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A new quantitative proteomics technology to characterize the SUMO-proteome. A) Method to identify and quantify sumoylated proteins using SILAC and MS. Untagged cells and HF-SUMO cells were combined for purification of sumoylated proteins. Ulp1 was used to elute sumoylated proteins for MS analysis. B) Scatter plot of the identified proteins based on two replicate experiments. The majority of the proteins show a large abundance ratio between HF-SUMO purification versus mock purification, indicating a highly purified sample. Candidate SUMO targets are identified based on at least 10-fold abundance ratio between HF-SUMO and mock sample in both replicate experiments. C) Western blot analysis of several SUMO targets confirmed them being sumoylated. Ulp1 treatment was used to treat half of the anti-HA immunoprecipitated sample. doi:10.1371/journal.pgen.1003670.g001
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The human genome contains many “at-risk” sequences that are prone to mutations, including diverse repeated sequences, segmental duplications and regions of copy number variations. Such repetitive sequence elements can cause genome rearrangements through non-allelic homologous recombination (NAHR) and many human diseases are caused by...
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... explore the roles of the SUMO E3 ligases in genome stability, we introduced deletions of SIZ1 and SIZ2 and two alleles of the essential MMS21 gene, mms21-11 and mms21-CH, into strains to measure the rates of accumulating GCRs. We tested the mutations in two strain backgrounds; strains containing the yel068c::CAN1/URA3 assay monitor GCRs formed using single- copy sequences, whereas strains containing the yel072w::CAN1/ URA3 assay monitor GCRs formed both by a segmental duplication and by single-copy sequences (Table 1 and Figure S1) [5]. Neither the siz1D mutation nor the siz2D mutation caused an appreciable defect in suppressing GCR formed in either assay. ...
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... order to monitor sumoylation on a proteome-wide scale, we developed a quantitative proteomics method to identify and quantify sumoylated proteins ( Figure 1A). We first integrated a 66HIS-36FLAG (HF) tag at the 59-end of the SMT3 gene at the chromosomal locus so that HF-SUMO and proteins covalently modified by HF-SUMO could be isolated by a tandem-affinity purification using Ni-NTA and anti-Flag columns. ...
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... performed two replicate experiments with the HF-SUMO cells labeled by light lysine and arginine in the first experiment and by heavy lysine and arginine in the second experiment. We identified 653 proteins in the first and 695 proteins in the second experiment, and 306 proteins were identified in both replicates ( Figure 1B). Among them, 176 proteins were strongly enriched (.10-fold) from HF- SUMO cells in both experiments (Table 2, Tables S1 and S2). ...
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... then lysed cultures and purified the tagged protein via anti-HA immunoprecipitation. Part of the immuno- precipitate was treated by recombinant Ulp1, and treated and untreated immunopreciptates were analyzed by anti-HA and anti- FLAG Western blots ( Figure 1C). The anti-FLAG antibody, which recognizes HF-SUMO, detected a species that was eliminated by Ulp1 treatment. ...
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... same methods were used to analyze the relative abundance of SUMO targets between wild- type and various mutants (see text for details). Figure S1 Patch analysis of the roles of Siz1, Siz2, Mms21, Esc2 and Slx5 in suppressing duplication-mediated GCRs. A) Patch analysis of siz1D, siz2D and siz1D siz2D mutants in duplication- mediated GCR strain background. ...
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... We explored this further by creating a panel of E3 ligase deletions between Siz1, Siz2, and Mms21 owing to their redundancy between substrates. While siz1Δ and siz2Δ caused no change, cells harboring the Mms21-CH mutation 40 , both on their own or combined with siz1Δ or siz2Δ caused a reduction in Rpd3 INQ foci (Fig. 3b). Strikingly, reduction in Rpd3 INQ formation in these mutants coincided with an increase in cytoplasmic inclusion formation, suggesting that SUMOylation could be an important INQ sorting signal (Fig. 3b). ...
