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We show that central components of the Fanconi anemia (FA) DNA repair pathway, the tumor suppressor proteins FANCI and FANCD2 (the ID complex), are SUMOylated in response to replication fork stalling. The ID complex is SUMOylated in a manner that depends on the ATR kinase, the FA ubiquitin ligase core complex, and the SUMO E3 ligases PIAS1/PIAS4 an...
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Elucidating the individual and collaborative functions of genome maintenance factors is critical for understanding how genome duplication is achieved. Here, we investigate a conserved scaffold in budding yeast, Rtt107, and its three partners: a SUMO E3 complex, a ubiquitin E3 complex, and Slx4. Biochemical and genetic findings show that Rtt107 inte...
High-resolution imaging shows that persistent DNA damage in budding yeast localizes in distinct perinuclear foci for repair. The signals that trigger DNA double-strand break (DSB) relocation or determine their destination are unknown. We show here that DSB relocation to the nuclear envelope depends on SUMOylation mediated by the E3 ligases Siz2 and...
Posttranslational modifications (PTMs) of proteins include enzymatic changes by covalent addition of cellular regulatory determinants such as ubiquitin (Ub) and small ubiquitin-like modifier (SUMO) moieties. These modifications are widely used by eukaryotic cells to control the functional repertoire of proteins. Over the last decade, it became appa...
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Streszczenie: Białko UBC9 jest jedynym enzymem koniugującym E2 w potranslacyjnej modyfik-acji białek, zwanej sumoilacją. Modyfikacja ta polega na przyłączeniu niewielkiego, podobnego do ubikwityny białka o nazwie SUMO. Badania ostatnich lat wskazują, że sumoilacja odgrywa ważną rolę w regulacji ekspresji genów i utrzymaniu stabilności genomowej zac...
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... Regarding the role of SUMOylation in genome stability, SUMO1 and SUMO2/3 can be detected in IR-induced foci (IRIF), laser-induced damage, DSBs, and can be precipitated from damaged chromatin, with SUMOylation known to play a role in HR and NHEJ pathways [158,[240][241][242][243]. Several targets are known, such as MDC1 [244], RPA [242,245], FANCI-D2 [246,247], with the outcome of the target SUMOylation often to produce an altered recruitment platform, such as for SIM-containing proteins or STUbLs like RNF4, the latter highlighting the interplay between Ub and SUMO signalling in genome stability [248]. Indeed, activation of RNF4 through binding to polySUMO chains via its tandem SIMs can result in the formation of Ub-SUMO hybrids that is coupled to VCP/p97-mediated protein extraction from damaged chromatin and often resulting in proteasome-mediated degradation [249][250][251]. ...
... As described above, ATR-mediated phosphorylation and FA core complex-mediated ubiquitylation enables regulated recruitment of FANCI-D2 to sites of ICL damage. To enable proficient repair, the FANCI-D2 clamp needs to be removed or extracted from chromatin, and this is enabled through PIAS4-mediated polySUMO2/3 chain formation leading to RNF4 recruitment and subsequent extraction via VCP/p97 [246]. SENP6 depletion reduces the chromatin retention of FANCI-D2 and may lead to premature removal of FANCI-D2 via RNF4, reducing efficiency of ICL repair. ...
Maintaining stability of the genome requires dedicated DNA repair and signalling processes that are essential for the faithful duplication and propagation of chromosomes. These DNA damage response (DDR) mechanisms counteract the potentially mutagenic impact of daily genotoxic stresses from both exogenous and endogenous sources. Inherent to these DNA repair pathways is the activity of protein factors that instigate repair processes in response to DNA lesions. The regulation, coordination, and orchestration of these DDR factors is carried out, in a large part, by post-translational modifications, such as phosphorylation, ubiquitylation, and modification with ubiquitin-like proteins (UBLs). The importance of ubiquitylation and UBLylation with SUMO in DNA repair is well established, with the modified targets and downstream signalling consequences relatively well characterised. However, the role of dedicated erasers for ubiquitin and UBLs, known as deubiquitylases (DUBs) and ubiquitin-like proteases (ULPs) respectively, in genome stability is less well established, particularly for emerging UBLs such as ISG15 and UFM1. In this review, we provide an overview of the known regulatory roles and mechanisms of DUBs and ULPs involved in genome stability pathways. Expanding our understanding of the molecular agents and mechanisms underlying the removal of ubiquitin and UBL modifications will be fundamental for progressing our knowledge of the DDR and likely provide new therapeutic avenues for relevant human diseases, such as cancer.
