Delineation of the SUMO-Modified Proteome Reveals Regulatory Functions Throughout Meiosis

Bhagwat, Nikhil R.; Owens, Shannon N; Ito, Masaru; Boinapalli, Jay V; Poa, Philip; Ditzel, Alexander; Kopparapu, Srujan; Mahalawat, Meghan; Davies, Owen R.; Collins, Sean R.; Johnson, Jeffrey R.; Krogan, Nevan J.; Hunter, Neil

doi:10.1101/828442

Cited by 3 publications

(4 citation statements)

References 141 publications

(134 reference statements)

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“…We proceeded to ask whether the SUMO binding of Nse5 contributes to Nse2-dependent sumoylation. Recently, mass spectrometry-based mapping showed that Smc5 and 6 are sumoylated at their coiled-coil regions (44). In our 3D model, these sites are located close to Nse2 and the Nse5-6 complex (Fig.…”

Section: Nse5 Sumo-interacting Motifs Affect Smc5 and 6 Sumoylationmentioning

confidence: 55%

“…(C) Smc5 and Smc6 sumoylation sites in the 3D model of the Smc5/6-Nse2/5/6 complex. The Smc5 and 6 sumoylation sites, including Smc5 K323 and 327 and Smc6 K343 and 350 as mapped by Bhagwat et al (44) are marked on our 3D model as green balls and indicated by arrows. (D) The sumoylation levels of Smc5 and Smc6, but not Nse4, were reduced in nse5-sim6,9 mutant cells.…”

Section: Discussionmentioning

confidence: 99%

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Integrative analysis reveals unique features of the Smc5/6 complex

Ser

et al. 2021

Preprint

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Structural maintenance of chromosomes (SMC) complexes are critical chromatin modulators. In eukaryotes, the cohesin and condensin SMC complexes organize chromatin, while the Smc5/6 complex directly regulates DNA replication and repair. The molecular basis for Smc5/6’s distinct functions is currently poorly understood. Here, we report an integrative structural study of the budding yeast Smc5/6 complex using electron microscopy, cross-linking mass spectrometry, and computational modeling. We show that while the complex shares a similar overall organization with other SMC complexes, it possesses several unique features. In contrast to the reported folded-arm structures of cohesin and condensin, our data suggest that Smc5 and Smc6 arm regions do not fold back on themselves. Instead, these long filamentous regions interact with subunits uniquely acquired by the Smc5/6 complex, namely the Nse2 SUMO ligase and the Nse5-Nse6 subcomplex. Further, we show that Nse5-Nse6 subcomplex adopts a novel structure with an extensive dimerization interface and multiple domains contacting other subunits of the Smc5/6 complex. We also provide evidence that the Nse5-Nse6 module uses its SUMO-binding motifs to contribute to Nse2-mediated sumoylation. Collectively, our integrative multi-scale study identifies distinct structural features of the Smc5/6 complex and functional cooperation amongst its co-evolved unique subunits.

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Section: Nse5 Sumo-interacting Motifs Affect Smc5 and 6 Sumoylationmentioning

confidence: 55%

Section: Discussionmentioning

confidence: 99%

Integrative analysis reveals unique features of the Smc5/6 complex

Ser

et al. 2021

Preprint

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“…In this regard, it will be of interest to determine the full set of factors that Mms21 acts through to exert its functions. We note that a recent analysis of the yeast meiotic SUMO proteome found SUMO conjugated to many recombination-associated proteins, including several subunits of the Smc5-Smc6-Mms21 complex (Bhagwat et al 2019).…”

Section: Discussionmentioning

confidence: 76%

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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Section: Discussionmentioning

confidence: 77%

C-terminal HA tags compromise function and exacerbate phenotypes ofSaccharomyces cerevisiaeBloom’s helicase homolog Sgs1 SUMOylation-associated mutants

Lichten

Cohen

2020

Preprint

View full text Add to dashboard Cite

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-Mms21mediated SUMOylation plays a minor role in the regulation of Sgs1-Top3-Rmi1 during recombination.

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Delineation of the SUMO-Modified Proteome Reveals Regulatory Functions Throughout Meiosis

Cited by 3 publications

References 141 publications

Integrative analysis reveals unique features of the Smc5/6 complex

Integrative analysis reveals unique features of the Smc5/6 complex

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

C-terminal HA tags compromise function and exacerbate phenotypes ofSaccharomyces cerevisiaeBloom’s helicase homolog Sgs1 SUMOylation-associated mutants

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