Sister chromatid cohesion halts DNA loop expansion.

Eukaryotic genomes are folded into DNA loops mediated by structural maintenance of chromosomes (SMC) complexes such as cohesin, condensin, and Smc5/6. This organization regulates different DNA-related processes along the cell cycle, such as transcription, recombination, segregation, and DNA repair. During the G2 stage, SMC-mediated DNA loops coexist with cohesin complexes involved in sister chromatid cohesion (SCC). However, the articulation between the establishment of SCC and the formation of SMC-mediated DNA loops along the chromatin remains unknown. Here, we show that SCC is indeed a barrier to cohesin-mediated DNA loop expansion along G2/M Saccharomyces cerevisiae chromosomes.

Evolutionary conservation of the structure and function of meiotic Rec114-Mei4 and Mer2 complexes.

Meiosis-specific Rec114-Mei4 and Mer2 complexes are thought to enable Spo11-mediated DNA double-strand break (DSB) formation through a mechanism that involves DNA-dependent condensation. However, the structure, molecular properties, and evolutionary conservation of Rec114-Mei4 and Mer2 are unclear. Here, we present AlphaFold models of Rec114-Mei4 and Mer2 complexes supported by nuclear magnetic resonance (NMR) spectroscopy, small-angle X-ray scattering (SAXS), and mutagenesis. We show that dimers composed of the Rec114 C terminus form α-helical chains that cup an N-terminal Mei4 α helix, and that Mer2 forms a parallel homotetrameric coiled coil. Both Rec114-Mei4 and Mer2 bind preferentially to branched DNA substrates, indicative of multivalent protein-DNA interactions. Indeed, the Rec114-Mei4 interaction domain contains two DNA-binding sites that point in opposite directions and drive condensation. The Mer2 coiled-coil domain bridges coaligned DNA duplexes, likely through extensive electrostatic interactions along the length of the coiled coil. Finally, we show that the structures of Rec114-Mei4 and Mer2 are conserved across eukaryotes, while DNA-binding properties vary significantly. This work provides insights into the mechanism whereby Rec114-Mei4 and Mer2 complexes promote the assembly of the meiotic DSB machinery and suggests a model in which Mer2 condensation is the essential driver of assembly, with the DNA-binding activity of Rec114-Mei4 playing a supportive role.

Division of Labor between PCNA Loaders in DNA Replication and Sister Chromatid Cohesion Establishment.

Concomitant with DNA replication, the chromosomal cohesin complex establishes cohesion between newly replicated sister chromatids. Several replication-fork-associated "cohesion establishment factors," including the multifunctional Ctf18-RFC complex, aid this process in as yet unknown ways. Here, we show that Ctf18-RFC's role in sister chromatid cohesion correlates with PCNA loading but is separable from its role in the replication checkpoint. Ctf18-RFC loads PCNA with a slight preference for the leading strand, which is dispensable for DNA replication. Conversely, the canonical Rfc1-RFC complex preferentially loads PCNA onto the lagging strand, which is crucial for DNA replication but dispensable for sister chromatid cohesion. The downstream effector of Ctf18-RFC is cohesin acetylation, which we place toward a late step during replication maturation. Our results suggest that Ctf18-RFC enriches and balances PCNA levels at the replication fork, beyond the needs of DNA replication, to promote establishment of sister chromatid cohesion and possibly other post-replicative processes.

The Co-chaperone Cns1 and the Recruiter Protein Hgh1 Link Hsp90 to Translation Elongation via Chaperoning Elongation Factor 2.

The Hsp90 chaperone machinery in eukaryotes comprises a number of distinct accessory factors. Cns1 is one of the few essential co-chaperones in yeast, but its structure and function remained unknown. Here, we report the X-ray structure of the Cns1 fold and NMR studies on the partly disordered, essential segment of the protein. We demonstrate that Cns1 is important for maintaining translation elongation, specifically chaperoning the elongation factor eEF2. In this context, Cns1 interacts with the novel co-factor Hgh1 and forms a quaternary complex together with eEF2 and Hsp90. The in vivo folding and solubility of eEF2 depend on the presence of these proteins. Chaperoning of eEF2 by Cns1 is essential for yeast viability and requires a defined subset of the Hsp90 machinery as well as the identified eEF2 recruiting factor Hgh1.

