NBS1 promotes the endonuclease activity of the MRE11‐RAD50 complex by sensing CtIP phosphorylation
2019; Springer Nature; Volume: 38; Issue: 7 Linguagem: Inglês
10.15252/embj.2018101005
ISSN1460-2075
AutoresRoopesh Anand, Arti Jasrotia, Diana Bundschuh, Sean Howard, Lepakshi Ranjha, Manuel Stucki, Petr Ćejka,
Tópico(s)RNA modifications and cancer
ResumoArticle20 February 2019free access Transparent process NBS1 promotes the endonuclease activity of the MRE11-RAD50 complex by sensing CtIP phosphorylation Roopesh Anand Institute for Research in Biomedicine, Faculty of Biomedical Sciences, Università della Svizzera italiana (USI), Bellinzona, Switzerland Search for more papers by this author Arti Jasrotia Department of Gynecology, University of Zurich, Schlieren, Switzerland Search for more papers by this author Diana Bundschuh Department of Gynecology, University of Zurich, Schlieren, Switzerland Search for more papers by this author Sean Michael Howard Institute for Research in Biomedicine, Faculty of Biomedical Sciences, Università della Svizzera italiana (USI), Bellinzona, Switzerland Search for more papers by this author Lepakshi Ranjha Institute for Research in Biomedicine, Faculty of Biomedical Sciences, Università della Svizzera italiana (USI), Bellinzona, Switzerland Search for more papers by this author Manuel Stucki Department of Gynecology, University of Zurich, Schlieren, Switzerland Search for more papers by this author Petr Cejka Corresponding Author [email protected] orcid.org/0000-0002-9087-032X Institute for Research in Biomedicine, Faculty of Biomedical Sciences, Università della Svizzera italiana (USI), Bellinzona, Switzerland Department of Biology, Institute of Biochemistry, Eidgenössische Technische Hochschule (ETH), Zürich, Switzerland Search for more papers by this author Roopesh Anand Institute for Research in Biomedicine, Faculty of Biomedical Sciences, Università della Svizzera italiana (USI), Bellinzona, Switzerland Search for more papers by this author Arti Jasrotia Department of Gynecology, University of Zurich, Schlieren, Switzerland Search for more papers by this author Diana Bundschuh Department of Gynecology, University of Zurich, Schlieren, Switzerland Search for more papers by this author Sean Michael Howard Institute for Research in Biomedicine, Faculty of Biomedical Sciences, Università della Svizzera italiana (USI), Bellinzona, Switzerland Search for more papers by this author Lepakshi Ranjha Institute for Research in Biomedicine, Faculty of Biomedical Sciences, Università della Svizzera italiana (USI), Bellinzona, Switzerland Search for more papers by this author Manuel Stucki Department of Gynecology, University of Zurich, Schlieren, Switzerland Search for more papers by this author Petr Cejka Corresponding Author [email protected] orcid.org/0000-0002-9087-032X Institute for Research in Biomedicine, Faculty of Biomedical Sciences, Università della Svizzera italiana (USI), Bellinzona, Switzerland Department of Biology, Institute of Biochemistry, Eidgenössische Technische Hochschule (ETH), Zürich, Switzerland Search for more papers by this author Author Information Roopesh Anand1, Arti Jasrotia2, Diana Bundschuh2, Sean Michael Howard1, Lepakshi Ranjha1, Manuel Stucki2 and Petr Cejka *,1,3 1Institute for Research in Biomedicine, Faculty of Biomedical Sciences, Università della Svizzera italiana (USI), Bellinzona, Switzerland 2Department of Gynecology, University of Zurich, Schlieren, Switzerland 3Department of Biology, Institute of Biochemistry, Eidgenössische Technische Hochschule (ETH), Zürich, Switzerland *Corresponding author. Tel: +41 91 820 03 61; E-mail: [email protected] EMBO J (2019)38:e101005https://doi.org/10.15252/embj.2018101005 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 DNA end resection initiates DNA double-strand break repair by homologous recombination. MRE11-RAD50-NBS1 and phosphorylated CtIP perform the first resection step via MRE11-catalyzed endonucleolytic DNA cleavage. Human NBS1, more than its homologue Xrs2 in Saccharomyces cerevisiae, is crucial for this process, highlighting complex mechanisms that regulate the MRE11 nuclease in higher eukaryotes. Using a reconstituted system, we show here that NBS1, through its FHA and BRCT