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10.1080/19491034.2024.2398450
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Review Article
Review
Multifaceted roles of SUMO in DNA metabolism
Y. M. THU
NUCLEUS
https://orcid.org/0000-0002-9608-7336
Thu Yee Mon
Department of Biology, Colby College , Waterville, ME, USA
CONTACT Yee Mon Thu ythu@colby.edu Department of Biology, Colby College, 5700 Mayflower Hill Drive, Waterville, ME 04901, USA
17 9 2024
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ABSTRACT

Sumoylation, a process in which SUMO (small ubiquitin like modifier) is conjugated to target proteins, emerges as a post-translational modification that mediates protein−protein interactions, protein complex assembly, and localization of target proteins. The coordinated actions of SUMO ligases, proteases, and SUMO-targeted ubiquitin ligases determine the net result of sumoylation. It is well established that sumoylation can somewhat promiscuously target proteins in groups as well as selectively target individual proteins. Through changing protein dynamics, sumoylation orchestrates multi-step processes in chromatin biology. Sumoylation influences various steps of mitosis, DNA replication, DNA damage repair, and pathways protecting chromosome integrity. This review highlights examples of SUMO-regulated nuclear processes to provide mechanistic views of sumoylation in DNA metabolism.

KEYWORDS

Chromatin regulation
DNA damage
genome instability
post-translational modification
SUMO
Maine IDeA Network of Biomedical Research Excellence The author is supported by the Maine IDeA Network of Biomedical Research Excellence grant and Colby College.
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pmcMain text

Sumoylation is a post-translational modification that orchestrates diverse cellular processes under physiological conditions as well as stress conditions. The biological importance of sumoylation has been especially recognized in safeguarding the integrity of the genome. This review highlights how the nuclear organization of SUMO regulators influence different cellular processes, primarily in budding yeast (Saccharomyces cerevisiae) and in mammalian cells.

Brief overview of SUMO machinery and enzymes

Sumoylation refers to a process in which SUMO (small ubiquitin like modifier) peptide is covalently conjugated to target proteins. E1, E2, and E3 SUMO ligases participate in a cascade of biochemical reactions to sumoylate a substrate. In budding yeast, SUMO protein is encoded by a single gene, SMT3 (suppressor of mif two 3), whereas, in vertebrates, up to five SUMO proteins (SUMO1–5) have been reported [1]. Target proteins can be modified with one SUMO peptide at one lysine residue (mono-sumoylation) or multiple SUMO moieties at different lysine residues (multi-sumoylation). Proteins can also be poly-sumoylated, a condition in which a chain of SUMO is linked to the substrate at a specific lysine residue.

SUMO is activated by a heterodimeric E1, Aos1 (activation of Smt3p 1)/Uba2 (ubiquitin activating 2) in S. cerevisiae and SAE1 (SUMO activating enzyme subunit 1)/SAE2 in human, resulting in SUMO covalently linked to the E1 enzyme via a thioester bond [1,2]. Activated SUMO is then transferred to the E2 conjugating enzyme, Ubc9 (ubiquitin conjugating 9). Ubc9 can sumoylate multiple substrates in vitro and in vivo without the contribution of E3 ligases by recognizing the consensus amino acid sequence for sumoylation [1,3]. In both mammals and yeast, only one E1 and one E2 have been identified [1]. In S. cerevisiae, Siz1 (SAP and mIZ-finger domain 1), Siz2, and Mms21 (methyl methanesulfonate sensitivity 21) are the three mitotic E3 SUMO ligases [3]. In human cells, the number of E3 SUMO ligases, such as PIAS1–4 (protein inhibitor of activated STAT 1–4), NSMCE2 (non‐SMC elements 2), and RanBP2 (RAN binding protein 2), identified to date is much higher than the number of E3 ligases in yeast [1]. Among the E3 SUMO ligases, Mms21 (NSMCE2 in human) functions as a subunit of the Smc5/6 (structural maintenance of chromosomes 5/6) complex, a cohesin-like complex involved in DNA replication and repair [4]. Discovery of specialized E3 enzymes suggests multiple layers of regulation in SUMO conjugation within the mammalian system. For example, ZNF451 (zinc finger protein 451) is recognized as a special E3 ligase that favors extension of SUMO chains over the initial conjugation of SUMO to the substrate protein [5]. Given that hundreds of proteins in yeast or thousands of proteins in mammals are sumoylated, the number of E3 SUMO ligases is surprisingly limited [1]. The general consensus is that E3 SUMO ligases confer substrate specificity.

SUMO can be covalently conjugated to the lysine residue within the context of the canonical consensus sequence, φKXE, where φ represents isoleucines, leucines or valines, and X represents any amino acid [1]. However, sumoylation is not restricted only to this consensus sequence. Other types of motifs such as inverted sumoylation motif and negatively charged amino acid-dependent sumoylation motif can also accommodate sumoylation [1,6]. In addition, phosphorylation can direct sumoylation of substrates. This co-modification occurs in the context of the sequence that contains the canonical sumoylation consensus sequence and a proline-directed phosphorylation site, separated by two amino acids (φKXEXXS/TP) [7,8]. Intriguingly, proline-directed phosphorylation, in which a serine or threonine immediately adjacent to a proline is phosphorylated, has been linked to cell cycle regulation such as regulation of mitosis [9]. In the case of phosphorylation-dependent sumoylation, proline-directed phosphorylation triggers sumoylation [7]. This co-regulatory system is indeed significant in genome stability – in mammalian cells, replication stress-activated ATM (ataxia telangiectasia mutated)/ATR (ataxia telangiectasia and Rad3-related) kinases regulate sumoylation of multiple DNA damage response proteins [10].

Sumoylation is a reversible reaction. SUMO chains from target proteins are deconjugated by a family of enzymes called, SUMO isopeptidases. In S. cerevisiae, Ulp1 (UbL-specific protease 1) and Ulp2 are enzymes responsible for this process [3]. Human isopeptidases include SENP1–3 (sentrin-specific protease 1–3), SENP5–7, DESI1 (desumoylating isopeptidase 1), DESI2, and USPL1 (ubiquitin specific peptidase like 1) [1]. Deconjugation of SUMO chains limits the effects poly-sumoylation has on target proteins. An additional mechanism, involving SUMO-targeted ubiquitin ligases (STUbLs), can also limit the consequences of poly-sumoylation. STUbLs are specialized E3 ubiquitin ligases that non-covalently bind poly-sumoylated proteins via SUMO-SUMO-interacting motif (SIM) interactions. Poly-SUMO chains covalently linked to the target proteins provide a scaffold where SIMs of the STUbL can bind [3]. This SUMO-SIM interaction promotes conjugation of ubiquitin chains on sumoylated proteins by the STUbL. In S. cerevisiae, the STUbL is a heterodimeric complex comprised of Slx5 (synthetic lethal of unknown X function 5) and Slx8 [3]. In mammalian cells, SUMO chains induce homodimerization of the mammalian STUbL, RNF4 (ring finger protein 4), and activate the function of RNF4 as a ubiquitin ligase [11]. STUbL targets are subsequently degraded by the proteosome [3]. In addition to RNF4 and Slx5/8, other STUbLs contribute to genome stability. These include Uls1 in budding yeast and Arkadia/RNF111 in human cells [12–17]. Recently, TOPORS (TOP1 binding arginine/serine rich protein) has been identified as a STUbL responsible for removing DNA−protein crosslinks [18,19].

