Ubiquitin-dependent recruitment of the Bloom Syndrome helicase upon replication stress is required to suppress homologous recombination
2013; Springer Nature; Volume: 32; Issue: 12 Linguagem: Inglês
10.1038/emboj.2013.117
ISSN1460-2075
AutoresShweta Tikoo, Vinoth Madhavan, Mansoor Hussain, Edward S. Miller, Prateek Arora, Anastasia Zlatanou, Priyanka Modi, Kelly Townsend, Grant S. Stewart, Sagar Sengupta,
Tópico(s)Telomeres, Telomerase, and Senescence
ResumoArticle24 May 2013free access Source Data Ubiquitin-dependent recruitment of the Bloom Syndrome helicase upon replication stress is required to suppress homologous recombination Shweta Tikoo Shweta Tikoo National Institute of Immunology, Aruna Asaf Ali Marg, New Delhi, India Search for more papers by this author Vinoth Madhavan Vinoth Madhavan National Institute of Immunology, Aruna Asaf Ali Marg, New Delhi, India Search for more papers by this author Mansoor Hussain Mansoor Hussain National Institute of Immunology, Aruna Asaf Ali Marg, New Delhi, India Search for more papers by this author Edward S Miller Edward S Miller School of Cancer Sciences, College of Medical and Dental Sciences, University of Birmingham, Birmingham, UK Search for more papers by this author Prateek Arora Prateek Arora National Institute of Immunology, Aruna Asaf Ali Marg, New Delhi, India Search for more papers by this author Anastasia Zlatanou Anastasia Zlatanou School of Cancer Sciences, College of Medical and Dental Sciences, University of Birmingham, Birmingham, UK Search for more papers by this author Priyanka Modi Priyanka Modi National Institute of Immunology, Aruna Asaf Ali Marg, New Delhi, India Search for more papers by this author Kelly Townsend Kelly Townsend School of Cancer Sciences, College of Medical and Dental Sciences, University of Birmingham, Birmingham, UK Search for more papers by this author Grant S Stewart Corresponding Author Grant S Stewart School of Cancer Sciences, College of Medical and Dental Sciences, University of Birmingham, Birmingham, UK Search for more papers by this author Sagar Sengupta Corresponding Author Sagar Sengupta National Institute of Immunology, Aruna Asaf Ali Marg, New Delhi, India Search for more papers by this author Shweta Tikoo Shweta Tikoo National Institute of Immunology, Aruna Asaf Ali Marg, New Delhi, India Search for more papers by this author Vinoth Madhavan Vinoth Madhavan National Institute of Immunology, Aruna Asaf Ali Marg, New Delhi, India Search for more papers by this author Mansoor Hussain Mansoor Hussain National Institute of Immunology, Aruna Asaf Ali Marg, New Delhi, India Search for more papers by this author Edward S Miller Edward S Miller School of Cancer Sciences, College of Medical and Dental Sciences, University of Birmingham, Birmingham, UK Search for more papers by this author Prateek Arora Prateek Arora National Institute of Immunology, Aruna Asaf Ali Marg, New Delhi, India Search for more papers by this author Anastasia Zlatanou Anastasia Zlatanou School of Cancer Sciences, College of Medical and Dental Sciences, University of Birmingham, Birmingham, UK Search for more papers by this author Priyanka Modi Priyanka Modi National Institute of Immunology, Aruna Asaf Ali Marg, New Delhi, India Search for more papers by this author Kelly Townsend Kelly Townsend School of Cancer Sciences, College of Medical and Dental Sciences, University of Birmingham, Birmingham, UK Search for more papers by this author Grant S Stewart Corresponding Author Grant S Stewart School of Cancer Sciences, College of Medical and Dental Sciences, University of Birmingham, Birmingham, UK Search for more papers by this author Sagar Sengupta Corresponding Author Sagar Sengupta National Institute of Immunology, Aruna Asaf Ali Marg, New Delhi, India Search for more papers by this author Author Information Shweta Tikoo1, Vinoth Madhavan1, Mansoor Hussain1, Edward S Miller2, Prateek Arora1, Anastasia Zlatanou2, Priyanka Modi1, Kelly Townsend2, Grant S Stewart 2 and Sagar Sengupta 1 1National Institute of Immunology, Aruna Asaf Ali Marg, New Delhi, India 2School of Cancer Sciences, College of Medical and Dental Sciences, University of Birmingham, Birmingham, UK *Corresponding authors. IBR West Extension, First Floor, School of Cancer Sciences, College of Medical and Dental Sciences, University of Birmingham, Vincent Drive, Edgbaston, Birmingham B15 2TT, UK. Tel:+44 (0)121 414 9168; Fax:+44 (0)121 414 4486; E-mail: [email protected] Institute of Immunology, Aruna Asaf Ali Marg, JNU Campus, New Delhi 110067, India. Tel.:+91 11 2670 3786; Fax:+91 11 2616 2125; E-mail: [email protected] The EMBO Journal (2013)32:1778-1792https://doi.org/10.1038/emboj.2013.117 PDFDownload PDF of article text and main figures. Peer ReviewDownload a summary of the editorial decision process including editorial decision letters, reviewer comments and author responses to feedback. ToolsAdd to favoritesDownload CitationsTrack CitationsPermissions ShareFacebookTwitterLinked InMendeleyWechatReddit Figures & Info 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. Introduction The mammalian genome codes for five related RECQ-like 3′–5′ DNA helicases: RECQL1, BLM, WRN, RECQL4 and RECQL5, all of which have been demonstrated to act upon a number of topologically different DNA structures in vitro. The most extensively studied of these helicases is BLM, which has been shown to unwind a variety of DNA substrates that include 3'-tailed duplexes, bubble structures, forked duplexes, G-quadruplex structures, DNA displacement loops and four-way Holliday Junctions (HJs). In addition to its conventional helicase activity, BLM can also promote ATP-dependent branch migration of the HJs, a process that may be involved in replication fork restoration (reviewed in Monnat, 2010 and Hickson and Mankouri, 2011). BLM is mutated in Bloom syndrome (BS); a rare autosomal recessive disorder that is typified by proportional dwarfism, sun-sensitive facial erythema, skin pigmentation abnormalities, immunodeficiency, infertility and an increased predilection to develop both lymphoid and epithelial-derived tumours (Ellis and German, 1996). Cells from BS patients characteristically exhibit increased chromosomal instability with increased numbers of chromatid gaps and breaks, as well as chromosome structural rearrangements, including symmetrical quadra-radials, telomere associations, anaphase bridges and lagging chromosomes being frequently observed. A unique cellular feature, commonly used in the molecular diagnosis of BS, is an ∼10-fold increase in the frequency of sister chromatid exchanges (SCEs), which is thought to arise as a consequence of uncontrolled homologous recombination (HR) during S and G2 phases of the cell cycle. The exact role of BLM is complicated by the fact that it can act to both promote and suppress HR repair (HRR) depending on the cellular context and the type of DNA damage. Following exposure to ionizing radiation, cells that have incurred DSBs in late S or G2 phase of the cell cycle undergo MRN- and CtIP-dependent break end-resection to promote HR through the generation of ssDNA. While the MRN complex in association with CtIP initiates this resection, it was recently shown that the progressive nucleolytic degradation of the DNA end is coordinated by the actions of DNA2, Exo1 and BLM (Gravel et al, 2008; Nimonkar et al, 2008, 2011). In contrast, following the generation of stalled replication forks caused by the exposure of cells to hydroxyurea (HU), 53BP1 in association with BLM is recruited to sites of aberrant fork structures where it functions to suppress rather than promote HR. This may in part be achieved by the ability of 53BP1 and BLM to directly bind to pro-recombinogenic core HR proteins like RAD51 (Bischof et al, 2001; Sengupta et al, 2003; Tripathi et al, 2007) and RAD54 (Srivastava et al, 2009), and to disrupt RAD51 nucleoprotein filaments (Bugreev et al, 2007; Tripathi et al, 2007). Therefore, the role played by BLM in dictating the type of DNA repair depends on the nature of the DNA damage and the presence of critical key regulatory proteins such as 53BP1. Despite our understanding of the enzymatic functions of BLM, very little is known about the mechanisms that trigger its relocalization to different DNA structures or how this is influenced by post-translational modifications. BLM seems