domains, functions as a sensor of CtIP phosphorylation. NBS1 then activates the MRE11-RAD50 nuclease through direct physical interactions with MRE11. In the absence of NBS1, MRE11-RAD50 exhibits a weaker nuclease activity, which requires CtIP but not strictly its phosphorylation. This identifies at least two mechanisms by which CtIP augments MRE11: a phosphorylation-dependent mode through NBS1 and a phosphorylation-independent mode without NBS1. In support, we show that limited DNA end resection occurs in vivo in the absence of the FHA and BRCT domains of NBS1. Collectively, our data suggest that NBS1 restricts the MRE11-RAD50 nuclease to S-G2 phase when CtIP is extensively phosphorylated. This defines mechanisms that regulate the MRE11 nuclease in DNA metabolism. Synopsis The crucial contribution of NBS1, the accessory subunit in the mammalian MRN complex, for initiating end resection of DNA double-strand breaks is defined by in vitro reconstitution to lie in sensing of CDK-dependent CtIP phosphorylation, providing a link to cell cycle stage. NBS1 senses CtIP phosphorylation via its FHA and BRCT domains. NBS1 stimulates the MRE11-RAD50 nuclease by directly interacting with MRE11. NBS1 restricts the nuclease of MRE11-RAD50 to conditions when CtIP is phosphorylated. In absence of NBS1, phosphorylation of CtIP is not strictly required to promote the nuclease of MRE11-RAD50. Introduction The maintenance of genomic integrity is critically important as cells are constantly exposed to various genotoxic agents. DNA double-strand breaks (DSBs) are difficult to repair due to the loss of genetic information in both DNA strands. While unrepaired DSBs may result in cell death, their inaccurate repair can lead to mutagenesis and inappropriate chromosomal translocations (Ranjha et al, 2018). Cells possess two main mechanisms for DSB repair. This includes end-joining pathways including Ku-dependent non-homologous end-joining (NHEJ) and Ku-independent microhomology-mediated end-joining (MMEJ), which are functional in any phase of the cell cycle and do not require a DNA template (Ranjha et al, 2018). The second mechanism is template-dependent homologous recombination (HR), which is generally restricted to the S-G2 phase of the cell cycle. The sister chromatid serves as an HR template in most cases in vegetative cells, explaining why recombination can only function in the cell cycle phases when sister chromatids are available (Ranjha et al, 2018). This is achieved by a regulatory mechanism involving cyclin-dependent kinases (CDKs), which phosphorylate key factors that function in the first step of the HR pathway termed DNA end resection (Huertas et al, 2008; Huertas & Jackson, 2009). Resection involves nucleolytic degradation of the 5′-terminated DNA strands at DSBs, leading to 3′ overhangs that are essential for the downstream steps in the recombination pathway. While limited DNA end resection may be involved in MMEJ, DNA that underwent extended resection is no longer ligatable and therefore unsuitable for end-joining. The decision whether and to which extent to resect DNA ends therefore regulates the pathway choice in DSB repair (Ranjha et al, 2018). The MRE11-RAD50-NBS1 (MRN, in human cells) or Mre11-Rad50-Xrs2 (MRX, in budding yeast) complex has multiple key evolutionarily conserved functions to initiate and coordinate the repair of DSBs (Paull, 2010; Stracker & Petrini, 2011). This includes roles in both end-joining and HR pathways (Moore & Haber, 1996; Carney et al, 1998; Paull & Gellert, 1998, 1999; de Jager et al, 2001; Rass et al, 2009). Additionally, MRN promotes DNA end tethering and is required to signal the presence of DSBs via the ATM kinase (Carney et al, 1998; Stewart et al, 1999; de Jager et al, 2001; Usui et al, 2001; Lee & Paull, 2005; Williams et al, 2008). This is achieved by a conserved interaction between the MRN subunit NBS1 (Xrs2 in Saccharomyces cerevisiae) and ATM (Tel1 in yeast). ATM in turn phosphorylates hundreds of protein targets that regulate the response to DSBs. Beyond ATM, NBS1 also interacts with MDC1, which binds phosphorylated proteins, including H2AX (γH2AX), to amplify the DNA damage signaling beyond the vicinity of the DSB (Goldberg et al, 2003). In recombination, the MRN complex has a