Non-covalent interactions between SUMO and SIM containing proteins are not restricted only to the regulation involving STUbLs. SUMO−SIM interactions also regulate sumoylation and desumoylation. For instance, in budding yeast, SIM or SIM-like motifs found within Siz1 or Mms21 interact with SUMO linked to Ubc9 as well as the free SUMO non-covalently bound to Ubc9 [20,21]. These interactions promote sumoylation of substrates by the E3 SUMO ligases [20,21]. Budding yeast Ulp2 also contains SIMs, which interact with its poly-sumoylated substrates [22]. Likewise, in mammalian cells, SIMs within SENP6 and SENP7 interact with poly-SUMO chains on their substrates to promote desumoylation [23,24]. In addition, SUMO-SIM interactions are fundamental to functional protein complex assembly and recruitment of proteins to subcellular locations in many of the examples discussed below. SIMs are characterized by short stretches of hydrophobic residues (isoleucines, leucines, valines) surrounded by acidic amino acids [1]. Phosphorylation can alter the dynamic of SUMO−SIM interactions. For example, serine residues adjacent to the SIM of PIAS1 are phosphorylated and phospho-SIM enhances the interaction between PIAS1 and SUMO1/2 [25]. More than one SIM are often found in proteins that interact with SUMO [26]. Proteomic identification of SUMO interacting proteins in mammalian cells show that SUMO−SIM interactions are central to multiple cellular processes such as RNA processing, DNA damage repair, and microtubule organization [27,28].

SUMO-like domain (SLD) containing proteins add to the complexity of regulation in the SUMO pathway. In lower eukaryotes, budding yeast Esc2 (establishment of silent chromatin 2) as well as fission yeast Rad60 interact with Ubc9 through their SLDs [29,30]. The interaction between these SLD-containing proteins and Ubc9 stimulates sumoylation of substrates by Mms21 or Nse2 (fission yeast Mms21) and is crucial for genome stability [29,30]. Similarly, a recent study reports that the mammalian ortholog, NFATC2IP (nuclear factor of activated T-cells, cytoplasmic 2-Interacting)/NIP45 (NFAT-interacting protein 45), also interacts with Ubc9 to stimulate Mms21-dependent sumoylation [31]. An additional study suggests that NIP45 may act as a specialized E3 SUMO ligase in conjunction with Ubc9 to support genome stability [32].

General regulation of SUMO enzymes via their localization

Immunofluorescent microscopy in budding yeast reveals cellular localization of SUMO enzymes in cycling cells under unchallenged conditions [33]. Budding yeast Ubc9 is localized broadly in the cytoplasm as well as in the nucleus [33]. Siz2 and Ulp2 are localized to the whole nuclear compartment whereas, Siz1’s location excludes nucleolus [33]. The budding yeast Smc5/6 complex, which Mms21 is a part of, has been observed in nucleolar and telomeric regions [34]. Budding yeast Ulp1 is located primarily within the nuclear pore complex, a structure localized to the nuclear envelope that controls transport in and out of the nucleus [33,35–37]. In mammalian cells, Ubc9, RANBP2, SENP1, and SENP2 are also shown to position at the nuclear pore complex [38–41].

Although SUMO conjugating enzymes may occupy a defined subcellular domain, evidence suggests that different physiological conditions can alter the physical proximity of E3 SUMO ligases and their substrates. For instance, in mammalian cells, a comprehensive SUMO proteomics study demonstrates that sumoylation status of many proteins change throughout normal cell cycle [42]. Two possible scenarios explain the changes in sumoylation status of proteins. In the first scenario, local concentrations of substrates may change while the localization of the enzyme remains relatively constant. For example, budding yeast Mms21 as a part of the Smc5/6 is associated with chromatin under unchallenged conditions, and it was proposed that this chromatin association may be necessary for its repair function [43]. In this scenario, different protein substrates may be recruited to the proximity of the chromatin-bound Smc5/6 complex depending on the nature of DNA breaks. In the second scenario, the enzyme is re-localized to execute its function. In budding yeast, nuclear Siz1 travels to the bud-neck (the constriction between the mother and daughter cells) prior to mitosis to sumoylate septins and this process regulates cell cycle [44,45]. Likewise, the budding yeast Smc5/6 complex can be actively loaded onto chromatin at sites of repair [43]. This notion is also corroborated by biochemical studies demonstrating that the Smc5/6 complex can associate with different DNA structures, such as single-stranded, double-stranded, and single-stranded and double-stranded junctions [46]. In this model, Mms21 is recruited to the sites where its substrates aggregate.

Regardless of the mechanisms, E3 SUMO ligases can be somewhat promiscuous in targeting their substrates. Substrates which are in the vicinity of the E3 SUMO enzyme and have appropriate modification sites can be sumoylated [47]. For instance, when Siz1 was experimentally localized to the bud-neck, the ability of Siz1 to sumoylate septin proteins was improved as long as the substrates contain SUMO consensus sequences [48]. Similarly, artificially targeting DNA repair proteins to chromatin leads to sumoylation of these proteins by chromatin-associated Siz2 [47]. These examples imply that the physical proximity between the E3 SUMO ligase and substrates determine the status of sumoylation. Since proteins that belong to the same functional complex are in the same microenvironment, they tend to get sumoylated synchronously in response to a stimulus [47]. This phenomenon has been observed in yeast as well as in mammalian cells [3,42,47]. For example, many double-strand break repair proteins are sumoylated in the vicinity of double-strand breaks [47]. However, some examples suggest that sumoylation can target specific proteins as well. For instance, budding yeast PCNA (proliferating cells nuclear antigen) is a SUMO target and specific interactions between Ubc9/Siz1 and PCNA ensure enzyme−substrate selectivity [21,49].

