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• W2897635597 abstract "Article10 October 2018free access Source DataTransparent process Local activation of mammalian separase in interphase promotes double-strand break repair and prevents oncogenic transformation Susanne Hellmuth Chair of Genetics, University of Bayreuth, Bayreuth, Germany Search for more papers by this author Cristina Gutiérrez-Caballero Centro de Investigación del Cáncer (CSIC-Universidad de Salamanca), Salamanca, Spain Search for more papers by this author Elena Llano Centro de Investigación del Cáncer (CSIC-Universidad de Salamanca), Salamanca, Spain Departamento de Fisiología, Universidad de Salamanca, Salamanca, Spain Search for more papers by this author Alberto M Pendás Corresponding Author [email protected] orcid.org/0000-0001-9264-3721 Centro de Investigación del Cáncer (CSIC-Universidad de Salamanca), Salamanca, Spain Search for more papers by this author Olaf Stemmann Corresponding Author [email protected] orcid.org/0000-0003-3044-2515 Chair of Genetics, University of Bayreuth, Bayreuth, Germany Search for more papers by this author Susanne Hellmuth Chair of Genetics, University of Bayreuth, Bayreuth, Germany Search for more papers by this author Cristina Gutiérrez-Caballero Centro de Investigación del Cáncer (CSIC-Universidad de Salamanca), Salamanca, Spain Search for more papers by this author Elena Llano Centro de Investigación del Cáncer (CSIC-Universidad de Salamanca), Salamanca, Spain Departamento de Fisiología, Universidad de Salamanca, Salamanca, Spain Search for more papers by this author Alberto M Pendás Corresponding Author [email protected] orcid.org/0000-0001-9264-3721 Centro de Investigación del Cáncer (CSIC-Universidad de Salamanca), Salamanca, Spain Search for more papers by this author Olaf Stemmann Corresponding Author [email protected] orcid.org/0000-0003-3044-2515 Chair of Genetics, University of Bayreuth, Bayreuth, Germany Search for more papers by this author Author Information Susanne Hellmuth1, Cristina Gutiérrez-Caballero2, Elena Llano2,3, Alberto M Pendás *,2 and Olaf Stemmann *,1 1Chair of Genetics, University of Bayreuth, Bayreuth, Germany 2Centro de Investigación del Cáncer (CSIC-Universidad de Salamanca), Salamanca, Spain 3Departamento de Fisiología, Universidad de Salamanca, Salamanca, Spain *Corresponding author. Tel: +34 923 294809; E-mail: [email protected] *Corresponding author. Tel: +49 921 552701; E-mail: [email protected] EMBO J (2018)37:e99184https://doi.org/10.15252/embj.201899184 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 Abstract Separase halves eukaryotic chromosomes in M-phase by cleaving cohesin complexes holding sister chromatids together. Whether this essential protease functions also in interphase and/or impacts carcinogenesis remains largely unknown. Here, we show that mammalian separase is recruited to DNA double-strand breaks (DSBs) where it is activated to locally cleave cohesin and facilitate homology-directed repair (HDR). Inactivating phosphorylation of its NES, arginine methylation of its RG-repeats, and sumoylation redirect separase from the cytosol to DSBs. In vitro assays suggest that DNA damage response-relevant ATM, PRMT1, and Mms21 represent the corresponding kinase, methyltransferase, and SUMO ligase, respectively. SEPARASE heterozygosity not only debilitates HDR but also predisposes primary embryonic fibroblasts to neoplasia and mice to chemically induced skin cancer. Thus, tethering of separase to DSBs and confined cohesin cleavage promote DSB repair in G2 cells. Importantly, this conserved interphase function of separase protects mammalian cells from oncogenic transformation. Synopsis Cohesin complexes hold sister chromatids together until their mitotic segregation, but also have interphase roles in the repair of DNA double strand breaks (DSBs). Such repair is now found to also require local activation of separase, the protease that cleaves cohesin