direct role in DNA end resection mediated by the MRE11 nuclease in the S-G2 phase of the cell cycle. MRE11 is likely the first nuclease to resect DSBs, which is particularly important for the processing of breaks with secondary DNA structures or protein adducts such as stalled topoisomerases or Ku (Shibata et al, 2014; Anand et al, 2016; Deshpande et al, 2016; Wang et al, 2017; Reginato et al, 2018). The current models posit that resection by MRE11 is initiated by endonucleolytic DNA cleavage internal to the DSB past the protein block, followed by 3′→5′ exonucleolytic degradation back toward the DNA end (Keeney & Kleckner, 1995; Neale et al, 2005; Garcia et al, 2011; Cannavo & Cejka, 2014; Shibata et al, 2014). The endonucleolytic cleavage sites also represent entry sites for processive DNA end resection nucleases that function downstream of MRN in the 5′→3′ direction, including EXO1 and DNA2 (Gravel et al, 2008; Mimitou & Symington, 2008; Zhu et al, 2008; Nimonkar et al, 2011). The MRN nuclease furthermore likely functions to promote MMEJ independently of the cell cycle, which to date remains very poorly defined (Ma et al, 2003; Rahal et al, 2010; Taylor et al, 2010; Truong et al, 2013; Deng et al, 2014; Sharma et al, 2015). The processing of protein-blocked DSBs is an evolutionarily conserved capacity of MRE11 homologues and their co-factors. In bacteria, the MRE11-RAD50-like SbcC-SbcD complex endonucleolytically cleaves DNA near blocked DNA ends (Connelly et al, 2003). Eukaryotic cells possess a third member of the MRE11-RAD50 complex, NBS1/Xrs2, as well as CtIP/Sae2, which function as co-factors of the MRE11 nuclease in resection (Carney et al, 1998; Sartori et al, 2007). In S. cerevisiae, Sae2 phosphorylated by CDK and other kinases has a critical function to promote the Mre11-Rad50 endonuclease near protein blocks (Huertas et al, 2008; Cannavo & Cejka, 2014; Wang et al, 2017; Reginato et al, 2018), while Xrs2 per se is not essential for the DNA clipping reaction in vitro (Oh et al, 2016), but may have a stimulatory function (Wang et al, 2017). It has been demonstrated that Xrs2 is responsible for the nuclear import of the MRX complex (Carney et al, 1998; Tsukamoto et al, 2005). When this function was bypassed by placing the nuclear localization signal on Mre11, Xrs2 became partially dispensable for the Mre11-Rad50-dependent functions in resection also in vivo (Oh et al, 2016). In contrast, Xrs2 per se was strictly required for the DNA damage signaling function of the MRX complex (Oh et al, 2016). In humans, both CtIP, when phosphorylated by CDK, and NBS1 promote the MRE11-RAD50 endonuclease (Anand et al, 2016; Deshpande et al, 2016). The comparatively stronger requirement for NBS1 in human cells with respect to budding yeast likely reflects the need for more complex regulatory mechanisms to control the MRE11 nuclease in high eukaryotes. How NBS1 performs this function, however, remains poorly characterized. Beyond DSB processing, unscheduled DNA degradation by MRE11 at stalled DNA replication forks may be responsible for the toxicity associated with defects in BRCA1 or BRCA2, further highlighting the importance to understand how MRE11 nuclease is regulated in human cells (Schlacher et al, 2011; Ray Chaudhuri et al, 2016; Feng & Jasin, 2017; Mijic et al, 2017). Human NBS1 consists of 754 amino acids. The N-terminus contains a forkhead-associated domain (FHA) and tandem BRCA1 C-terminal (BRCT) motifs (BRCT1 and BRCT2), which bind phosphorylated proteins, including CtIP and MDC1 (Spycher et al, 2008; Williams et al, 2009; Wang et al, 2013). At its C-terminal part, NBS1 contains ATM and MRE11 interaction sites, as well as three potential nuclear localization signals that promote the nuclear import of MRN (Carney et al, 1998; Nakada et al, 2003; Falck et al, 2005; Tsukamoto et al, 2005; You et al, 2005). The FHA domain of S. cerevisiae Xrs2 binds phosphorylated Sae2, although this capacity appears to be partially dispensable for DNA end resection in vitro and in vivo (Liang et al, 2015; Oh et al, 2016). Structural and biochemical characterization of MRN in Schizosaccharomyces pombe revealed that it also binds phosphorylated Ctp1, an ortholog of CtIP/Sae2 in S. pombe, through the FHA motif of Nbs1 (Lloyd et