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Background Colony growth on solid media is a simple and effective measure for high-throughput genomic experiments such as yeast two-hybrid, synthetic dosage lethality and Synthetic Physical Interaction screens. The development of robotic pinning tools has facilitated the experimental design of these assays, and different imaging software can be used to automatically measure colony sizes on plates. However, comparison to control plates and statistical data analysis is often laborious and pinning issues or plate specific growth effects can lead to the detection of false-positive growth defects. Results We have developed ScreenGarden, a shinyR application, to enable easy, quick and robust data analysis of plate-based high throughput assays. The code allows comparisons of different formats of data and different sized arrays of colonies. A comparison of ScreenGarden with previous analysis tools shows that it performs, at least, equivalently. The software can be run either via a website or offline via the RStudio program; the code is available and can be modified by expert uses to customise the analysis. Conclusions ScreenGarden provides a simple, fast and effective tool to analyse colony growth data from genomic experiments.

Most cell divisions are asymmetric with some cellular components distributed preferentially to one of the two nascent daughter cells. These asymmetries are typically important for the developmental fate of the resulting daughter cells. Herrero et al. describe ....., The polarized partitioning of proteins in cells underlies asymmetric cell division, which is an important driver of development and cellular diversity. The budding yeast Saccharomyces cerevisiae divides asymmetrically, like many other cells, to generate two distinct progeny cells. A well-known example of an asymmetric protein is the transcription factor Ace2, which localizes specifically to the daughter nucleus, where it drives a daughter-specific transcriptional network. We screened a collection of essential genes to analyze the effects of core cellular processes in asymmetric cell division based on Ace2 localization. This screen identified mutations that affect progression through the cell cycle, suggesting that cell cycle delay is sufficient to disrupt Ace2 asymmetry. To test this model, we blocked cells from progressing through mitosis and found that prolonged metaphase delay is sufficient to disrupt Ace2 asymmetry after release, and that Ace2 asymmetry is restored after cytokinesis. We also demonstrate that members of the evolutionarily conserved facilitates chromatin transcription (FACT) chromatin-reorganizing complex are required for both asymmetric and cell cycle-regulated localization of Ace2, and for localization of the RAM network components.

Hsp70 interactions are critical for cellular viability and the response to stress. Previous attempts to characterize Hsp70 interactions have been limited by their transient nature and the inability of current technologies to distinguish direct versus bridged interactions. We report the novel use of cross-linking mass spectrometry (XL-MS) to comprehensively characterize the Saccharomyces cerevisiae (budding yeast) Hsp70 protein interactome. Using this approach, we have gained fundamental new insights into Hsp70 function, including definitive evidence of Hsp70 self-association as well as multipoint interaction with its client proteins. In addition to identifying a novel set of direct Hsp70 interactors that can be used to probe chaperone function in cells, we have also identified a suite of posttranslational modification (PTM)-associated Hsp70 interactions. The majority of these PTMs have not been previously reported and appear to be critical in the regulation of client protein function. These data indicate that one of the mechanisms by which PTMs contribute to protein function is by facilitating interaction with chaperones. Taken together, we propose that XL-MS analysis of chaperone complexes may be used as a unique way to identify biologically important PTMs on client proteins.

SummaryHsp70 interactions are critical for cellular viability and the response to stress. Previous attempts to characterize Hsp70 interactions have been limited by their transient nature and inability of current technologies to distinguish direct vs bridged interactions. We report the novel use of cross-linking mass spectrometry (XL-MS) to comprehensively characterize the budding yeast Hsp70 protein interactome. Using this approach, we have gained fundamental new insights into Hsp70 function, including definitive evidence of Hsp70 self-association as well as multi-point interaction with its client proteins. In addition to identifying a novel set of direct Hsp70 interactors which can be used to probe chaperone function in cells, we have also identified a suite of PTM-associated Hsp70 interactions. The majority of these PTMs have not been previously reported and appear to be critical in the regulation of client protein function. These data indicate that one of the mechanisms by which PTMs contribute to protein function is by facilitating interaction with chaperones. Taken together, we propose that XL-MS analysis of chaperone complexes may be used as a unique way to identify biologically-important PTMs on client proteins.In vivo confirmation of Hsp70 dimerizationComprehensive direct interactome of Hsp70Multi-domain interactions between Hsp70 and client proteinsIdentification of novel biologically-important client protein PTMs