during mitosis. Human separase accumulates at DSBs in G2- but not G1-phase. Human separase supports error-free homology-directed repair (HDR) but not error-prone non-homologous end joining (NHEJ). DSB-tethered separase is activated to locally cleave cohesin. Phosphorylation, arginine methylation and SUMOylation of separase are required to target it to DSBs and support DNA damage repair. SEPARASE heterozygosity weakens HDR and predisposes to Ras-induced neoplasia in MEFs and chemically-induced skin cancer in mice. Introduction DNA double-strand breaks (DSBs) pose an enormous threat to genome integrity because they frequently lead to cancer-associated translocations. Therefore, DSBs trigger a DNA damage response (DDR) leading to checkpoint-mediated cell cycle arrest followed by DSB repair or apoptosis (Polo & Jackson, 2011). DDR involves hierarchical recruitment and diverse posttranslational modifications (PTMs) of proteins at DSBs. Early steps are phosphorylation of histone variant H2AX at Ser139 (resulting in γH2AX) and Arg-methylation-dependent recognition and resection of DSBs by the MRE11-RAD50-NBS1 (MRN) complex (Polo & Jackson, 2011; Thandapani et al, 2013). One branch of the subsequent DNA damage signaling cascade consists of MRN-mediated recruitment and activation of ATM kinase which—together with MDC1, 53BP1, and other mediators—phosphorylates the effector kinase Chk2 (Polo & Jackson, 2011). Non-homologous end joining (NHEJ) and homology-directed repair (HDR) represent the two major pathways of downstream DSB repair. The error-free HDR occurs primarily in S- and G2-phase cells because it usually requires the undamaged sister chromatid as a homologous template (Polo & Jackson, 2011). The cohesin complex, whose Smc1, Smc3, and Rad21/Scc1 subunits form a 40-nm tripartite ring, also plays an important role in DSB repair (Sjogren & Nasmyth, 2001). However, its canonical functions lie in sister chromatid cohesion and chromosome separation (Nasmyth & Haering, 2009). Loading of cohesin onto chromatin in telophase (higher eukaryotic cells) or late G1 (yeast) is catalyzed by an Scc2-Scc4 complex, known as kollerin, and may involve transient detachment of Smc1 from Smc3 (Nasmyth & Haering, 2009). Concomitant to replication in S-phase, the two arising sister chromatids of each chromosome are then entrapped within cohesin rings and, thus, paired. Cohesion is stabilized by Esco1/2-dependent acetylation of Smc3 and binding of sororin and counteracted by the anti-cohesive factor Wapl (Nasmyth & Haering, 2009). Phosphorylation-dependent inactivation of sororin in early mitosis enables Wapl to somehow open the Smc3-Rad21 gate, thereby displacing cohesin from chromosome arms (Buheitel & Stemmann, 2013; Nishiyama et al, 2013). Centromeric cohesin/sororin is stabilized by Sgo1-PP2A-dependent dephosphorylation and removed only at the metaphase-to-anaphase transition when degradation of securin liberates separase to proteolytically cleave Rad21 (Uhlmann et al, 2000; Liu et al, 2013). Interestingly, a single DSB in yeast triggers the replication-independent, genome-wide enforcement of sister chromatid cohesion by de novo loading of cohesin. This recruitment was most profound at the break and occurred in a γH2AX-, MRE11-, and kollerin-dependent manner (Strom et al, 2004, 2007; Unal et al, 2004, 2007). On the other hand, Aragón et al reported a decrease in cohesin from DSBs when re-synthesis of Rad21 was inhibited (McAleenan et al, 2013). This decrease coincided with the damage-induced formation of Rad21 fragments and the impairment of DSB repair by expression of a separase-resistant Rad21 variant, observations which had previously been reported also for Schizosaccharomyces pombe and which suggested activation of separase during DDR in postreplicative yeast cells (Nagao et al, 2004; McAleenan et al, 2013). Cohesin accumulates at DSBs also in human cells (Kim et al, 2002; Potts et al, 2006). However, whether mammalian separase is