SUMO regulation in mitosis

Multiple mutants in the SUMO pathway exhibit defects in mitosis, implying their functional contributions to this cell cycle stage. Ulp1 in budding yeast, for instance, was identified as a protease that arrested cells in G2/M phase when its function was lost [50]. Budding yeasts lacking Ulp2 also show mitotic defects, suggesting that this protease regulates crucial mechanisms required for chromosome segregation [51,52]. Likewise, SMT3, budding yeast SUMO gene, was identified as a gene necessary for chromosome segregation along with genes crucial for mitosis such as, CSE4 (chromosome segregation 4) and IPL1 (increase in ploidy 1) [53]. Cells that are deficient in Siz1 and Siz2 exhibit large-budded phenotypes, suggesting that they experience mitotic delays [44]. (A small bud starts to emerge when a budding yeast cell initiates S-phase. By the time it reaches G2/M, the bud, which represents the daughter cell, has grown in size. Thus, large-budded cells represent cells at G2/M.) Similarly, depletion of SLX5 and SLX8 as well as their human counterpart RNF4 results in chromosome mis-segregation [54].

The dynamics of sumoylation in mitosis further solidify the notion that this post-translation modification plays a pivotal role in chromosome segregation. SUMO proteomic profiling of mammalian mitotic chromosomes identified approximately 150 proteins, underscoring the importance of SUMO in this cell cycle stage [55]. In mammalian cells, SUMO-1 is identified on mitotic spindles and in spindle midzone whereas, SUMO-2/3 localize in the centromeric region, suggesting that SUMO regulation influences more than one stage of mitosis [56]. In both budding yeast and humans, many sumoylated mitotic proteins are identified at the interface of centromeres and kinetochores [57,58]. This can be explained partly by localization of SUMO ligases to the same regions. For example, the budding yeast Smc5/6 complex has been shown to localize to centromeric and peri-centromeric regions and sumoylate kinetochore proteins [43,59]. Likewise, a similar pericentromeric localization of Mms21 was observed in mice [60]. These examples underscore the biological significance of the SUMO machinery in coordinating different aspects of chromosome segregation.

Here, I highlight selected examples to illustrate mechanisms by which SUMO regulates distinct processes of mitosis. For an extensive review of the role of sumoylation in mitosis, please refer to additional reviews [57,58,61].

Regulation at centromeric regions

In preparation for mitosis, cells must first establish centromeric regions where kinetochores can be assembled. Kinetochores provide a platform for the mitotic spindles to connect to the chromatids. Correct establishment of centromeric regions is determined by a centromeric histone, Cse4 in budding yeast and CENP-A (centromere protein-A) in mammalian cells. Cse4 is sumoylated by Siz1 and Siz2 and subsequently targeted by Slx5/8 for degradation [62,63]. This SUMO-mediated degradation is crucial to prevent mislocalization of the yeast centromeric histone to non-centromeric regions of the chromosome [62,63]. In human cells, SUMO-dependent degradation regulates stable association of the centromeric histone at centromeres. CENP-A maintenance at centromeres via sumoylation depends on the activity of SENP6 SUMO isopeptidase and RNF4. These enzymes indirectly impact the stability of CENP-A at centromeres by regulating the stability of the CCAN (constitutive centromere-associated network) complex. SENP6 desumoylates the CENP-H/I/K subunits, a part of the CCAN complex, at the inner kinetochore and prevents them from RNF4-dependent proteasomal degradation [64]. A proteomics study and a genetic screen, along with a recent study, independently confirm that SENP6’s role in stabilizing centromeric localization of CENP-A is via the CCAN complex [65–67]. This SENP6 regulation of the CCAN complex may be conserved in budding yeast although the detailed mechanism has not been illustrated yet. Ulp2, SENP6 homolog in S. cerevisiae, is recruited to the kinetochore via its interaction with a CCAN complex subunit [68,69].

Regulation of kinetochore-microtubule attachment

Sumoylation influences microtubule attachment to kinetochores by altering protein-protein interactions or protein localization. For instance, a mammalian kinesin microtubule motor protein CENP-E non-covalently interacts with SUMO chains on BubR1 (budding uninhibited by benzimidazole-related 1) and this interaction brings CENP-E to the kinetochore [56]. Since CENP-E is critical for kinetochore-microtubule attachment, BubR1 mutants that cannot be sumoylated exhibit chromosome mis-segregation defects, presumably due to the loss of interaction between BubR1 and CENP-E [70,71]. In addition to BubR1, poly-sumoylation of another CENP-E interacting protein, Nuf2 (Nuf2 component of NDC80 kinetochore complex) mediates association of CENP-E with kinetochores during mitosis [72]. Sumoylation regulates additional mammalian mitotic proteins responsible for kinetochore-microtubule attachment. For instance, SUMO-1 conjugated RanGAP1 (Ran GTPase activating protein 1) and RanBP2 E3 SUMO ligase localize near kinetochores and facilitate stable microtubule attachment to kinetochores [73,74]. In another example, sumoylation of Aurora A promotes its interaction with its upstream activator [75]. As a result, the kinase activity of Aurora A in mitosis is dampened by SENP3 and SENP5, enzymes that deconjugate SUMO from Aurora A, and this mechanism ensures correct spindle assembly [75].

Regulating metaphase to anaphase transition

Tight regulation of kinetochore-microtubule attachment is necessary to achieve chromosome biorientation, a condition in which sister chromatids are stably attached to microtubules emanating from opposite poles [57,76]. Steps leading to chromosome biorientation include an ‘error correction’ process, which destabilizes erroneous kinetochore-microtubule attachments [77]. Destabilization of kinetochore-microtubule attachment during the error correction activates the spindle assembly checkpoint (SAC) activation and prevents entry into anaphase [77,78]. This process safeguards against chromosome mis-segregation. If biorientation is achieved, error correction activities are diminished and SAC is deactivated so that chromosome segregation can ensue [77,78]. The CPC complex (chromosomal passenger complex) contributes to the error correction process and SAC activation [78]. The CPC includes Bir1 (Baculoviral IAP repeat-containing protein 1), Sli15 (synthetically lethal with Ipl1 15), Nbl1 (N-terminal-Borealin like protein 1), and Ipl1 in budding yeast [78]. In mammalian cells, the CPC includes Survivin, INCENP (inner centromere protein), Borealin, and Aurora B [78].

Sumoylation of CPC subunits has been reported in multiple systems [59,79–83]. Among other regulations, this modification contributes to the function of the CPC in metaphase to anaphase transition. Sumoylation of Bir1 and Sgo1 (Shugoshin 1) (a protein that interacts with Bir1) by Siz1 and Siz2 limits the availability of these proteins in the vicinity of centromere-kinetochores, diminishing their error−correction activities, which will otherwise destabilize microtubule attachment to kinetochores [82]. This mechanism promotes sister kinetochore biorientation and subsequent transitioning into anaphase [82]. SUMO-dependent removal of Bir1 in promoting metaphase to anaphase transition is also observed in DNA damage-induced SAC activation. Sumoylated Bir1 is subjected to Slx5-mediated proteasomal degradation as a mechanism to overcome DNA damage-induced SAC [84]. In mammalian cells, sumoylation regulates the centromeric localization of another CPC subunit, Aurora B. Lack of this modification displaces the protein kinase and another CPC subunit, INCENP, from the centromeric region, which would otherwise localize to this region during prometaphase [83]. Since sumoylation changes the dynamics of Aurora B localization, the authors of the study hypothesized that sumoylation might be responsible for extraction of Aurora B from chromatin during prometaphase and metaphase-to-anaphase transition [83].

