Topoisomerase I-Dependent Viability Loss in <i>Saccharomyces cerevisiae</i> Mutants Defective in Both SUMO Conjugation and DNA Repair

Chen, Xiaole L.; Silver, Hannah R.; Xiong, Lei; Belichenko, Irina; Adegite, Caroline; Johnson, Erica S.

doi:10.1534/genetics.107.074708

Cited by 39 publications

(53 citation statements)

References 72 publications

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“…This link between WSS1 and genome maintenance may be related to the fact that wss1⌬ mutants display severe synthetic-fitness defects in the absence of the Sgs1 DNA helicase (37,49). The notion that SUMO plays a role in DNA damage repair is not new, given that the loss of a number of general SUMO regulators results in sensitivity to DNA-damaging agents (4,8,9,26,31). However, unlike ulp2⌬ mutants, for example, wss1⌬ mutants do not display obvious growth defects or a large number of synthetic-fitness interactions.…”

Section: Discussionmentioning

confidence: 99%

Wss1 Is a SUMO-Dependent Isopeptidase That Interacts Genetically with the Slx5-Slx8 SUMO-Targeted Ubiquitin Ligase

Mullen

Chen

Brill

2010

Molecular and Cellular Biology

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Protein sumoylation plays an important but poorly understood role in controlling genome integrity. In Saccharomyces cerevisiae, the Slx5-Slx8 SUMO-targeted Ub ligase appears to be needed to ubiquitinate sumoylated proteins that arise in the absence of the Sgs1 DNA helicase. WSS1, a high-copy-number suppressor of a mutant SUMO, was implicated in this pathway because it shares phenotypes with SLX5-SLX8 mutants, including a wss1⌬ sgs1⌬ synthetic-fitness defect. Here we show that Wss1, a putative metalloprotease, physically binds SUMO and displays in vitro isopeptidase activity on poly-SUMO chains. Like that of SLX5, overexpression of WSS1 suppresses sgs1⌬ slx5⌬ lethality and the ulp1ts growth defect. Interestingly, although Wss1 is relatively inactive on ubiquitinated substrates and poly-Ub chains, it efficiently deubiquitinates a Ub-SUMO isopeptide conjugate and a Ub-SUMO fusion protein. Wss1 was further implicated in Ub metabolism on the basis of its physical association with proteasomal subunits. The results suggest that Wss1 is a SUMO-dependent isopeptidase that acts on sumoylated substrates as they undergo proteasomal degradation.Protein modification by SUMO is implicated in a wide variety of cellular processes (19,20). Well known for its ability to control subcellular localization (29, 57), sumoylation has been shown to compete with ubiquitination (15), to mediate proteinprotein interactions (38, 39), and to affect protein turnover through SUMO-targeted ubiquitin (Ub) ligases (23,40,47,48,51,53,54). Indeed, modification by SUMO is so common that the number of proteins targeted for sumoylation in Saccharomyces cerevisiae has been estimated to be over 500 (28). Genetic studies of budding yeast have revealed a role for sumoylation in recombination-mediated DNA repair, although the role of SUMO in this process is not completely understood (4,5,16,56).Similar to ubiquitination, the conjugation of SUMO (Smt3 in budding yeast) to substrate proteins requires an ATP-dependent E1-activating enzyme (Aos1/Uba2), an E2-conjugating enzyme (Ubc9), and one of several E3 ligases (e.g., Siz1 or Siz2). The product of this reaction is an isopeptide linkage between the C-terminal glycine residue of mature SUMO and the ε-amino group of lysine residues in target proteins. In vivo, this bond is unstable, as it is subject to hydrolysis by a family of SUMO-specific cysteine proteases known as Ulps in yeast (Ulp1 and Ulp2) and SENPs (six members) in mammals (24,26,33).SUMO-specific proteases regulate SUMO metabolism at multiple steps. In yeast, Ulp1 carries out the essential role of processing the C terminus of the Smt3 precursor [Smt3(Y101)] into its mature form [Smt3(G98)] by removing its 3 terminal amino acids (aa) (24). Both Ulp1 and Ulp2 are responsible for deconjugating SUMO from target proteins, while Ulp2 and its human homologs, SENP6/7, suppress the accumulation of poly-SUMO chains (6, 27, 33). Ulp1 and Ulp2 are primarily located in the nucleus, although Ulp1, which concentrates at nuclear pores, has cytoplasmic targets (22,25...