... RNF4 shows SIM-dependent accumulation at DNA damage sites, which are also loci of SUMO-dependent signaling 27,28 . Two SUMOdependent targets identified by BioE3, Fanconi Anemia group I protein, FANCI and FANCD2, were shown to be SUMOylated on damaged chromatin and regulated through ubiquitination by RNF4 to allow cell survival after DNA damage 29 . MDC1 also participates in DNA repair and was previously shown to be a SUMO-dependent target of RNF4 30 . ...
Hundreds of E3 ligases play a critical role in recognizing specific substrates for modification by ubiquitin (Ub). Separating genuine targets of E3s from E3-interactors remains a challenge. We present BioE3, a powerful approach for matching substrates to Ub E3 ligases of interest. Using BirA-E3 ligase fusions and bioUb, site-specific biotinylation of Ub-modified substrates of particular E3s facilitates proteomic identification. We show that BioE3 identifies both known and new targets of two RING-type E3 ligases: RNF4 (DNA damage response, PML bodies), and MIB1 (endocytosis, autophagy, centrosome dynamics). Versatile BioE3 identifies targets of an organelle-specific E3 (MARCH5) and a relatively uncharacterized E3 (RNF214). Furthermore, BioE3 works with NEDD4, a HECT-type E3, identifying new targets linked to vesicular trafficking. BioE3 detects altered specificity in response to chemicals, opening avenues for targeted protein degradation, and may be applicable for other Ub-likes (UbLs, e.g., SUMO) and E3 types. BioE3 applications shed light on cellular regulation by the complex UbL network.
... This DNA damage-induced SUMOylation triggers RNF4-dependent ubiquitylation of the ID complex. The SUMO-dependent polyubiquitin chains on the ID complex guide the DVC1-p97 ubiquitin-selective segregase complex for the active removal of the ID complex from chromatin (Gibbs-Seymour et al., 2015). Notably, recent clinical research on FA patients has shown that FANCA undergoes SUMOylation and subsequent RNF4-dependent ubiquitination and proteasome-mediated degradation in the absence of FAAP20 binding (Xie et al., 2015). ...
Various post-translational modifications (PTMs) collaboratively fine-tune protein activities. SUMO-targeted ubiquitin E3 ligases (STUbLs) emerge as specialized enzymes that recognize SUMO-modified substrates through SUMO-interaction motifs and subsequently ubiquitinate them via the RING domain, thereby bridging the SUMO and ubiquitin signaling pathways. STUbLs participate in a wide array of molecular processes, including cell cycle regulation, DNA repair, replication, and mitosis, operating under both normal conditions and in response to challenges such as genotoxic stress. Their ability to catalyze various types of ubiquitin chains results in diverse proteolytic and non-proteolytic outcomes for target substrates. Importantly, STUbLs are strategically positioned in close proximity to SUMO proteases and deubiquitinases (DUBs), ensuring precise and dynamic control over their target proteins. In this review, we provide insights into the unique properties and indispensable roles of STUbLs, with a particular emphasis on their significance in preserving genome integrity in humans.
... The best-characterized mammalian STUbLs include RNF4 and RNF111 13 . Many DDR factors are directly or indirectly regulated by RNF4, including BRCA1-BARD1, MDC1, RPA70 and the Fanconi Anemia ID complex (FANCI and FANDC2) [14][15][16][17][18] . ...
... Similarly, in another recent proteomics screen, SENP6 was identified as a key regulator of sister chromatid cohesion and chromatin residency of proteins involved in the ATR-Chk1 DNA damage checkpoint 24 . In addition, SENP6 was described to counteract SUMOylation of the DDR factors EXO1, RPA70, and the Fanconi Anemia ID complex 16,17,25 . ...
... In cellular fractionation assays, SENP6 was found in both the chromatin and nucleoplasmic fraction of untreated and aphidicolin-treated cells 24 . A catalytic dead variant of HA-tagged SENP6 was found to localize to FANCI foci in response to MMC treatment, but not in response to laser micro-irradiation, in a transient overexpression system 16 . We first investigated whether endogenous SENP6 localizes to and accumulates at DNA double-strand breaks after IR. ...