Regulating linkage between sister chromatids

Two sister chromatids are held together by the cohesin complex loaded onto chromosomes prior to S-phase [76]. The cohesin complex contains three subunits Smc1, Smc3, and Scc1/Mcd1/Rad21 (sister chromatid cohesion 1/mitotic chromosome determinant 1/Rad21 radiation sensitive 21) (Mcd1 hereafter) [76]. Cohesins provide cohesion between two sisters until the Mcd1 subunit of the cohesin complex is cleaved by separase enzyme at the correct time of mitosis [76]. At the onset of anaphase, an E3 ubiquitin ligase, the anaphase promoting complex (APC/C), degrades securin, a protein that inhibits separase’s activity, allowing separase to break down cohesin between sister chromatids [76].

Cohesion establishment and disruption between sister chromatids are regulated by the SUMO pathway. Remarkably, all budding yeast cohesin subunits are sumoylated in unperturbed S-phase as well as under DNA damage conditions [85,86]. Siz1, Siz2, and Mms21 E3 SUMO ligases have all been shown to sumoylate cohesin subunits [85]. Sumoylation of Mcd1 in S-phase promotes cohesion establishment between two sister chromatids, working in parallel with acetylation-mediated closure of the cohesin ring [86]. Although Mms21-dependent sumoylation of Mcd1 promotes cohesion in S-phase, poly-sumoylation of Mcd1 has to be limited to prevent premature loss of cohesion (Figure 1a) [87]. Pds5 (precocious dissociation of sisters 5), a cohesin associated protein, restricts extension of poly-SUMO chains on Mcd1 by another SUMO ligase Siz2 and protects Mcd1 from SUMO-mediated proteasomal degradation (Figure 1a) [87]. If Pds5’s function fails, sister chromatids are separated prematurely [87]. These data suggest that the SUMO-dependent degradation regulates Mcd1 degradation in parallel with separase. In addition to regulating sumoylation of Mcd1, Pds5 itself is sumoylated and this sumoylation attenuates cohesion between sister chromatids [88]. Sumoylation of Top2 (DNA topoisomerase II) similarly fine tunes sister chromatid separation by modulating the dynamics of cohesion [89]. In mammalian cells, sumoylation of one subunit of the APC/C complex controls timely entry into anaphase, suggesting that sumoylation might influence APC/C’s activity [90]. Figure 1. Examples of the effects of sumoylation in mitosis, DNA replication, DNA repair, and DNA damage response. Yellow diamonds represent SUMO. a) In S. cerevisiae, Pds5 limits the extension of SUMO chains on Mcd1 by Siz2. Sumoylation of Mcd1 primes the protein for sumo-dependent proteasomal degradation by Slx5/Slx8. Mcd1 degradation promotes sister chromatid separation. b) In S. cerevisiae, DDK’s activity facilitates pre-initiation complex formation. Ulp2 counteracts extension of SUMO chains on DDK by Siz1 and Siz2 and protects DDK from sumo-mediated proteasomal degradation. Thus, sumoylation controls replication initiation by modulating the availability of DDK. c) in S. cerevisiae, assembly of the STR complex (Sgs1-Top3-Rmi1) is enhanced by sumoylation. Sumoylation of Sgs1 by Mms21 promotes association between subunits. The STR complex dissolves DNA intermediates resulting from recombination or similar processes. d) in human cells, MDC1 binds to γH2A.X deposited around double-strand breaks and recruits other repair proteins such as RNF8. Degradation of sumoylated MDC1 by RNF4 promotes recruitment of additional repair proteins.

It is illustrates sumoylation, desumoylation, SUMO-dependent degradation of proteins, and biological processes resulting from SUMO modifications with forward and reverse arrows. (A) Displays two sister chromatids with cohesion complexes in between. The figure hones in on Mcd1 as part of the cohesion complex. (B) Portrays the pre-replication complex (Mcm2-7 and ORC) bound to DNA converted to the pre-initiation complex (CMG complex and ORC) by DDK and CDK (C) Illustrates Sgs1 and Top3 and Rmi1 forming a complex. (D) displays a double-strand break with H2A.X wrapped around the DNA and MDC1 and RNF8 complex bound to H2A.X.

All budding yeast SMC complexes (cohesin, condensin, and Smc5/6 complex) are subjected to SUMO-mediated proteasomal degradation, which limits their stability and function. Budding yeast Ulp2 SUMO protease prevents polysumoylation of SMC complexes so that they are not channeled into SUMO-mediated degradation catalyzed by Slx5/Slx8 [91]. The role of Ulp2 in regulating all SMC complexes explains the chromosome instability phenotype observed in ulp2 mutants [51].

SUMO control of cohesion is not only important under unperturbed conditions but also crucial for establishment of cohesion under damage conditions. In budding yeast, sumoylation of Mcd1 promotes cohesions on chromatin so that the use of sister chromatids as template is favored during double-strand break repair [85]. In mammalian cells, the Smc5/6 complex is positioned around double-strand breaks and this complex recruits cohesin to establish cohesion around the breaks [92]. Mms21-mediated sumoylation of SCC1 (mammalian Mcd1) favors the use of sister chromatids by antagonizing the activity of Wapl (wings apart-like protein), which would otherwise destabilize cohesin chromatin association [93].

In addition to its role in cohesion-mediated linkage between sister chromatids, SUMO machinery also controls DNA-mediated linkages to ensure that sister chromatids separate without errors. This type of linkages is especially prevalent when replication fails to complete or sister chromatid junctions resulting from homologous recombination (discussed below) are unresolved prior to mitosis. In such circumstances, the budding yeast Smc5/6 complex and Mms21 is responsible for disentangling sister chromatids to minimize chromosome segregation errors [94]. Another example of SUMO-mediated removal of DNA linkages during mitosis includes SUMO-dependent recruitment of Yen1, a structure specific endonuclease that processes recombination intermediates, to sites of where its activity is needed. Budding yeast Yen1 that cannot bind to SUMO chains is not able to process DNA intermediates, resulting in chromosome segregation errors [95].