Oncogene-induced replication stress is a major driver of genomic instability in cancer cells, with a central role in both cancer initiation and progression (1). Despite its critical role in cancer development, the mechanisms that lay at the basis of oncogene-induced replication stress remains poorly understood. Here, we investigate the mechanism of c-Myc-induced replication stress. Our data shows that c-Myc induces replication stress by increasing the amount of cohesins bound to chromatin in the G1 phase of the cell cycle. This is independent of previously suggested mechanisms involving deregulation of replication initiation and transcriptional interference. Restoring the amount of chromatin-bound cohesins to control levels, or preventing the accumulation of cohesins at CTCF sites, in cells experiencing oncogenic c-Myc activity prevents replication stress. Increased cohesins chromatin occupancy correlates with a c-Myc-dependent increase in the levels of the cohesion loader Mau2. Preventing c-Myc-induced increase in Mau2 reduces oncogene-induced replication stress. Together our data support a novel mechanism for oncogene-induced replication stress. Since c-Myc activation is a crucial event in many human cancers (2), identifying the mechanisms through which this oncogene promotes replication stress provides critical insights into cancer biology.

Evolutionarily conserved polo-like kinase, Cdc5 (Plk1 in humans), associates with kinetochores during mitosis; however, the role of cell cycle–dependent centromeric ( CEN) association of Cdc5 and its substrates that exclusively localize to the kinetochore have not been characterized. Here we report that evolutionarily conserved CEN histone H3 variant, Cse4 (CENP-A in humans), is a substrate of Cdc5, and that the cell cycle–regulated association of Cse4 with Cdc5 is required for cell growth. Cdc5 contributes to Cse4 phosphorylation in vivo and interacts with Cse4 in mitotic cells. Mass spectrometry analysis of in vitro kinase assays showed that Cdc5 phosphorylates nine serine residues clustered within the N-terminus of Cse4. Strains with cse4-9SA exhibit increased errors in chromosome segregation, reduced levels of CEN-associated Mif2 and Mcd1/Scc1 when combined with a deletion of MCM21. Moreover, the loss of Cdc5 from the CEN chromatin contributes to defects in kinetochore integrity and reduction in CEN-associated Cse4. The cell cycle–regulated association of Cdc5 with Cse4 is essential for cell viability as constitutive association of Cdc5 with Cse4 at the kinetochore leads to growth defects. In summary, our results have defined a role for Cdc5-mediated Cse4 phosphorylation in faithful chromosome segregation.

Faithful chromosome segregation maintains chromosomal stability as errors in this process contribute to chromosomal instability (CIN), which has been observed in many diseases including cancer. Epigenetic regulation of kinetochore proteins such as Cse4 (CENP-A in humans) plays a critical role in high-fidelity chromosome segregation. Here we show that Cse4 is a substrate of evolutionarily conserved Cdc7 kinase, and that Cdc7-mediated phosphorylation of Cse4 prevents CIN. We determined that Cdc7 phosphorylates Cse4 in vitro and interacts with Cse4 in vivo in a cell cycle-dependent manner. Cdc7 is required for kinetochore integrity as reduced levels of CEN-associated Cse4, a faster exchange of Cse4 at the metaphase kinetochores, and defects in chromosome segregation, are observed in a cdc7-7 strain. Phosphorylation of Cse4 by Cdc7 is important for cell survival as constitutive association of a kinase-dead variant of Cdc7 (cdc7-kd) with Cse4 at the kinetochore leads to growth defects. Moreover, phospho-deficient mutations of Cse4 for consensus Cdc7 target sites contribute to CIN phenotype. In summary, our results have defined a role for Cdc7-mediated phosphorylation of Cse4 in faithful chromosome segregation.

The kinetochore is a mega-dalton protein assembly that forms within centromeric regions of chromosomes and directs their segregation during cell division. Here we review cell cycle-mediated phosphorylation events at the kinetochore, with a focus on the budding yeast Saccharomyces cerevisiae and the insight gained from forced associations of kinases and phosphatases. The point centromeres found in the budding yeast S. cerevisiae are one of the simplest such structures found in eukaryotes. The S. cerevisiae kinetochore comprises a single nucleosome, containing a centromere-specific H3 variant Cse4CENP-A, bound to a set of kinetochore proteins that connect to a single microtubule. Despite the simplicity of the budding yeast kinetochore, the proteins are mostly homologous with their mammalian counterparts. In some cases, human proteins can complement their yeast orthologs. Like its mammalian equivalent, the regulation of the budding yeast kinetochore is complex: integrating signals from the cell cycle, checkpoints, error correction, and stress pathways. The regulatory signals from these diverse pathways are integrated at the kinetochore by post-translational modifications, notably phosphorylation and dephosphorylation, to control chromosome segregation. Here we highlight the complex interplay between the activity of the different cell-cycle kinases and phosphatases at the kinetochore, emphasizing how much more we have to understand this essential structure.