activated during DDR to cleave cohesin at DSBs remains an unresolved issue. Overexpression of separase results in aneuploidy and tumorigenesis in the mouse model, occurs in several human cancers, and is associated with poor clinical outcome (Zhang et al, 2008; Meyer et al, 2009; Finetti et al, 2014; Mukherjee et al, 2014a,b). Conversely, SEPARASE heterozygosity also causes genomic instability and cancer in zebrafish and p53 null mice (Shepard et al, 2007; Mukherjee et al, 2011) arguing that SEPARASE is both an oncogene and a tumor suppressor gene at the same time and that separase's proteolytic activity needs tight control. Whether separase's role in tumorigenesis is due to its role in sister chromatid segregation and/or a hitherto unknown function still needs clarification. Here, we show that separase associates with DSBs in postreplicative cells, where it is activated to locally cleave cohesin and facilitate HDR. Its recruitment to damaged DNA requires three PTMs of separase: inhibitory phosphorylation of a nuclear export sequence (NES), Arg-methylation of a conserved RG-repeat motif, and sumoylation of Lys1034. Importantly, SEPARASE heterozygosity simultaneously results the reduced DDR and heightened predisposition to oncogenic transformation. Results Separase is recruited to DSBs Previous yeast studies implied a role of separase in DDR (Nagao et al, 2004; McAleenan et al, 2013). To address whether human separase might have a similar function, we adopted a system to induce site-specific DSBs (Iacovoni et al, 2010; Caron et al, 2012). Upon addition of 4-hydroxytamoxifen (OHT), the restriction endonuclease AsiSI fused to an estrogen receptor (ER) re-locates from the cytosol into the nucleus to cleave DNA at 8-bp recognition sites (around 200 per cell; Chailleux et al, 2014), thereby triggering formation of γH2AX- and phosphoChk2-positive foci (Fig EV1A). When ER-AsiSI was expressed in a transgenic HEK293 line that inducibly produces Myc-tagged separase in response to doxycycline (Dox) addition (Boos et al, 2008), separase formed nuclear foci that co-localized with γH2AX—but only when both OHT and Dox were present (Fig EV1B). Similarly, overexpressed separase localized to γH2AX-positive foci when the topoisomerase II inhibitor doxorubicin (DRB) was used to inflict DSBs (Fig EV1C). Importantly, endogenous separase also co-localized with γH2AX to sites of DNA damage as judged by immunofluorescence microscopy (IFM) of DRB-treated HEK293 cells. Demonstrating the specificity of the DRB-induced separase foci, they were absent in separase-depleted and undamaged cells (Figs 1A and EV1D). To independently confirm recruitment of endogenous separase to damaged sites, we conducted chromatin immunoprecipitation (ChIP) experiments followed by multiplex qPCR or qPCR (see Fig EV1E for length and positions of PCR products). These analyses consistently revealed that, much like an anti-γH2AX and unlike non-specific IgG, a separase antibody precipitated DNA close to two different AsiSI sites if and only if OHT was added, while a region that was more than 2.2 Mbp away from the nearest AsiSI site did not co-purify (Fig 1B and C). The specific recruitment of separase to DSBs was further enhanced by induced overexpression of the protease (Fig EV1F). Click here to expand this figure. Figure EV1. Separase localizes to DSBs A system for induced introduction of site-specific DSBs. Transgenic HEK293 cells constitutively expressing FLAG-tagged AsiSI-ER were treated with OHT or carrier solvent and then analyzed by IFM as indicated. Scale bar = 5 μm. Transgenic HEK293 cells treated with Dox to induce expression of Myc-separase-WT and/or with OHT to induce nuclear accumulation of ER-AsiSI and DSBs were analyzed by IFM as indicated. Scale bar = 5 μm. γH2AX- and separase-positive foci formation in response to DNA damage by DRB. Prior to their analysis by IFM, transgenic HEK293 cells in G2-phase were Dox- and/or DRB-treated to induce the expression