Regulating spindle dynamics in anaphase

During anaphase, mitotic spindles need to be reorganized so that chromatids can be segregated faithfully. At this stage, many proteins are transported to the spindle midzone to regulate spindle stability, elongation, and subsequent dismantling of the spindles [76]. Sumoylation controls repositioning of proteins involved in this process. In budding yeast, sumoylation of Ndc10 (nuclear division cycle 10) is required for localization of Ndc10 along the anaphase spindle and spindle midzone and this modification regulates anaphase spindle elongation [80]. In addition to Ndc10, CPC subunits re-localize from centromere−kinetochore region to the spindle midzone [76,78]. Sumoylation of budding yeast Mcm21 (minichromosome maintenance 21) functions as a scaffold to re-localize CPC proteins to the spindle midzone to facilitate timely spindle disassembly [96]. In C. elegans, Ulp4 (SENP6/7 in mammalian) localizes to the spindle midzone and promotes relocation of Air2 (Aurora B in mammalian) to this region [81].

SUMO regulators in DNA replication

Regulation of normal replication

Faithful duplication of the genome requires accurate spatial and temporal coordination among multiple steps of the replication process. First, origin recognition complex (ORC) must be assembled along with Cdc6 (cell division cycle 6) and Cdt1 (chromatin licensing and DNA replication factor 1) at origins during G1 phase, followed by loading of double-hexamers Mcm2-7 helicase [97,98]. During G1-to-S-phase transition, S-CDK (S-phase cyclin-dependent kinase) and DDK (Dbf4-dependent kinase Cdc7) facilitate recruitment of additional replication factors, including DNA polymerase ε and co-activators needed for a functional helicase [97,98]. In subsequent steps, more replisome proteins, including PCNA sliding clamp and DNA polymerase δ, associate with DNA to progress the replication fork [97–99]. Here, I highlight selected examples in which key steps of DNA replication are modulated by sumoylation. For a more extensive review of SUMO in DNA replication, readers may refer to other reviews [100–102].

Sumoylation of multiple replication proteins underscores the importance of sumoylation in this process [103–106]. Sumoylation of ORC subunits has been reported in yeast and humans [104,107]. In budding yeast, hypersumoylation of ORC reduces the recruitment of the MCM helicase to origins [108]. In humans, inability to sumoylate ORC leads to DNA re-replication, suggesting that sumoylation controls the timing of replication [107]. Likewise, DNA helicase subunits are sumoylated in lower and higher eukaryotes [103,104,109,110]. In S. cerevisiae, sumoylation of Mcm3 is necessary for efficient loading of the MCM helicase at origins [111]. Desumoylation of the helicase subunits enables DNA unwinding activity of the helicase to commence G1 to S-phase transition [105]. An additional SUMO-dependent regulation of G1-to-S-phase transition includes DDK. Siz1 and Siz2 E3 SUMO ligases sumoylate Cdc7 and Dbf4 (dumbbell former 4) (subunits of the DDK complex) (Figure 1b) [112]. Ulp2 limits SUMO chain elongation on these proteins and protects them from polysumoylation-dependent proteasomal degradation managed by Slx5/Slx8 and Cdc48 (Figure 1b) [112]. Thus, SUMO-mediated degradation controls replication initiation by altering the availability of functional DDK (Figure 1b) [112].

In addition to early stages of replication, sumoylation contributes to replication fork progression. For example, the budding yeast Smc5/6 complex sumoylates DNA polymerase ε and this modification is necessary for faithful replication of the genome [106]. The role of the Smc5/6 complex in DNA synthesis makes sense given its chromatin localization during unperturbed replication [43]. In human cells, the effect of SUMO on replication fork has been explored at a large-scale level. Proteomic studies in human cells have uncovered that replisomes, molecular machineries comprised of proteins crucial for DNA synthesis, are rich in sumoylation and low in ubiquitination [113,114]. The SUMO deubiquitinase USP7 (ubiquitin specific peptidase 7) cleaves ubiquitin moieties from SUMO and maintain the SUMO-high and ubiquitin-low environment for replisomes [115–117]. Given that SUMO-rich conditions are maintained at mammalian replication forks, it is conceivable that additional mechanisms that fine tune replication are likely to depend on SUMO regulation. For example, sumoylation of BLM helicase in mammalian cells regulates the speed at which the replication fork progresses and the stability of the fork [118].

Sumoylation of the sliding clamp PCNA exemplifies the importance of this modification in regulating the integrity of the replication fork. Sumoylation of PCNA has been observed in multiple species but is the most well understood in budding yeast [49,119]. In budding yeast, sumoylation of PCNA can be observed during unperturbed S-phase as well as in cells undergoing replication stress [120]. Budding yeast PCNA is sumoylated at two distinct lysine residues: K164 and K127. K164 of PCNA is sumoylated by Siz1, whereas sumoylation of K127 depends on both Siz1 and Siz2 [120]. In budding yeast, sumoylated PCNA preferentially interacts with Srs2 (suppressor of rad six 2) to prevent illegitimate recombination at replication forks [119,121]. Likewise, sumoylation of PCNA in mammalian cells also contributes to anti-recombinogenic activity at replication forks [122,123].

Counteracting challenges in replication

An ongoing replication fork can encounter various challenges. Genomes contain regions that are repetitive in nature, making it hard to replicate [124]. Template DNA may carry DNA lesions introduced by exogenous damaging agents. If these lesions are left unrepaired until S-phase, they impede the progression of replication forks [124]. Any condition in which normal replication is interrupted is referred to as replication stress. Regulators of the SUMO pathway, especially the Smc5/6 complex and Slx5/Slx8 (RNF4 in mammalian), orchestrate pathways cells employ to remedy replication stress.

In S. cerevisiae, the Smc5/6 complex is localized to the natural pause sites to resolve challenges imposed at regions of chromosomes that are naturally difficult to replicate [34,125,126]. These regions include ribosomal DNA (rDNA) regions and regions with late origins of replication [34,125,126]. Likewise, Mms21 is required to counteract replication stress induced by genotoxic agents as well as genetic defects in replication proteins [84,127–129].

The ability of Mms21 and the Smc5/6 complex to resist replication stress may be attributed to its diverse roles in ensuring the integrity of replication fork in more than one mechanism. During replication fork stalling, Mms21 sumoylates a subunit of the DNA polymerase, Polε, in a checkpoint dependent manner [130]. Sumoylation of the DNA polymerase under this circumstance modulates the activity of the replisome to appropriately respond to fork stalling [130]. In addition, the Smc5/6 complex coordinates with Rtt107 (regulator of Ty1 transposition 107) to promote replication progression under replication stress conditions [131].

One way for cells to ameliorate replication fork stalling is via fork regression [132]. During fork regression, specialized DNA helicases such as Mph1 (mutator phenotype 1) and Rad5 disengage newly synthesized DNA strands from the template strands. This process facilitates annealing of two daughter DNA strands to each other as a way to reverse the fork [132–134]. The Smc5/6 complex and Mms21 regulates Mph1-dependent fork regression in both yeast and mammalian cells [133–135]. Additionally, the budding yeast Smc5/6 complex has been identified at collapsed forks and is associated with repair at these structures [43,136].