The transition from mitosis into the first gap phase of the cell cycle in budding yeast is controlled by the Mitotic Exit Network (MEN). The network interprets spatiotemporal cues about the progression of mitosis and ensures that release of Cdc14 phosphatase occurs only after completion of key mitotic events. The MEN has been studied intensively; however, a unified understanding of how localisation and protein activity function together as a system is lacking. In this paper, we present a compartmental, logical model of the MEN that is capable of representing spatial aspects of regulation in parallel to control of enzymatic activity. We show that our model is capable of correctly predicting the phenotype of the majority of mutants we tested, including mutants that cause proteins to mislocalise. We use a continuous time implementation of the model to demonstrate that Cdc14 Early Anaphase Release (FEAR) ensures robust timing of anaphase, and we verify our findings in living cells. Furthermore, we show that our model can represent measured cell–cell variation in Spindle Position Checkpoint (SPoC) mutants. This work suggests a general approach to incorporate spatial effects into logical models. We anticipate that the model itself will be an important resource to experimental researchers, providing a rigorous platform to test hypotheses about regulation of mitotic exit., This study presents a logical compartmental model of the yeast mitotic exit network (MEN) that represents spatial aspects of regulation. The model correctly predicts the majority of published MEN phenotypes and reveals both the role of the Cdc14 early anaphase release and a link between mitosis length and checkpoint competence.

1AbstractThe transition from mitosis into the first gap phase of the cell cycle in budding yeast is controlled by the Mitotic Exit Network (MEN). The network interprets spatio-temporal cues about the progression of mitosis and ensures that release of Cdc14 phosphatase occurs only after completion of key mitotic events. The MEN has been studied intensively however a unified understanding of how localization and protein activity function together as a system is lacking. In this paper we present a compartmental, logical model of the MEN that is capable of representing spatial aspects of regulation in parallel to control of enzymatic activity. Through optimization of the model, we reveal insights into role of Cdc5 in Cdc15 localization and the importance of Lte1 regulation in control of Bfa1. We show that our model is capable of correctly predicting the phenotype of∼80% of mutants we tested, including mutants representing mislocalizing proteins. We use a continuous time implementation of the model to demonstrate the role of Cdc14 Early Anaphase Release (FEAR) to ensure robust timing of anaphase and verify our findings in living cells. We show that our model can represent measured cell-cell variation in Spindle Position Checkpoint (SPoC) mutants. Finally, we use the model to predict the impact of forced localization of MEN proteins and validate these predictions experimentally. This model represents a unified view of the mechanism of mitotic exit control.

Background The creation of arrays of yeast strains each encoding a different protein with constant tags is a powerful method for understanding how genes and their proteins control cell function. As genetic tools become more sophisticated there is a need to create custom libraries encoding proteins fused with specialised tags to query gene function. These include protein tags that enable a multitude of added functionality, such as conditional degradation, fluorescent labelling, relocalization or activation and also DNA and RNA tags that enable barcoding of genes or their mRNA products. Tools for making new libraries or modifying existing ones are becoming available, but are often limited by the number of strains they can be realistically applied to or by the need for a particular starting library. Results We present a new recombination-based method, CATS – Cas9-Assisted Tag Switching, that switches tags in any existing library of yeast strains. This method employs the reprogrammable RNA guided nuclease, Cas9, to both introduce endogenous double strand breaks into the genome as well as liberating a linear DNA template molecule from a plasmid. It exploits the relatively high efficiency of homologous recombination in budding yeast compared with non-homologous end joining. Conclusions The method takes less than 2 weeks, is cost effective and can simultaneously introduce multiple genetic changes, thus providing a rapid, genome-wide approach to genetic modification.

Fluorescence lifetime imaging (FLIM) provides a means to contrast different molecular species and to map variations in the local fluorophore molecular environment, including to read out Forster resonant energy transfer (FRET), e.g. to assay protein interactions or genetically expressed FRET biosensors. We have implemented wide-field time-gated FLIM in a modular open automated microscopy platform for high content analysis (HCA). To demonstrate its relevance to drug discovery, we have demonstrated the capability of our openFLIM HCA platform to assay interactions of low copy number endogenous proteins in yeast cells labelled with fluorescent proteins. We have also demonstrated the capability of multiwell plate FLIM assays to provide readouts of a FRET biosensor in 2-D and 3-D cell cultures.

