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• W2804370147 abstract "Article16 May 2018free access Source DataTransparent process DNA activates the Nse2/Mms21 SUMO E3 ligase in the Smc5/6 complex Nathalia Varejão Institut de Biotecnologia i de Biomedicina (IBB), Department of de Bioquímica i Biologia Molecular, Universitat Autònoma de Barcelona, Bellaterra, Spain Search for more papers by this author Eva Ibars Institut de Recerca Biomèdica de Lleida (IRBLLEIDA), Department of Ciències Mèdiques Bàsiques, Universitat de Lleida, Lleida, Spain Search for more papers by this author Jara Lascorz Institut de Biotecnologia i de Biomedicina (IBB), Department of de Bioquímica i Biologia Molecular, Universitat Autònoma de Barcelona, Bellaterra, Spain Search for more papers by this author Neus Colomina Institut de Recerca Biomèdica de Lleida (IRBLLEIDA), Department of Ciències Mèdiques Bàsiques, Universitat de Lleida, Lleida, Spain Search for more papers by this author Jordi Torres-Rosell Corresponding Author [email protected] orcid.org/0000-0003-1308-6926 Institut de Recerca Biomèdica de Lleida (IRBLLEIDA), Department of Ciències Mèdiques Bàsiques, Universitat de Lleida, Lleida, Spain Search for more papers by this author David Reverter Corresponding Author [email protected] orcid.org/0000-0002-5347-0992 Institut de Biotecnologia i de Biomedicina (IBB), Department of de Bioquímica i Biologia Molecular, Universitat Autònoma de Barcelona, Bellaterra, Spain Search for more papers by this author Nathalia Varejão Institut de Biotecnologia i de Biomedicina (IBB), Department of de Bioquímica i Biologia Molecular, Universitat Autònoma de Barcelona, Bellaterra, Spain Search for more papers by this author Eva Ibars Institut de Recerca Biomèdica de Lleida (IRBLLEIDA), Department of Ciències Mèdiques Bàsiques, Universitat de Lleida, Lleida, Spain Search for more papers by this author Jara Lascorz Institut de Biotecnologia i de Biomedicina (IBB), Department of de Bioquímica i Biologia Molecular, Universitat Autònoma de Barcelona, Bellaterra, Spain Search for more papers by this author Neus Colomina Institut de Recerca Biomèdica de Lleida (IRBLLEIDA), Department of Ciències Mèdiques Bàsiques, Universitat de Lleida, Lleida, Spain Search for more papers by this author Jordi Torres-Rosell Corresponding Author [email protected] orcid.org/0000-0003-1308-6926 Institut de Recerca Biomèdica de Lleida (IRBLLEIDA), Department of Ciències Mèdiques Bàsiques, Universitat de Lleida, Lleida, Spain Search for more papers by this author David Reverter Corresponding Author [email protected] orcid.org/0000-0002-5347-0992 Institut de Biotecnologia i de Biomedicina (IBB), Department of de Bioquímica i Biologia Molecular, Universitat Autònoma de Barcelona, Bellaterra, Spain Search for more papers by this author Author Information Nathalia Varejão1, Eva Ibars2, Jara Lascorz1, Neus Colomina2, Jordi Torres-Rosell *,2 and David Reverter *,1 1Institut de Biotecnologia i de Biomedicina (IBB), Department of de Bioquímica i Biologia Molecular, Universitat Autònoma de Barcelona, Bellaterra, Spain 2Institut de Recerca Biomèdica de Lleida (IRBLLEIDA), Department of Ciències Mèdiques Bàsiques, Universitat de Lleida, Lleida, Spain *Corresponding author. Tel: +34 973 702438; E-mail: [email protected] *Corresponding author. Tel: +34 935 868955; E-mail: [email protected] EMBO J (2018)37:e98306https://doi.org/10.15252/embj.201798306 See also: A Pichler (June 2018) 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 Modification of chromosomal proteins by conjugation to SUMO is a key step to cope with DNA damage and to maintain the integrity of the genome. The recruitment of SUMO E3 ligases to chromatin may represent one layer of control on protein