Species-specific protein-protein interactions govern the humanization of the 20S proteasome in yeast.

Yeast and humans share thousands of genes despite a billion years of evolutionary divergence. While many human genes can functionally replace their yeast counterparts, nearly half of the tested shared genes cannot. For example, most yeast proteasome subunits are "humanizable," except subunits comprising the ß-ring core, including ß2c (HsPSMB7, a constitutive proteasome subunit). We developed a high-throughput pipeline to humanize yeast proteasomes by generating a large library of Hsß2c mutants and screening them for complementation of a yeast ß2 (ScPup1) knockout. Variants capable of replacing ScPup1 included (1) those impacting local protein-protein interactions (PPIs), with most affecting interactions between the ß2c C-terminal tail and the adjacent ß3 subunit, and (2) those affecting ß2c proteolytic activity. Exchanging the full-length tail of human ß2c with that of ScPup1 enabled complementation. Moreover, wild-type human ß2c could replace yeast ß2 if human ß3 was also provided. Unexpectedly, yeast proteasomes bearing a catalytically inactive HsPSMB7-T44A variant that blocked precursor autoprocessing were viable, suggesting an intact propeptide stabilizes late assembly intermediates. In contrast, similar modifications in human ß2i (HsPSMB10), an immunoproteasome subunit and the co-ortholog of yeast ß2, do not enable complementation in yeast, suggesting distinct interactions are involved in human immunoproteasome core assembly. Broadly, our data reveal roles for specific PPIs governing functional replaceability across vast evolutionary distances.

Rapid, scalable, combinatorial genome engineering by marker-less enrichment and recombination of genetically engineered loci in yeast.

A major challenge to rationally building multi-gene processes in yeast arises due to the combinatorics of combining all of the individual edits into the same strain. Here, we present a precise and multi-site genome editing approach that combines all edits without selection markers using CRISPR-Cas9. We demonstrate a highly efficient gene drive that selectively eliminates specific loci by integrating CRISPR-Cas9-mediated double-strand break (DSB) generation and homology-directed recombination with yeast sexual assortment. The method enables marker-less enrichment and recombination of genetically engineered loci (MERGE). We show that MERGE converts single heterologous loci to homozygous loci at ∼100% efficiency, independent of chromosomal location. Furthermore, MERGE is equally efficient at converting and combining multiple loci, thus identifying compatible genotypes. Finally, we establish MERGE proficiency by engineering a fungal carotenoid biosynthesis pathway and most of the human α-proteasome core into yeast. Therefore, MERGE lays the foundation for scalable, combinatorial genome editing in yeast.

Humanized yeast to model human biology, disease and evolution.

For decades, budding yeast, a single-cellular eukaryote, has provided remarkable insights into human biology. Yeast and humans share several thousand genes despite morphological and cellular differences and over a billion years of separate evolution. These genes encode critical cellular processes, the failure of which in humans results in disease. Although recent developments in genome engineering of mammalian cells permit genetic assays in human cell lines, there is still a need to develop biological reagents to study human disease variants in a high-throughput manner. Many protein-coding human genes can successfully substitute for their yeast equivalents and sustain yeast growth, thus opening up doors for developing direct assays of human gene function in a tractable system referred to as 'humanized yeast'. Humanized yeast permits the discovery of new human biology by measuring human protein activity in a simplified organismal context. This Review summarizes recent developments showing how humanized yeast can directly assay human gene function and explore variant effects at scale. Thus, by extending the 'awesome power of yeast genetics' to study human biology, humanizing yeast reinforces the high relevance of evolutionarily distant model organisms to explore human gene evolution, function and disease.

Discovery of new vascular disrupting agents based on evolutionarily conserved drug action, pesticide resistance mutations, and humanized yeast.

Thiabendazole (TBZ) is an FDA-approved benzimidazole widely used for its antifungal and antihelminthic properties. We showed previously that TBZ is also a potent vascular disrupting agent and inhibits angiogenesis at the tissue level by dissociating vascular endothelial cells in newly formed blood vessels. Here, we uncover TBZ's molecular target and mechanism of action. Using human cell culture, molecular modeling, and humanized yeast, we find that TBZ selectively targets only 1 of 9 human ß-tubulin isotypes (TUBB8) to specifically disrupt endothelial cell microtubules. By leveraging epidemiological pesticide resistance data and mining chemical features of commercially used benzimidazoles, we discover that a broader class of benzimidazole compounds, in extensive use for 50 years, also potently disrupt immature blood vessels and inhibit angiogenesis. Thus, besides identifying the molecular mechanism of benzimidazole-mediated vascular disruption, this study presents evidence relevant to the widespread use of these compounds while offering potential new clinical applications.

