCE. Representative metaphase spreads showing SCEs from wild‐type and SLFN11‐deficient SF268 cells (red arrows) (A). SCEs were scored for 50 metaphase spreads for each cell line (B). Each point represents the total number of SCEs in a single metaphase spread, and black bars indicate the average value for all spreads. Statistical significance was determined using the Mann–Whitney U‐test (***P < 0.0001).

  2. C–E

    Overexpression of exogenous SLFN11 in U2OS cells results in decreased HR‐directed DNA repair. A U2OS DR‐GFP cell line stably expressing Flag‐tagged SLFN11 was generated (C, D). The resulting cells were electroporated with an I‐SceI expression plasmid. Forty‐eight hours after electroporation, cells were harvested and assayed for GFP expression by FACS analysis. Error bars represent SD; n = 3 (E).

Source data are available online for this figure.
Figure 6
Figure 6. SLFN11 promotes the destabilization of RPA–ssDNA complex
  1. A, B

    Loss of SLFN11 in SF268 cells delays the decline of RPA foci. Wild‐type or SLFN11‐deficient SF268 cells were treated with CPT (1 μM) for 1 h. Cells were then washed, shifted to fresh medium (time 0), and processed at the indicated time points for immunofluorescence by using anti‐RPA2 antibody. Representative RPA2 foci were shown (A). Scale bar, 10 μm. Quantification of RPA2 foci formation using NIH ImageJ software (B). Error bars represent SD; n = 3.

  2. C, D

    SLFN11 depletion in DU145 cells delays the decline of RPA foci. Wild‐type or SLFN11‐depleted DU145 cells were treated with CPT (1 μM) for 1 h. Cells were then washed, shifted to fresh medium (time 0), and processed at the indicated time points for immunofluorescence by using anti‐RPA2 antibody. Representative RPA2 foci were shown (C). Scale bar, 10 μm. Quantification of RPA2 foci formation using NIH ImageJ software (D). Error bars represent SD; n = 3.

  3. E

    SLFN11‐deficient SF268 cells exhibited a substantial increase in chromatin‐bound RPA after CPT withdrawal. Wild‐type or SLFN11‐deficient SF268 cells were treated with CPT (1 μM) for 1 h. Cells were then washed, shifted to fresh medium (time 0), and harvested at the indicated time points for immunoblotting with the indicated antibodies.

  4. F, G

    Loss of SLFN11 in SF268 cells delays the decline of BrdU foci. SLFN11 wild‐type or SLFN11‐deficient SF268 cells were labeled with BrdU and 24 h later were treated with CPT (1 μM) for 1 h. Cells were then washed, shifted to fresh medium (time 0), and processed at the indicated time points for immunofluorescence by using anti‐BrdU antibody under non‐denaturing conditions. Representative BrdU foci were shown (G). Scale bar, 10 μm. Quantification of BrdU foci formation using NIH ImageJ software (F). Error bars represent SD; n = 3.

Source data are available online for this figure.
Figure EV3
Figure EV3. Loss of SLFN11 in SF268 cells delays the decline of RPA foci
  1. A, B

    Wild‐type or SLFN11‐deficient SF268 cells were treated with IR (10 Gy) and allowed to recover for the indicated times. Cells were then fixed and processed for immunofluorescence by using anti‐RPA2 antibody (A). Scale bar, 10 μm. Quantification of RPA2 foci formation using NIH ImageJ software (B). Error bars represent SD; n = 3.

Figure EV4
Figure EV4. The ATPase activity of SLFN11 is not required for its function in the DNA damage response
  1. A, B

    SDS–PAGE profile of purified wild type and mutant of SLFN11 (A). Wild‐type SLFN11, but not the K605A/D668M mutant, possesses ATP hydrolysis activity (B). ATPase assays were carried out as described in the Materials and Methods section. Error bars represent SD; n = 3.

  2. C, D

    A SLFN11‐deficient SF268 cell line to express Flag‐tagged wild‐type SLFN11, the K605M/D668A mutant, or the Δ5 mutant was generated. Cells were treated with various doses of IR (C) or UV (D). The medium was replaced 24 h later, and cells were then permitted to grow for 14 days before staining. Error bars represent SD; n = 3.

Figure 7
Figure 7. The RPA1‐binding ability of SLFN11 is required for its function in DNA damage response
  1. A–C

    A SLFN11‐deficient SF268 cell line to express Flag‐tagged wild‐type SLFN11, the K605M/D668A mutant, or the Δ5 mutant was generated. The resulting cell line was treated with CPT (1 μM) for 1 h. Cells were then washed and shifted to fresh medium. Twenty‐four hours later, cells were fixed and processed for RPA2 or RAD51 immunofluorescence. Scale bar, 10 μm. Error bars represent SD; n = 3.

  2. D

    The ability of SLFN11‐deficient SF268 cells to maintain DNA damage checkpoint was disrupted by the re‐expression of wild‐type SLFN11 and the K605M/D668A mutant, but not the Δ5 mutant. Cells were treated with CPT (1 μM) for 1 h, then washed and shifted to fresh medium. Twenty‐four hours later, cells were harvested for immunoblotting with the indicated antibodies.

  3. E

    Re‐introduction of wild‐type SLFN11 and the K605M/D668A mutant, but not the Δ5 mutant, re‐sensitized the SLFN11‐deficient SF268 cells to DNA‐damaging agents. Cells were treated with various doses of CPT for 24 h, then shifted to fresh medium and permitted to grow for 14 days before staining. Error bars represent SD; n = 3.

  4. F

    A proposed model for the role of SLFN11 in the regulation of DNA damage response. Please refer to the main text for details.

Source data are available online for this figure.
See this image and copyright information in PMC

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