Another major contribution of the Smc5/6 complex to alleviate challenges in replication is through its functional connection with the template switching pathway. During this process, the daughter strand that encounters the lesion in the template switches to anneal an undamaged strand as a way to circumvent the impediment [132,137]. Results of the template switching pathway are DNA intermediates, that can compromise genome integrity if left unresolved. In budding yeast, the Smc5/6 complex is responsible for processing these DNA intermediates to minimize genome instability (discussed more below) [138–141].

In addition to Mms21, the STUbL is a key regulator of replication stress. The mammalian RNF4 is crucial for regulating stalled replication forks. One such mechanism involves the Fanconi anemia (FA) pathway subunits, FANCI and FAND2 [142]. Sumoylation of FANCI and FAND2 triggers the activity of RNF4, which facilitates the removal of the FA proteins from sites of damage, potentially to limit or fine tune the activity FA proteins [142]. RNF4 also prevents excessive replication fork reversal by degrading sumoylated DNA topoisomerase II alpha [143]. Prolonged replication forks or unprotected forks may result in replication fork collapse [124]. RNF4 removes sumoylated BLM (BLM RecQ like helicase) from collapsed replication forks and this process is necessary for resumption of replication [144]. In cells that are unable to stabilize replication forks due to deficiency in ATR activity, RNF4-dependent removal of chromatin bound proteins stalled replication forks facilitate conversion of this structure into double-strand breaks by a structure-specific endonuclease [145]. Different roles of RNF4 in replication fork stalling and collapse explain the need for RNF4 or Slx5/Slx8 in mutants that experience replication stress [84,146]. In addition to these mechanisms, the STUbL enables rescue of collapsed replication forks by relocating to the nuclear periphery and these mechanisms are discussed below.

Responding to different DNA structures that can compromise genome stability

Facilitating removal of aberrant DNA intermediates or DNA-protein crosslinks

DNA intermediates, referred to as joint molecules, can be generated by more than one DNA processing mechanism, such as the template switching pathway and homologous recombination. Although these aberrant DNA structures are a result of a mechanism that alleviates genome instability, if left unprocessed, they can also compromise the integrity of genetic materials and cellular survival. Thus, mechanisms that untangle DNA intermediates are crucial to function downstream of the template switching or homologous recombination. The STR complex, composed of Sgs1 (slow growth suppressor 1) helicase, Top3 (topoisomerase 3) DNA topoisomerase, and Rmi1 (RecQ mediated genome instability 1), is responsible for dissolving recombinogenic DNA structures [147]. In this dissolution pathway, Sgs1 associates with the Smc5/6 complex to be in the proximity of Mms21 [148,149]. Sumoylation of Sgs1 by Mms21 augments the assembly of the functional STR complex, sumoylation of Top3/Rmi1, and stimulates dissolution of DNA intermediates (Figure 1c) [148–150]. These mechanistic insights explain the observations that Ubc9, Mms21, Smc5/6, and Sgs1 limit structures arising from recombination [138,139]. In addition to its role in mitotic cells, Mms21 limits inappropriate recombination intermediates during meiosis [151].

In parallel to the dissolution pathway by the STR complex, structure-selective endonucleases can resolve the DNA intermediates arising from recombination or replication fork processing. These nucleases include Slx1/4-Mms4/Mus81 (MMS and UV sensitive 81) nuclease complex and Yen1 [147,152–154]. As a scaffold protein, Slx4 has diverse roles depending on its interacting partners [155]. Thus, SUMO regulation of Slx4 discussed here may be applicable to other mechanisms beyond the activities of the nuclease-mediated resolution pathway. In human cells, Slx4 complexed with Ubc9 has been proposed to function as an E3 SUMO ligase [156]. Human Slx4 localization to sites of damage is mediated by interaction of its SIMs with SUMO [157,158]. Unlike Slx4, evidence on the SUMO-regulation of Mms4-Mus81 and Yen1 in resolution of DNA intermediates is more direct. In S. cerevisiae, Mms4-Mus81 and Yen1 nuclease activities are optimal only in mitosis [154]. Restriction of nuclease activities to mitosis is a mechanism to prioritize the STR-dependent dissolution and limits crossover events resulting from the activity of these structure-selective endonucleases [154]. These nucleases become more active at G2/M transition so that they can resolve the DNA intermediates right before chromosome segregation [154]. Sumoylation regulates the temporal activities of Mms4/Mus81 and Yen1 nucleases. For instance, Mms4 is a substrate of the Slx5/8 complex [159]. The absence of SUMO-mediated degradation makes active chromatin-bound Mms4 available in G1 phase of the cell cycle [159]. Similarly, Siz1 and Siz2 sumoylate Yen1 upon DNA damage to stimulate its activity but the Slx5/8 complex-dependent degradation of Yen1 ensures that Yen1 is not active in S-phase [160].

In addition to DNA intermediates, DNA-protein crosslinks (DPCs) can compromise genome stability. DPCs arise when DNA topoisomerases or other proteins are covalently conjugated to DNA molecules [161]. Persistent DPCs, which should have been short-lived, pose a major risk to genome stability [161]. Sumoylation and SUMO-mediated proteasomal degradation resolve these aberrant structures in different eukaryotes [162–167]. A recent study further supports the role of sumoylation in removing DPCs. In human cells, Slx4 oligomers provides scaffolds for multiple protein-protein interactions, some of which are mediated by SUMO−SIM interactions [168]. These interactions form ‘Slx4 condensates,’ where RNF4 is recruited to remove DPCs [168].

Regulation at genomic loci characterized by DNA repeats: telomeres and rDNA

SUMO enzymes play a key role in ensuring chromosomal integrity at eukaryotic genomic loci that contain repetitive DNA sequences. These regions include telomeres and rDNA loci.

Telomeres preserve the structural integrity of chromosomes. With each round of cell cycle, telomere length becomes shorter if no active mechanism is in place to maintain telomere length. Telomere erosion can be prevented by the telomerase enzyme that can maintain or extend the length of telomeres. However, if telomere maintenance is needed in the absence of the telomerase activity, cells resort to an alternative pathway that relies on recombination. In budding yeast and human cells, Mms21 and the Smc5/6 complex are associated with the recombination-dependent telomere maintenance pathway [169,170]. In yeast, chromatin immunoprecipitation data locate the Smc5/6 complex at the telomeric regions [43]. Yeast cells lacking the function of Mms21 exhibit defects in regulation of telomere lengths [171,172]. The telomere length regulation by the Mms21/Smc5/6 complex may be attributed to its ability to prevent aberrant recombination intermediates between telomeres [170]. In addition to Mms21, budding yeast Siz1 and Siz2 regulate nuclear pore complex-dependent regulation of telomeres [173]. Siz1 and Siz2 sumoylate telomere-bound proteins, which subsequently associate with Slx5/Slx8 [173]. The Slx5/Slx8 complex sequesters damaged telomeres to the NPC by interaction with Nup84 (nuclear pore 84) [173]. This process facilitates recombination-dependent, telomerase-independent restoration of telomere length [173].

