DNA bending facilitates the error-free DNA damage tolerance pathway and upholds genome integrity

Abstract DNA replication is sensitive to damage in the template. To bypass lesions and complete replication, cells activate recombination-mediated (error-free) and translesion synthesis-mediated (error-prone) DNA damage tolerance pathways. Crucial for error-free DNA damage tolerance is template switching, which depends on the formation and resolution of damage-bypass intermediates consisting of sister chromatid junctions. Here we show that a chromatin architectural pathway involving the high mobility group box protein Hmo1 channels replication-associated lesions into the error-free DNA damage tolerance pathway mediated by Rad5 and PCNA polyubiquitylation, while preventing mutagenic bypass and toxic recombination. In the process of template switching, Hmo1 also promotes sister chromatid junction formation predominantly during replication. Its C-terminal tail, implicated in chromatin bending, facilitates the formation of catenations/hemicatenations and mediates the roles of Hmo1 in DNA damage tolerance pathway choice and sister chromatid junction formation. Together, the results suggest that replication-associated topological changes involving the molecular DNA bender, Hmo1, set the stage for dedicated repair reactions that limit errors during replication and impact on genome stability

Cmr1/WDR76 defines a nuclear genotoxic stress body linking genome integrity and protein quality control

DNA replication stress is a source of genomic instability. Here we identify ​changed mutation rate 1 (​Cmr1) as a factor involved in the response to DNA replication stress in Saccharomyces cerevisiae and show that ​Cmr1—together with ​Mrc1/​Claspin, ​Pph3, the chaperonin containing ​TCP1 (CCT) and 25 other proteins—define a novel intranuclear quality control compartment (INQ) that sequesters misfolded, ubiquitylated and sumoylated proteins in response to genotoxic stress. The diversity of proteins that localize to INQ indicates that other biological processes such as cell cycle progression, chromatin and mitotic spindle organization may also be regulated through INQ. Similar to ​Cmr1, its human orthologue ​WDR76 responds to proteasome inhibition and DNA damage by relocalizing to nuclear foci and physically associating with CCT, suggesting an evolutionarily conserved biological function. We propose that ​Cmr1/​WDR76 plays a role in the recovery from genotoxic stress through regulation of the turnover of sumoylated and phosphorylated proteins

Targeting of SUMO substrates to a Cdc48-Ufd1-Npl4 segregase and STUbL pathway in fission yeast

In eukaryotes, the conjugation of proteins to the small ubiquitin-like modifier (SUMO) regulates numerous cellular functions. A proportion of SUMO conjugates are targeted for degradation by SUMO-targeted ubiquitin ligases (STUbLs) and it has been proposed that the ubiquitin-selective chaperone Cdc48/p97-Ufd1-Npl4 facilitates this process. However, the extent to which the two pathways overlap, and how substrates are selected, remains unknown. Here we address these questions in fission yeast through proteome-wide analyses of SUMO modification sites. We identify over a thousand sumoylated lysines in a total of 468 proteins and quantify changes occurring in the SUMO modification status when the STUbL or Ufd1 pathways are compromised by mutations. The data suggest the coordinated processing of several classes of SUMO conjugates, many dynamically associated with centromeres or telomeres. They provide new insights into subnuclear organization and chromosome biology, and, altogether, constitute an extensive resource for the molecular characterization of SUMO function and dynamics

The S phase checkpoint promotes the Smc5/6 complex dependent SUMOylation of Pol2, the catalytic subunit of DNA polymerase ε

Replication fork stalling and accumulation of single-stranded DNA trigger the S phase checkpoint, a signalling cascade that, in budding yeast, leads to the activation of the Rad53 kinase. Rad53 is essential in maintaining cell viability, but its targets of regulation are still partially unknown. Here we show that Rad53 drives the hyper-SUMOylation of Pol2, the catalytic subunit of DNA polymerase ε, principally following replication forks stalling induced by nucleotide depletion. Pol2 is the main target of SUMOylation within the replisome and its modification requires the SUMO-ligase Mms21, a subunit of the Smc5/6 complex. Moreover, the Smc5/6 complex co-purifies with Pol ε, independently of other replisome components. Finally, we map Pol2 SUMOylation to a single site within the N-terminal catalytic domain and identify a SUMO-interacting motif at the C-terminus of Pol2. These data suggest that the S phase checkpoint regulate Pol ε during replication stress through Pol2 SUMOylation and SUMO-binding abilit

Control of Nuclear Activities by Substrate-Selective and Protein-Group SUMOylation