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Section: Discussionmentioning

confidence: 99%

Wss1 Is a SUMO-Dependent Isopeptidase That Interacts Genetically with the Slx5-Slx8 SUMO-Targeted Ubiquitin Ligase

Mullen

Chen

Brill

2010

Molecular and Cellular Biology

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“…These results suggest that the increased levels of Top1 upon copper induction are a consequence of protein stability rather than increased gene expression. Since Top1 is known to be sumoylated under certain conditions (Mao et al 2000;Chen et al 2007) and SUMO modification can stabilize proteins (de Cristofaro et al 2009), we explored whether SUMO post-translational modification plays any role in Top1 stability in ESCRT mutants.…”

Section: The Escrt Complexmentioning

confidence: 99%

“…To explore the effect of Top1 SUMOylation at natural acceptor sites on the protein, we expressed an active-site tyrosine substitution allele (top1-Y 727 F-top1-YF), since SUMOylated levels of this mutant protein are elevated (Chen et al 2007). Chen and colleagues also found that much of the SUMOylation occurs on three lysine residues (K65, K91, K92).…”

Section: The Escrt Complexmentioning

confidence: 99%

Selective ploidy ablation, a high-throughput plasmid transfer protocol, identifies new genes affecting topoisomerase I–induced DNA damage

et al. 2010

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We have streamlined the process of transferring plasmids into any yeast strain library by developing a novel mating-based, high-throughput method called selective ploidy ablation (SPA). SPA uses a universal plasmid donor strain that contains conditional centromeres on every chromosome. The plasmid-bearing donor is mated to a recipient, followed by removal of all donor-strain chromosomes, producing a haploid strain containing the transferred plasmid. As proof of principle, we used SPA to transfer plasmids containing wild-type and mutant alleles of DNA topoisomerase I (TOP1) into the haploid yeast genedisruption library. Overexpression of Top1 identified only one sensitive mutation, rpa34, while overexpression of top1-T 722 A allele, a camptothecin mimetic, identified 190 sensitive gene-disruption strains along with rpa34. In addition to known camptothecin-sensitive strains, this set contained mutations in genes involved in the Rpd3 histone deacetylase complex, the kinetochore, and vesicle trafficking. We further show that mutations in several ESCRT vesicle trafficking components increase Top1 levels, which is dependent on SUMO modification. These findings demonstrate the utility of the SPA technique to introduce plasmids into the haploid gene-disruption library to discover new interacting pathways.

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“…We recently reported that a siz1⌬ siz2⌬ rad52⌬ strain is nearly inviable, but that its growth defect is suppressed by deleting the gene encoding topoisomerase I (TOP1; Chen et al, 2007). This phenotype is independent of 2 m and is observed in strains both containing (cir ϩ ) and lacking (cir°) 2 m. However, this set of mutations also showed a separate effect on 2 m accumulation.…”

Section: Hrr Is Required For 2 M Hyperamplification In Siz1⌬ Siz2⌬mentioning

confidence: 99%

“…Ulp1 is also required for maturation of the SUMO precursor and so participates in both SUMO conjugation and deconjugation. Mutants in certain nuclear pore complex (NPC) and nuclear envelope proteins also have sumoylation defects because they mislocalize Ulp1 (Zhao et al, 2004;Chen et al, 2007;Lewis et al, 2007;Palancade et al, 2007). Recently, a factor that acts downThis article was published online ahead of print in MBC in Press (http://www.molbiolcell.org/cgi/doi/10.1091/mbc.E08 -06 -0659) on December 24, 2008. stream of sumoylation has been identified: a heterodimer consisting of Slx5/Hex3, and Slx8 acts as a ubiquitin ligase that targets sumoylated proteins for ubiquitin-dependent proteolysis (Ii et al, 2007a;Prudden et al, 2007;Sun et al, 2007;Uzunova et al, 2007;Xie et al, 2007).…”