al, 2009; Williams et al, 2009). A point mutation that affects this interaction resulted in hypersensitivity to ionizing radiation and camptothecin, as well as impaired Ctp1 enrichment at DSBs (Williams et al, 2009). Similarly in human cells, CtIP phosphorylated by CDK at multiple sites in the center of the protein binds the FHA-BRCT domains of NBS1, which is important for DNA end resection (Wang et al, 2013). However, this CDK-dependent phosphorylation was also important for the subsequent modification of T859 by ATM that is required for resection in vivo. Phosphomimetic T859E could partially bypass the requirement for the phosphorylation of CtIP at the CDK sites at the center of the protein that mediate its interaction with the FHA-BRCT domains of NBS1. This raised questions whether modification of these central CDK sites is required for resection per se, or only serves as a platform to help phosphorylate CtIP at T859 (Wang et al, 2013). Furthermore, mutations in FHA-BRCT abrogate the interaction of NBS1 with MDC1, which may have an indirect effect on resection as a result of disrupted signaling or MRN recruitment (Hari et al, 2010). Because of the multiple functions of the MRN complex and potential pleiotropic phenotypes associated with NBS1 defects, the interpretation of cell-based resection assays with NBS1 variants is challenging. Here, we primarily employ in vitro reconstituted reactions to define the function of NBS1 in DNA end resection by MRN-CtIP. We show that both FHA and BRCT domains of NBS1 promote resection by MRE11 through interactions with phosphorylated CtIP. When NBS1 senses that CtIP is phosphorylated, it promotes resection by a mechanism that is dependent on its interaction with MRE11. This is in agreement with a recent study showing that an NBS1 fragment containing the MRE11 binding site but not the FHA-BRCT domains rescues the inviability of NBS1-deficient mouse embryonic fibroblasts (Kim et al, 2017). Importantly, we identify an NBS1-independent DNA cleavage activity of MRE11-RAD50 and CtIP. Although less efficient than the resection capacity of the MRN-CtIP holocomplex, the NBS1-independent activity is promoted by CtIP but surprisingly does not strictly require its phosphorylation. Accordingly, we find limited CtIP- and MRE11-dependent but NBS1-independent DSB resection activity in vivo. These results suggest that a mechanism that interferes with the function of NBS1 might allow limited resection in the absence of CDK-dependent modification of CtIP, which might be relevant for understanding MRE11 nuclease functions in G1. Results NBS1 in trans promotes endonucleolytic cleavage by MRE11-RAD50 and phosphorylated CtIP Previously, we showed that phosphorylated CtIP (pCtIP) promotes the clipping of the 5′-terminated DNA strand near protein-blocked DSBs by the MRE11 nuclease within the MRE11-RAD50-NBS1 (MRN) complex (Anand et al, 2016). This reaction is believed to initiate DNA end resection by MRN. The dsDNA clipping efficiency of MRE11-RAD50 (MR) was strongly reduced compared to MRN, showing that NBS1 has an important function to stimulate this activity (Anand et al, 2016; Deshpande et al, 2016). To define the function of NBS1 in the regulation of the MRE11 nuclease, we purified NBS1, as well as MR, MRN, and pCtIP from baculovirus-infected Spodoptera frugiperda 9 (Sf9) insect cells (Fig EV1A–D). Click here to expand this figure. Figure EV1. Preparation and analysis of NBS1 constructs A representative 10% polyacrylamide gel showing purification of recombinant MBP-NBS1-his. The gel was stained with Coomassie brilliant blue. Amylose flowthrough and eluate, flowthrough and eluate from amylose resin; Ni-NTA flowthrough and eluate, flowthrough and eluate from nickel-nitrilotriacetic acid (Ni-NTA). * indicates truncated products. Samples from a representative purification of the MR complex were analyzed by 10% polyacrylamide gel electrophoresis. The gel was stained with Coomassie brilliant blue. FLAG flowthrough and eluate, flowthrough and eluate from anti-FLAG affinity resin. Samples from a representative purification of the MRN complex analyzed by 10% polyacrylamide gel electrophoresis. Samples from a representative purification of phosphorylated CtIP (expressed and purified with phosphatase inhibitors) analyzed by 10% polyacrylamide gel electrophoresis. MBP, maltose binding protein; PP, PreScission protease. Polyacrylamide gel stained with Coomassie brilliant blue showing the partial cleavage of MBP-NBS1-his by PreScission protease (PP). The PP recognition site is in between the MBP tag and NBS1. 