sumoylation. However, we currently do not understand how cells upregulate the activity of E3 ligases on chromatin. Here we show that the Nse2 SUMO E3 in the Smc5/6 complex, a critical player during recombinational DNA repair, is directly stimulated by binding to DNA. Activation of sumoylation requires the electrostatic interaction between DNA and a positively charged patch in the ARM domain of Smc5, which acts as a DNA sensor that subsequently promotes a stimulatory activation of the E3 activity in Nse2. Specific disruption of the interaction between the ARM of Smc5 and DNA sensitizes cells to DNA damage, indicating that this mechanism contributes to DNA repair. These results reveal a mechanism to enhance a SUMO E3 ligase activity by direct DNA binding and to restrict sumoylation in the vicinity of those Smc5/6-Nse2 molecules engaged on DNA. Synopsis A novel mechanism based on DNA contacts with a sensor patch on Smc5 enhances the activity of the SUMO E3 ligase Nse2 in the Smc5/6 complex after chromatin loading, contributing to increased sumoylation on chromatin and yeast cell survival after DNA damage. DNA binding to a positively charged DNA sensor region in the arm domain of Smc5 stimulatesSUMO E3 activity of Nse2. Electrostatic interactions of DNA with the Smc5 DNA sensor trigger a conformational change in the Nse2-Smc5 subcomplex. The Smc5 DNA sensor promotes Nse2-dependent sumoylation in vivo and is required for yeast cell viability in response to DNA damage. Introduction Genome integrity is under constant surveillance. External insults, the metabolism of the cell, and most chromosome transactions can damage the genome, resulting in deleterious mutations or cell death. Cells have therefore developed DNA repair and DNA damage tolerance mechanisms, which often require post-translational modifications to recruit and activate repair factors. Post-translational modification by conjugation to the small ubiquitin-like modifier SUMO actively participates in maintaining the integrity of the genome: SUMO plays pivotal roles during chromosome replication and segregation and in virtually all DNA repair mechanisms (Bergink & Jentsch, 2009). In fact, the extensive sumoylation of many DNA repair and replication factors constitutes an integral part of the DNA damage response (Cremona et al, 2012). SUMOylation involves the formation of an isopeptide bond between the ε-amino group of a lysine residue and the C-terminus of the SUMO protein. This reaction requires the participation of an E1 activating enzyme (Uba2/Aos1) that passes the SUMO proteins to an E2 conjugating enzyme (Ubc9). Once charged with SUMO, Ubc9 transfers SUMO to the target protein. This can occur directly, by recognition of a SUMO consensus motif (Bernier-Villamor et al, 2002; Yunus & Lima, 2006), but most often requires the participation of an E3 SUMO ligase enzyme. Budding yeast codes for three mitotic SUMO E3 ligases, including two members of the PIAS family (Siz1 and Siz2) and the Siz/PIAS-related Nse2 protein (Johnson & Gupta, 2001; Zhao & Blobel, 2005). Since the E1 and E2 enzymes in the SUMO pathway lack known DNA binding domains, modification of chromosome-bound proteins seems to rely on recruitment of SUMO E3 ligases to chromatin (Ulrich, 2014). This can occur by direct binding of the E3 ligase to DNA, by interaction of the E3 with chromosome-associated proteins, or by granting access of the E3 to DNA lesions via previous phosphorylation and ubiquitination events at damaged sites. For example, an N-terminal SAP domain localize Siz2 on DNA, where it promotes sumoylation of homologous recombination factors (Psakhye & Jentsch, 2012); additionally, Siz2 binds ssDNA through interaction with the ssDNA binding replication protein A (RPA; Chung & Zhao, 2015). The Nse2 SUMO ligase, a subunit of the Smc5/6 complex, has also been shown to play key roles in the maintenance of chromosome integrity (Zhao & Blobel, 2005; Ampatzidou et al, 2006; Branzei et al, 2006; Potts et al, 2006; Pebernard et al, 2008b; Behlke-Steinert et al, 2009; Chavez et al, 2010; Bermúdez-López et al, 2015). As Nse2 lacks DNA binding domains, its DNA repair functions require its stable docking onto the Smc5 protein (Duan et al, 2009; Bermúdez-López et al, 2015), and the subsequent association of the Smc5/6 complex with damaged sites (De Piccoli et al, 2006; Lindroos et al, 2006; Tapia-Alveal & O'Connell, 2011; Bustard et al, 2012). Structural Maintenance of Chromosomes (SMC) complexes are topologically closed molecules formed by the heterodimerization of two elongated SMC subunits and by a distinct number of associated non-SMC elements (Uhlmann, 2016). SMC proteins contain three different domains: an ATPase head structurally related to that of ABC transporters (hereafter named “HEAD”), an extended coiled coil region (“ARM”), and a heterodimerization or hinge domain (“HINGE”). While hinge heterodimerization closes the molecule at one end, a kleisin subunit connects the two ATPase heads at the other end, defining an inner compartment delimited mainly by two long coiled coils. Each SMC complex has specific and essential roles: Cohesin maintains connections between sister chromatids, condensin compacts chromosomes, and Smc5/6 promotes chromosome disjunction. Despite these seemingly disparate functions, all SMC complexes share a common property, which is to organize chromosomes by topologically embracing DNA inside their ring-shaped structure. This function requires the ATPase activity of the head domains, which regulates the entry and release of DNA fibers inside the SMC ring. In prokaryotes, the ATPase-dependent conformational changes in the head domain can affect the architecture of the coiled coil domains, altering the ability of SMC molecules to embrace DNA (Bürmann et al, 2017). Once loaded onto chromatin, Smc5/6 participates in critical chromosome transactions during DNA replication and repair. The Smc5 and Smc6 subunits can bind strongly to DNA in vitro through several binding regions located in the hinge, the head and the arm regions (Roy & D'Amours, 2011; Roy et al, 2011, 2015; Alt et al, 2017). In vivo, the affinity of the SMC core for DNA is regulated by the other six non-SMC subunits (Nse proteins). Nse4, the kleisin subunit in the Smc5/6 complex, associates with Nse1 and Nse3 to constitute a stable subcomplex. The Nse1-Nse3 pair contains a patch of positively charged residues that acts as a DNA-binding surface and mediates Smc5/6 loading onto chromatin (Zabrady et al, 2016). Although no specific function has been attributed to the Nse5 and Nse6 subunits, their alleged functional homologues in vertebrates (SLF1 and SLF2) have been proposed to promote recruitment of the Smc5/6 complex to interstrand cross-links (Räschle et al, 2015). The Nse2 SUMO ligase has an undefined role in sister chromatid recombination and chromosome disjunction by promoting the sumoylation of several targets including subunits in cohesin (Potts et al, 2006; Almedawar et al, 2012; McAleenan et al, 2012), Smc5/6 (Bermúdez-López et al, 2015) and STR complexes (Bermúdez-López et al, 2016; Bonner et al, 2016). To reach its substrates, the E3 domain must first dock onto the central part of the ARM region of Smc5 through a long N-terminal helical domain (Duan et al, 2009). While docking of Nse2 onto Smc5 through the N-terminal domain is essential for viability (Duan et al, 2009; Bermúdez-López et al, 2015), the C-terminal RING domain, which codes for the E3 SUMO ligase activity, is