of Myc-separase and/or inflict DSBs, respectively. Scale bar = 5 μm. Western blot analysis of experiment shown in Fig 1A. Position and sizes of PCR fragments from the ChIP–multiplex PCR and ChIP-qPCR experiments. Schematic is not drawn to scale. Transgenic HEK293 cells supplemented with Dox to induce expression of Myc-separase-WT were mock- or OHT-treated in G2-phase and then subjected to ChIP-qPCR. Shown are averages (bars) of three independent experiments (dots). Download figure Download PowerPoint Figure 1. Human separase localizes to DSBs in postreplicative cells A. Following transfection of given siRNAs and synchronization in G2-phase, Hek293 cells were DRB- or mock-treated and then subjected to IFM using the indicated antibodies. Lower panels display a threefold magnification of the boxed area shown above. Scales bars correspond to 5 and 1 μm, respectively. See Fig EV1D for corresponding immunoblot. B. HEK293 cells were thymidine-arrested for 20 h, mock- or OHT-treated to induce DSBs by nuclear accumulation of ER-AsiSI, and then subjected to ChIP–multiplex PCR. C. ChIP samples from (B) were analyzed by qPCR. Shown are averages (bars) of three independent experiments (dots). D, E. Separase interacts with γH2AX in DSB-containing G2 but not G1 cells. Transgenic HEK293 cells treated with Dox to induce expression of Myc-separase-WT and with OHT to induce nuclear accumulation of ER-AsiSI and infliction of DSBs were synchronized in G1- or G2-phase and analyzed by IP–Western blotting (D) and by IFM for γH2AX- and Myc-separase-positive foci (see Fig EV2E). The quantification of the IFM in (E) shows averages (bars) of three independent experiments (dots) counting ≥ 100 cells each. Download figure Download PowerPoint Separase functions in HDR but not NHEJ Is separase of functional importance for DNA damage repair? Indicating that this might indeed be the case, depletion of separase but not mock treatment strongly compromised the ability of HEK293 cells to form colonies in the presence of camptothecin, a topoisomerase I inhibitor that causes replication-dependent DNA single- and double-strand breaks (Fig EV2A and B). Click here to expand this figure. Figure EV2. Depletion of Separase by RNAi compromises HDR as judged by a GFP-based in vivo assay A, B. Depletion of separase renders human cells hypersensitive to camptothecin. Twelve hours after transfection of the indicated siRNAs, 100 HEK293 cells each were plated onto 10-cm petri dishes. Another 12 h later, camptothecin (0.25 nM end concentration) or carrier solvent (DMSO) was added. Colonies were stained by crystal violet 12 days thereafter and photographed (A). For each condition, three independent experiments were quantified by ImageJ (dots) and averaged (bars) (B). C. Schematic of the experiment shown in Figs 1D and E, and EV2D and E. D. Dox- and OHT-treated HEK293 cells synchronized in G1- or G2-phase were propidium iodide (PI)-stained and analyzed by DNA content by flow cytometry. E. Representative IFM images of Dox- and OHT-treated HEK293 cells synchronized in G1- or G2-phase. Scale bar = 5 μm. F, G. U2OS DR-GFP (HDR reporter) cells were transfected with GL2 or one of five different SEPARASE-directed siRNAs. Following a second transfection to express ER-tagged I-SceI and addition of OHT to induce nuclear accumulation of the homing endonuclease, cells were subjected to flow cytometry (G) and immunoblotting (F). Download figure Download PowerPoint We noted that not all γH2AX-positive interphase cells showed Myc-separase foci and therefore pre-synchronized ER-AsiSI-expressing cells in either G1- or G2-phase prior to OHT addition and analyses (see Fig EV2C for timeline of the experiment). Successful synchronization was confirmed by measurement of cyclin A2 and DNA contents by immunoblotting and flow cytometry, respectively (Figs 1D and EV2D). Lysates from these different cell populations were benzonase-treated to digest DNA and then subjected to IP using anti-Myc