Systematic Humanization of the Yeast Cytoskeleton Discerns Functionally Replaceable from Divergent Human Genes.

Many gene families have been expanded by gene duplications along the human lineage, relative to ancestral opisthokonts, but the extent to which the duplicated genes function similarly is understudied. Here, we focused on structural cytoskeletal genes involved in critical cellular processes, including chromosome segregation, macromolecular transport, and cell shape maintenance. To determine functional redundancy and divergence of duplicated human genes, we systematically humanized the yeast actin, myosin, tubulin, and septin genes, testing ∼81% of human cytoskeletal genes across seven gene families for their ability to complement a growth defect induced by inactivation or deletion of the corresponding yeast ortholog. In five of seven families-all but α-tubulin and light myosin, we found at least one human gene capable of complementing loss of the yeast gene. Despite rescuing growth defects, we observed differential abilities of human genes to rescue cell morphology, meiosis, and mating defects. By comparing phenotypes of humanized strains with deletion phenotypes of their interaction partners, we identify instances of human genes in the actin and septin families capable of carrying out essential functions, but failing to fully complement the cytoskeletal roles of their yeast orthologs, thus leading to abnormal cell morphologies. Overall, we show that duplicated human cytoskeletal genes appear to have diverged such that only a few human genes within each family are capable of replacing the essential roles of their yeast orthologs. The resulting yeast strains with humanized cytoskeletal components now provide surrogate platforms to characterize human genes in simplified eukaryotic contexts.

Humanization of yeast genes with multiple human orthologs reveals functional divergence between paralogs.

Despite over a billion years of evolutionary divergence, several thousand human genes possess clearly identifiable orthologs in yeast, and many have undergone lineage-specific duplications in one or both lineages. These duplicated genes may have been free to diverge in function since their expansion, and it is unclear how or at what rate ancestral functions are retained or partitioned among co-orthologs between species and within gene families. Thus, in order to investigate how ancestral functions are retained or lost post-duplication, we systematically replaced hundreds of essential yeast genes with their human orthologs from gene families that have undergone lineage-specific duplications, including those with single duplications (1 yeast gene to 2 human genes, 1:2) or higher-order expansions (1:>2) in the human lineage. We observe a variable pattern of replaceability across different ortholog classes, with an obvious trend toward differential replaceability inside gene families, and rarely observe replaceability by all members of a family. We quantify the ability of various properties of the orthologs to predict replaceability, showing that in the case of 1:2 orthologs, replaceability is predicted largely by the divergence and tissue-specific expression of the human co-orthologs, i.e., the human proteins that are less diverged from their yeast counterpart and more ubiquitously expressed across human tissues more often replace their single yeast ortholog. These trends were consistent with in silico simulations demonstrating that when only one ortholog can replace its corresponding yeast equivalent, it tends to be the least diverged of the pair. Replaceability of yeast genes having more than 2 human co-orthologs was marked by retention of orthologous interactions in functional or protein networks as well as by more ancestral subcellular localization. Overall, we performed >400 human gene replaceability assays, revealing 50 new human-yeast complementation pairs, thus opening up avenues to further functionally characterize these human genes in a simplified organismal context.

Erratum: Correction Notice: Single-step Precision Genome Editing in Yeast Using CRISPR-Cas9.

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The Many Nuanced Evolutionary Consequences of Duplicated Genes.