The yeast centrosome or Spindle Pole Body (SPB) is an organelle situated in the nuclear membrane, where it nucleates spindle microtubules and acts as a signaling hub. Various studies have explored the effects of forcing individual proteins to interact with the yeast SPB, however no systematic study has been performed. We used synthetic physical interactions to detect proteins that inhibit growth when forced to associate with the SPB. We found the SPB to be especially sensitive to relocalization, necessitating a novel data analysis approach. This novel analysis of SPI screening data shows that regions of the cell are locally more sensitive to forced relocalization than previously thought. Furthermore, we found a set of associations that result in elevated SPB number and, in some cases, multi-polar spindles. Since hyper-proliferation of centrosomes is a hallmark of cancer cells, these associations point the way for the use of yeast models in the study of spindle formation and chromosome segregation in cancer.

The kinetochore, a multi-protein complex assembled on centromeres, is essential to segregate chromosomes during cell division. Deficiencies in kinetochore function can lead to chromosomal instability and aneuploidy—a hallmark of cancer cells. Kinetochore function is controlled by recruitment of regulatory proteins, many of which have been documented, however their function often remains uncharacterized and many are yet to be identified. To identify candidates of kinetochore regulation we used a proteome-wide protein association strategy in budding yeast and detected many proteins that are involved in post-translational modifications such as kinases, phosphatases and histone modifiers. We focused on the Polo-like kinase, Cdc5, and interrogated which cellular components were sensitive to constitutive Cdc5 localization. The kinetochore is particularly sensitive to constitutive Cdc5 kinase activity. Targeting Cdc5 to different kinetochore subcomplexes produced diverse phenotypes, consistent with multiple distinct functions at the kinetochore. We show that targeting Cdc5 to the inner kinetochore, the constitutive centromere-associated network (CCAN), increases the levels of centromeric RNA via an SPT4 dependent mechanism., Author summary During cell division, replicated chromosomes must be equally divided between the two daughter cells. This is achieved in part by a multi-protein structure called the kinetochore. Errors during chromosome segregation can lead to inheritance of abnormal chromosome number. This situation is referred to as aneuploidy—a hallmark of cancer cells. Kinetochore function is tightly regulated by recruitment of regulatory proteins under certain conditions and at specific times during the cell cycle. Kinetochore regulators are often mis-regulated in many types of cancers. The major cell-cycle regulator, Polo-like kinase (Plk1), is one such regulator. Plk1 is overexpressed in many cancers and is thus considered an important target in cancer therapeutics. To understand its function in kinetochore regulation, we manipulated the Plk1 homolog in budding yeast, Cdc5, in both space and time. Our data suggest that Cdc5 has multiple different functions at the yeast kinetochore in agreement with studies in human cells. Furthermore, we show that constitutive Cdc5 localization at the inner kinetochore can disrupt the function of the whole kinetochore, perturb mitotic progression and increase the levels of centromeric RNA. These results have implications for human disease, since mis-regulation of centromeric transcription has been observed in stressed, aging and cancerous cells.

The location of proteins within eukaryotic cells is often critical for their function and relocation of proteins forms the mainstay of regulatory pathways. To assess the importance of protein location to cellular homeostasis, we have developed a methodology to systematically create binary physical interactions between a query protein and most other members of the proteome. This method allows us to rapidly assess which of the thousands of possible protein interactions modify a phenotype. As proof of principle we studied the kinetochore, a multiprotein assembly that links centromeres to the microtubules of the spindle during cell division. In budding yeast, the kinetochores from the 16 chromosomes cluster together to a single location within the nucleus. The many proteins that make up the kinetochore are regulated through ubiquitylation and phosphorylation. By systematically associating members of the proteome to the kinetochore, we determine which fusions affect its normal function. We identify a number of candidate kinetochore regulators, including the phosphatase Cdc14. We examine where within the kinetochore Cdc14 can act and show that the effect is limited to regions that correlate with known phosphorylation sites, demonstrating the importance of serine phospho-regulation for normal kinetochore homeostasis.