2 μg recombinant MBP-NBS1-his was incubated for 1 h at 4°C with 1 μg of PreScission protease. Nuclease assays as in Fig 1A but with 3′-end-labeled 70-bp dsDNA. Nuclease assays with MR and pCtIP and either uncleaved (left panel) or partially cleaved MBP-NBS1 (right panel, ˜ 50% cleaved) with PreScission protease. 5′-end-labeled 70-bp-long dsDNA was used as a substrate. Polyacrylamide gel stained with Coomassie brilliant showing purified recombinant MBP tag. Nuclease assays with MR, pCtIP, MBP-NBS1, or MBP, as indicated. 5′-end-labeled 70-bp-long dsDNA was used as a substrate. MBP tag does not affect the nuclease reactions. Quantitation of assays such as in (I). Averages shown; n = 3; error bars, SEM. Polyacrylamide gel stained with Coomassie brilliant showing purified recombinant MBP-NBS1-his and FLAG-NBS1-his. * indicates truncated product. Nuclease assays with MR and pCtIP and either MBP-NBS1-his (left panel) or FLAG-NBS1 (right panel). 3′-end-labeled 70-bp-long dsDNA was used a substrate. Download figure Download PowerPoint NBS1 was purified by affinity chromatography by utilizing maltose binding protein (MBP) and 10× histidine (his) tags, located at the N- and C-termini, respectively. As the removal of the MBP tag by PreScission protease cleavage was not very efficient (Fig EV1E), the MBP tag was retained during the final purification (Fig EV1A). MBP-NBS1-his added in trans promoted dsDNA clipping by MR and pCtIP (Fig 1A and B). This was almost as efficient as DNA cleavage by pCtIP and MRN purified as a complex, where NBS1 was untagged (Figs 1A and B, and EV1F). Therefore, the affinity tags did not notably impair the stimulatory function of NBS1 on dsDNA clipping by MR and pCtIP in vitro. To further confirm this, we note that treatment of MBP-NBS1 with PreScission protease, which cleaves ~ 50% of the MBP tag off NBS1, did not affect dsDNA clipping efficiency (Fig EV1E and G). The MBP tag, expressed separately, did not affect the cleavage capacity of MR and pCtIP (Fig EV1H–J). Finally, FLAG-NBS1-his construct promoted DNA cleavage similarly as MBP-NBS1-his (Fig EV1K and L). In summary, we conclude that MBP-NBS1-his (hereafter NBS1 for brevity) added in trans promotes the capacity of MR and pCtIP ensemble to clip 5′-terminated DNA in the vicinity of protein blocks. Figure 1. NBS1 in trans with MR and pCtIP cleaves DNA similarly as MRN-pCtIP A representative nuclease assay with MR, MBP-NBS1-his (denoted MBP-NBS1), MRN, and pCtIP on 5′-end-labeled 70-bp dsDNA with all ends blocked with streptavidin. Samples were separated on 15% denaturing polyacrylamide gel. Quantitation of nuclease assays such as in (A). Averages shown; n ≥ 3; error bars, SEM. Download figure Download PowerPoint FHA and BRCT domains of NBS1 are important for the processing of protein-blocked DSBs, while its interaction with MRE11 is essential NBS1 mediates physical interactions of the MRN complex with ATM, BRCA1, BLM, MDC1, CtIP, and possibly other factors (Carney et al, 1998; Stewart et al, 1999; Wang et al, 2000, 2013; Goldberg et al, 2003; Chen et al, 2008; Spycher et al, 2008; Williams et al, 2008; Hari et al, 2010). Out of these, the interactions with CtIP and MDC1 are at least in part phosphorylation-dependent. To specifically define the functional interaction of NBS1 with MR and pCtIP in resection, we employed our in vitro reconstituted system (Anand et al, 2016). To this point, we prepared NBS1 variants lacking particular domains or carrying mutations specifically affecting known interactions with the MR and pCtIP ensemble (Figs 2A and EV2A), and assayed them in nuclease assays to determine their effect on the MRE11 endonuclease. Figure 2. FHA and BRCT domains of NBS1 are important for MR-pCtIP stimulation while MRE11-NBS1 interaction is essential A schematic representation of purified recombinant wild-type NBS1 and variants. All constructs were MBP tagged at the N- and his-tagged at the C-terminus. The effect of mutations