In mammalian cells, recombination-dependent telomere maintenance pathway, known as the ALT (alternative lengthening of telomeres), can occur in subnuclear structures called PML (promyelocytic leukemia protein) bodies, where protein complexes aggregate to regulate multiple cellular processes such as DNA repair and transcriptional regulation [174]. The human Smc5/6 complex is found in ALT-associated PML bodies (APBs), specialized PML bodies where telomeres and DNA repair factors are localized. The Smc5/6 complex is responsible for localizing telomeres to APBs and sumoylating telomere binding proteins to facilitate APB formation [169,175]. Consistent with the observation, the Smc5/6 complex localizes temporarily at telomeric DNA during PML body formation and its SUMO ligase activity maintains telomere length [169,176]. In addition, another mammalian SUMO ligase, PIAS4, also regulates APB-dependent telomere synthesis [177].

SUMO enzymes regulate another form of repetitive DNA sequences within the genome–rDNA. All three E3 ligases in yeast are crucial for maintaining rDNA stability [126]. If a double-strand break occurs within the rDNA, recombination may be necessary to repair the damage. To facilitate this process, rDNA must be first released from the nucleolus where it is protected from unregulated recombination. Sumoylation of the CLIP (chromosome linkage INM protein)-cohibin complex, which tethers rDNA to the nucleolus, disrupts the complex and thus promotes the nucleolar release of rDNA to permit DNA repair in the nucleoplasm [178]. The budding yeast Smc5/6 complex also regulates rDNA downstream of this release – the complex limits DNA intermediates resulting from recombination at rDNA loci [34].

SUMO regulation in double-strand break repair

DNA double-strand breaks are the result of genotoxic stresses, such as irradiation, reactive oxygen species as well as replication fork collapse [179,180]. Regardless of the source of double-strand breaks, cells utilize two major pathways of double-strand break repair. Homologous recombination (HR) relies on a template strand that shares considerable homology with the broken strand. The template may be sister chromatids or a homologous chromosome. In contrast, non-homologous end joining (NHEJ) does not rely on the template strand. Successful completion of both types of double-strand break repair requires recruitment of repair proteins to the sites of damage, stable assembly of protein complexes, and timely removal of proteins from the damage sites to ensure completion of the repair process. Dynamics of sumoylation and desumoylation in the vicinity of double-strand breaks facilitate these processes.

In S. cerevisiae, sumoylation plays a central role in protein complex assembly. DNA resection around the double-strand breaks and subsequent generation of single-strand DNA trigger sumoylation of repair proteins [47,181]. The single-strand DNA binding protein, Rfa2 (replication factor A 2) (a subunit of the RPA complex), recruits Siz2 to double-strand breaks, where Siz2 sumoylates repair proteins for protein complex assembly [47,181]. Functionally, sumoylation of HR proteins such as Rad52 and Rad59 promotes double-strand break repair [47]. In addition to Siz2, the Smc5/6 complex is recruited to double-strand breaks where it facilitates HR between sister chromatids [182]. This observation is in agreement with DNA-dependent regulation of Mms21’ SUMO ligase activity. DNA association of Smc5, a direct binding partner of Mms21, stimulates Mms21’s enzymatic activity, strongly suggesting a role of Mms21 in DNA damage repair (and other DNA processing activities discussed in other sections) [183]. Yeast NHEJ pathway is also regulated by Siz SUMO ligases – sumoylation of Lif1 (ligase interacting factor 1), a subunit of the DNA ligase IV complex, reduces its self-association and NHEJ activities [184]. Although the mechanisms are currently unknown, another SUMO regulator, Slx5, localizes to double-strand breaks, suggesting its role in this process [185].

Similar to S. cerevisiae, sumoylation plays a prominent role in mammalian double-strand break repair pathways. Sumoylation can influence the chromatin status to indirectly affect DNA repair. For example, SENP7-mediated desumoylation of the chromatin repressive KAP1 (KRAB-associated protein 1) relaxes the chromatin structure to facilitate double-strand break repair by HR [23]. In addition, multiple studies have demonstrated direct involvement of sumoylation in regulating double-strand break repair proteins. SUMO chains can facilitate the initiation of HR by serving as a binding platform for proteins crucial for this repair pathway. One strategy cells utilize to extend SUMO chains is by diminishing the activity of isopeptidases. In a specific example, dissociation of SENP6 from RPA1 (replication protein A 1) (a subunit of RPA) permits accumulation of SUMO chains on RPA1 [186]. Sumoylated RPA1 recruits RAD51 recombinase to initiate HR [186]. Likewise, sumoylation of BLM helicase at stalled replication forks facilitates recruitment of RAD51, RAD52, and BRCA2 (breast cancer gene 2) so that HR pathway can commence [187]. Likewise, PIAS1 and PIAS4 E3 SUMO ligase-dependent sumoylation in the vicinity of double-strand breaks is important for recruitment of 53BP1 (p53-binding protein 1), BRCA1, and RNF168 [188].

Beyond protein recruitment and assembly, sumoylation regulates turnover of repair proteins at the breaks to facilitate transitioning from one step to the next in the repair process. RNF4 promotes SUMO-dependent degradation of RPA1 to encourage loading of the homologous recombination proteins BRCA2 and RAD51 on resected DNA for HR [189]. Likewise, RNF4-mediated turnover of MDC1 (mediator of DNA damage checkpoint 1) is proposed to accommodate other repair protein recruitment to the double-strand breaks so that subsequent repair steps can proceed (Figure 1d) [189]. In parallel with RNF4, SENP2 controls the level of sumoylated MDC1 [190]. Upon double-strand break induction by irradiation, SENP2-MDC1 interaction is interrupted [190]. As a result, polysumoylated MDC1 becomes destabilized by RNF4 [190]. These data suggest that counterbalancing actions of a SUMO isopeptidase and the STUbL intricately controls the coordinated recruitment and clearance of repair proteins at double-strand breaks. A proteomics study identifying potential SENP6 targets in human cells further supports this model. The activity of SENP6 limits polysumoylation of DNA damage response proteins, preventing them from RNF4-dependent degradation [191].

