Ubiquitin-dependent recruitment of the Bloom syndrome helicase upon replication stress is required to suppress homologous recombination

Abstract

Limiting the levels of homologous recombination (HR) that occur at sites of DNA damage is a major role of BLM helicase. However, very little is known about the mechanisms dictating its relocalization to these sites. Here, we demonstrate that the ubiquitin/SUMO-dependent DNA damage response (UbS-DDR), controlled by the E3 ligases RNF8/RNF168, triggers BLM recruitment to sites of replication fork stalling via ubiquitylation in the N-terminal region of BLM and subsequent BLM binding to the ubiquitin-interacting motifs of RAP80. Furthermore, we show that this mechanism of BLM relocalization is essential for BLM's ability to suppress excessive/uncontrolled HR at stalled replication forks. Unexpectedly, we also uncovered a requirement for RNF8-dependent ubiquitylation of BLM and PML for maintaining the integrity of PML-associated nuclear bodies and as a consequence the localization of BLM to these structures. Lastly, we identified a novel role for RAP80 in preventing proteasomal degradation of BLM in unstressed cells. Taken together, these data highlight an important biochemical link between the UbS-DDR and BLM-dependent pathways involved in maintaining genome stability.

Figure 1

Recruitment of BLM to the sites of stalled replication depends on RNF8 and RNF168. (A, B) BLM colocalizes with PML-NBs and ubiquitin in the absence of DNA damage. GFP-BLM cells were either grown under asynchronous conditions or treated with HU (+HU). GFP-BLM cells were co-stained with (A) anti-PML antibody and (B) anti-ubiquitin FK2 antibody. Nuclei are stained by DAPI. Scale 5 μM. (C) Lack of E3 ligases leads to lack of BLM recruitment after HU treatment. GFP-BLM cells were transfected with either the control siRNA or siRNAs against Ubc5, Ubc13, RNF8 or RNF168. The cells were treated with HU. GFP-BLM was visualized along with 53BP1. Scale 5 μM. (D) BLM accumulates in the nucleolus in the absence of RNF8. Same as (C) except only siRNA Control or siRNA RNF8 #1 was used. GFP-BLM cells were stained with anti-RNF8 antibodies. Nuclei visualized by DAPI. Scale 5 μM. (E) RNF168 colocalizes with BLM after HU treatment. RIDDLE cells complemented with HA-tagged RNF168 were grown in the presence of HU (+HU) or in the postwash condition (+HU/PW). One of the cells under either conditions is zoomed. Immunofluorescence was carried out with anti-BLM (A300–110A) and anti-HA antibodies. Scale 5 μM.

Figure 2

BLM is ubiquitylated by RNF8/RNF168 in vitro. (A) Ubc13/RNF8 leads to the poly-ubiquitylation of BLM. In vitro ubiquitylation reactions were carried out using recombinant full-length BLM and RNF8 as the E3 ligase. The ubiquitylated BLM was detected by an anti-BLM antibody (A300-120A). A parallel reaction was also carried out using K63R ubiquitin mutant. (B) BLM is ubiquitylated by RNF168/Ubc13. Ubiquitylation reactions were carried out with BLM using Ubc13 as the E2 and RNF168 as the E3 ligase. Westerns were carried out with antibody against BLM (A300-120A). The Coomassie stained gel for purified BLM used for ubiquitylation is shown at the bottom. (C) Poly-ubiquitylation of BLM mediated by Ubc13/RNF168 is K63 linked. In vitro ubiquitylation reactions using BLM as the substrate were carried out using either wild-type ubiquitin or ubiquitin mutant (K63R). Western analysis was carried out with antibody against (top) BLM (A300-120A) or (bottom) ubiquitin (P4D1). (D) The RING domain of RNF8 and RNF168 enhances the Ubc13-/RNF168-mediated ubiquitylation of BLM. In vitro BLM ubiquitylation reactions were carried out using either wild-type RNF8/RNF168 or their RING deleted/disrupted counterpart. The ubiquitylated forms of BLM were detected by using anti-BLM antibody (A300-120A) (for RNF168, left) or anti-Ubiquitin (P4D1) (for RNF8, right). For RNF8-dependent ubiquitylation, the Coomassie stained gel for purified BLM used for ubiquitylation is shown at the bottom. (E) BLM is ubiquitylated by RNF168/Ubc5a. Ubiquitylation reactions were carried out with BLM using Ubc5a as the E2 and RNF168 as the E3 ligase. Westerns were carried out with antibody against ubiquitin (P4D1). (F) Both Ubc5a and Ubc13 can act as the E2 conjugating enzyme for BLM. (F) Same as (A, E), except parallel ubiquitylation reactions were carried out using either Ubc5a or Ubc13. Western analysis was carried out with antibodies against ubiquitin-P4D1 (top) or FK1 (middle). The blots were further probed with anti-BLM antibody (A300-110A). Source data for this figure is available on the online supplementary information page.