Spatial compartmentalization is a key facet of protein quality control that serves to store disassembled or non-native proteins until triage to the refolding or degradation machinery can occur in a regulated manner. Yeast cells sequester nuclear proteins at intranuclear quality control bodies (INQ) in response to various stresses, although the regulation of this process remains poorly understood. Here we reveal the SUMO modification of the small heat shock protein Btn2 under DNA damage and place Btn2 SUMOylation in a pathway promoting protein clearance from INQ structures. Along with other chaperones, and degradation machinery, Btn2-SUMO promotes INQ clearance from cells recovering from genotoxic stress. These data link small heat shock protein post-translational modification to the regulation of protein sequestration in the yeast nucleus.
... For instance, segmental duplication can mediate GCR formation via homologous recombination, and specific genetic pathways prevent these duplication-mediated GCRs (dGCRs) [6]. In particular, enzymes that catalyze reversible sumoylation, including the Mms21 SUMO E3 ligase, have a specific role in preventing dGCRs [7,8]. In the same vein, mutations affecting essential chromosomal DNA replication factors cause similar accumulations of dGCRs [9]. ...
... Existing studies have not identified specific sumoylation sites or events that prevent the accumulations of GCRs. For instance, though sumoylation-deficient mutants of PCNA and DNA polymerase ε accumulate modest GCRs [17,18], SUMO E3 ligase mutants accumulate dGCRs at significantly higher rates [8], suggesting that sumoylation of other DNA replication proteins is crucial for genome maintenance. Prior studies suggested that SUMO modifications of MCM and Dbf4-dependent kinase (DDK) might inhibit DNA replication initiation [19,20]. ...
... This could cause DSBs to form GCRs. Consistent with this idea, the dGCR rate is elevated 9-fold in mcm3-K767R,~100-fold in mcm3-K768R, and over 200-fold in mcm3-2KR (Fig 6B and S1 Table). This is comparable to the rates of dGCRs accumulation in the SUMO E3 ligase-null mutants [7,8] and MCM loading mutants such as cdc6-1. In contrast, DNA replication initiation mutants such as cdc7 and dbf4 have significantly lower dGCR rates [9], suggesting that the level of MCM loaded at origins plays a more important role in suppressing dGCR than the origin-firing activity. ...
Timely completion of eukaryotic genome duplication requires coordinated DNA replication initiation at multiple origins. Replication begins with the loading of the Mini-Chromosome Maintenance (MCM) complex, proceeds by the activation of the Cdc45-MCM-GINS (CMG) helicase, and ends with CMG removal after chromosomes are fully replicated. Post-translational modifications on the MCM and associated factors ensure an orderly transit of these steps. Although the mechanisms of CMG activation and removal are partially understood, regulated MCM loading is not, leaving an incomplete understanding of how DNA replication begins. Here we describe a site-specific modification of Mcm3 by the Small Ubiquitin-like MOdifier (SUMO). Mutations that prevent this modification reduce the MCM loaded at replication origins and lower CMG levels, resulting in impaired cell growth, delayed chromosomal replication, and the accumulation of gross chromosomal rearrangements (GCRs). These findings demonstrate the existence of a SUMO-dependent regulation of origin-bound MCM and show that this pathway is needed to prevent genome rearrangements.
... Of note, Smc5/6 and RNAP I show some interesting connections. First, both of them bind to the rDNA array (Torres-Rosell et al, 2005) (Russell & Zomerdijk, 2006); besides, RNAP I is SUMOylated in an Nse2-dependent manner (Albuquerque et al, 2013); in addition, inactivation of RNAP I partially relieves the rDNA missegregation phenotype of smc5/6 mutants (Torres-Rosell et al, 2007). ...
Ubiquitination controls numerous cellular processes, and its deregulation is associated to many pathologies. The Nse1 subunit in the Smc5/6 complex contains a RING domain with ubiquitin E3 ligase activity and important functions in genome integrity. However, Nse1-dependent ubiquitin targets remain largely unknown. Here, we use label-free quantitative proteomics to analyse the nuclear ubiquitinome of nse1-C274A RING mutant cells. Our results show that Nse1 impacts on the ubiquitination of several proteins involved in DNA damage tolerance, ribosome biogenesis and metabolism that, importantly, extend beyond canonical functions of the Smc5/6 complex in chromosome segregation. In addition, our analysis uncovers an unexpected connection between Nse1 and RNA polymerase I (RNAP I) ubiquitination. Specifically, Nse1 promotes the ubiquitination of K408 and K410 in A190, the largest subunit of RNAP I, to induce its degradation. We propose that this mechanism contributes to Smc5/6-dependent rDNA disjunction in response to transcriptional elongation defects.
