Correction: Distinct SUMO Ligases Cooperate with Esc2 and Slx5 to Suppress Duplication-Mediated Genome Rearrangements.
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ABSTRACT:
SUBMITTER: Albuquerque CP
PROVIDER: S-EPMC5006978 | biostudies-literature |
REPOSITORIES: biostudies-literature
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Project description:Suppression of duplication-mediated gross chromosomal rearrangements (GCRs) is essential to maintain genome integrity in eukaryotes. Here we report that SUMO ligase Mms21 has a strong role in suppressing GCRs in Saccharomyces cerevisiae, while Siz1 and Siz2 have weaker and partially redundant roles. Understanding the functions of these enzymes has been hampered by a paucity of knowledge of their substrate specificity in vivo. Using a new quantitative SUMO-proteomics technology, we found that Siz1 and Siz2 redundantly control the abundances of most sumoylated substrates, while Mms21 more specifically regulates sumoylation of RNA polymerase-I and the SMC-family proteins. Interestingly, Esc2, a SUMO-like domain-containing protein, specifically promotes the accumulation of sumoylated Mms21-specific substrates and functions with Mms21 to suppress GCRs. On the other hand, the Slx5-Slx8 complex, a SUMO-targeted ubiquitin ligase, suppresses the accumulation of sumoylated Mms21-specific substrates. Thus, distinct SUMO ligases work in concert with Esc2 and Slx5-Slx8 to control substrate specificity and sumoylation homeostasis to prevent GCRs.
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Project description:RecQ-like helicases are a highly conserved protein family that functions during DNA repair and, when mutated in humans, is associated with cancer and/or premature aging syndromes. The budding yeast RecQ-like helicase Sgs1 has important functions in double-strand break (DSB) repair of exogenously induced breaks, as well as those that arise endogenously, for example during DNA replication. To further investigate Sgs1's regulation, we analyzed the subcellular localization of a fluorescent fusion of Sgs1 upon DNA damage. Consistent with a role in DSB repair, Sgs1 recruitment into nuclear foci in asynchronous cultures increases after ionizing radiation (IR) and after exposure to the alkylating agent methyl methanesulfonate (MMS). Yet, despite the importance of Sgs1 in replicative damage repair and in contrast to its elevated protein levels during S-phase, we find that the number of Sgs1 foci decreases upon nucleotide pool depletion by hydroxyurea (HU) treatment and that this negative regulation depends on the intra S-phase checkpoint kinase Mec1. Importantly, we identify the SUMO-targeted ubiquitin ligase (STUbL) complex Slx5-Slx8 as a negative regulator of Sgs1 foci, both spontaneously and upon replicative damage. Slx5-Slx8 regulation of Sgs1 foci is likely conserved in eukaryotes, since expression of the mammalian Slx5-Slx8 functional homologue, RNF4, restores Sgs1 focus number in slx8 cells and furthermore, knockdown of RNF4 leads to more BLM foci in U-2 OS cells. Our results point to a model where RecQ-like helicase subcellular localization is regulated by STUbLs in response to DNA damage, presumably to prevent illegitimate recombination events.
| S-EPMC4308569 | biostudies-literature
Project description: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.
| S-EPMC7891725 | biostudies-literature
Project description:The small ubiquitin related modifier (SUMO)-mediated posttranslational protein modification is widely conserved among eukaryotes. Similar to ubiquitination, SUMO modifications are attached to the substrate protein through three reaction steps by the E1, E2 and E3 enzymes. To date, multiple families of SUMO E3 ligases have been reported in yeast and animals, but only two types of E3 ligases have been identified in Arabidopsis: SAP and Miz 1 (SIZ1) and Methyl Methanesulfonate-Sensitivity protein 21 (MMS21)/HIGH PLOIDY 2 (HPY2), hereafter referred to as HPY2. Both proteins possess characteristic motifs termed Siz/PIAS RING (SP-RING) domains, and these motifs are conserved throughout the plant kingdom. Previous studies have shown that loss-of-function mutations in HPY2 or SIZ1 cause dwarf phenotypes and that the phenotype of siz1-2 is caused by the accumulation of salicylic acid (SA). However, we demonstrate here that the phenotype of hpy2-1 does not depend on SA accumulation. Consistently, the expression of SIZ1 driven by the HPY2 promoter does not complement the hpy2-1 phenotypes, indicating that they are not functional homologs. Lastly, we show that the siz1-2 and hpy2-1 double mutant results in embryonic lethality, supporting the hypothesis that they have non-overlapping roles during embryogenesis. Together, these results suggest that SIZ1 and HPY2 function independently and that their combined SUMOylation is essential for plant development.
| S-EPMC3466189 | biostudies-literature
Project description:We identify the SUMO-Targeted Ubiquitin Ligase (STUbL) family of proteins and propose that STUbLs selectively ubiquitinate sumoylated proteins and proteins that contain SUMO-like domains (SLDs). STUbL recruitment to sumoylated/SLD proteins is mediated by tandem SUMO interaction motifs (SIMs) within the STUbLs N-terminus. STUbL-mediated ubiquitination maintains sumoylation pathway homeostasis by promoting target protein desumoylation and/or degradation. Thus, STUbLs establish a novel mode of communication between the sumoylation and ubiquitination pathways. STUbLs are evolutionarily conserved and include: Schizosaccharomyces pombe Slx8-Rfp (founding member), Homo sapiens RNF4, Dictyostelium discoideum MIP1 and Saccharomyces cerevisiae Slx5-Slx8. Cells lacking Slx8-Rfp accumulate sumoylated proteins, display genomic instability, and are hypersensitive to genotoxic stress. These phenotypes are suppressed by deletion of the major SUMO ligase Pli1, demonstrating the specificity of STUbLs as regulators of sumoylated proteins. Notably, human RNF4 expression restores SUMO pathway homeostasis in fission yeast lacking Slx8-Rfp, underscoring the evolutionary functional conservation of STUbLs. The DNA repair factor Rad60 and its human homolog NIP45, which contain SLDs, are candidate STUbL targets. Consistently, Rad60 and Slx8-Rfp mutants have similar DNA repair defects.