Artificially tethering two proteins or protein fragments together is a powerful method to query molecular mechanisms. However, this approach typically relies upon a prior understanding of which two proteins, when fused, are most likely to provide a specific function and is therefore not readily amenable to large-scale screening. Here, we describe the Synthetic Physical Interaction (SPI) method to create proteome-wide forced protein associations in the budding yeast Saccharomyces cerevisiae. This method allows thousands of protein-protein associations to be screened for those that affect either normal growth or sensitivity to drugs or specific conditions. The method is amenable to proteins, protein domains, or any genetically encoded peptide sequence.

Artificially tethering two proteins or protein fragments together is a powerful method to query molecular mechanisms. However, this approach typically relies upon a prior understanding of which two proteins, when fused, are most likely to provide a specific function and is therefore not readily amenable to large-scale screening. Here, we describe the Synthetic Physical Interaction (SPI) method to create proteome-wide forced protein associations in the budding yeast Saccharomyces cerevisiae. This method allows thousands of protein-protein associations to be screened for those that affect either normal growth or sensitivity to drugs or specific conditions. The method is amenable to proteins, protein domains, or any genetically encoded peptide sequence.

Styles et al. develop an optimized method that combines high-content microscopy and automated phenotypic analysis with genome-wide yeast genetics to identify genes in DNA damage repair.

Kinetochores serve both a structural role linking chromosomes to the mitotic spindle and a regulatory role, controlling the timing of mitosis via the spindle assembly checkpoint. To identify proteins that regulate the kinetochore we used a genome-wide fluorescence microscopy approach. We combined an array of mutants of either non-essential gene deletions or essential temperature-sensitive alleles with fluorescently tagged spindle pole bodies (centrosome) and outer kinetochores. Quantitative and qualitative analysis revealed mutants that affect the levels and distribution of kinetochores respectively. These mutants are enriched for those involved in mRNA processing, chromatin organization, DNA replication/repair and mitosis. Our data show that the Pkc1 kinase maintains the kinetochore focus via its ability to prevent cell stress and this phenotype is rescued by an osmotic stabilizer. These data support the notion that kinetochore and microtubule homeostasis are perturbed by the stress response pathways. Hence this observation provides a candidate mechanism for extracellular stress leading to chromosome segregation defects.

The polarized partitioning of proteins in cells underlies asymmetric cell division, which is an important driver of development and cellular diversity. Like most cells, the budding yeastSaccharomyces cerevisiaedivides asymmetrically to give two distinct daughter cells. This asymmetry mimics that seen in metazoans and the key regulatory proteins are conserved from yeast to human. A well-known example of an asymmetric protein is the transcription factor Ace2, which localizes specifically to the daughter nucleus, where it drives a daughter-specific transcriptional network. We performed a reverse genetic screen to look for regulators of asymmetry based on the Ace2 localization phenotype. We screened a collection of essential genes in order to analyze the effect of core cellular processes in asymmetric cell division. This identified a large number of mutations that are known to affect progression through the cell cycle, suggesting that cell cycle delay is sufficient to disrupt Ace2 asymmetry. To test this model we blocked cells from progressing through mitosis and found that prolonged cell cycle arrest is sufficient to disrupt Ace2 asymmetry after release. We also demonstrate that members of the evolutionary conserved FACT chromatin-remodeling complex are required for both asymmetric and cell cycle-regulated localization of Ace2.

To understand the function of eukaryotic cells, it is critical to understand the role of protein-protein interactions and protein localization. Currently, we do not know the importance of global protein localization nor do we understand to what extent the cell is permissive for new protein associations – a key requirement for the evolution of new protein functions. To answer this question, we fused every protein in the yeast Saccharomyces cerevisiae with a partner from each of the major cellular compartments and quantitatively assessed the effects upon growth. This analysis reveals that cells have a remarkable and unanticipated tolerance for forced protein associations, even if these associations lead to a proportion of the protein moving compartments within the cell. Furthermore, the interactions that do perturb growth provide a functional map of spatial protein regulation, identifying key regulatory complexes for the normal homeostasis of eukaryotic cells. DOI: http://dx.doi.org/10.7554/eLife.13053.001, eLife digest Our actions often depend on who we interact with: parents, teachers, friends, colleagues. So it is for proteins in the cell: their function depends on which other proteins they work with. If a protein interacts with new partners or ends up in a new neighborhood of the cell, it can perform an entirely unexpected role, rewiring how that cell works. There are millions of possible protein-protein interactions, but it is not known how cells behave if their proteins are forced into new associations. For example, how many of these associations affect how well the cell can grow? Using budding yeast, Berry et al. were able to associate every protein in the cell with proteins from each of the major areas of the cell such as the nucleus, cell membrane or mitochondria. These new associations and relocations were then examined to see how many of them caused problems, slowing the cell’s growth or killing it. Unexpectedly, most forced associations had no detectable effect, indicating that the cell is remarkably tolerant of new protein-protein interactions. This contradicts a common idea that proteins are very fussy about their partner proteins, and will not work properly if they are forced into new interactions. The associations that do cause a growth defect are often between proteins that normally work together, indicating that their association is normally carefully controlled during the normal growth of cells. In some cases these forced associations identified previously unknown regulators of cell behavior. Proteins that interact with the wrong partners or are in the wrong place within cells cause a number of diseases. Future forced association experiments will allow us to examine such interactions and possibly search for drugs that will correct the problem. DOI: http://dx.doi.org/10.7554/eLife.13053.002