... As we did not find any role for Top1-SUMO, we attempted to identify other candidate SUMO targets by quantitative SUMO proteomics (Albuquerque et al., 2013). Global sumoylation changes were examined in two sets of mutants, (1) tdp1-AID wss1Δ (+) auxin versus (−) auxin and (2) ulp1-ΔN tdp1-AID wss1Δ (+) auxin versus tdp1-AID wss1Δ (+) auxin, as summarized in Figure S3A. ...
... The enrichment of sumoylated proteins in preparation for quantitative mass spectrometry (SILAC-MS) analysis was performed as previously described (Albuquerque et al., 2013) but with the following exceptions: each mutant strain was grown in 1-l of synthetic media containing either light or heavy stable isotope-labeled lysine and arginine until an OD 600 of 0.3, at which point auxin (1 mM final concentration) was added to deplete Tdp1-AID*-6HA. Cells were harvested following 6 hours of auxin treatment and lysed under denaturing condition as previously described (Albuquerque et al., 2013). ...
... The enrichment of sumoylated proteins in preparation for quantitative mass spectrometry (SILAC-MS) analysis was performed as previously described (Albuquerque et al., 2013) but with the following exceptions: each mutant strain was grown in 1-l of synthetic media containing either light or heavy stable isotope-labeled lysine and arginine until an OD 600 of 0.3, at which point auxin (1 mM final concentration) was added to deplete Tdp1-AID*-6HA. Cells were harvested following 6 hours of auxin treatment and lysed under denaturing condition as previously described (Albuquerque et al., 2013). Following lysis, protein concentrations were determined by Bradford Reagent (Bio-Rad) and were normalized prior to mixing and subsequent purification of sumoylated proteins. ...
Endogenous metabolites, environmental agents, and therapeutic drugs promote formation of covalent DNA-protein crosslinks (DPCs). Persistent DPCs compromise genome integrity and are eliminated by multiple repair pathways. Aberrant Top1-DNA crosslinks, or Top1ccs, are processed by Tdp1 and Wss1 functioning in parallel pathways in Saccharomyces cerevisiae. It remains obscure how cells choose between diverse mechanisms of DPC repair. Here, we show that several SUMO biogenesis factors (Ulp1, Siz2, Slx5, and Slx8) control repair of Top1cc or an analogous DPC lesion. Genetic analysis reveals that SUMO promotes Top1cc processing in the absence of Tdp1 but has an inhibitory role if cells additionally lack Wss1. In the tdp1Δ wss1Δ mutant, the E3 SUMO ligase Siz2 stimulates sumoylation in the vicinity of the DPC, but not SUMO conjugation to Top1. This Siz2-dependent sumoylation inhibits alternative DPC repair mechanisms, including Ddi1. Our findings suggest that SUMO tunes available repair pathways to facilitate faithful DPC repair.
... Indeed, Slx5 and Slx8 mutants accumulate Rts1 at centromeres during metaphase and display variable distance between sister chromatids, potentially experiencing a loss of centromeric tension [205]. In fact, inactivating Slx5 prevents the degradation of SUMOylated Mcd1, a cohesin subunit, and causes an accumulation of condensin subunits (Table 2) [118,206]. Furthermore, the budding yeast homolog of securin, Pds1, is stabilized in mcm10-1 slx5∆ mutants, confirming that cohesion cleavage is perturbed and responsible for prolonged mitotic arrest [22]. Additionally, Slx5/Slx8 degrades CPC components, Bir1 and Sli15; thus, several STUbL substrates reside at the kinetochore, coinciding with Slx5 localization (Table 2) [22,205]. ...
... In slx5∆ mutants, 75% of GCRs and in slx8∆ mutants, 53% were de novo telomere additions [193]. Moreover, methyl methanesulfonate sensitivity 21 (Mms21)-dependent SUMOylation that acts upstream of Slx5/Slx8 largely suppresses duplication-mediated GCRs [206]. Whether these GCRs arise from deficiencies in DSB repair, elevated replication stress, impaired cohesion cleavage, or other kinetochore-related substrates remains ambiguous. ...