Gene duplication is seen as a major source of structural and functional divergence in genome evolution. Under the conventional models of sub or neofunctionalization, functional changes arise in one of the duplicates after duplication. However, we suggest here that the presence of a duplicated gene can result in functional changes to its interacting partners. We explore this hypothesis by in silico evolution of a heterodimer when one member of the interacting pair is duplicated. We examine how a range of selection pressures and protein structures leads to differential patterns of evolutionary divergence. We find that a surprising number of distinct evolutionary trajectories can be observed even in a simple three member system. Further, we observe that selection to correct dosage imbalance can affect the evolution of the initial function in several unexpected ways. For example, if a duplicate is under selective pressure to avoid binding its original binding partner, this can lead to changes in the binding interface of a nonduplicated interacting partner to exclude the duplicate. Hence, independent of the fate of the duplicate, its presence can impact how the original function operates. Additionally, we introduce a conceptual framework to describe how interacting partners cope with dosage imbalance after duplication. Contextualizing our results within this framework reveals that the evolutionary path taken by a duplicate's interacting partners is highly stochastic in nature. Consequently, the fate of duplicate genes may not only be controlled by their own ability to accumulate mutations but also by how interacting partners cope with them.

Single-step Precision Genome Editing in Yeast Using CRISPR-Cas9.

Genome modification in budding yeast has been extremely successful largely due to its highly efficient homology-directed DNA repair machinery. Several methods for modifying the yeast genome have previously been described, many of them involving at least two-steps: insertion of a selectable marker and substitution of that marker for the intended modification. Here, we describe a CRISPR-Cas9 mediated genome editing protocol for modifying any yeast gene of interest (either essential or nonessential) in a single-step transformation without any selectable marker. In this system, the Cas9 nuclease creates a double-stranded break at the locus of choice, which is typically lethal in yeast cells regardless of the essentiality of the targeted locus due to inefficient non-homologous end-joining repair. This lethality results in efficient repair via homologous recombination using a repair template derived from PCR. In cases involving essential genes, the necessity of editing the genomic lesion with a functional allele serves as an additional layer of selection. As a motivating example, we describe the use of this strategy in the replacement of HEM2, an essential yeast gene, with its corresponding human ortholog ALAD.

Systematic bacterialization of yeast genes identifies a near-universally swappable pathway.

Eukaryotes and prokaryotes last shared a common ancestor ~2 billion years ago, and while many present-day genes in these lineages predate this divergence, the extent to which these genes still perform their ancestral functions is largely unknown. To test principles governing retention of ancient function, we asked if prokaryotic genes could replace their essential eukaryotic orthologs. We systematically replaced essential genes in yeast by their 1:1 orthologs from Escherichia coli. After accounting for mitochondrial localization and alternative start codons, 31 out of 51 bacterial genes tested (61%) could complement a lethal growth defect and replace their yeast orthologs with minimal effects on growth rate. Replaceability was determined on a pathway-by-pathway basis; codon usage, abundance, and sequence similarity contributed predictive power. The heme biosynthesis pathway was particularly amenable to inter-kingdom exchange, with each yeast enzyme replaceable by its bacterial, human, or plant ortholog, suggesting it as a near-universally swappable pathway.

Efforts to make and apply humanized yeast.

Despite a billion years of divergent evolution, the baker's yeast Saccharomyces cerevisiae has long proven to be an invaluable model organism for studying human biology. Given its tractability and ease of genetic manipulation, along with extensive genetic conservation with humans, it is perhaps no surprise that researchers have been able to expand its utility by expressing human proteins in yeast, or by humanizing specific yeast amino acids, proteins or even entire pathways. These methods are increasingly being scaled in throughput, further enabling the detailed investigation of human biology and disease-specific variations of human genes in a simplified model organism.

An Overview of Tyrosine Site-specific Recombination: From an Flp Perspective.

Tyrosine site-specific recombinases (YRs) are widely distributed among prokaryotes and their viruses, and were thought to be confined to the budding yeast lineage among eukaryotes. However, YR-harboring retrotransposons (the DIRS and PAT families) and DNA transposons (Cryptons) have been identified in a variety of eukaryotes. The YRs utilize a common chemical mechanism, analogous to that of type IB topoisomerases, to bring about a plethora of genetic rearrangements with important physiological consequences in their respective biological contexts. A subset of the tyrosine recombinases has provided model systems for analyzing the chemical mechanisms and conformational features of the recombination reaction using chemical, biochemical, topological, structural, and single molecule-biophysical approaches. YRs with simple reaction requirements have been utilized to bring about programmed DNA rearrangements for addressing fundamental questions in developmental biology. They have also been employed to trace the topological features of DNA within high-order DNA interactions established by protein machines. The directed evolution of altered specificity YRs, combined with their spatially and temporally regulated expression, heralds their emergence as vital tools in genome engineering projects with wide-ranging biotechnological and medical applications.