dispensable; however, mutations in the SUMO ligase domain render cells sensitive to DNA damage, highlighting its relevance in genome maintenance. It is currently unclear whether all Smc5/6-Nse2 molecules are SUMO-active, potentially targeting soluble proteins, or whether their activity is restrained to those Smc5/6-Nse2 molecules directly engaged on DNA, promoting the modification of chromosome-associated targets only. Here we show that DNA stimulates the SUMO ligase activity of Nse2 via a direct and non-specific interaction with a positively charged patch in the ARM region of Smc5. This interaction probably induces a conformational change in the Nse2 molecule that ultimately enhances its SUMO E3 ligase activity, thereby promoting DNA damage repair. Overall, our findings define a new mechanism to activate a SUMO ligase on site, which does not merely rely on recruitment of the E3 to DNA, but on local upregulation of its activity after loading onto chromatin. Results DNA binding enhances the SUMO conjugation activity of Nse2 Despite most known SUMO-targets of Nse2 are chromosomal proteins (Andrews et al, 2005; Zhao & Blobel, 2005; Potts et al, 2006; Potts & Yu, 2007; Pebernard et al, 2008a; Almedawar et al, 2012; McAleenan et al, 2012; Yong-Gonzales et al, 2012; Albuquerque et al, 2013), it is currently unknown whether chromatin loading of Smc5/6 molecules modulates its E3 ligase activity. We therefore tested whether the SUMO conjugation activity of Nse2 could be directly affected by the presence of DNA. In vitro assays using recombinant full-length Smc5 in complex with Nse2 show a substantial increase in SUMO conjugation in the presence of single-stranded DNA (Figs 1A and EV1). The SUMO E3 ligase activity of Nse2 is strikingly enhanced by the presence of ssDNA, as observed after 30 min in multiple turnover reactions. The C-terminal kleisin domain of Nse4 (cNse4—from Ile246 to Asp402) was used as a model substrate in our in vitro reactions, although SUMO conjugation can also be observed internally on lysine residues of Smc5 (Figs 1A and EV1). These results suggest that DNA binds to the Smc5-Nse2 heterodimer and stimulates the E3 SUMO ligase activity of Nse2. It is important to note that in addition to target selectivity, E3 ligases also optimize catalysis, by placing the functional groups in an optimal orientation for Ub/Ubl transfer between the E2-thioester and the target substrate (Reverter & Lima, 2005; Deshaies & Joazeiro, 2009; Plechanovová et al, 2012; Scott et al, 2014; Buetow et al, 2015; Streich & Lima, 2016). Figure 1. Stimulation of the SUMO E3 ligase activity of the Smc5-Nse2 complex upon binding to DNA Time-course SUMO conjugation reaction in the presence or absence of ssDNA (virion ϕx174) using recombinant full-length Smc5-Nse2 complex. The substrate utilized was the C-terminal kleisin domain of Nse4 (cNse4). Reaction was run at 30°C and stopped at indicated minutes by adding SDS-loading buffer. Labels indicate the bands in the SDS–PAGE of the proteins in the reaction mixture (N-S2, cNse4-SUMO2; N-2S2, cNse4-2SUMO2; and N-3S2, cNse4-3SUMO2). Left, SYPRO-stained and Western blot (anti-SUMO2) of the SUMO conjugation reaction by Smc5-Nse2 complex in the presence of ssDNA (virion ϕx174) and dsDNA (pET-DUET-1) at either 1 or 10 nM. Reactions were run at 30°C and stopped at 60 min by adding SDS-loading buffer. Right, bar diagram comparison of the relative SUMO conjugated cNse4 substrate in the presence of either ssDNA (virion ϕx174) or dsDNA (pET-DUET-1) at indicated concentrations. Straight line shows the basal cNse4-SUMO conjugation in the absence of DNA. Data values are mean ± s.e.m. and n = 3 technical replicates. Left, Western blot of the SUMO conjugation reaction in the presence of 50b