Gcn2 structurally mimics and functionally repurposes the HisRS enzyme for the integrated stress response.

Protein kinase Gcn2 attenuates protein synthesis in response to amino acid starvation while stimulating translation of a transcriptional activator of amino acid biosynthesis. Gcn2 activation requires a domain related to histidyl-tRNA synthetase (HisRS), the enzyme that aminoacylates tRNAHis. While evidence suggests that deacylated tRNA binds the HisRS domain for kinase activation, ribosomal P-stalk proteins have been implicated as alternative activating ligands on stalled ribosomes. We report crystal structures of the HisRS domain of Chaetomium thermophilum Gcn2 that reveal structural mimicry of both catalytic (CD) and anticodon-binding (ABD) domains, which in authentic HisRS bind the acceptor stem and anticodon loop of tRNAHis. Elements for forming histidyl adenylate and aminoacylation are lacking, suggesting that Gcn2HisRS was repurposed for kinase activation, consistent with mutations in the CD that dysregulate yeast Gcn2 function. Substituting conserved ABD residues well positioned to contact the anticodon loop or that form a conserved ABD-CD interface impairs Gcn2 function in starved cells. Mimicry in Gcn2HisRS of two highly conserved structural domains for binding both ends of tRNA-each crucial for Gcn2 function-supports that deacylated tRNAs activate Gcn2 and exemplifies how a metabolic enzyme is repurposed to host new local structures and sequences that confer a novel regulatory function.

Beyond RNA binding domains: determinants of protein-RNA binding.

RNA binding proteins (RBPs) are composed of RNA binding domains (RBDs) often linked via intrinsically disordered regions (IDRs). Structural and biochemical analysis have shown that disordered linkers contribute to RNA binding by orienting the adjacent RBDs and also characterized certain disordered repeats that directly contact the RNA. However, the relative contribution of IDRs and predicted RBDs to the in-vivo binding pattern is poorly explored. Here, we upscaled the RNA tagging method to map the transcriptome-wide binding of sixteen RBPs in budding yeast. We then performed extensive sequence mutations to distinguish binding determinants within predicted RBDs and the surrounding IDRs in eight of these. The majority of the predicted RBDs tested were not individually essential for mRNA binding, while multiple IDRs that lacked predicted RNA binding potential appeared essential for binding affinity or specificity. Our results provide new insights into the function of poorly studied RBPs and emphasize the complex and distributed encoding of RBP-RNA interaction in-vivo.

Systematic Gene-to-Phenotype Arrays: A High-Throughput Technique for Molecular Phenotyping.

We have developed a highly parallel strategy, systematic gene-to-phenotype arrays (SGPAs), to comprehensively map the genetic landscape driving molecular phenotypes of interest. By this approach, a complete yeast genetic mutant array is crossed with fluorescent reporters and imaged on membranes at high density and contrast. Importantly, SGPA enables quantification of phenotypes that are not readily detectable in ordinary genetic analysis of cell fitness. We benchmark SGPA by examining two fundamental biological phenotypes: first, we explore glucose repression, in which SGPA identifies a requirement for the Mediator complex and a role for the CDK8/kinase module in regulating transcription. Second, we examine selective protein quality control, in which SGPA identifies most known quality control factors along with U34 tRNA modification, which acts independently of proteasomal degradation to limit misfolded protein production. Integration of SGPA with other fluorescent readouts will enable genetic dissection of a wide range of biological pathways and conditions.

Sterol Oxidation Mediates Stress-Responsive Vms1 Translocation to Mitochondria.