Figure 3

BLM is ubiquitylated by RNF8 and RNF168 in vivo. (A) Loss of RNF8 leads to decreased K63-linked ubiquitylation of BLM in vivo. U2OS shRNF8 cells were grown in the absence or presence of Doxycycline (Dox), without or with HU co-treatment. (Top) Nuclear extracts were probed with antibodies against RNF8, BLM (A300-110A) and Lamin A/C. (Middle and bottom) Immunoprecipitations were carried out with anti-Ubiquitin K63-linkage specific antibody (or the corresponding IgG). The immunoprecipitates were probed with antibodies against BLM (A300-120A). (B, C). Loss of RNF168 leads to decreased K63-linked ubiquitylation of BLM in vivo after HU treatment. Same as (A) except U2OS shRNF168 cells were used in (B) and RIDDLE syndrome cells complemented with either empty vector or HA-tagged RNF168 cells were used in (C). U2OS shRNF168 cells were grown in the absence or presence of Doxycycline (Dox). Both U2OS shRNF168 and RIDDLE syndrome cells were treated with HU. The direct westerns are on top while immunoprecipitation with anti-Ub (K63) antibodies (or the corresponding IgG) followed by anti-BLM (A300-120A) westerns is shown at the bottom. Source data for this figure is available on the online supplementary information page.

Figure 4

Ubiquitylation of BLM at 105, 225 and 259 is required for BLM recruitment to the sites of stalled replication. (A) BLM undergoes K63-linked ubiquitylation at lysines residues 105, 225 and 259. (Left) Coomassie gel demonstrating the expression of GST-tagged BLM (WT) or BLM (K105R), BLM (K225R), BLM (K259R). (Right) In vitro ubiquitylation reactions were carried out using equal amounts wild-type BLM or the three BLM mutants (K105R, K225R and K259R). Western blots were carried out with antibodies against BLM (A300-120A). Two different exposures are shown to demonstrate the differential ubiquitylation. (B) Complete abrogation of RNF8-/RNF168-mediated ubiquitylation in BLM (3K-R) mutant. Same as (A) except BLM (3K-R) mutant was used. Expression of wild-type GST-tagged BLM and BLM (3K-R) mutant via Coomassie staining is shown on the left. RNF8 or RNF168 was used as the E3 ligase in parallel reactions. Two different exposures are shown to demonstrate the differential ubiquitylation. (C) Mutation of lysines at 105, 225 and 259 on BLM leads to loss of BLM poly-ubiquitylation after DNA damage. Wild-type BLM or (3K-R) mutant was overexpressed in 293T cells and subsequently treated with HU. (Top) The expression levels were determined by western analysis using antibodies against BLM (A300-110A) and hsp90. (Bottom) Immunoprecipitations were carried out using anti-K63-linked ubiquitin antibody. Immunoprecipitates were probed with antibodies against GFP or the corresponding IgG. Equal amount of antibody used for immunoprecipitation is demonstrated by comparing the IgG level. (D) Ubiquitylation of BLM at lysines 105, 225 and 259 is required for its recruitment to the stalled replication forks. 293T cells were transfected with EGFP-tagged wild-type BLM or BLM (K105R), BLM (K225R), BLM (K259R). Post-transfection the cells were treated with HU for 24 h. Transfected cells were tracked. Nuclei are stained by DAPI. Scale 5 μM. (E) Mutation of lysines at 105, 225 and 259 on BLM leads to enhanced nucleolar accumulation of BLM after HU treatment. (Top) Same as (D) except after transfection with either BLM (WT) or BLM (3K-R), the transfected cells were tracked. Nuclei are stained by DAPI. Scale 5 μM. (Bottom) Quantitation of (D) and (E, top). Source data for this figure is available on the online supplementary information page.