The SUMO protease SENP6 maintains genomic stability, but mechanistic understanding of this process remains limited. We find that SENP6 deconjugates SUMO2/3 polymers on a group of DNA damage response proteins, including BRCA1-BARD1, 53BP1, BLM and ERCC1-XPF. SENP6 maintains these proteins in a hypo-SUMOylated state under unstressed conditions and counteracts their polySUMOylation after hydroxyurea-induced stress. Co-depletion of RNF4 leads to a further increase in SUMOylation of BRCA1, BARD1 and BLM, suggesting that SENP6 antagonizes targeting of these proteins by RNF4. Functionally, depletion of SENP6 results in uncoordinated recruitment and persistence of SUMO2/3 at UVA laser and ionizing radiation induced DNA damage sites. Additionally, SUMO2/3 and DNA damage response proteins accumulate in nuclear bodies, in a PML-independent manner driven by multivalent SUMO-SIM interactions. These data illustrate coordinated regulation of SUMOylated DNA damage response proteins by SENP6, governing their timely localization at DNA damage sites and nuclear condensation state.
... STUbLs also cooperate with cell cycle defective protein 48 (Cdc48, or p97), a molecular segregase that liberates proteins from higher-order complexes or chromatin [37,38]. Their cooperation ensures proper DNA interstrand cross-link (ICL) repair and limits illegitimate processing of replication-associated recombination intermediates [39,40]. Although several individual targets of human RNF4 or yeast Slx5/Slx8 have been identified, a comprehensive understanding of their function in DNA replication has not been systematically explored. ...
DNA replication requires precise regulation achieved through post-translational modifications, including ubiquitination and SUMOylation. These modifications are linked by the SUMO-targeted E3 ubiquitin ligases (STUbLs). Ring finger protein 4 (RNF4), one of only two mammalian STUbLs, participates in double-strand break repair and resolving DNA-protein cross-links. However, its role in DNA replication has been poorly understood. Using CRISPR/Cas9 genetic screens, we discovered an unexpected dependency of RNF4 mutants on ubiquitin specific peptidase 7 (USP7) for survival in TP53-null retinal pigment epithelial cells. TP53-/-/RNF4-/-/USP7-/- triple knockout (TKO) cells displayed defects in DNA replication that cause genomic instability. These defects were exacerbated by the proteasome inhibitor bortezomib, which limited the nuclear ubiquitin pool. A shortage of free ubiquitin suppressed the ataxia telangiectasia and Rad3-related (ATR)-mediated checkpoint response, leading to increased cell death. In conclusion, RNF4 and USP7 work cooperatively to sustain a functional level of nuclear ubiquitin to maintain the integrity of the genome.
... Many clients are cytoplasmic but recent evidence has also identified nuclear targets, e.g., Aurora B is removed by p97 during mitosis, required to maintaining mitotic fidelity (Ramadan et al., 2007;Dobrynin et al., 2011). While most commonly targeted via ubiquitin signals, p97 has also been shown to be targeted via SUMO binding adapters (Yeung et al., 2008;Bergink et al., 2013;Gibbs-Seymour et al., 2015). ...
Centromeres are unique chromosomal loci that form the anchorage point for the mitotic spindle during mitosis and meiosis. Their position and function are specified by a unique chromatin domain featuring the histone H3 variant CENP-A. While typically formed on centromeric satellite arrays, CENP-A nucleosomes are maintained and assembled by a strong self-templated feedback mechanism that can propagate centromeres even at non-canonical sites. Central to the epigenetic chromatin-based transmission of centromeres is the stable inheritance of CENP-A nucleosomes. While long-lived at centromeres, CENP-A can turn over rapidly at non-centromeric sites and even erode from centromeres in non-dividing cells. Recently, SUMO modification of the centromere complex has come to the forefront as a mediator of centromere complex stability, including CENP-A chromatin. We review evidence from different models and discuss the emerging view that limited SUMOylation appears to play a constructive role in centromere complex formation, while polySUMOylation drives complex turnover. The deSUMOylase SENP6/Ulp2 and the proteins segregase p97/Cdc48 constitute the dominant opposing forces that balance CENP-A chromatin stability. This balance may be key to ensuring proper kinetochore strength at the centromere while preventing ectopic centromere formation.