to have a role both in sensing DNA damage and in transmission of the damage signal to downstream effector proteins (reviewed in Tikoo and Sengupta, 2010). BLM is phosphorylated via the Chk1/ATR pathway in response to replication stress (Davies et al, 2004; Sengupta et al, 2004). However, while ATR-dependent BLM phosphorylation is not required for its localization to sites of stalled replication forks, it is essential for S-phase checkpoint recovery (Davies et al, 2004). In contrast, dephosphorylation of Serine-646 has been shown to be essential for BLM to be recruited to the sites of damage (Kaur et al, 2010). Additionally, it has been suggested that modification of BLM by SUMO influences its cellular localization to PML-containing nuclear bodies (PML-NBs) as well as its ability to stimulate RAD51 recruitment to damaged replication forks (Eladad et al, 2005; Ouyang et al, 2009). Hence, phosphorylation, SUMOylation and possibly ubiquitylation of BLM may play important roles not only in regulating its dynamic movement between the PML-NBs and damaged DNA, but also its decision to promote or suppress RAD51-dependent HRR. Interestingly, following the induction of DSBs both the ubiquitin and SUMO conjugation pathways function in concert to stimulate repair of damaged DNA. This ubiquitin/SUMO-dependent DDR (UbS-DDR) is mediated by an E3 ubiquitin/SUMO ligase cascade composed of RNF8, HERC2, RNF168, PIAS1 and PIAS4, which promotes alterations in the local chromatin architecture surrounding the break to allow the efficient recruitment of essential repair factors such as the BRCA1-A complex and 53BP1 (Kolas et al, 2007; Mailand et al, 2007; Wang et al, 2007; Doil et al, 2009; Galanty et al, 2009; Stewart et al, 2009; Bekker-Jensen et al, 2010; Mattiroli et al, 2012). Given the link between BLM cellular localization regulation and the SUMO system (Eladad et al, 2005; Ouyang et al, 2009), it is conceivable that ubiquitin may also play a key role in regulating BLM recruitment to sites of DNA damage. Here, we demonstrate that following replication stress, the RNF8/RNF168-dependent ubiquitylation of BLM directs its recruitment to sites of stalled replication forks via an interaction with the UIM domains of RAP80. In addition, we have identified a role for RNF8-dependent BLM ubiquitylation in undamaged cells that functions to maintain its localization to PML-NBs and prevents its sequestration into nucleoli. We also provide evidence that BLM binds constitutively to RAP80 in the absence of DNA damage in a ubiquitin-independent manner and this is required to maintain BLM stability within the cell. Lastly, we show that this ubiquitin-mediated relocalization of BLM is required for its ability to suppress excessive/uncontrolled HR. Results BLM is recruited to sites of replication stress in a ubiquitin-dependent manner It has been demonstrated that the BLM helicase and 53BP1 together function to limit HR by suppressing the RAD51 response (Tripathi et al, 2007, 2008). Given the biochemical association between BLM, 53BP1 and the UbS-DDR, we hypothesized that the UbS-DDR may regulate the accumulation of BLM in response to replication stress. Hence, cells stably expressing GFP-tagged BLM were either mock treated or exposed to HU, fixed, stained with antibodies directed against PML or poly-ubiquitin chains (FK2). BLM is known to primarily reside in PML-NBs in undamaged cells (Yankiwski et al, 2000). Following treatment with HU, BLM relocalizes from PML-NBs to sites of stalled replication forks (Figure 1A). Consistent with the potential for the UbS-DDR to regulate the nuclear recruitment of BLM, HU-treated cells exhibited a significant colocalization of BLM with sites of ubiquitylation (Figure 1B). 