Small ubiquitin-like modifier (SUMO)-targeted E3 ubiquitin ligases (STUbLs) are specialized enzymes that recognize SUMOylated proteins and attach ubiquitin to them. They therefore connect the cellular SUMOylation and ubiquitination circuits. STUbLs participate in diverse molecular processes that span cell cycle regulated events, including DNA repair, replication, mitosis, and transcription. They operate during unperturbed conditions and in response to challenges, such as genotoxic stress. These E3 ubiquitin ligases modify their target substrates by catalyzing ubiquitin chains that form different linkages, resulting in proteolytic or non-proteolytic outcomes. Often, STUbLs function in compartmentalized environments, such as the nuclear envelope or kinetochore, and actively aid in nuclear relocalization of damaged DNA and stalled replication forks to promote DNA repair or fork restart. Furthermore, STUbLs reside in the same vicinity as SUMO proteases and deubiquitinases (DUBs), providing spatiotemporal control of their targets. In this review, we focus on the molecular mechanisms by which STUbLs help to maintain genome stability across different species.
... These pathways involve genes that act during DNA replication and repair, and genes that are involved in post-translational protein modifications. Among the latter, we found that modifications by Small Ubiquitin-like MOdifer (SUMO) play a highly specific and significant role in suppressing duplication mediated gross chromosomal rearrangements (dGCRs) [4,5]. Saccharomyces cerevisiae expresses three mitotic SUMO E3 ligases, Siz1, Siz2 and Mms21 [6,7]. ...
... Inactivating all three SUMO E3 ligases results in lethality [8], like the deletions of SUMO (SMT3), the sole E1 (AOS1-UBA2) and E2 (UBC9) enzymes [9], suggesting that these SUMO E3 ligases are necessary for controlling intracellular sumoylation. Although deletion of SIZ1 and SIZ2 results in a relatively modest increase in dGCRs, inactivating the Mms21 E3 ligase through point mutations in its catalytic domain (mms21-CH) leads to a highly specific accumulation of dGCRs [4,5]. Moreover, combining mms21-CH with either siz1Δ or siz2Δ leads to a further increase in the rate of accumulating dGCRs, indicating the partially redundant roles of these SUMO E3 ligases [4]. ...
... Although deletion of SIZ1 and SIZ2 results in a relatively modest increase in dGCRs, inactivating the Mms21 E3 ligase through point mutations in its catalytic domain (mms21-CH) leads to a highly specific accumulation of dGCRs [4,5]. Moreover, combining mms21-CH with either siz1Δ or siz2Δ leads to a further increase in the rate of accumulating dGCRs, indicating the partially redundant roles of these SUMO E3 ligases [4]. This raised key questions that have yet to be understood. ...
Protein sumoylation, especially when catalyzed by the Mms21 SUMO E3 ligase, plays a major role in suppressing duplication-mediated gross chromosomal rearrangements (dGCRs). How Mms21 targets its substrates in the cell is insufficiently understood. Here, we demonstrate that Esc2, a protein with SUMO-like domains (SLDs), recruits the Ubc9 SUMO conjugating enzyme to specifically facilitate Mms21-dependent sumoylation and suppress dGCRs. The D430R mutation in Esc2 impairs its binding to Ubc9 and causes a synergistic growth defect and accumulation of dGCRs with mutations that delete the Siz1 and Siz2 E3 ligases. By contrast, esc2-D430R does not appreciably affect sensitivity to DNA damage or the dGCRs caused by the catalytically inactive mms21-CH . Moreover, proteome-wide analysis of intracellular sumoylation demonstrates that esc2-D430R specifically down-regulates sumoylation levels of Mms21-preferred targets, including the nucleolar proteins, components of the SMC complexes and the MCM complex that acts as the catalytic core of the replicative DNA helicase. These effects closely resemble those caused by mms21-CH , and are relatively unaffected by deleting Siz1 and Siz2. Thus, by recruiting Ubc9, Esc2 facilitates Mms21-dependent sumoylation to suppress the accumulation of dGCRs independent of Siz1 and Siz2.