... Since dsDNA has been shown to protect against both FANCI and FANCD2 deubiquitination, ubiquitinated ID2 complexes may need to be disengaged from DNA to be more effectively deubiquitinated. This could be achieved through the action of the DVC1-p97 ubiquitin segregase, which has been shown to be responsible for removal of ID2 from sites of DNA damage, once ID2 has been SUMOylated and subsequently polyubiquitinated on SUMO (Gibbs-Seymour et al, 2015). Another possibility would be that ubiquitinated ID2 and/or USP1/UAF1 are modulated (by factors and in ways that are yet unknown) for effective cleavage of the conjugated ubiquitins in the presence of DNA. ...
Di-monoubiquitination of the FANCI-FANCD2 (ID2) complex is a
central and crucial step for the repair of DNA interstrand crosslinks
via the Fanconi anaemia pathway. While FANCD2 ubiquitination
precedes FANCI ubiquitination, FANCD2 is also deubiquitinated
at a faster rate than FANCI, which can result in a FANCIubiquitinated
ID2 complex (IUbD2). Here, we present a 4.1 A cryo-
EM structure of IUbD2 complex bound to double-stranded DNA. We
show that this complex, like ID2Ub and IUbD2Ub, is also in the closed
ID2 conformation and clamps on DNA. The target lysine of FANCD2
(K561) becomes fully exposed in the IUbD2-DNA structure and is
thus primed for ubiquitination. Similarly, FANCI’s target lysine
(K523) is also primed for ubiquitination in the ID2Ub-DNA complex.
The IUbD2-DNA complex exhibits deubiquitination resistance, conferred
by the presence of DNA and FANCD2. ID2Ub-DNA, on the
other hand, can be efficiently deubiquitinated by USP1-UAF1,
unless further ubiquitination on FANCI occurs. Therefore, FANCI
ubiquitination effectively maintains FANCD2 ubiquitination in two
ways: it prevents excessive FANCD2 deubiquitination within an
IUbD2Ub-DNA complex, and it enables re-ubiquitination of FANCD2
within a transient, closed-on-DNA, IUbD2 complex.
... For example, RNF4 targets SUMO-modified MDC1 for poly-ubiquitination and subsequent degradation, thereby regulating the assembly/disassembly of DNA damage response (DDR) proteins at DNA damage sites (34)(35)(36)(37). In addition to MDC1, several other DDR factors, such as FANCD2/FANCI, RPA, CtIP and BRCA1 have also been reported to be substrates of RNF4 (35,(37)(38)(39). However, although several lines of evidence have revealed the importance of RNF4 in maintaining genomic integrity, whether and how RNF4 contributes to the stabilization of stalled replication forks are currently poorly understood. ...
... RNF4 contains four functional SIM regions in its N terminus and a RING domain at its C-terminal region ( Figure 1A). A previous study demonstrated that the catalytically-inactive mutant of RNF4 (RNF4-CS; substitution of cysteines 173 and 176 with serine residues) exhibited much more stable interactions with its substrates than its wild-type counterpart, and may be employed as a means to trap and identify novel RNF4 substrates (38). To isolate physiological substrates of RNF4, we utilized this 'substrate-trapping' approach in our tandem affinity purification-mass spectrometry (TAP-MS) experimentation. ...
... To date, only a few STUBLs have been identified, including Slx5-Slx8 in budding yeast, Rfp1-2/Slx8 in fission yeast, and RNF4 and RNF111/Arkadia in mammalian cells (27)(28)(29)(45)(46)(47). Previous studies by others and ourselves have uncovered important regulatory roles of the mammalian STUbLs RNF4 and RNF111/Arkadia in facilitating the extraction of a series of DNA damage response proteins, including CtIP, MDC1, FANCD2/FANCI, RPA, BRCA1 and XPC, from sites of DNA damage (35)(36)(37)(38)(39)(45)(46)(47). However, whether these STUBLs also have crucial roles in protecting stalled replication forks is not clearly defined. ...