in FHA and/or BRCT domains of NBS1 on the nuclease of MR and pCtIP. Quantitation of experiments such as shown in Fig EV2D. Averages shown; n ≥ 4, error bars, SEM. Statistical significance denotations represent the analysis between wild-type full-length NBS1 (in black) and the corresponding concentrations of the NBS1 variants; ns (P > 0.05, not significant), *(P < 0.05), **(P < 0.01), ***(P < 0.001), two-tailed t-test. Analysis of pCtIP binding to NBS1 variants. Anti-CtIP antibody was immobilized on protein G agarose, bound to pCtIP, and tested for interactions with the indicated NBS1 variants (see cartoon at the top). Western blot of eluates (right part) was performed with anti-CtIP and anti-NBS1 antibodies. Left part (input) indicates that the anti-NBS1 antibody recognizes the NBS1 constructs to a similar level. Analysis of phosphorylated CtIP (pCtIP, mock-treated) or λ phosphatase-treated CtIP (λCtIP) interactions with NBS1. The CtIP variants were immobilized on protein G agarose and incubated with NBS1 (see cartoon on the left). The Western blot was performed with anti-CtIP and anti-NBS1 antibodies. The effect of the MRE11 interaction region (MIR) of NBS1 on the nuclease of MR and pCtIP. Quantitation of experiments such as shown in Fig EV2E. Averages shown, n ≥ 3, error bars, SEM. The quantitation of wild-type NBS1 (NBS1 WT, in black) is the same as in panel (B) and is shown again for reference. Statistical significance denotations represent the analysis between wild-type full-length NBS1 (in black) and the corresponding concentrations of the NBS1 variants; ns (P > 0.05, not significant), ***(P < 0.001), two-tailed t-test. Download figure Download PowerPoint Click here to expand this figure. Figure EV2. Stimulation of MR-pCtIP by NBS1 A representative polyacrylamide gel (4–15%) stained with Coomassie brilliant blue showing purified NBS1 variants. All constructs were MBP tagged at the N- and his-tagged at the C-terminus. A representative nuclease assay with MR, pCtIP, and various concentrations of MBP-NBS1-his (labeled NBS1) on 3′-end-labeled dsDNA. Quantitation of experiments such as shown in (B). Averages shown; n = 5; error bars, SEM. Representative nuclease assays with MR, pCtIP, and various MBP-NBS1-his fragments (10 nM) containing MRE11 interaction region (MIR), using 3′-end-labeled dsDNA as a substrate. Representative nuclease assays with MR, pCtIP, and various NBS1 fragments (10 nM) lacking MRE11 interaction region (MIR) on 3′-end-labeled dsDNA. Representative nuclease assays with MR, pCtIP, and various concentrations of MBP-NBS1∆MIR, using 3′-end-labeled dsDNA as a substrate. A representative 10% polyacrylamide gel stained with Coomassie brilliant blue showing purified recombinant MRE11-his. Interaction of MRE11 with NBS1. MRE11 was immobilized on antibody-coupled protein G agarose and incubated with MBP-NBS1-his or MBP-NBS1∆MIR-his (see cartoon). Bound proteins were analyzed by Western blot with anti-his antibodies. Nuclease assay with NBS1 variants, showing that these proteins alone do not possess any nuclease activity. Download figure Download PowerPoint We first varied the concentrations of wild-type NBS1 in reactions with MR and pCtIP. While weak NBS1-independent DNA cleavage was observed, we found that less than equimolar concentrations of NBS1, compared to the MR complex, were sufficient for maximal DNA clipping activity (Fig EV2B and C). We next tested various NBS1 fragments lacking FHA, BRCT, or both domains (Figs 2B and EV2D). As phosphorylation of CtIP is required for resection (Huertas & Jackson, 2009), and the MRN complex primarily binds phosphorylated CtIP via the FHA and BRCT domains of NBS1 (Fig 2C and D; Wang et al, 2013; Williams et al, 2009), a strong defect in resection was anticipated. As shown in Fig 2B, a deletion of either FHA or BRCT domain in NBS1 had only a minor effect on resection in vitro, while elimination of both domains significantly reduced, but did not eliminate resection. Similarly to the elimination of both FHA and BRCT domains, a stronger inhibitory effect was observed with the NBS1 RRHK variant, carrying point mutations in FHA-BRCT (R28A, R43A, and H45A in FHA and K160M in BRCT1, Fig 2A and B; Wang et al, 2013). The RRHK mutations decreased the