Figure 5

Destabilization of PML-NBs in the absence of RNF8 leads to the accumulation of BLM in nucleolus. (A, B) RNF8 knockdown destabilizes PML nuclear bodies. (A) GFP-BLM cells were transfected with either siRNA Control or siRNA RNF8 #1 and the cells were grown in asynchronous (Asyn.) conditions. Whole cell lysates were probed with antibodies against PML (PG-M3), BLM (A300-110A) and hsp90. (B) Same as (A), except GFP-BLM was tracked with PML-NBs by carrying out immunofluorescence with anti-PML (PG-M3) antibody. Nuclei are stained by DAPI. Scale 5 μM. (C) Ubc13/RNF8 leads to the poly-ubiquitylation of PML. In vitro ubiquitylation reaction was carried out using recombinant full-length PML III (left) or PML IV (right) and RNF8 as the E3 ligase. Poly-ubiquitylated forms of PML isoforms were detected by anti-ubiquitin antibody (FK2). Parallel reactions were also carried out using K63R ubiquitin mutant. The Coomassie-stained gels for purified PML III and IV used for ubiquitylation are shown. (D) PML knockdown relocated BLM to the nucleolus. Same as (B) except the GFP-BLM cells were transfected with either siRNA Control or siRNA PML. Post-transfection the cells were grown in asynchronous conditions. Source data for this figure is available on the online supplementary information page.

Figure 6

RAP80 regulates BLM stability. (A, B) BLM and RAP80 interact in vivo. Immunoprecipitation of BLM was carried out in (A) BS/A-15 cells or only in (B) A-15 cells using either (A) anti-RAP80 or (B) anti-BLM (NB 100–161) antibody or their corresponding IgG. Cells were grown in three conditions, namely Asynchronous (Asyn.), after HU treatment (+HU) and in postwash (+HU/PW) condition. Western blotting on the immunoprecipitates was carried out with antibodies against anti-BLM (NB 100-161) and anti-RAP80. (C) BLM and RAP80 colocalize after replication arrest. GFP-BLM cells were either grown under asynchronous conditions or treated with HU (+HU). GFP-BLM cells were co-stained with anti-RAP80 antibody. Scale 5 μM. (D) RAP80 interacts with BLM via its UIM after exposure to HU. 293T cells were transfected with either EGFP-tagged wild-type RAP80 or its ΔUIM mutant. (Left) Whole cell lysates were probed with antibodies against BLM (A300-110A) or GFP. CR band represents a cross-reactive band. (Right) Immunoprecipitations were carried out with antibodies against RAP80 or IgG control. Immunoprecipitates were probed with antibodies against BLM (A300-110A) and RAP80. IgG acted as a control for the usage of equal amount of antibody during immunoprecipitation. (E) Overexpression of RAP80 stabilizes BLM. 293T cells were either left untransfected or transfected with either Flag-BLM or GFP-RAP80 or both. Whole cell lysates were probed with antibodies against BLM (NB 100-161), GFP and hsp90. (*) is a cross contaminating band. (F) Ablation of RAP80 destabilizes BLM. GFP-BLM cells were transfected with siRNA control, siRNA BLM, siRNA RAP80 or siRNA BLM and siRNA RAP80. Whole cell lysates were probed with antibodies against BLM (A300-110A), RAP80 and hsp90. (G, H) Destabilization of BLM level after ablation of RAP80 is reversed after treatment with proteasome inhibitors. GFP-BLM cells were either transfected with either siRNA control, or siRNA RAP80 and grown in the absence or presence of (G) LLnL or (H) MG132. Whole cell lysates were probed with antibodies against BLM (A300-110A), RAP80 and hsp90. (I) BLM physically interacts with RAP80. (Top) Glutathione-Sepharose bound GST or GST-BLM (1–1417) was incubated with equal amounts of the lysates from 293T cells transfected with GFP-RAP80. The bound GFP-RAP80 was probed with antibody against GFP. (Bottom) Coomassie gel indicating purified GST and GST-BLM (1–1417). (J) Interaction between GST-BLM and the internal deletions and end fusion proteins of RAP80. (Left) Western blot depicting the expression of the wild-type RAP80 and the RAP80 internal deletions and the end fusion constructs as detected after probing with antibodies against GFP. (Right) Bead bound purified GST-BLM was incubated with lysates expressing EGFP-tagged RAP80 mutants. Bound RAP80 was detected by probing with anti-GFP antibody. (*) represents the EGFP-RAP80 fragments, which interact with GST-BLM. Source data for this figure is available on the online supplementary information page.