or unspecific IgG. Interestingly, γH2AX specifically co-purified with Myc-separase from OHT-treated G2- but not G1-phase cells (Fig 1D, lower part). Consistently, IFM revealed that 80% of DSBs containing G2 cells exhibited γH2AX- and Myc-separase-positive foci as compared to merely 10% in the G1-enriched pool (Figs 1E and EV2E). Due to imperfect synchronization of the HEK293 cells (Fig EV2D), this probably represents an underestimation. These data therefore imply that separase is recruited to DSBs exclusively in postreplicative cells. Because HDR, but not NHEJ, requires the undamaged sister chromatid as a homologous template, the above result indicates a possible role of separase in HDR rather than NHEJ. To resolve this issue, we adopted in vivo assays for HDR versus NHEJ (Pierce et al, 1999; Gunn & Stark, 2012). Herein, the homing endonuclease I-SceI in corresponding transgenic U2OS cells introduces a single DSB, the repair of which creates a functional GFP expression cassette only if it occurs by HDR in one reporter line and by NHEJ in the other. Following transfection of a SEPARASE-directed (SEP-1) or control siRNA (GL2), the corresponding cells were again transfected to express I-SceI in fusion with an estrogen receptor (ER) and synchronized in early S-phase. Six hours after release from thymidine arrest, cells were supplemented with OHT to induce nuclear accumulation of the homing endonuclease (or with carrier solvent as a negative control). Two days thereafter, cells were harvested and analyzed for protein content by immunoblotting and for GFP fluorescence and DNA content by flow cytometry. The OHT-induced GFP expression was unaffected by separase in the NHEJ reporter line (Fig 2A and B). However, in the HDR reporter line depletion of separase resulted in a markedly reduced GFP Western signal (Fig 2A) and 2.6-fold less GFP-positive cells (Fig 2B). At the time of analysis, OHT-treated HDR reporter cells lacking separase also displayed slightly enhanced γH2AX and cyclin A2 signals and a modest accumulation in G2/M (Fig 2A and C; see also Fig EV2F). This is consistent with a compromised repair proficiency and consequent DNA damage checkpoint-dependent delay of mitotic entry. To exclude the possibility that compromised HDR in separase-depleted reporter cells was due to an off-target effect, we tested four additional SEPARASE-directed siRNAs (SEP-2-5) in the same assay. All reduced the number of GFP-positive cells relative to the GL2 control, and all but one (SEP-3) did so to a similar extent as did SEP-1 (Fig EV2F and G). Consistent with SEPARASE being the relevant target, the weaker effect of SEP-3 correlated with its weaker knock-down efficiency (Fig EV2F). In summary, these results demonstrate that human separase is required for proper HDR but dispensable for NHEJ. Figure 2. Separase supports HDR but not NHEJ A–C. Separase is required for proper HDR but dispensable for NHEJ. U2OS DR-GFP (HDR reporter) and U2OS EJ5-GFP (NHEJ reporter) cells were separase-depleted by RNAi (SEP-1) or control-treated with GL2 siRNA, transfected to express HA-tagged ER-I-SceI, and then supplemented with OHT in G2-phase to induce nuclear accumulation of the homing endonuclease. Ethanol-supplemented samples served as negative controls. Two days later, cells were subjected to immunoblotting (A) and flow cytometry to quantify the percentage of GFP-positive cells (B) and PI-stained cellular DNA (C). The GFP quantification in (B) displays averages (bars) of three independent experiments (dots). D, E. Co-depletion of Wapl and separase has a synergistic effect on HDR. U2OS DR-GFP cells were transfected with the indicated siRNAs, treated as in (A + B) and analyzed by GFP flow cytometry (D) and immunoblotting (E). Shown in (D) are averages (bars) of two to three independent experiments (dots). Download figure Download PowerPoint A role of cohesin in HDR is well established (Kim et al, 2002; Strom et al, 2004; Unal et al, 2004; Potts et al, 