Vms1 translocates to damaged mitochondria in response to stress, whereupon its binding partner, Cdc48, contributes to mitochondrial protein homeostasis. Mitochondrial targeting of Vms1 is mediated by its conserved mitochondrial targeting domain (MTD), which, in unstressed conditions, is inhibited by intramolecular binding to the Vms1 leucine-rich sequence (LRS). Here, we report a 2.7 Å crystal structure of Vms1 that reveals that the LRS lies in a hydrophobic groove in the autoinhibited MTD. We also demonstrate that the oxidized sterol, ergosterol peroxide, is necessary and sufficient for Vms1 localization to mitochondria, through binding the MTD in an interaction that is competitive with binding to the LRS. These data support a model in which stressed mitochondria generate an oxidized sterol receptor that recruits Vms1 to support mitochondrial protein homeostasis.

The Plasticity of the Hsp90 Co-chaperone System.

The Hsp90 system in the eukaryotic cytosol is characterized by a cohort of co-chaperones that bind to Hsp90 and affect its function. Although progress has been made regarding the underlying biochemical mechanisms, how co-chaperones influence Hsp90 client proteins in vivo has remained elusive. By investigating the effect of 12 Hsp90 co-chaperones on the activity of different client proteins in yeast, we find that deletion of co-chaperones can have a neutral or negative effect on client activity but can also lead to more active clients. Only a few co-chaperones are active on all clients studied. Closely related clients and even point mutants can depend on different co-chaperones. These effects are direct because differences in client-co-chaperone interactions can be reconstituted in vitro. Interestingly, some co-chaperones affect client conformation in vivo. Thus, co-chaperones adapt the Hsp90 cycle to the requirements of the client proteins, ensuring optimal activation.

AID and Reactive Oxygen Species Can Induce DNA Breaks within Human Chromosomal Translocation Fragile Zones.

DNA double-strand breaks (DSBs) occurring within fragile zones of less than 200 base pairs account for the formation of the most common human chromosomal translocations in lymphoid malignancies, yet the mechanism of how breaks occur remains unknown. Here, we have transferred human fragile zones into S. cerevisiae in the context of a genetic assay to understand the mechanism leading to DSBs at these sites. Our findings indicate that a combination of factors is required to sensitize these regions. Foremost, DNA strand separation by transcription or increased torsional stress can expose these DNA regions to damage from either the expression of human AID or increased oxidative stress. This damage causes DNA lesions that, if not repaired quickly, are prone to nuclease cleavage, resulting in DSBs. Our results provide mechanistic insight into why human neoplastic translocation fragile DNA sequences are more prone to enzymes or agents that cause longer-lived DNA lesions.

Trade-offs between the instantaneous growth rate and long-term fitness: Consequences for microbial physiology and predictive computational models.

Microbial systems biology has made enormous advances in relating microbial physiology to the underlying biochemistry and molecular biology. By meticulously studying model microorganisms, in particular Escherichia coli and Saccharomyces cerevisiae, increasingly comprehensive computational models predict metabolic fluxes, protein expression, and growth. The modeling rationale is that cells are constrained by a limited pool of resources that they allocate optimally to maximize fitness. As a consequence, the expression of particular proteins is at the expense of others, causing trade-offs between cellular objectives such as instantaneous growth, stress tolerance, and capacity to adapt to new environments. While current computational models are remarkably predictive for E. coli and S. cerevisiae when grown in laboratory environments, this may not hold for other growth conditions and other microorganisms. In this contribution, we therefore discuss the relationship between the instantaneous growth rate, limited resources, and long-term fitness. We discuss uses and limitations of current computational models, in particular for rapidly changing and adverse environments, and propose to classify microbial growth strategies based on Grimes's CSR framework.

Observation of DNA intertwining along authentic budding yeast chromosomes.