Replication fork reversal occurs via a two-step process that entails reversal initiation and reversal extension. DNA topoisomerase IIalpha (TOP2A) facilitates extensive fork reversal, on one hand through resolving the topological stress generated by the initial reversal, on the other hand via its role in recruiting the SUMO-targeted DNA translocase PICH to stalled forks in a manner that is dependent on its SUMOylation by the SUMO E3 ligase ZATT. However, how TOP2A activities at stalled forks are precisely regulated remains poorly understood. Here we show that, upon replication stress, the SUMO-targeted ubiquitin E3 ligase RNF4 accumulates at stalled forks and targets SUMOylated TOP2A for ubiquitination and degradation. Downregulation of RNF4 resulted in aberrant activation of the ZATT–TOP2A–PICH complex at stalled forks, which in turn led to excessive reversal and elevated frequencies of fork collapse. These results uncover a previously unidentified regulatory mechanism that regulates TOP2A activities at stalled forks and thus the extent of fork reversal.
... In support of this observation, ectopic expression of SENP6 increased the resistance of SU-DHL-5 cells to olaparib treatment in comparison to SU-DHL-5 EV control cells (Figs. 7g and S13e, f). We anticipated that the phenotypes and PARPi sensitivity of SENP6-deficient BCL cells are at least partially caused by unscheduled activation of the RNF4 pathway resulting from unrestricted polySUMOylation 23,25,38,39 . Given the abundant expression of Rnf4 in MYC-driven lymphomas ( Supplementary Fig. 14a), we hypothesized that co-depletion of RNF4 and SENP6 may impair PARPi sensitivity. ...
SUMOylation is a post-translational modification of proteins that regulates these proteins’ localization, turnover or function. Aberrant SUMOylation is frequently found in cancers but its origin remains elusive. Using a genome-wide transposon mutagenesis screen in a MYC-driven B-cell lymphoma model, we here identify the SUMO isopeptidase (or deconjugase) SENP6 as a tumor suppressor that links unrestricted SUMOylation to tumor development and progression. Notably, SENP6 is recurrently deleted in human lymphomas and SENP6 deficiency results in unrestricted SUMOylation. Mechanistically, SENP6 loss triggers release of DNA repair- and genome maintenance-associated protein complexes from chromatin thereby impairing DNA repair in response to DNA damages and ultimately promoting genomic instability. In line with this hypothesis, SENP6 deficiency drives synthetic lethality to Poly-ADP-Ribose-Polymerase (PARP) inhibition. Together, our results link SENP6 loss to defective genome maintenance and reveal the potential therapeutic application of PARP inhibitors in B-cell lymphoma.
... It is recruited to sites of DNA damage via sumoylated MDC1 and is required for exonucleolytic processing of DSBs preceding HR-mediated repair (Galanty et al., 2012;Luo et al., 2012;Yin et al., 2012). RNF4-mediated ubiquitylation facilitates the extraction of proteins from DNA repair sites through recruitment of the Cdc48/p97 segregase (Nie et al., 2012), it regulates FANCI/ FANCD2 turnover at stalled forks (Gibbs-Seymour et al., 2015), and it mediates the release of FAAP20 from sumoylated FANCA during interstrand crosslink repair (Xie et al., 2015). Although the function of RNF4 in DSB repair has been studied by many laboratories, it's role in responding to fork collapse has not been well characterized in mammalian cells. ...
Sumoylation is an important enhancer of responses to DNA replication stress and the SUMO-targeted ubiquitin E3 ligase RNF4 regulates these responses by ubiquitylation of sumoylated DNA damage response factors. The specific targets and functional consequences of RNF4 regulation in response to replication stress, however, have not been fully characterized. Here we demonstrated that RNF4 is required for the restart of DNA replication following prolonged hydroxyurea (HU)-induced replication stress. Contrary to its role in repair of γ-irradiation-induced DNA double-strand breaks (DSBs), our analysis revealed that RNF4 does not significantly impact recognition or repair of replication stress-associated DSBs. Rather, using DNA fiber assays, we found that the firing of new DNA replication origins, which is required for replication restart following prolonged stress, was inhibited in cells depleted of RNF4. We also provided evidence that RNF4 recognizes and ubiquitylates sumoylated Bloom syndrome DNA helicase BLM and thereby promotes its proteosome-mediated turnover at damaged DNA replication forks. Consistent with it being a functionally important RNF4 substrate, co-depletion of BLM rescued defects in the firing of new replication origins observed in cells depleted of RNF4 alone. We concluded that RNF4 acts to remove sumoylated BLM from collapsed DNA replication forks, which is required to facilitate normal resumption of DNA synthesis after prolonged replication fork stalling and collapse.