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. Download figure Download PowerPoint To investigate whether the RNF8/RNF168-dependent E3 ubiquitin ligase cascade functioned during replication stress to promote the focal relocalization of BLM, cells expressing GFP-BLM were treated with either a control siRNA or siRNA directed against UbcH5a, Ubc13, RNF8 or RNF168. Following exposure to HU, immunofluorescence (IF) was used to determine the localization of BLM. Depletion of RNF8 or RNF168 (using two different siRNA sequences) resulted in a failure of cells to properly recruit BLM to sites of DNA damage (Figure 1C; Supplementary Figure 1A and C). These observations were also recapitulated in cells derived from a RIDDLE syndrome patient (Supplementary Figure 1B). Taken together, this indicates that BLM functions downstream of RNF8 and RNF168 during the DDR invoked by exposure to HU. Indeed we observed both RNF8/RNF168 relocate to sites of replication damage that contained BLM (Figure 1D and E) but not when the DNA damage has been repaired (compare +HU and +HU/PW conditions). Interestingly, BLM was predominantly localized to the nucleoli of cells lacking RNF8 (Figure 1D; Supplementary Figure 1C), suggesting that a basal level of RNF8- but not RNF168-dependent ubiquitylation is required for the normal localization of BLM within the nucleus even in undamaged cells. This is supported by the observation that BLM colocalized at sites of ubiquitylation in asynchronously growing cells (Figure 1B). Interestingly, depletion of either Ubc13 or UbcH5a alone did not affect BLM relocalization after HU treatment (Figure 1C; Supplementary Figure 1C). However, the combined loss of Ubc13 and UbcH5a completely abrogated the formation of BLM foci induced by replication stress comparable to a lack of RNF168 (Supplementary Figure 1C), possibly indicating functional redundancy between these two E2 enzymes. BLM is targeted by RNF8 and RNF168 for ubiquitylation in vitro and in vivo Based on the above observations, it is conceivable that the requirement for RNF8/RNF168 to facilitate BLM relocalization after HU treatment is mediated through their ability to target it for ubiquitylation. To investigate whether BLM is a substrate for RNF8/RNF168, in vitro ubiquitylation assays were carried out using recombinant BLM in combination with purified RNF8/RNF168 (Supplementary Figure 2A) in the presence of Ubc13/UbcH5a as the E2 conjugating enzyme. Both E3 ligases strongly stimulated the K63-dependent poly-ubiquitylation of BLM in vitro in the presence of Ubc13 (Figure 2A–C). Moreover, we demonstrated that this reaction was dependent on the active RING domain of the two E3 ligases (Figure 2D). Though UbcH5a could also be utilized as an E2 to catalyse BLM poly-ubiquitylation (Figure 2E), BLM ubiquitylation by Ubc13 was more robust (Figure 2F), indicating that the RNF8/RNF168 preferentially utilize Ubc13 to poly-ubiquitylate BLM in vitro. 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. Source Data for Figure 2 [embj2013117-sup-0001-SourceData-S1.pdf] Download figure Download PowerPoint To ascertain whether BLM could also be ubiquitylated in vivo in a DNA damage-inducible manner, U2OS cells expressing doxycycline (Dox) inducible shRNA to either RNF8 or RNF168 were used. These cells were either left asynchronous or treated with HU in the presence or absence of Dox. Immunoprecipitations were carried out using an antibody to K63-linked poly-ubiquitin chains and western blotted for the presence of BLM. BLM poly-ubiquitylation was significantly induced following exposure to HU. However, the cells lacking either RNF8 or RNF168 failed to efficiently promote K63-linked ubiquitylation of BLM (Figure 3A and B). The dependency for RNF168 to mediate BLM ubiquitylation in vivo was confirmed in RIDDLE syndrome cells complemented with WT RNF168 but not an empty vector (Figure 3C). Interestingly BLM was mono-ubiquitylated in the absence of HU, which was compromised in the absence of RNF8 (Figure 3A). These data demonstrate that BLM is mono-ubiquitylated in the absence of DNA damage and poly-ubiquitylated following exposure to DNA damage in a RNF8- and RNF168-dependent manner. 