DNA replication of circular genomes generates physically interlinked or catenated sister DNAs. These are resolved through transient DNA fracture by type II topoisomerases to permit chromosome segregation during cell division. Topoisomerase II is similarly required for linear chromosome segregation, suggesting that linear chromosomes also remain intertwined following DNA replication. Indeed, chromosome resolution defects are a frequent cause of chromosome segregation failure and consequent aneuploidies. When and where intertwines arise and persist along linear chromosomes are not known, owing to the difficulty of demonstrating intertwining of linear DNAs. Here, we used excision of chromosomal regions as circular "loop outs" to convert sister chromatid intertwines into catenated circles. This revealed intertwining at replication termination and cohesin-binding sites, where intertwines are thought to arise and persist but not to a greater extent than elsewhere in the genome. Intertwining appears to spread evenly along chromosomes but is excluded from heterochromatin. We found that intertwines arise before replication termination, suggesting that replication forks rotate during replication elongation to dissipate torsion ahead of the forks. Our approach provides previously inaccessible insight into the topology of eukaryotic chromosomes and illuminates a process critical for successful chromosome segregation.

Mcm10 regulates DNA replication elongation by stimulating the CMG replicative helicase.

Activation of the Mcm2-7 replicative DNA helicase is the committed step in eukaryotic DNA replication initiation. Although Mcm2-7 activation requires binding of the helicase-activating proteins Cdc45 and GINS (forming the CMG complex), an additional protein, Mcm10, drives initial origin DNA unwinding by an unknown mechanism. We show that Mcm10 binds a conserved motif located between the oligonucleotide/oligosaccharide fold (OB-fold) and A subdomain of Mcm2. Although buried in the interface between these domains in Mcm2-7 structures, mutations predicted to separate the domains and expose this motif restore growth to conditional-lethal MCM10 mutant cells. We found that, in addition to stimulating initial DNA unwinding, Mcm10 stabilizes Cdc45 and GINS association with Mcm2-7 and stimulates replication elongation in vivo and in vitro. Furthermore, we identified a lethal allele of MCM10 that stimulates initial DNA unwinding but is defective in replication elongation and CMG binding. Our findings expand the roles of Mcm10 during DNA replication and suggest a new model for Mcm10 function as an activator of the CMG complex throughout DNA replication.

Dissecting structure and function of the monovalent cation/H+ antiporters Mdm38 and Ylh47 in Saccharomyces cerevisiae.

Saccharomyces cerevisiae Mdm38 and Ylh47 are homologs of the Ca2+/H+ antiporter Letm1, a candidate gene for seizures associated with Wolf-Hirschhorn syndrome in humans. Mdm38 is important for K+/H+ exchange across the inner mitochondrial membrane and contributes to membrane potential formation and mitochondrial protein translation. Ylh47 also localizes to the inner mitochondrial membrane. However, knowledge of the structures and detailed transport activities of Mdm38 and Ylh47 is limited. In this study, we conducted characterization of the ion transport activities and related structural properties of Mdm38 and Ylh47. Growth tests using Na+/H+ antiporter-deficient Escherichia coli strain TO114 showed that Mdm38 and Ylh47 had Na+ efflux activity. Measurement of transport activity across E. coli-inverted membranes showed that Mdm38 and Ylh47 had K+/H+, Na+/H+, and Li+/H+ antiport activity, but unlike Letm1, they lacked Ca2+/H+ antiport activity. Deletion of the ribosome-binding domain resulted in decreased Na+ efflux activity in Mdm38. Structural models of Mdm38 and Ylh47 identified a highly conserved glutamic acid in the pore-forming membrane-spanning region. Replacement of this glutamic acid with alanine, a non-polar amino acid, significantly impaired the ability of Mdm38 and Ylh47 to complement the salt sensitivity of E. coli TO114. These findings not only provide important insights into the structure and function of the Letm1-Mdm38-Ylh47 antiporter family but by revealing their distinctive properties also shed light on the physiological roles of these transporters in yeast and animals. IMPORTANCE: The inner membrane of mitochondria contains numerous ion transporters, including those facilitating H+ transport by the electron transport chain and ATP synthase to maintain membrane potential. Letm1 in the inner membrane of mitochondria in animals functions as a Ca2+/H+ antiporter. However, this study reveals that homologous antiporters in mitochondria of yeast, Mdm38 and Ylh47, do not transport Ca2+ but instead are selective for K+ and Na+. Additionally, the identification of conserved amino acids crucial for antiporter activity further expanded our understanding of the structure and function of the Letm1-Mdm38-Ylh47 antiporter family.