oligonucleotide and the 5 kb virion ϕx174 at the indicated concentrations. Reactions were run at 30°C and stopped after 60 min by adding SDS-loading buffer. Right, bar diagram comparison of the relative SUMO conjugated cNse4 substrate in the presence of 25, 34, or 50 bases oligonucleotides at indicated concentrations. Straight line shows the basal cNse4-SUMO conjugation in the absence of DNA. Data values are mean ± s.e.m. and n = 3 technical replicates. Bar diagrams calculation was generated using ImageJ software (Schneider et al, 2012). Download figure Download PowerPoint Click here to expand this figure. Figure EV1. Enhancement of the SUMO E3 ligase activity of Smc5-Nse2 upon binding to DNA Time-course conjugation reaction of SUMO1 (left) or Smt3 (right) in the presence or absence of ssDNA (virion ϕx174) at 8 nM using full-length Smc5-Nse2 complex. The substrate utilized was the C-terminal kleisin domain of Nse4 (cNse4). Reactions using human or yeast E1 and E2 enzymes, for SUMO1 and Smt3, respectively, were run at 30°C and stopped at indicated minutes by adding SDS-loading buffer. Labels indicate the bands in the SDS–PAGE of the proteins in the reaction mixture (N-S1, cNse4-SUMO1; N-2S1, cNse4-2SUMO1; N-Smt3, cNse4-Smt3; N-2Smt3, cNse4-2Smt3). SYPRO-stained SDS–PAGE of one of the triplicate SUMO conjugation reactions used to calculate the ssDNA vs. dsDNA plot in Fig 1B. The reactions were run for 60 min at 30°C and stopped by adding SDS-loading buffer. SYPRO-stained SDS–PAGE of one of the triplicate SUMO conjugation reactions used to calculate the oligonucleotide plot in Fig 1C. The reactions were run for 60 min at 30°C and stopped by adding SDS-loading buffer. Download figure Download PowerPoint Most of our in vitro assays utilize single-stranded DNA (ssDNA, 5 kb virion ϕx174), which seems to produce a higher increase in SUMO conjugation than a double-stranded DNA plasmid of a similar length (Fig 1B), although both types of DNA molecules can produce a substantial increment in SUMO conjugation (Figs 1B and EV1). Single-stranded DNA has been reported to bind Smc5 and Smc6 molecules with higher affinity than double-stranded DNA through multiple binding sites, including parts of the coiled coil ARM domain (Roy et al, 2011, 2015). Additionally, shorter ssDNA molecules, such as random oligonucleotides of 20, 34, and 50 nucleotides, can also enhance SUMO conjugation when used at higher concentrations (μM) than the virion plasmid (nM) in a dose-dependent manner (Figs 1C and EV1). Comparative activity assays using different types of ssDNA molecules at similar nucleotide concentration reveal an equal stimulation of the SUMO conjugation for either oligonucleotides of 50b or long ssDNA molecules (5 kb virion ϕx174 ssDNA; Fig 1C). Small oligonucleotides (25b) can also enhance SUMO conjugation when used at higher concentrations (Fig 1C). These results suggest a non-specific dose-dependent binding of DNA to the Smc5/6 complex resulting in a stimulation of the E3 ligase activity of Nse2. A minimal Smc5 ARM domain is sufficient for upregulation of DNA-dependent SUMO conjugation Nse2 has weaker SUMO conjugation activity in the absence of Smc5, with no significant increase in the presence of ssDNA (Fig 2B and E, and Appendix Fig S1). This observation indicates that the Smc5 protein might directly interact with DNA to enhance sumoylation. Smc5 has been reported to bind DNA through multiple binding sites (Roy et al, 2011, 2015). Therefore, we mapped the regions in Smc5 responsible for sensing DNA that could enhance SUMO conjugation. We prepared two truncations of Smc5 in complex with Nse2: one lacking the dimerization HINGE domain (ΔHinge/Smc5), and another lacking the ATPase HEAD domain (ΔHead/Smc5). In both