... Alternatively, we hypothesized that the small subunits of condensin that actually slide around the DNA and make direct contact to it, would eventually still be associated during Smc2 depletion. Indeed, Ycs4 and Ycg1 are sumoylated by Siz1/Siz2 much like Yen1, and different form the Mms2-dependent sumoylation of the Smc subunits of the complex [539]. In asynchronous cells, even in conditions of induced DNA damage, co-localization of the two tagged proteins was observed but with no convincing accumulation of Ycs4 concomitant with Yen1 foci (figure 57 C). ...
... Furthermore, its binding to DNA introduces a slight distortion which could contribute to Yen1 accessing its substrates. Finally, Rap1 is known to be sumoylated and is targeted by Uls1 which removes sumo-Rap1 from telomeres [388,539,[542][543][544][545][546][547][548]. The actual function of Rap1 sumoylation is unknown, but similar to what we speculated for condensins, we thought that in the event of persistent junctions, Rap1 could stall branch-migration and then be a site of pausing of these DNA intermediates. ...
The repair of double-stranded DNA breaks (DSBs) by homologous recombination involves the formation of branched intermediates that can lead to crossovers following nucleolytic resolution. Ubiquitin and SUMO modification is commonplace amongst the DNA damage repair proteins. What is more, a number of DSB repair factors interact with each other when sumoylated, making use of SUMO interaction motifs (SIMs). The nuclease Yen1 is tightly controlled during the cell cycle to limit the extent of crossover formation and preserve genome integrity. In this manuscript we describe further regulation of Yen1 by ubiquitination, sumoylation and non-covalent interaction with SUMO through its newly characterized SIMs. Yen1 is sumoylated by Siz1 and Siz2 SUMO ligases, especially in conditions of DNA damage. Furthermore, Yen1 is a substrate of the Slx5-Slx8 ubiquitin ligase. Loss of Slx5-Slx8 stabilizes the sumoylated fraction of Yen1, and results in persistent localization of Yen1 in nuclear foci. Slx5-Slx8-dependent ubiquitination of Yen1 occurs mainly at K714 and mutation of this lysine increases crossover formation during DSB repair and suppresses chromosome segregation defects when other nucleases are unavailable. In addition, proper and timely nucleolytic processing from Yen1 is dependent on interactions mediated by non-covalent binding to sumoylated partners. Mutations in the motifs that allow SUMO-mediated recruitment of Yen1 leads to its mis-localization, decreasing Yen1’s ability to resolve DNA joint-molecule intermediates and resulting in increased genome instability and chromosome mis-segregation.
... However, unlike the vast array of ubiquitination enzymes, all organisms examined so far contain only a single SUMO E1 and E2 enzyme and a few SUMO E3s (Pichler et al. 2017). For example, in budding yeast, the Aos1-Uba2 heterodimeric E1, the Ubc9 E2, and three mitotic E3s (Siz1, Siz2, and Mms21) are responsible for sumoylating hundreds of proteins (Albuquerque et al. 2013). How a large number of proteins are specifically and efficiently modified by a small number of sumoylation enzymes is an outstanding question. ...
... Experiments were done and data are presented as in Figure 1A. genome stability during growth and enhance HJ removal in the presence of MMS (Sollier et al. 2009;Albuquerque et al. 2013). We first queried genome stability using the gross chromosomal rearrangement (GCR) assay (Putnam et al. 2009). ...
SUMO modification regulates diverse cellular processes by targeting hundreds of proteins. However, the limited number of sumoylation enzymes raises the question of how such a large number of substrates are efficiently modified. Specifically, how genome maintenance factors are dynamically sumoylated at DNA replication and repair sites to modulate their functions is poorly understood. Here, we demonstrate a role for the conserved yeast Esc2 protein in this process by acting as a SUMO E2 cofactor. Esc2 is required for genome stability and binds to Holliday junctions and replication fork structures. Our targeted screen found that Esc2 promotes the sumoylation of a Holliday junction dissolution complex and specific replisome proteins. Esc2 does not elicit these effects via stable interactions with substrates or their common SUMO E3. Rather, we show that a SUMO-like domain of Esc2 stimulates sumoylation by exploiting a noncovalent SUMO binding site on the E2 enzyme. This role of Esc2 in sumoylation is required for Holliday junction clearance and genome stability. Our findings thus suggest that Esc2 acts as a SUMO E2 cofactor at distinct DNA structures to promote the sumoylation of specific substrates and genome maintenance.