2006). Simultaneous requirement of its antagonist separase in the same repair pathway seems counterintuitive at first. To clarify this issue, we analyzed side by side the effects of cohesin and separase single and double depletions in the abovementioned GFP-based HDR assay. Flow cytometric quantification showed that RNAi of separase, Rad21, or another cohesin subunit, Smc1α, all reduced the amount of GFP-positive cells to a similar extent and at least by a factor of 2, thereby confirming the requirement of both cohesin and separase for proper HDR (Fig 2D, columns 1–3 and 5). Interestingly, simultaneous knock-down of separase together with Rad21 or Smc1α only marginally aggravated the HDR defect relative to the single depletions (compare columns 2–6). We believe that effective HDR requires heightening of both density and turnover of cohesin at DSBs (see Discussion). Co-depletion of separase will increase residual cohesion under conditions when cohesin becomes limiting, and this might compensate for the negative effect on HDR of reduced cohesin dynamics due to the sole absence of separase. We also investigated Wapl, another anti-cohesive factor with key function in proteolysis-independent cohesin removal (Kueng et al, 2006). While Wapl depletion alone reduced the GFP formation only mildly, simultaneous knock-down of Wapl and separase had a clear synergistic effect reducing the amount of GFP-positive cells in the individual depletions from 2.8 and 4.6%, respectively, to 1% in the double knock-down (Fig 2D, columns 2, 7, and 8). This suggests that total abrogation of cohesin dynamics by interference with both known anti-cohesive mechanisms leads to maximal impairment of HDR. Immunoblotting not only confirmed the successful and even knock-downs but also the flow cytometric quantification of GFP (Fig 2E). Moreover, it revealed increased levels of γH2AX and cyclin A2 in the absence of cohesin and/or separase indicating DSB persistence and checkpoint-mediated cell cycle arrest under these conditions (lower two panels). Separase is activated at DSBs where it locally cleaves cohesin Does human separase get activated to cleave cohesin in response to DSBs similar to the situation in yeast? To address this question, histone H2B-mCherry-Rad21107–268-eGFP was constitutively expressed in MYC-SEPARASE cells (Fig 3A, cartoon at bottom). Separase-dependent cleavage of this protease activity sensor causes mCherry- and eGFP-positive chromatin to selectively lose its eGFP signal. These doubly transgenic cells were transfected to transiently express also AsiSI-ER, treated with OHT or carrier solvent for different times, and analyzed by immunoblotting (Fig 3A and B). Indeed, this demonstrated DNA damage-dependent proteolysis of the sensor (Fig 3A, bottom panel). Enrichment by IP of its soluble cleavage products revealed that endogenous Rad21 is also cleaved and that these cleavage events increased with duration of DSB induction, i.e., OHT treatment (Fig 3B). Separase activity was only slightly enhanced by Dox-induced overexpression (Fig 3A and B), although this resulted in considerably more protease being recruited to sites of DNA damage as revealed by ChIP-qPCR (compare Figs 1C and EV1F). Under these conditions, the activation of separase therefore seems to be limiting. Figure 3. Human separase locally cleaves Rad21 at DSBs Transgenic HEK293 cells constitutively expressing a separase sensor (cartoon below) and inducibly expressing Myc-separase were transiently transfected to express Flag-AsiSI-ER, Dox- and/or OHT-treated in G2-phase, and analyzed by IP–Western blotting. Cells from (A) were analyzed by IP–Western blotting for cleavage of endogenous Rad21. As a control, in vitro-translated (IVT) Rad21 was incubated with hyperactive separase-SA or a protease-dead (PD) variant (Boos et al, 2008). Sensor-expressing cells were treated in G2-phase with DRB and nocodazole for 6 and 2 h, respectively, prior to chromosome spreading and IFM