Sumoylation of Smc5 Promotes Error-free Bypass at Damaged Replication Forks

Zapatka, Mariel; Pociño-Merino, Irene; Heluani‐Gahete, Hayat; Bermúdez-López, Marcelino; Tarrés, Marc; Ibars, Eva; Solé-Soler, Roger; Gutiérrez-Escribano, Pilar; Apostolova, Sonia; Casas, Cèlia; Aragón, Luís; Wellinger, Ralf Erik; Colomina, Neus; Torres-Rosell, Jordi

doi:10.1016/j.celrep.2019.10.123

Cited by 20 publications

(20 citation statements)

References 81 publications

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“…The Smc5/6 complex is one of four highly conserved structural maintenance of chromosomes (SMC) complexes found in eukaryotes and is best known for its role in DNA repair and overall genome stability (Aragón, 2018). It has been described that Smc5/6 sumoylation occurs as a regulatory consequence of collapsed replication forks and has a functional role in modulating replication associated repair and error free DNA bypass via the Mph1 helicase (Zapatka et al, 2019). The Smc5/6 complex also has been shown to interact with the E3 SUMO ligase Mms21 to promote the sumoylation of the STR helicase complex that acts in the removal of recombination intermediates (Bonner et al, 2016); however we did not find members of the complex Sgs1, Top3, or Rmi1 to undergo increased sumoylation in our MS analysis (Supplemental Table 1).…”

Section: Discussionmentioning

confidence: 99%

“…The Smc5-Smc6 complex is one of four highly conserved structural maintenance of chromosomes complexes found in eukaryotes and is best known for its role in DNA repair and overall genome stability ( Aragón, 2018 ). It has been described that Smc5 and Smc6 sumoylation occurs as a regulatory consequence of stalled replication forks and has a functional role in modulating replication-associated repair and error-free DNA bypass via the Mph1 helicase ( Zapatka et al. , 2019 ).…”

Section: Discussionmentioning

confidence: 99%

“…It has been reported that Smc5 and Smc6 are sumoylated after exposure to levels of methyl methanesulfonate (MMS) that induce DNA damage (Zapatka et al, 2019) . We wanted to verify that Smc5 and Smc6 were sumoylated during a time course of MMS exposure, and if the addition of ethanol with MMS treatment caused no effect or was additive.…”

Section: The E3 Sumo Ligase Mms21 Promotes the Sumoylation Of Smc5/6 During Acute Ethanol Stressmentioning

confidence: 99%

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Acute ethanol stress induces sumoylation of conserved chromatin structural proteins inSaccharomyces cerevisiae

Bradley

Marsh

Borror

et al. 2021

MBoC

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Stress is ubiquitous to life and can irreparably damage essential biomolecules and organelles in cells. To survive, organisms must sense and adapt to stressful conditions. One highly conserved adaptive stress response is through the post-translational modification of proteins by the small ubiquitin-like modifier (SUMO). Here, we examine the effects of acute ethanol stress on protein sumoylation in the budding yeast Saccharomyces cerevisiae . We found that cells exhibit a transient sumoylation response after acute exposure to ≤ 7.5% ethanol. By contrast, the sumoylation response becomes chronic at 10% ethanol exposure. Mass spectrometry analyses identified 18 proteins that are sumoylated after acute ethanol exposure, with 15 known to associate with chromatin. Upon further analysis, we found that the chromatin structural proteins Smc5 and Smc6 undergo ethanol-induced sumoylation that depends on the activity of the E3 SUMO ligase Mms21. Using cell-cycle arrest assays, we observed that Smc5 and Smc6 ethanol-induced sumoylation occurs during G1 and G2/M phases but not S phase. Acute ethanol exposure also resulted in the formation of Rad52 foci at levels comparable to Rad52 foci formation after exposure to the DNA alkylating agent methyl methanesulfonate (MMS). MMS exposure is known to induce the intra-S phase DNA damage checkpoint via Rad53 phosphorylation, but ethanol exposure did not induce Rad53 phosphorylation. Ethanol abrogated the effect of MMS on Rad53 phosphorylation when added simultaneously. From these studies, we propose that acute ethanol exposure induces a change in chromatin leading to sumoylation of specific chromatin-structural proteins.

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Section: Discussionmentioning

confidence: 99%

Section: Discussionmentioning

confidence: 99%

Section: The E3 Sumo Ligase Mms21 Promotes the Sumoylation Of Smc5/6 During Acute Ethanol Stressmentioning

confidence: 99%

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Acute ethanol stress induces sumoylation of conserved chromatin structural proteins inSaccharomyces cerevisiae

Bradley

Marsh

Borror

et al. 2021

MBoC

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“…A recent report ( Zapatka et al 2019 ) has proposed that Mms21 -mediated SUMOylation of Smc5 is involved in error-free bypass of damaged replication forks. It is worth noting that, in this study, mutations in SMC5 thought to interfere with its SUMOylation did not have strong phenotypes, but exhibited synthetic chromosome segregation defects when combined with mms4 ∆ .…”

Section: Discussionmentioning

confidence: 99%

C-Terminal HA Tags Compromise Function and Exacerbate Phenotypes ofSaccharomyces cerevisiaeBloom’s Helicase Homolog Sgs1 SUMOylation-Associated Mutants