cases, the coiled coil domain of Smc5 included the Nse2-binding region (Fig 2A). Full-length and Smc5 truncations displayed a comparable and remarkable increase in SUMO conjugation in the presence of DNA (Fig 2B and Appendix Fig S1). The ΔHinge/Smc5 construct displayed a lower fold increase in sumoylation, most probably due to its already higher activity than wild-type Smc5, even in the absence of DNA (Fig 2B and Appendix Fig S1). We speculate that the HINGE domain may exert an inhibitory role on Nse2-dependent sumoylation. As cNse4 does not interact with either ΔHead/Smc5 or Arm/Smc5 (Appendix Fig S2), these assays also indicate that the enhancement of the SUMO E3 ligase activity can occur either on external substrates (such as cNse4) or on internal lysines residues of Smc5 (Fig 2C and D, and Appendix Fig S1). The only common region in all Smc5 constructs corresponds to the ARM coiled coil region that docks Nse2 (from Asp302 to Thr366, and from Arg737 to Gln813), indicating that it might contain a minimal DNA binding region (DNA sensor) that promotes the stimulatory effect on the E3 ligase activity of Nse2. Figure 2. Enhancement of the SUMO E3 ligase activity upon DNA binding by Smc5-Nse2 truncation complexes Schematic representation of the domain composition of the heterodimeric full-length Smc5-Nse2 complex. Bar diagram representation of the relative SUMO conjugation activity of Nse2, full-length Smc5-Nse2, ΔHinge/Smc5-Nse2, ΔHead/Smc5-Nse2, and Arm/Smc5-Nse2 truncation constructs (schematic representation above). Orange bars indicate the presence of ssDNA (virion ϕx174), and red bars indicate absence of ssDNA. Reaction rates were performed at least in three different independent experiments (see Fig EV2). Data values are mean ± s.e.m.; and n = 3 technical replicates. Significance was measured by a two-tailed unpaired t-test relative to wild-type. **P < 0.01. SYPRO-stained (left) and Western blot (right) time-course SUMO conjugation reaction using and ΔHead/Smc5-Nse2 truncation construct in the presence of ssDNA. T7-tagged Smc5 and E1 were immunodetected by an anti-T7 antibody. Reactions were run at 30°C and stopped at indicated times by adding SDS-loading buffer. SYPRO-stained (left) and Western blot (right) time-course SUMO conjugation reaction using and ΔHinge/Smc5-Nse2 truncation construct in the presence of ssDNA. T7-tagged Smc5 and E1 were immunodetected by an anti-T7 antibody. Reactions were run at 30°C and stopped at indicated times by adding SDS-loading buffer. Western blot of the time-course SUMO conjugation reaction in the presence of ssDNA (virion ϕx174) using either Nse2 (left) or Arm/Smc5-Nse2 complex (right). The reactions were run in the presence or absence of cNse4 external substrate. Reaction was run at 30°C and stopped at indicated minutes by adding SDS-loading buffer (N-S2, cNse4-SUMO2; N-2S2, cNse4-2SUMO2; N-3S2, cNse4-3SUMO2; and pS2, poly-SUMO2). Download figure Download PowerPoint Therefore, we produced this Smc5 coiled coil ARM region (named Arm/Smc5) in complex with Nse2, based on the published crystal structure of Nse2-Smc5 (PDB 3HTK; Duan et al, 2009). Activity assays in the presence or absence of cNse4 also display a striking comparable enhancement in SUMO conjugation upon DNA binding to Arm/Smc5-Nse2, similarly to the other Smc5 long truncation constructs (Fig 2B and E). These results indicate that the DNA binding patch involved in the enhancement of the E3 ligase might be restricted to this ARM/Smc5 region in contact with Nse2. A positive-patch region of Smc5 ARM domain interacts with DNA Similarly to the full-length Smc5-Nse2 complex, different ssDNA molecules can also stimulate SUMO conjugation in the Arm/Smc5-Nse2 construct in a dose-dependent manner (Fig 