Section: Introductionmentioning

confidence: 99%

Deficient SUMO Attachment to Flp Recombinase Leads to Homologous Recombination-dependent Hyperamplification of the Yeast 2 μm Circle Plasmid

Xiong

Silver

Ahmed

et al. 2009

MBoC

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Many Saccharomyces cerevisiae mutants defective in the SUMO pathway accumulate elevated levels of the native 2 m circle plasmid (2 m). Here we show that accumulation of 2 m in the SUMO pathway mutants siz1⌬ siz2⌬, slx5⌬, and slx8⌬ is associated with formation of an aberrant high-molecular-weight (HMW) form of 2 m. Characterization of this species from siz1⌬ siz2⌬ showed that it contains tandem copies of the 2 m sequence as well as single-stranded DNA. Accumulation of this species requires both the 2 m-encoded Flp recombinase and the cellular homologous recombination repair (HRR) pathway. Importantly, reduced SUMO attachment to Flp is sufficient to induce formation of this species. Our data suggest a model in which Flp that cannot be sumoylated causes DNA damage, whose repair via HRR produces an intermediate that generates tandem copies of the 2 m sequence. This intermediate may be a rolling circle formed via break-induced replication (BIR), because mutants defective in BIR contain reduced levels of the HMW form. This work also illustrates the importance of using cir°strains when studying mutants that affect the yeast SUMO pathway, to avoid confusing direct functions of the SUMO pathway with secondary effects of 2 m amplification. INTRODUCTIONThe 2 m circle is a naturally occurring 6318-base pair plasmid (2 m) present in ϳ40 -60 copies in virtually all strains of Saccharomyces (Broach and Volkert, 1991). 2 m's only known activity is self-propagation, which is accomplished via two distinct plasmid maintenance mechanisms (Jayaram et al., 2004). One of these allows the plasmid to be amplified if its copy number drops and is mediated by the plasmidencoded Flp recombinase and two DNA target sites for Flp (FRT) that are present on opposite sides of the plasmid. Flp catalyzes an intramolecular recombination reaction between the two FRT sites, thereby inverting one side of the 2 m with respect to the other. Futcher (1986) has proposed a model that explains how this activity could allow multiple copies of 2 m to be made from a single origin firing: if recombination occurs during replication between the converging replication forks (RFs), it would generate a double rolling circle where both forks chase each other around the template in the same direction. The 2 m sequence would be rereplicated until a second Flp-dependent recombination event permits convergence of the RFs.Native levels of 2 m have little effect on the growth rate of wild-type (wt) yeast (Broach and Volkert, 1991). However, in several mutants defective in the SUMO (Smt3) pathway, accumulation of high copy numbers of 2 m is associated with slow, cold-sensitive growth, and cell cycle delays caused by activation of the DNA damage checkpoint (Zhao et al., 2004;Chen et al., 2005;Dobson et al., 2005;Burgess et al., 2007;Ii et al., 2007b). These strains are also heterogeneous and form irregularly shaped colonies, presumably because different lineages contain different levels of 2 m, leading to different degrees of growth inhibition. Removal of 2 m from these strai...

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Topoisomerase I-Dependent Viability Loss in Saccharomyces cerevisiae Mutants Defective in Both SUMO Conjugation and DNA Repair

Cited by 39 publications

References 72 publications

Wss1 Is a SUMO-Dependent Isopeptidase That Interacts Genetically with the Slx5-Slx8 SUMO-Targeted Ubiquitin Ligase

Wss1 Is a SUMO-Dependent Isopeptidase That Interacts Genetically with the Slx5-Slx8 SUMO-Targeted Ubiquitin Ligase

Selective ploidy ablation, a high-throughput plasmid transfer protocol, identifies new genes affecting topoisomerase I–induced DNA damage

Deficient SUMO Attachment to Flp Recombinase Leads to Homologous Recombination-dependent Hyperamplification of the Yeast 2 μm Circle Plasmid

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