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. Source Data for Figure 3 [embj2013117-sup-0002-SourceData-S2.pdf] Download figure Download PowerPoint Ubiquitylation of the BLM N-terminal region is required for its relocalization to sites of DNA damage In order to ascribe a biological function to the RNF8-/RNF168-dependent ubiquitylation of BLM, initially the sites of ubiquitylation needed to be identified. To narrow down the lysine residues within BLM potentially targeted for ubiquitylation, two independent ubiquitylation site prediction programs (UbPred and UbiPred) were used, which both highlighted lysines at 105, 225 and 259 (K105, K225 and K259) as being high confidence target residues. To investigate whether these lysine residues were ubiquitylated by RNF8/RNF168, recombinant BLM containing each individual lysine mutated to arginine or all three sites mutated in combination (3K-R) were purified, tested for their ability to perform helicase activity to an equal extent (Supplementary Figure 2B) and subsequently used as the substrates during in vitro ubiquitylation reactions. Loss of any of the three predicted lysine residues individually resulted in a reduction in the level of in vitro BLM poly-ubiquitylation (Figure 4A). The RNF8/RNF168-dependent ubiquitylation was completely abrogated in 3K-R BLM mutant (Figure 4B), indicating that RNF8/RNF168 can target multiple lysine residues within BLM for ubiquitin chain conjugation in vitro. 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. Source Data for Figure 4 [embj2013117-sup-0003-SourceData-S3.pdf] Download figure Download PowerPoint To determine whether the three sites of BLM ubiquitylation identified in vitro also mediated the conjugation of poly-ubiquitin chains in vivo, extracts derived from 293T cells transfected with either WT or mutant 3K-R BLM expression constructs were subjected to immunoprecipitation using an antibody specific for K63-linked poly-ubiquitin chains and western blotted for the presence of BLM. Loss of these three critical N-terminal lysine residues resulted in a significant decrease in the overall level of K63-linked BLM ubiquitylation after HU treatment (Figure 4C), supporting the notion that they represent the major sites of K63-linked BLM ubiquitylation in vivo. It is conceivable that the RNF8-/RNF168-dependent ubiquitylation of BLM at specific residues is required for the relocalization of BLM to sites of replication stress following HU exposure. To test this hypothesis, cells transfected with either a WT or 3K-R mutant EGFP-tagged BLM expression construct were treated with HU and the focal recruitment of BLM monitored by fluorescence microscopy. In stark contrast to the WT BLM, the single mutants substantially compromised the ability of the exogenous BLM to form HU-induced foci (Figure 4D and E; Supplementary Figure 2C). This defect was exacerbated when all three sites of ubiquitylation on BLM were lost (Figure 4E). A similar lack of BLM 3K-R localization was also observed after ionizing radiation (Supplementary Figure 2D). Combined, these data support the concept that RNF8-/RNF168-dependent ubiquitylation of BLM promotes its recruitment to sites of DNA damage. Each of the BLM lysine mutants exhibited a notable relocalization from PML-NBs into the nucleoli; a phenotype with striking similarity to that which we had previously observed for the nuclear localization of BLM in cells depleted of RNF8 (Figure 1C and D; Supplementary Figure 1C). Our previous observations have demonstrated that BLM was mono-ubiquitylated in undamaged cells in an RNF8-dependent manner (Figure 3A) and that in these cells BLM could be found colocalizing with sites of ubiquitin chain formation in PML-NBs (Figure 1A and B). This suggests that a basal level of mono-ubiquitylation of BLM by RNF8 may be required to maintain its normal nuclear localization and/or prevent its sequestration to the nucleoli. Interestingly, we noted that cells depleted of RNF8 exhibited a reduction in the number of PML-NBs as well as the overall level of PML expression (Figure 5A and B). This may be due to the K63-linked ubiquitylation-mediated stabilization of PML isoforms by Ubc13/RNF8 (Supplementary Figure 2A; Figure 5C). Hence, a lack of PML coupled with the loss of monoubiquitylation of BLM leads to the relocalization of BLM to the nucleolus in cells lacking RNF8 (Figure 5D). 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. Source Data for Figure 5 [embj2013117-sup-0004-SourceData-S4.pdf] Download figure Download PowerPoint Recruitment of ubiquitylated BLM is mediated by RAP80 One of the primary
Referência(s)