... Slx5 interacts with the SLD-containing protein Esc2 and both mediate the turnover of certain proteins, such as Srs2 26,27 . As Esc2 interacts directly with the Mms4-Mus81 endonuclease 28 , we next addressed whether Esc2 also participates in regulating the Mms4-P abundance. ...
The Mus81-Mms4 nuclease is activated in G2/M via Mms4 phosphorylation to allow resolution of persistent recombination structures. However, the fate of the activated phos-phorylated Mms4 remains unknown. Here we find that Mms4 is engaged by (poly) SUMOylation and ubiquitylation and targeted for proteasome degradation, a process linked to the previously described Mms4 phosphorylation cycle. Mms4 is a mitotic substrate for the SUMO-Targeted Ubiquitin ligase Slx5/8, the SUMO-like domain-containing protein Esc2, and the Mms1-Cul8 ubiquitin ligase. In the absence of these activities, phosphorylated Mms4 accumulates on chromatin in an active state in the next G1, subsequently causing abnormal processing of replication-associated recombination intermediates and delaying the activation of the DNA damage checkpoint. Mus81-Mms4 mutants that stabilize phosphorylated Mms4 have similar detrimental effects on genome integrity. Overall, our findings highlight a repli-cation protection function for Esc2-STUbL-Cul8 and emphasize the importance for genome stability of resetting phosphorylated Mms4 from one cycle to another.
... Changes in Nse2 activity may imply repositioning of the donor SUMO for optimal catalysis through an unknown mechanism, as occurs in other SUMO ligases [82,83]. Besides, the activity of Nse2 is modulated by ATR and PKA-dependent phosphorylation [84,85] and binding to members of the RENi (Rad60-Esc2-NIP45) family of SUMO-like domain proteins, which probably promote recruitment of the Smc5/6 complex to DNA lesions [86][87][88][89]. ...
... To this end, Nse2 targets several factors involved in chromosome organization, replication and DNA damage repair ( Figure 3 and Table 1). A first group of Nse2 targets are the three eukaryotic SMC complexes [86,91]. Sumoylation of cohesin promotes sister chromatid cohesion during a normal cell cycle and in response to DNA damage [92][93][94]. ...
... In budding yeast, this relocation has been reported in the repetitive rDNA locus, , and seems to require sumoylation of Rad52 [49]. Nse2 also targets two rDNA-specific proteins: the Fob1 protein, which is responsible for the polar arrest of replication forks, and the Rpa135 subunit of the RNA polymerase I complex [66,86], although the role of these modifications is currently unknown. Analogously to chromosome breaks, relocation of damaged replication forks to nuclear pores also relies on Nse2dependent sumoylation, by targeting the fork-associated proteins RPA, Smc5 and Rad59; STUbL recognizes their sumoylation, moving damaged forks to the nuclear periphery [102,105]. ...
The Smc5/6 complex plays essential roles in chromosome segregation and repair, by promoting disjunction of sister chromatids. The core of the complex is constituted by an heterodimer of Structural Maintenance of Chromosomes (SMC) proteins that use ATP hydrolysis to dynamically associate with and organize chromosomes. In addition, the Smc5/6 complex contains six non-SMC subunits. Remarkably, and differently to other SMC complexes, the Nse1 and Nse2 subunits contain RING-type domains typically found in E3 ligases, pointing to the capacity to regulate other proteins and complexes through ubiquitin-like modifiers. Nse2 codes for a C-terminal SP-RING domain with SUMO ligase activity, assisting Smc5/6 functions in chromosome segregation through sumoylation of several chromosome-associated proteins. Nse1 codes for a C-terminal NH-RING domain and, although it has been proposed to have ubiquitin ligase activity, no Smc5/6-dependent ubiquitylation target has been described to date. Here, we review the function of the two RING domains of the Smc5/6 complex in the broader context of SMC complexes as global chromosome organizers of the genome.