phosphorylation-dependent interaction of NBS1 with phosphorylated MDC1 (Hari et al, 2010), as well as with pCtIP (Fig 2C), in agreement with a dramatic DNA end resection defect in vivo, as scored by the single-strand annealing reporter assay (Wang et al, 2013). Despite the strong physical interaction between the FHA and BRCT domains of NBS1 and pCtIP (Fig 2C), which is dependent on CtIP phosphorylation (Fig 2D), we conclude that both domains of NBS1 are important, but not essential for the DNA clipping activity in vitro together with MR and pCtIP. Furthermore, NBS1 (335–754), which lacks FHA-BRCT but contains a central linker region, exhibited similar stimulatory activity to NBS1 (622–754) lacking the central region (Fig 2B and E). This result revealed that the central NBS1 region (residues 335–621) is largely dispensable for MR- and pCtIP-mediated resection, despite this region mediated residual interaction with pCtIP (Fig 2C). It has been demonstrated that the MRE11-RAD50 complex directly interacts with NBS1 via the MRE11 interaction region (MIR) within the C-terminal part of NBS1, with the most important motif located between residues 684–690 of NBS1 (Desai-Mehta et al, 2001; Schiller et al, 2012; Kim et al, 2017). This interaction facilitates MRN entry into the nucleus as only NBS1 contains the nuclear localization sequence. Therefore, in vivo experiments with mutated MIR of NBS1 cannot easily distinguish effects related to impaired nuclear entry from direct effects on the biochemical activities of the MR complex. Using our in vitro system, where any effects on nuclear import are irrelevant, we observed that in contrast to the FHA and BRCT domains, the MRE11 interaction region in NBS1 was absolutely essential for the stimulatory function of NBS1 on the MRE11-RAD50 endonuclease in conjunction with pCtIP (Figs 2E and EV2E). Specifically, the NBS1 (1–692) fragment lacking the very C-terminal region of NBS1, but possessing MIR, exhibited similar activity as full-length NBS1 (Figs 2E and EV2D). In contrast, the NBS1 (1–683) mutant lacking nine residues comprising MIR (residues 684–692) completely lost its stimulatory activity (Figs 2E and EV2E). Likewise, the internal deletion of MIR (residues 684–690) totally abolished NBS1 function in DNA clipping, even at high concentrations (Figs 2E, and EV2E and F). In accord with previous studies (Desai-Mehta et al, 2001; Schiller et al, 2012), we observed a dramatic reduction of the MRE11 and NBS1 physical interaction when NBS1 ∆MIR variant was used in pulldown experiments instead of wild-type NBS1 (Fig EV2G and H). None of the NBS1 variants used here had any DNA cleavage activity, as expected (Fig EV2I). In summary, the NBS1-dependent interaction with MRE11-RAD50, more than that with pCtIP, is important for the endonuclease activity of the MRE11-RAD50 and pCtIP nuclease ensemble. In the absence of pCtIP, NBS1 promotes MR cleavage independently of its FHA and BRCT domains Recently, it has been shown that NBS1 alone is capable of stimulating the endonucleolytic activity of MR on protein-blocked dsDNA, independently of CtIP (or pCtIP; Deshpande et al, 2016). Although CtIP-independent DNA end resection does not likely occur in vivo (Sartori et al, 2007), we used the in vitro assay to learn more about the function of NBS1. To this point, we employed our NBS1 mutants in an MR-dependent nuclease assay with streptavidin-blocked dsDNA. In contrast to the assays that included pCtIP, the reactions were incubated for 2 h instead of 30 min to compensate for the lower cleavage efficacy in the absence of pCtIP. We observed that all NBS1 fragments containing MIR stimulated the clipping activity of MR almost indistinguishably, irrespectively of FHA and BRCT domains (Fig 3A and B). Notably, one of these constructs, the short C-terminal NBS1 fragment containing 133 residues (NBS1 622–754), stimulated the endonucleolytic cleavage to the same extent as full-length wild-type NBS1. Conversely, NBS1 fragments lacking MIR did not at all promote MR (Fig 3A and B). Very similar results were obtained using circular ssDNA as a substrate, which forms a variety of secondary DNA structures (Fig EV3A and B). However, as noted previously, we believe th
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