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Project description:The Slx5-Slx8 complex is a ubiquitin ligase that preferentially ubiquitylates SUMOylated substrates, targeting them for proteolysis. Mutations in SLX5, SLX8, and other SUMO pathway genes were previously identified in our laboratory as genomic suppressors of a point mutation (mot1-301) in the transcriptional regulator MOT1 To further understand the links between the SUMO and ubiquitin pathways, a screen was performed for high-copy suppressors of mot1-301, yielding three genes (MOT3, MIT1, and ULS1). MOT3 and MIT1 have characteristics of prions, and ULS1 is believed to encode another SUMO-targeted ubiquitin ligase (STUbL) that functionally overlaps with Slx5-Slx8. Here we focus on ULS1, obtaining results suggesting that the relationship between ULS1 and SLX5 is more complex than expected. Uls1 interacted with Slx5 physically in to yeast two-hybrid and co-immunoprecipitation assays, a uls1 mutation that blocked the interaction between Uls1 and Slx5 interfered with ULS1 function, and genetic analyses indicated an antagonistic relationship between ULS1 and SLX5 Combined, our results challenge the assumption that Uls1 and Slx5 are simply partially overlapping STUbLs and begin to illuminate a regulatory relationship between these two proteins.
| S-EPMC3618364 | biostudies-literature
Project description: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.
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Project description:Post-translational modifications alter protein function to mediate complex hierarchical regulatory processes that are crucial to eukaryotic cellular function. The small ubiquitin-like modifier (SUMO) is an important post-translational modification that affects transcriptional regulation, nuclear localization, and the maintenance of genome stability. Nucleotide excision repair (NER) is a very versatile DNA repair system that is essential for protection against ultraviolet (UV) irradiation. The deficiencies in NER function remarkably increase the risk of skin cancer. Recent studies have shown that several NER factors are SUMOylated, which influences repair efficiency. However, how SUMOylation modulates NER has not yet been elucidated. In the present study, we performed RNAi knockdown of SUMO E3 ligases and found that, in addition to PIASy, the polycomb protein Pc2 affected the repair of cyclobutane pyrimidine dimers. PIAS1 affected both the removal of 6-4 pyrimidine pyrimidone photoproducts and cyclobutane pyrimidine dimers, whereas other SUMO E3 ligases did not affect the removal of either UV lesion.
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Project description:Post-translational modification by Small Ubiquitin-like Modifiers (SUMO) has been implicated in protein targeting, in the maintenance of genomic integrity and in transcriptional control. But the specific molecular effects of SUMO modification on many target proteins remain to be elucidated. Recent findings point at the importance of SUMO-mediated histone NAD-dependent deacetylase (HDAC) recruitment in transcriptional regulation.We describe the RENi family of SUMO-like domain proteins (SDP) with the unique feature of typically containing two carboxy-terminal SUMO-like domains. Using sequence analytic evidence, we collect family members from animals, fungi and plants, most prominent being yeast Rad60, Esc2 and mouse NIP45 http://mendel.imp.univie.ac.at/SEQUENCES/reni/. Different proteins of the novel family are known to interact directly with histone NAD-dependent deacetylases (HDACs), structural maintenance of chromosomes (SMC) proteins, and transcription factors. In particular, the highly non-trivial designation of the first of the two successive SUMO-domains in non-plant RENi provides a rationale for previously published functionally impaired mutant variants.Till now, SUMO-like proteins have been studied exclusively in the context of their covalent conjugation to target proteins. Here, we present the exciting possibility that SUMO domain proteins, similarly to ubiquitin modifiers, have also evolved in a second line - namely as multi-domain proteins that are non-covalently attached to their target proteins. We suggest that the SUMO stable fusion proteins of the RENi family, which we introduce in this work, might mimic SUMO and share its interaction motifs (in analogy to the way that ubiquitin-like domains mimic ubiquitin). This presumption is supported by parallels in the spectrum of modified or bound proteins e.g. transcription factors and chromatin-associated proteins and in the recruitment of HDAC-activity.
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Project description:Centrosomes consist of two centrioles embedded in pericentriolar material and function as the main microtubule organising centres in dividing animal cells. They ensure proper formation and orientation of the mitotic spindle and are therefore essential for the maintenance of genome stability. Centrosome function is crucial during embryonic development, highlighted by the discovery of mutations in genes encoding centrosome or spindle pole proteins that cause autosomal recessive primary microcephaly, including Cep63 and Cep152. In this study we show that Cep63 functions to ensure that centriole duplication occurs reliably in dividing mammalian cells. We show that the interaction between Cep63 and Cep152 can occur independently of centrosome localisation and that the two proteins are dependent on one another for centrosomal localisation. Further, both mouse and human Cep63 and Cep152 cooperate to ensure efficient centriole duplication by promoting the accumulation of essential centriole duplication factors upstream of SAS-6 recruitment and procentriole formation. These observations describe the requirement for Cep63 in maintaining centriole number in dividing mammalian cells and further establish the order of events in centriole formation.
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