Cohen

Lichten

2020

G3 Genes|Genomes|Genetics

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The Sgs1 helicase and Top3-Rmi1 decatenase form a complex that affects homologous recombination outcomes during the mitotic cell cycle and during meiosis. Previous studies have reported that Sgs1-Top3-Rmi1 function is regulated by SUMOylation that is catalyzed by the Smc5-Smc6-Mms21 complex. These studies used strains in which SGS1 was C-terminally tagged with three or six copies of a human influenza hemagglutinin-derived epitope tag (3HA and 6HA). They identified SGS1 mutants that affect its SUMOylation, which we will refer to as SGS1 SUMO-site mutants. In previous work, these mutants showed phenotypes consistent with substantial loss of Sgs1-Top3-Rmi1 function during the mitotic cell cycle. We find that the reported phenotypes are largely due to the presence of the HA epitope tags. Untagged SGS1 SUMO-site mutants show either wild-type or weak hypomorphic phenotypes, depending on the assay. These phenotypes are exacerbated by both 6HA and 3HA epitope tags in two different S. cerevisiae strain backgrounds. Importantly, a C-terminal 6HA tag confers strong hypomorphic or null phenotypes on an otherwise wild-type Sgs1 protein. Taken together, these results suggest that the HA epitope tags used in previous studies seriously compromise Sgs1 function. Furthermore, they raise the possibilities either that sufficient SUMOylation of the Sgs1-Top3-Rmi1 complex might still occur in the SUMO-site mutants isolated, or that Smc5-Smc6-Mms21-mediated SUMOylation plays a minor role in the regulation of Sgs1-Top3-Rmi1 during recombination.

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“…The Smc5/6 complex is one of four highly conserved s tructural m aintenance of c hromosomes (SMC) complexes found in eukaryotes and is best known for its role in DNA repair and overall genome stability (Aragón, 2018). It has been described that Smc5/6 sumoylation occurs as a regulatory consequence of collapsed replication forks and has a functional role in modulating replication associated repair and error free DNA bypass via the Mph1 helicase (Zapatka et al, 2019). The Smc5/6 complex also has been shown to interact with the E3 SUMO ligase Mms21 to promote the sumoylation of the STR helicase complex that acts in the removal of recombination intermediates (Bonner et al, 2016); however we did not find members of the complex Sgs1, Top3, or Rmi1 to undergo increased sumoylation in our MS analysis (Supplemental Table 1).…”

Section: Discussionmentioning

confidence: 99%

Acute ethanol stress induces sumoylation of conserved chromatin structural proteins inSaccharomyces cerevisiae

Bradley

Marsh

Borror

et al. 2020

Preprint

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Stress is a ubiquitous part of life that disrupts cellular function and, if unresolved, can irreparably damage essential biomolecules and organelles. All organisms can experience stress in the form of unfavorable environmental conditions including exposure to extreme temperatures, hypoxia, reactive oxygen species, alcohol, or shifts in osmolarity. To survive, organisms must sense these changes then react and adapt. One highly conserved adaptive response to stress is through protein sumoylation, which is a post-translational modification by the small ubiquitin-like modifier (SUMO) protein. In this study, we examine the effects of acute ethanol stress on protein sumoylation in the budding yeast Saccharomyces cerevisiae. Although ethanol induces protein sumoylation, the targets and roles of sumoylation are largely unknown. Here, we found that cells exhibit a transient sumoylation response after exposure of cells to ≤ 7.5% volume/volume ethanol. The response peaks at 15 minutes and resolves by 60 minutes, indicating that cells have an adaptive response to low concentrations of ethanol. By contrast, the sumoylation response becomes chronic at 10% ethanol stress with no resolution by 60 minutes. To identify key targets of ethanol-induced sumoylation, we performed mass spectrometry analyses and identified 18 proteins with increased sumoylation after acute ethanol exposure, with 15 identified as known chromatin-associated proteins. Two of these proteins are the chromatin structural proteins Smc5 and Smc6, which are sumoylated by the activity of the SUMO ligase Mms21. Ethanol-induced Smc5/6 sumoylation occurs during G1 and G2M phases of the cell cycle but is abrogated during S phase despite the fact that other proteins are sumoylated during this phase. Acute ethanol exposure leads to formation of Rad52 foci indicating DNA damage similar to that observed with the addition of methyl methanesulfonate (MMS), which is an alkylating agent that damages DNA. Whereas MMS exposure induces the intra-S phase DNA damage checkpoint as observed by Rad53 phosphorylation, ethanol exposure does not induce the intra-S phase checkpoint and prevents Rad53 phosphorylation when added with MMS. From these results, we propose that ethanol induces a structural change in chromatin, possibly through DNA damage, and this causes sumoylation of conserved chromatin-associated proteins, including Smc5 and Smc6.

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Sumoylation of Smc5 Promotes Error-free Bypass at Damaged Replication Forks

Cited by 20 publications

References 81 publications

Acute ethanol stress induces sumoylation of conserved chromatin structural proteins inSaccharomyces cerevisiae

Acute ethanol stress induces sumoylation of conserved chromatin structural proteins inSaccharomyces cerevisiae

C-Terminal HA Tags Compromise Function and Exacerbate Phenotypes ofSaccharomyces cerevisiaeBloom’s Helicase Homolog Sgs1 SUMOylation-Associated Mutants

Acute ethanol stress induces sumoylation of conserved chromatin structural proteins inSaccharomyces cerevisiae

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