Hexapeptides that inhibit processing of branched DNA structures induce a dynamic ensemble of Holliday junction conformations.

Holliday junctions are critical intermediates in DNA recombination, repair, and restart of blocked replication. Hexapeptides have been identified that bind to junctions and inhibit various junction-processing enzymes, and these peptides confer anti-microbial and anti-tumor properties. Earlier studies suggested that inhibition results from stabilization of peptide-bound Holliday junctions in the square planar conformation. Here, we use single molecule fluorescence resonance energy transfer (smFRET) and two model junctions, which are AT- or GC-rich at the branch points, to show that binding of the peptide KWWCRW induces a dynamic ensemble of junction conformations that differs from both the square planar and stacked X conformations. The specific features of the conformational distributions differ for the two peptide-bound junctions, but both junctions display greatly decreased Mg(2+) dependence and increased conformational fluctuations. The smFRET results, complemented by gel mobility shift and small angle x-ray scattering analyses, reveal structural effects of peptides and highlight the sensitivity of smFRET for analyzing complex mixtures of DNA structures. The peptide-induced conformational dynamics suggest multiple stacking arrangements of aromatic amino acids with the nucleobases at the junction core. This conformational heterogeneity may inhibit DNA processing by increasing the population of inactive junction conformations, thereby preventing the binding of processing enzymes and/or resulting in their premature dissociation.

Stereospecific suppression of active site mutants by methylphosphonate substituted substrates reveals the stereochemical course of site-specific DNA recombination.

Tyrosine site-specific recombinases, which promote one class of biologically important phosphoryl transfer reactions in DNA, exemplify active site mechanisms for stabilizing the phosphate transition state. A highly conserved arginine duo (Arg-I; Arg-II) of the recombinase active site plays a crucial role in this function. Cre and Flp recombinase mutants lacking either arginine can be rescued by compensatory charge neutralization of the scissile phosphate via methylphosphonate (MeP) modification. The chemical chirality of MeP, in conjunction with mutant recombinases, reveals the stereochemical contributions of Arg-I and Arg-II. The SP preference of the native reaction is specified primarily by Arg-I. MeP reaction supported by Arg-II is nearly bias-free or RP-biased, depending on the Arg-I substituent. Positional conservation of the arginines does not translate into strict functional conservation. Charge reversal by glutamic acid substitution at Arg-I or Arg-II has opposite effects on Cre and Flp in MeP reactions. In Flp, the base immediately 5' to the scissile MeP strongly influences the choice between the catalytic tyrosine and water as the nucleophile for strand scission, thus between productive recombination and futile hydrolysis. The recombinase active site embodies the evolutionary optimization of interactions that not only favor the normal reaction but also proscribe antithetical side reactions.

Evolution. Systematic humanization of yeast genes reveals conserved functions and genetic modularity.

To determine whether genes retain ancestral functions over a billion years of evolution and to identify principles of deep evolutionary divergence, we replaced 414 essential yeast genes with their human orthologs, assaying for complementation of lethal growth defects upon loss of the yeast genes. Nearly half (47%) of the yeast genes could be successfully humanized. Sequence similarity and expression only partly predicted replaceability. Instead, replaceability depended strongly on gene modules: Genes in the same process tended to be similarly replaceable (e.g., sterol biosynthesis) or not (e.g., DNA replication initiation). Simulations confirmed that selection for specific function can maintain replaceability despite extensive sequence divergence. Critical ancestral functions of many essential genes are thus retained in a pathway-specific manner, resilient to drift in sequences, splicing, and protein interfaces.

The partitioning and copy number control systems of the selfish yeast plasmid: an optimized molecular design for stable persistence in host cells.