Figure 7

Targeting of BLM to the chromatin in the absence of RNF8/RNF168 decreases HR. (A, E) Expression of chromatin targeted BLM fusions. (A) Untagged BLM (WT), H2AX-BLM (WT) or MDC1 FHA-BLM (WT) (cloned in pIRES vector) (E) AcGFP-tagged BLM (WT), H2AX-BLM (WT) or MDC1 FHA-BLM (WT) (cloned in AcGFP-N1 vector) were expressed in 293T cells. Western analysis was carried out with antibodies against (A) BLM (A300-110A) and hsp90 and (E) GFP and hsp90. (B, C) Chromatin targeted BLM rescues the hyper-recombinogenic phenotype of cells not expressing either RNF8 or RNF168. (B) U2OS shRNF8 or (C) U2OS shRNF168 cells were grown with or without Dox treatment. The cells were either transfected with pBHRF construct alone or along with BLM, MDC1 FHA-BLM or H2AX-BLM. Forty eight hours post transfection, the host cell reactivation assays were carried out after treatment with HU for the final 16 h. The GFP/BFP ratio was determined as the readout for the extent of HR. (D) Lack of BLM ubiquitylation prevents its anti-recombinogenic function. A-15/BS cells were transfected with pBHRF. In BS cells, co-transfection was carried out with either wild-type BLM or the BLM (3K-R) mutant. Forty eight hours post transfection, the host cell reactivation assays were carried out after treatment with HU for the final 16 h. The GFP/BFP ratio was determined as the readout for the extent of HR. (F) Direct chromatin targeting BLM rescues the loss of ubiquitylation machinery. (Top) BLM fusions (MDC1 FHA-EGFP-BLM and H2AX-EGFP-BLM) were transiently transfected into U2OS shRNF8 cells in the presence of Dox. Transfection was also carried out in parallel with BLM (WT). The cells were treated with HU. EGFP-BLM expression of the fusion proteins was tracked. Nuclei were stained by DAPI. Scale 5 μM. (Bottom) The percentage of cells with nucleolar BLM was determined from the above experiment. Source data for this figure is available on the online supplementary information page.

Figure 8

Schematic diagram depicting the recruitment of BLM to the chromatin after DNA damage. In the absence of DNA damage, BLM remains in the PML nuclear bodies (PML-NBs) complexed with RAP80. Absence of RNF8 prevents the mono-ubiquitylation of BLM and poly-ubiquitylation of PML-NBs. These ubiquitylation events together result in the disruption of the PML-NBs and accumulation of BLM in the nucleolus. Ablation of RAP80 leads to proteasomal degradation of BLM. In the presence of replication stress, BLM is poly-ubiquitylated in the PML-NBs by RNF8. The poly-ubiquitylated BLM interacts with the ubiquitin-interacting motifs (UIMs) of RAP80, which recruits BLM to the site of damage on the chromatin. At the site of the lesion, BLM is acted on sequentially by RNF8 and RNF168. These two E3 ligases amplify the ubiquitylation signal and retain BLM at the site of the damage, so that BLM can subsequently carry out its functions during HRR.