3A). Additionally, single turnover reactions using this minimal Arm/Smc5-Nse2 construct display a strong enhancement of the E2-thioester SUMO discharge in the presence of a 50b ssDNA oligonucleotide, indicating the role of the DNA binding promoting the isopeptidic bond formation by the stimulation of the E3 ligase (Fig 3B). Figure 3. A positive-patch region on the surface of Smc5 ARM domain interacts with DNA Western blot of the SUMO conjugation reaction in the presence of different oligonucleotides using the minimal Smc5/ARM-Nse2 construct. Reactions were run at 30°C and stopped after 60 min by adding SDS-loading buffer. 25b (μM), 34b (μM), and 50b (μM), stands for a 25, 34, and 50 bases oligonucleotides, respectively, and the indicated concentration is in μM units. 5 kb (nM) stands for the virion ϕx174, and the indicated concentration is in nM units (N-S1, cNse4-SUMO1; N-2S1, cNse4-2SUMO1; and pS1, poly-SUMO1) (*overexposed chemiluminescent signal). Left, Ubc9-thioester formation in the presence of E1, E2 enzymes, Alexa488-SUMO1, and ATP. Right, single turnover reaction of the SUMO conjugation reaction in the presence or absence of ssDNA (50b) using Arm/Smc5-Nse2 as E3. Samples were run in the presence (below) or absence of β-mercaptoethanol (above). Ribbon representation of the complex between the ARM domain of Smc5 (yellow and orange) and Nse2 (pink) (PDB 3HTK; Duan et al, 2009). Lysine residues forming the positive-charged patch in the surface of the coiled coil Smc5 ARM are labeled and shown in stick representation (blue). Zinc atom in the Nse2 RING domain is depicted as a yellow sphere. Bar diagram representation of the SUMO conjugation rates of activity assays of Arm/Smc5-Nse2 KE mutants in the presence (orange bars) or absence (red bars) of ssDNA (virion ϕx174), relative to wild type (set to 1). Reaction rates were performed at least in three different independent experiments. Data values are mean ± s.e.m. and n = 3 technical replicates. Significance was measured by a two-tailed unpaired t-test relative to wild-type. *P < 0.05, **P < 0.01, ***P < 0.001. DNA binding properties of wild-type, K337E/K344E/K764E and K743E/K745E Arm/Smc5-Nse2 mutants, were determined by electrophoretic mobility shift assays (EMSA) saturation experiments. Protein complexes were incubated for 30 min at 30°C before loading the agarose gel electrophoresis. Numbers above gel indicate the molar ratio (×103) of protein over ssDNA (virion ϕx174) in each lane. Download figure Download PowerPoint Our next goal was to uncover the DNA binding regions on the surface of the minimal Smc5 ARM domain that can stimulate the activity of Nse2. Interestingly, the crystal structure of the Smc5-Nse2 complex revealed positive-charged patch regions in the Smc5 coiled coil surface of Arm/Smc5-Nse2 that could fulfill non-specific interaction to DNA (Fig 3C). To check this electrostatic interaction, we produced several Arm/Smc5-Nse2 constructs with different combinations of lysine to glutamic acid mutants to counter-charge binding to phosphate groups of DNA. All tested Arm/Smc5-Nse2 KE mutants show a comparable activity in the absence of DNA (Figs 3D and EV2), indicating in all cases that the mutagenesis has not compromised either the structure or the catalytic properties of the enzyme. However, in the presence of DNA, all single point, double, and K337E/K344E/K764E mutants reduce SUMO conjugation at different levels, reaching an almost complete loss of enhancement in the K743E/K745E mutant (Fig 3D). Interestingly, the most relevant lysine residues locate in the region next to the RING domain, reducing the effect as they move away from that region. We attribute this decrease in the SUMO E3 ligase activity of Nse2 to an electrostatic perturbation