Saccharomyces cerevisiae Δ9-desaturase Ole1 forms a supercomplex with Slc1 and Dga1.

Biosynthesis of the various lipid species that compose cellular membranes and lipid droplets depends on the activity of multiple enzymes functioning in coordinated pathways. The flux of intermediates through lipid biosynthetic pathways is regulated to respond to nutritional and environmental demands placed on the cell necessitating that there be flexibility in pathway activity and organization. This flexibility can in part be achieved through the organization of enzymes into metabolon supercomplexes. However, the composition and organization of such supercomplexes remain unclear. Here, we identified protein-protein interactions between acyltransferases Sct1, Gpt2, Slc1, Dga1, and the Δ9 acyl-CoA desaturase Ole1 in Saccharomyces cerevisiae. We further determined that a subset of these acyltransferases interact with each other independent of Ole1. We show that truncated versions of Dga1 lacking the carboxyl-terminal 20 amino acid residues are nonfunctional and unable to bind Ole1. Furthermore, charged-to-alanine scanning mutagenesis revealed that a cluster of charged residues near the carboxyl terminus was required for the interaction with Ole1. Mutation of these charged residues disrupted the interaction between Dga1 and Ole1 but allowed Dga1 to retain catalytic activity and to induce lipid droplet formation. These data support the formation of a complex of acyltransferases involved in lipid biosynthesis that interacts with Ole1, the sole acyl-CoA desaturase in S. cerevisiae, that can channel unsaturated acyl chains toward phospholipid or triacylglycerol synthesis. This desaturasome complex may provide the architecture that allows for the necessary flux of de novo-synthesized unsaturated acyl-CoA to phospholipid or triacylglycerol synthesis as demanded by cellular requirements.

Expanded in vivo substrate profile of the yeast N-terminal acetyltransferase NatC.

N-terminal acetylation is a conserved protein modification among eukaryotes. The yeast Saccharomyces cerevisiae is a valuable model system for studying this modification. The bulk of protein N-terminal acetylation in S. cerevisiae is catalyzed by the N-terminal acetyltransferases NatA, NatB, and NatC. Thus far, proteome-wide identification of the in vivo protein substrates of yeast NatA and NatB has been performed by N-terminomics. Here, we used S. cerevisiae deleted for the NatC catalytic subunit Naa30 and identified 57 yeast NatC substrates by N-terminal combined fractional diagonal chromatography analysis. Interestingly, in addition to the canonical N-termini starting with ML, MI, MF, and MW, yeast NatC substrates also included MY, MK, MM, MA, MV, and MS. However, for some of these substrate types, such as MY, MK, MV, and MS, we also uncovered (residual) non-NatC NAT activity, most likely due to the previously established redundancy between yeast NatC and NatE/Naa50. Thus, we have revealed a complex interplay between different NATs in targeting methionine-starting N-termini in yeast. Furthermore, our results showed that ectopic expression of human NAA30 rescued known NatC phenotypes in naa30Δ yeast, as well as partially restored the yeast NatC Nt-acetylome. Thus, we demonstrate an evolutionary conservation of NatC from yeast to human thereby underpinning future disease models to study pathogenic NAA30 variants. Overall, this work offers increased biochemical and functional insights into NatC-mediated N-terminal acetylation and provides a basis for future work to pinpoint the specific molecular mechanisms that link the lack of NatC-mediated N-terminal acetylation to phenotypes of NatC deletion yeast.

Replisome-cohesin interactions provided by the Tof1-Csm3 and Mrc1 cohesion establishment factors.