Similar to S. cerevisiae, SUMO regulation in mammalian is not only restricted to homologous recombination. PIAS1, PIAS4, SENP2 and RNF4 all regulate both HR and NHEJ repair pathways [188–190,192]. Multiple NHEJ proteins have been identified in SUMO proteomics studies [8,193]. Mechanistically, recruitment and stabilization of the protein complex crucial for the NHEJ repair [XRCC4 (X-ray repair cross complementing 4), DNA-PKcs (DNA-dependent protein kinase catalytic subunit) and Ligase IV] is regulated by SUMO-SIM non-covalent interactions [27]. Non-conventional SIMs also contribute to SUMO-mediated protein-protein interactions during NHEJ. XRCC4 has been shown to interact with SUMO2 via a non-conventional SIM and this interaction functionally regulates NHEJ [194]. Due to the diversity of interactions, this study proposed that individual SIMs/non-conventional SIMs as well as different combinations of these may contribute to various aspects of NHEJ [194].

Diverting damaged DNA to nuclear periphery

Association of chromatin with the nuclear envelope is crucial for multiple DNA metabolic processes such as transcriptional regulation, global organization of chromatin, and genome stability mechanisms [195]. SUMO enzymes are responsible for regulating some of these processes, most prominently contributing to genome integrity functions. Siz2 E3 ligase and Ulp1 isopeptidase are responsible for spatial and temporal organization of a specialized chromatin region in the vicinity of the nuclear envelope [196]. Intriguingly, Nup60, a nuclear pore complex subunit, is sumoylated upon genotoxic stress [197]. Sumoylation of Nup60 is crucial for resisting genotoxic agents, suggesting that this modification functionally contributes to DNA damage response [197].

Multiple studies have demonstrated a highly regulated pathway in which damaged DNA that is difficult to repair is relocated to the nuclear periphery in a SUMO-dependent manner. Nuclear periphery associated DNA repair is utilized for persistent double-strand break repair, heterochromatin repair, and restart of collapsed replication forks [198]. Sequestering the broken chromosome to the nuclear periphery confines the damaged DNA to a subcellular compartment where repair can take place. This compartmentalization minimizes aberrant recombination events between non-allelic DNA [198]. This is especially important for regions of the genome with multiple repetitive DNA [198]. SUMO regulators play prominent roles in regulating this process in collaboration with DNA repair proteins.

Strong evidence comes from genetic interaction studies that identify functionally related genes based on their genetic interaction similarity profiles in S. cerevisiae [199]. This unbiased approach reveals Slx5/Slx8 to be functionally similar to a subset of nuclear pore complex proteins, such as Nup60, Nup84, and Nup133 [199]. Mechanisms by which damaged DNA is shunted to the nuclear pore complex depend on activities of different SUMO regulators. These mechanisms were uncovered in a genetic model of replication fork collapse [200]. In this model, replication forks frequently collapse within the DNA regions containing long CAG repeats. Due to the repeats that can complementary base pair with each other, secondary DNA structures are common within these regions, stalling replication forks and making them susceptible to collapse [201]. Mms21 sumoylates proteins such as RPA, Rad52, and Rad59, that are associated with collapsed forks [200]. Slx5/Slx8 binds to these sumoylated proteins via its SIMs and relocate the damaged structure to the nuclear pore complex [200]. The SUMO-dependent, nuclear periphery-associated genome maintenance mechanism is not only restricted to relocating broken forks resulting from repetitive DNA sequences. In Schizosaccharomyces pombe, stalled replication forks associated with non-repetitive regions are relocated to the nuclear pore complex in a manner dependent on Pli1 (Siz1/Siz2 homolog) and the STUbL [202]. Removal of SUMO by the isopeptidase Ulp1 associated with the nuclear pore complex is proposed to facilitate replication fork restart [202]. Specifically, nuclear pore-associated fission yeast Ulp1 contributes primarily to the initiation of replication restart while the proteasome enriched at the nuclear pore affects the progression of restarted forks [203].

Similar to stalled and collapsed forks, persistent double-strand breaks are also known to localize to the nuclear envelope in S. cerevisiae [204,205]. SUMO-mediated relocation of persistent double-strand breaks to the nuclear pore complex depends on both Siz2 and Mms21 [204]. Similarly, Slx5/Slx8 is responsible for relocating the breaks to nuclear pore regions via its SIMs interacting with sumoylated proteins around the breaks [204]. Ulp1 is also implicated in nuclear pore complex-mediated double-strand break repair [206]. Physically, Ulp1 is tethered to the nuclear pore complex [35,207–209]. Nuclear pore complex proteins are required for Ulp1 localization at the nuclear periphery so that appropriate level of SUMO-mediated DNA repair can be accomplished [206]. Intriguingly, SUMO-mediated nuclear periphery-associated repair is highly conserved. In Drosophila, double-strand breaks that arise within heterochromatic regions are relocated to the nuclear periphery [210]. Heterochromatin is typically composed of repetitive DNA sequences and thus, are susceptible to aberrant HR upon double-strand break formation. Sequestering heterochromatic double-strand breaks to the nuclear periphery minimizes this undesirable outcome [198]. In Drosophila, Nse2 (Mms21 in flies) and dPIAS (Siz1/2 in S. cerevisiae and PIAS1–4 in mammals) sumoylate proteins at heterochromatic double-strand breaks and SUMO chains allow these breaks to be anchored at the nuclear periphery by interacting with Slx5/Slx8 [210].

Conclusions

As highlighted by the examples reviewed above, sumoylation plays influential roles in multiple steps of DNA-replication, mitosis, DNA damage repair, and specialized processes that safeguard the integrity of the genome, especially in special regions with repetitive sequences. The ability of the E3 SUMO ligases to sumoylate multiple substrates in their vicinity allow a regulatory system in which subunits of a complex or proteins that belong to the same pathway to be modulated synchronously. This phenomenon, well accepted as ‘protein group modification,’ has been observed in both lower and higher eukaryotes [1,47]. However, sumoylation is not exclusively for protein group modification. Evidence suggests that some SUMO substrates are selectively modified by the SUMO ligases, as discussed above for PCNA.

Due to SUMO−SIM interactions, sumoylation can initiate protein complex assembly. Furthermore, sumoylation of a protein may alter its function in a manner that does not necessarily depend on protein-protein interactions. The activity of STUbLs can restrict functions or events promoted by sumoylation while SUMO proteases can protect proteins from STUbL-mediated SUMO-dependent degradation. As a result, the interplay between SUMO ligases, proteases, and STUbLs provides versatility in regulation of many target proteins. Depending on which enzyme is recruited to the local environment, the SUMO-dependent biological activity may be enhanced, slowed down, or halted.

Disclosure statement

No potential conflict of interest was reported by the author(s).

Data availability statement

Data sharing is not applicable to this article as no new data were created or analyzed in this study.
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