The multi-copy 2 micron plasmid of Saccharomyces cerevisiae, a resident of the nucleus, is remarkable for its high chromosome-like stability. The plasmid does not appear to contribute to the fitness of the host, nor does it impose a significant metabolic burden on the host at its steady state copy number. The plasmid may be viewed as a highly optimized selfish DNA element whose genome design is devoted entirely towards efficient replication, equal segregation and copy number maintenance. A partitioning system comprised of two plasmid coded proteins, Rep1 and Rep2, and a partitioning locus STB is responsible for equal or nearly equal segregation of plasmid molecules to mother and daughter cells. Current evidence supports a model in which the Rep-STB system promotes the physical association of the plasmid with chromosomes and thus plasmid segregation by a hitchhiking mechanism. The Flp site-specific recombination system housed by the plasmid plays a critical role in maintaining steady state plasmid copy number. A decrease in plasmid population due to rare missegregation events is rectified by plasmid amplification via a recombination induced rolling circle replication mechanism. Appropriate plasmid amplification, without runaway increase in copy number, is ensured by positive and negative regulation of FLP gene expression by plasmid coded proteins and by the control of Flp level/activity through host mediated post-translational modification(s) of Flp. The Flp system has been successfully utilized to understand mechanisms of site-specific recombination, to bring about directed genetic alterations for addressing fundamental problems in biology, and as a tool in biotechnological applications.

Yeast cells expressing the human mitochondrial DNA polymerase reveal correlations between polymerase fidelity and human disease progression.

Mutations in the human mitochondrial polymerase (polymerase-γ (Pol-γ)) are associated with various mitochondrial disorders, including mitochondrial DNA (mtDNA) depletion syndrome, Alpers syndrome, and progressive external opthamalplegia. To correlate biochemically quantifiable defects resulting from point mutations in Pol-γ with their physiological consequences, we created "humanized" yeast, replacing the yeast mtDNA polymerase (MIP1) with human Pol-γ. Despite differences in the replication and repair mechanism, we show that the human polymerase efficiently complements the yeast mip1 knockouts, suggesting common fundamental mechanisms of replication and conserved interactions between the human polymerase and other components of the replisome. We also examined the effects of four disease-related point mutations (S305R, H932Y, Y951N, and Y955C) and an exonuclease-deficient mutant (D198A/E200A). In haploid cells, each mutant results in rapid mtDNA depletion, increased mutation frequency, and mitochondrial dysfunction. Mutation frequencies measured in vivo equal those measured with purified enzyme in vitro. In heterozygous diploid cells, wild-type Pol-γ suppresses mutation-associated growth defects, but continuous growth eventually leads to aerobic respiration defects, reduced mtDNA content, and depolarized mitochondrial membranes. The severity of the Pol-γ mutant phenotype in heterozygous diploid humanized yeast correlates with the approximate age of disease onset and the severity of symptoms observed in humans.

Functional analysis of the genes encoding diaminopropionate ammonia lyase in Escherichia coli and Salmonella enterica serovar Typhimurium.

Diaminopropionate ammonia lyase (DAPAL) is a pyridoxal-5'phosphate (PLP)-dependent enzyme that catalyzes the conversion of diaminopropionate (DAP) to pyruvate and ammonia and plays an important role in cell metabolism. We have investigated the role of the ygeX gene of Escherichia coli K-12 and its ortholog, STM1002, in Salmonella enterica serovar Typhimurium LT2, presumed to encode DAPAL, in the growth kinetics of the bacteria. While Salmonella Typhimurium LT2 could grow on dl-DAP as a sole carbon source, the wild-type E. coli K-12 strain exhibited only marginal growth on dl-DAP, suggesting that DAPAL is functional in S. Typhimurium. The expression of ygeX in E. coli was low as detected by reverse transcriptase PCR (RT-PCR), consistent with the poor growth of E. coli on dl-DAP. Strains of S. Typhimurium and E. coli with STM1002 and ygeX, respectively, deleted showed loss of growth on dl-DAP, confirming that STM1002 (ygeX) is the locus encoding DAPAL. Interestingly, the presence of dl-DAP caused a growth inhibition of the wild-type E. coli strain as well as the knockout strains of S. Typhimurium and E. coli in minimal glucose/glycerol medium. Inhibition by dl-DAP was rescued by transforming the strains with plasmids containing the STM1002 (ygeX) gene encoding DAPAL or supplementing the medium with Casamino Acids. Growth restoration studies using media lacking specific amino acid supplements suggested that growth inhibition by dl-DAP in the absence of DAPAL is associated with auxotrophy related to the inhibition of the enzymes involved in the biosynthetic pathways of pyruvate and aspartate and the amino acids derived from them.