Reversible modification of proteins by SUMO (small ubiquitin-like modifier) affects a large number of cellular processes. In striking contrast to the related ubiquitin pathway, only a few enzymes participate in the SUMO system, although this pathway has numerous substrates as well. Emerging evidence suggests that SUMOylation frequently targets entire groups of physically interacting proteins rather than individual proteins. Protein-group SUMOylation appears to be triggered by recruitment of SUMO ligases to preassembled protein complexes. Because SUMOylation typically affects groups of proteins that bear SUMO-interaction motifs (SIMs), protein-group SUMOylation may foster physical interactions between proteins through multiple SUMO-SIM interactions. Individual SUMO modifications may act redundantly or additively, yet they may mediate dedicated functions as well. In this review, we focus on the unorthodox principles of this pathway and give examples for SUMO-controlled nuclear activities. We propose that collective SUMOylation is typical for nuclear assemblies and argue that SUMO serves as a distinguishing mark for functionally engaged protein fractions

Protein Group Modification and Synergy in the SUMO Pathway as Exemplified in DNA Repair

Protein modification by SUMO affects a wide range of protein substrates. Surprisingly, although SUMO pathway mutants display strong phenotypes, the function of individual SUMO modifications is often enigmatic, and SUMOylation-defective mutants commonly lack notable phenotypes. Here, we use DNA double-strand break repair as an example and show that DNA damage triggers a SUMOylation wave, leading to simultaneous multisite modifications of several repair proteins of the same pathway. Catalyzed by a DNA-bound SUMO ligase and triggered by single-stranded DNA, SUMOylation stabilizes physical interactions between the proteins. Notably, only wholesale elimination of SUMOylation of several repair proteins significantly affects the homologous recombination pathway by considerably slowing down DNA repair. Thus, SUMO acts synergistically on several proteins, and individual modifications only add up to efficient repair. We propose that SUMOylation may thus often target a protein group rather than individual proteins, whereas localized modification enzymes and highly specific triggers ensure specificity

Identification of Substrates of Protein-Group SUMOylation

Protein modification by conjugation to the ubiquitin-related protein SUMO (SUMOylation) regulates numerous cellular functions and is reversible. However, unlike typical posttranslational modifications, SUMOylation often targets and regulates proteins of functionally and physically linked protein groups, rather than individual proteins. Functional studies of protein-group SUMOylation are thus particularly challenging, as they require the identification of ideally all members of a modified protein group. Here, we describe mass spectrometric approaches to detect SUMOylated protein groups in Saccharomyces cerevisiae, yet the protocols can be readily adapted for studies of SUMOylation in mammalian cells

Autophagic Clearance of PolyQ Proteins Mediated by Ubiquitin-Atg8 Adaptors of the Conserved CUET Protein Family

Selective ubiquitin-dependent autophagy plays a pivotal role in the elimination of protein aggregates, assemblies, or organelles and counteracts the cytotoxicity of proteins linked to neurodegenerative diseases. Following substrate ubiquitylation, the cargo is delivered to autophagosomes involving adaptors like human p62 that bind ubiquitin and the autophagosomal ubiquitin-like protein Atg8/LC3; however, whether similar pathways exist in lower eukaryotes remained unclear. Here, we identify by a screen in yeast a new class of ubiquitin-Atg8 adaptors termed CUET proteins, comprising the ubiquitin-binding CUE-domain protein Cue5 from yeast and its human homolog Tollip. Cue5 collaborates with Rsp5 ubiquitin ligase, and the corresponding yeast mutants accumulate aggregation-prone proteins and are vulnerable to polyQ protein expression. Similarly, Tollip depletion causes cytotoxicity toward polyQ proteins, whereas Tollip overexpression clears human cells from Huntington's disease-linked polyQ proteins by autophagy. We thus propose that CUET proteins play a critical and ancient role in autophagic clearance of cytotoxic protein aggregates

Failed mitochondrial import and impaired proteostasis trigger SUMOylation of mitochondrial proteins

Modification by the ubiquitin-like protein SUMO affects hundreds of cellular substrate proteins and regulates a wide variety of physiological processes. While the SUMO system appears to predominantly target nuclear proteins and, to a lesser extent, cytosolic proteins, hardly anything is known about the SUMOylation of proteins targeted to membrane-enclosed organelles. Here, we identify a large set of structurally and functionally unrelated mitochondrial proteins as substrates of the SUMO pathway in yeast. We show that SUMO modification of mitochondrial proteins does not rely on mitochondrial targeting and, in fact, is strongly enhanced upon import failure, consistent with the modification occurring in the cytosol. Moreover, SUMOylated forms of mitochondrial proteins particularly accumulate in HSP70- and proteasome-deficient cells, suggesting that SUMOylation participates in cellular protein quality control. We therefore propose that SUMO serves as a mark for nonfunctional mitochondrial proteins, which only sporadically arise in unstressed cells but strongly accumulate upon defective mitochondrial import and impaired proteostasis. Overall, our findings provide support for a role of SUMO in the cytosolic response to aberrant proteins