The chromosomal cohesin complex establishes sister chromatid cohesion during S phase, which forms the basis for faithful segregation of DNA replication products during cell divisions. Cohesion establishment is defective in the absence of either of three non-essential Saccharomyces cerevisiae replication fork components Tof1-Csm3 and Mrc1. Here, we investigate how these conserved factors contribute to cohesion establishment. Tof1-Csm3 and Mrc1 serve known roles during DNA replication, including replication checkpoint signaling, securing replication fork speed, as well as recruiting topoisomerase I and the histone chaperone FACT. By modulating each of these functions independently, we rule out that one of these known replication roles explains the contribution of Tof1-Csm3 and Mrc1 to cohesion establishment. Instead, using purified components, we reveal direct and multipronged protein interactions of Tof1-Csm3 and Mrc1 with the cohesin complex. Our findings open the possibility that a series of physical interactions between replication fork components and cohesin facilitate successful establishment of sister chromatid cohesion during DNA replication.

Growth-dependent Gene Expression Variation Influences the Strength of Codon Usage Biases.

The most highly expressed genes in microbial genomes tend to use a limited set of synonymous codons, often referred to as "preferred codons." The existence of preferred codons is commonly attributed to selection pressures on various aspects of protein translation including accuracy and/or speed. However, gene expression is condition-dependent and even within single-celled organisms transcript and protein abundances can vary depending on a variety of environmental and other factors. Here, we show that growth rate-dependent expression variation is an important constraint that significantly influences the evolution of gene sequences. Using large-scale transcriptomic and proteomic data sets in Escherichia coli and Saccharomyces cerevisiae, we confirm that codon usage biases are strongly associated with gene expression but highlight that this relationship is most pronounced when gene expression measurements are taken during rapid growth conditions. Specifically, genes whose relative expression increases during periods of rapid growth have stronger codon usage biases than comparably expressed genes whose expression decreases during rapid growth conditions. These findings highlight that gene expression measured in any particular condition tells only part of the story regarding the forces shaping the evolution of microbial gene sequences. More generally, our results imply that microbial physiology during rapid growth is critical for explaining long-term translational constraints.

The Hob proteins are novel and conserved lipid-binding proteins at ER-PM contact sites.

Membrane contact sites are critical junctures for organelle signaling and communication. Endoplasmic reticulum-plasma membrane (ER-PM) contact sites were the first membrane contact sites to be described; however, the protein composition and molecular function of these sites is still emerging. Here, we leverage yeast and Drosophila model systems to uncover a novel role for the Hobbit (Hob) proteins at ER-PM contact sites. We find that Hobbit localizes to ER-PM contact sites in both yeast cells and the Drosophila larval salivary glands, and this localization is mediated by an N-terminal ER membrane anchor and conserved C-terminal sequences. The C-terminus of Hobbit binds to plasma membrane phosphatidylinositols, and the distribution of these lipids is altered in hobbit mutant cells. Notably, the Hobbit protein is essential for viability in Drosophila, providing one of the first examples of a membrane contact site-localized lipid binding protein that is required for development.

Ultrastructure expansion microscopy reveals the cellular architecture of budding and fission yeast.

The budding and fission yeasts Saccharomyces cerevisiae and Schizosaccharomyces pombe have served as invaluable model organisms to study conserved fundamental cellular processes. Although super-resolution microscopy has in recent years paved the way to a better understanding of the spatial organization of molecules in cells, its wide use in yeasts has remained limited due to the specific know-how and instrumentation required, contrasted with the relative ease of endogenous tagging and live-cell fluorescence microscopy. To facilitate super-resolution microscopy in yeasts, we have extended the ultrastructure expansion microscopy (U-ExM) method to both S. cerevisiae and S. pombe, enabling a 4-fold isotropic expansion. We demonstrate that U-ExM allows imaging of the microtubule cytoskeleton and its associated spindle pole body, notably unveiling the Sfi1p-Cdc31p spatial organization on the appendage bridge structure. In S. pombe, we validate the method by monitoring the homeostatic regulation of nuclear pore complex number through the cell cycle. Combined with NHS-ester pan-labelling, which provides a global cellular context, U-ExM reveals the subcellular organization of these two yeast models and provides a powerful new method to augment the already extensive yeast toolbox. This article has an associated First Person interview with Kerstin Hinterndorfer and Felix Mikus, two of the joint first authors of the paper.