in DNA binding. Also, in contrast to the KE mutants, the SUMO conjugation enhancement of the K743R/K745R double mutant is similar to the wild-type form (Fig 3D), confirming the role of the electrostatic charge of this interface in the DNA binding. Additionally, electrophoretic mobility shift assays (EMSA) using two different Arm/Smc5-Nse2 KE mutants, K337E/K344E/K764E and K743E/K745E, showed a significant reduction in the DNA binding in comparison with the wild-type form, confirming the perturbation of the binding between DNA and the Arm/Smc5-Nse2 complex (Fig 3E). These EMSA experiments were conducted at higher DNA:protein ratios in comparison with the in vitro activity assays, denoting the unspecific electrostatic binding between ARM/Smc5 and the DNA molecule. Click here to expand this figure. Figure EV2. Mutagenesis analysis of the SUMO E3 ligase activity of the Smc5-Nse2 constructs upon binding to DNA SYPRO-stained (left) and Western blot (right) of the time-course reaction of SUMO conjugation in the presence or absence of ssDNA (virion ϕx174) at 8 nM, using either wild-type Arm/Smc5-Nse2 or K337E/K344E/K764E mutant. The reactions were run at 30°C with in the presence of the C-terminal kleisin domain of Nse4 as a substrate. (N-S2, cNse4-SUMO2; N-2S2, cNse4-2SUMO2; N-3S2, cNse4-3SUMO2; and pS2, poly-SUMO2). SYPRO-stained SDS–PAGE of SUMO conjugation reactions of wild-type Arm/Smc5-Nse2 at indicated NaCl concentrations in the presence or absence of ssDNA (virion ϕx174) at 8 nM (N-S2, cNse4-SUMO2; N-2S2, cNse4-2SUMO2; N-3S2, cNse4-3SUMO2; and pS2, poly-SUMO2). SYPRO-stained SDS–PAGE of SUMO conjugation reactions of wild-type and 7KE mutant of Δhinge/Smc5-Nse2 and Δhead/Smc5-Nse2 in the presence or absence of 50b ssDNA. Download figure Download PowerPoint DNA binding to Smc5-Nse2 triggers a conformational change The enhancement of the SUMO conjugation upon DNA binding could derive from the structural modification in the Nse2 E3 ligase to stimulate its enzymatic activity. To test this idea, we used circular dichroism spectroscopy, which measures the differential absorption of the circularly polarized light. In the far ultraviolet region, this variation arises mainly from changes in the secondary structure elements and is highly sensitive to conformational changes of proteins (Kelly et al, 2005). The circular dichroism analysis of the Arm/Smc5-Nse2 complex displays the spectra of a well-folded α-helical rich protein, with two characteristic ellipticity minimals at 210 and 222 nm, respectively (Fig 4 and Appendix Fig S3). The structural integrity of the complex was confirmed by temperature denaturation after incubation at 100°C, which resulted in a total loss of the circular dichroism signal (Fig 4A). Interestingly, increasing concentrations of DNA (ϕx174) produced a dose-dependent change of the circular dichroism spectra, which might indicate a DNA-induced structural change. Interestingly, the variation of ellipticity displayed by the wild-type spectra was reduced significantly at different levels when four different types of Arm/Smc5-Nse2 KE mutants were used (Fig 4A and Appendix Fig S3). In all cases, the change in the molar ellipticity for the Arm/Smc5-Nse2 KE mutants did not reach the saturation levels displayed by t" @default.
• W2804370147 created "2018-06-01" @default.
• W2804370147 creator A5003813851 @default.
• W2804370147 creator A5004223415 @default.
• W2804370147 creator A5014871766 @default.
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• W2804370147 date "2018-05-16" @default.
• W2804370147 modified "2023-10-16" @default.
• W2804370147 title "DNA activates the Nse2/Mms21 SUMO E3 ligase in the Smc5/6 complex" @default.
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