using Hec1 and γH2AX antibodies. The separase sensor was detected based on autofluorescence of eGFP and mCherry, while Hec1 and γH2AX antibodies were detected with corresponding Cy5- and marina blue-labeled secondary antibodies, respectively. Note that sizes of spread chromosomes vary greatly with buffer conditions, which is why no scale bar is shown. Download figure Download PowerPoint When sensor-expressing cells were treated in G2-phase with DRB and the spindle toxin nocodazole, subsequent chromatin spreads displayed some mitotic figures with γH2AX-positive foci on condensed chromosomes, suggesting that the corresponding cells had slipped from the DNA damage into the mitotic checkpoint arrest (Fig 3C). Notably, γH2AX foci coincided with regions of decreased eGFP fluorescence (or relative increase in mCherry over eGFP in an overlay) indicative of local separase-dependent sensor cleavage. This net loss of sensor from DSBs does not contradict the reported overall accumulation of cohesin at DSBs because loading mechanisms are fundamentally different for authentic cohesin versus the histone-based sensor. Together, these results strongly indicate that separase is activated at sites of DSBs to locally cleave cohesin during DDR. DNA damage- and phosphorylation-dependent inactivation of separase's NES In undamaged interphase cells, separase is excluded from the nucleus due to the presence of a nuclear export sequence (NES) centered around position 1665 (Sun et al, 2006). Therefore, its NES might be inactivated in response to DSBs to retain separase in the nucleus. Interestingly, an NES in p53 is inhibited by DNA damage-induced phosphorylation (Zhang & Xiong, 2001) and separase's NES is immediately flanked by a serine at position 1660. When Myc-separase-WT was immunoprecipitated from cyclin A2-positive, DRB-treated G2-phase cells, separase was detected in an immunoblot by a pan-specific phosphoSer antibody (Fig 4A). This signal was greatly diminished in the absence of DNA damage or when samples were phosphatase-treated prior to SDS–PAGE. Importantly, the phosphoSer signal was also missing when a Ser-1660 to Ala variant instead of separase-WT was immunoprecipitated from DRB-treated G2 cells. At the same time, γH2AX co-purified with separase-WT but not separase-S1660A from benzonase-treated cell lysates (Fig 4A), suggesting that the phosphorylation site variant is unable to associate with DSBs (see below). Figure 4. NES phosphorylation and RG-repeat methylation of separase in response to DSBs A–C. Ser1660 is phosphorylated in response to DNA damage and required for the interaction of separase with γH2AX. HEK293 cells were arrested in G2-phase by sequential thymidine and RO-3306 treatment, DRB- (+) or mock-treated (−), and then analyzed as indicated. (A and B) Myc-separase-WT or Myc-separase-S1660A-expressing cells were subjected to IP–Western blotting using, among others, a pan-specific antibody against phosphorylated serine (A, pan-pS) or a separase antibody specific for phosphorylated Ser1660 (B, pS1660). (C) DNA damage-induced Ser1660 phosphorylation of endogenous separase is largely blocked by ATM inhibition. G2-enriched HEK293 cells were treated with KU-55933 (0.3 μM) and/or DRB and analyzed by IP–Western blotting 12 h thereafter using the indicated antibodies. D. Preventing NES phosphorylation spoils nuclear localization of separase in response to DSBs. HeLaK cells expressing N-terminally NLS-eGFP-tagged separase variants were treated with DRB or carrier solvent (− DRB) for 4 h and then subjected to IFM using anti-γH2AX and anti-Nup153 to visualize sites of DNA damage and nuclear pore complexes, respectively. Transgenic separase was detected based on the eGFP autofluorescence. Note that due to their relatively high nuclear concentration, co-localization of separase-WT and separase-S1660D with γH2AX foci is not discernable. Scale bar = 5 μm. E. In vitro phosphorylation of separase on Ser1660 by ATM kinase. Incubation of GST-p53 (amino acids 9–22), GST, separase-WT, or separase-S1660A with recombinant ATM-WT, ATM-KD (kinase dead), and/or KU-55933 in the presence of [γ-33P]-ATP was followed by immunoblotting and autoradiography. F. Arg-methylation of RG-repeats mediates recruitment of separase to DSB-containing chromatin. Myc-separase-WT- or Myc-separase-KG-expressing cells were treated with DRB as indicated and analyzed by IP–Western blotting and Coomassie staining. G. In vitro Arg-methylation of separase's RG-repeats by PRMT1. Incubation of histone H4, separase-WT, or separase-KG with recombinant PRMT1 or reference buffer in the presence of S-adenosyl-L-[methyl-3H]-methionine was followed by Coomassie staining and autoradiography. Download figure Download PowerPoint We then raised an antibody that specifically recognized phosphorylated Ser-1660 of separase. Confirming our earlier interpretations, this anti-pS1660 strongly reacted with full-length Myc-separase-WT from DSB-containing cells (Fig 4B). In contrast, Myc-separase-S1660A from accordingly DRB-treated cells and separase-WT from undamaged G2-arrested control cells were hardly recognized. Importantly, this tool also allowed us to detect the NES phosphorylation of endogenous separase upon infliction of DSBs in untransfected, G2-arrested HEK293T cells (Fig 4C). Visible nuclear accumulation of separase (in the absence of DNA damage) requires both mutational inactivation of the NES and fusion of the protease with a nuclear localization sequence (NLS; Sun et al, 2006). Transiently expressed NLS-eGFP-separase accumulated in nuclei not only when bulky hydrophobic residues within the NES were replaced by alanines (NESmut) but also when DSBs were inflicted" @default.
• W2897635597 created "2018-10-26" @default.
• W2897635597 creator A5019334692 @default.
• W2897635597 creator A5030456765 @default.
• W2897635597 creator A5050407648 @default.
• W2897635597 creator A5058515239 @default.
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• W2897635597 date "2018-10-10" @default.
• W2897635597 modified "2023-10-12" @default.
• W2897635597 title "Local activation of mammalian separase in interphase promotes double‐strand break repair and prevents oncogenic transformation" @default.
• W2897635597 cites W1966900987 @default.
• W2897635597 cites W1969158226 @default.
• W2897635597 cites W1970151860 @default.
• W2897635597 cites W1975092906 @default.
• W2897635597 cites W1980236727 @default.
• W2897635597 cites W1982367970 @default.
• W2897635597 cites W1982548286 @default.
• W2897635597 cites W1998550531 @default.
• W2897635597 cites W2004304977 @default.
• W2897635597 cites W2012211545 @default.
• W2897635597 cites W2022431828 @default.
• W2897635597 cites W2026100351 @default.
• W2897635597 cites W2026347115 @default.
• W2897635597 cites W2027849611 @default.
• W2897635597 cites W2036579213 @default.
• W2897635597 cites W2036607675 @default.
• W2897635597 cites W2037233550 @default.
• W2897635597 cites W2041656651 @default.
• W2897635597 cites W2043925902 @default.
• W2897635597 cites W2049020274 @default.
• W2897635597 cites W2054782539 @default.
• W2897635597 cites W2063394866 @default.
• W2897635597 cites W2064805607 @default.
• W2897635597 cites W2064888996 @default.
• W2897635597 cites W2074615071 @default.
• W2897635597 cites W2078427400 @default.
• W2897635597 cites W2078577401 @default.
• W2897635597 cites W2084212184 @default.
• W2897635597 cites W2085037886 @default.
• W2897635597 cites W2088509777 @default.
• W2897635597 cites W2096951683 @default.
• W2897635597 cites W2098848421 @default.
• W2897635597 cites W2100701860 @default.
• W2897635597 cites W2101548325 @default.
• W2897635597 cites W2103770333 @default.
• W2897635597 cites W2110691960 @default.
• W2897635597 cites W2116045064 @default.
• W2897635597 cites W2118411078 @default.
• W2897635597 cites W2127442777 @default.
• W2897635597 cites W2127806929 @default.
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