Asymmetric cell division is of fundamental importance in biology as it allows for the establishment of separate cell lineages during the development of multicellular organisms. Although microbial systems, including the yeast Saccharomyces cerevisiae , are excellent models of asymmetric cell division, this phenotype occurs in all cell divisions; consequently, models of lineage-specific segregation patterns in these systems do not exist. Here, we report the first example of lineage-specific asymmetric division in yeast. We used fluorescent tags to show that components of the yeast kinetochore, the protein complex that anchors chromosomes to the mitotic spindle, divide asymmetrically in a single postmeiotic lineage. This phenotype is not seen in vegetatively dividing haploid or diploid cells. This kinetochore asymmetry suggests a mechanism for the selective segregation of sister centromeres to daughter cells to establish different cell lineages or fates. These results provide a mechanistic link between lineage-defining asymmetry of metazoa with unicellular eukaryotes.

In 2007, we published the results of a genome-wide screen for ORFs that affect the frequency of Rad52 foci in yeast. That paper was published within the constraints of conventional online publishing tools, and it provided only a glimpse into the actual screen data. New tools in the JCB DataViewer now show how these data can—and should—be shared.

The type I restriction and modification (R-M) enzyme from Salmonella enterica serovar kaduna ( Sty SKI) recognises the DNA sequence 5'-CGAT(N)7GTTA, an unusual target for a type I R-M system in that it comprises two tetranucleotide components. The amino target recognition domain (TRD) of Sty SKI recognises 5'-CGAT and shows 36% amino acid identity with the carboxy TRD of Eco R124I which recognises the complementary, but degenerate, sequence 5'-RTCG. Current models predict that the amino and carboxy TRDs of the specificity subunit are in inverted orientations within a structure with 2-fold rotational symmetry. The complementary target sequences recognised by the amino TRD of Sty SKI and the carboxy TRD of Eco R124I are consistent with the predicted inverted positions of the TRDs. Amino TRDs of similar amino acid sequence have been shown to recognise the same nucleotide sequence. The similarity reported here, the first example of one between amino and carboxy TRDs, while consistent with a conserved mechanism of target recognition, offers additional flexibility in the evolution of sequence specificity by increasing the potential diversity of DNA targets for a given number of TRDs. Sty SKI identifies the first member of the IB family in Salmonella species.

The accurate segregation of chromosomes during cell division is achieved by attachment of chromosomes to the mitotic spindle via the kinetochore, a large multi-protein complex that assembles on centromeres. The budding yeast kinetochore comprises more than 60 different proteins. Although the structure and function of many of these proteins has been investigated, we have little understanding of the steady state regulation of kinetochores. The primary model of kinetochore homeostasis suggests that kinetochores assemble hierarchically from the centromeric DNA via the inclusion of a centromere-specific histone into chromatin. We tested this model by trying to perturb kinetochore protein levels by overexpressing an outer kinetochore gene, MTW1. This increase in protein failed to change protein recruitment, consistent with the hierarchical assembly model. However, we find that deletion of Psh1, a key ubiquitin ligase that is known to restrict inner kinetochore protein loading, does not increase levels of outer kinetochore proteins, thus breaking the normal kinetochore stoichiometry. This perturbation leads to chromosome segregation defects, which can be partially suppressed by mutation of Ubr2, a second ubiquitin ligase that normally restricts protein levels at the outer kinetochore. Together these data show that Psh1 and Ubr2 synergistically control the amount of proteins at the kinetochore., Author Summary As cells divide, their replicated chromosomes must be correctly allocated to the two nascent daughter cells. This is achieved by the kinetochore, which provides a physical link between the chromosomes and the microtubules that drive their movement. If chromosome separation fails, the resulting cells have an abnormal number of chromosomes. This state is called aneuploidy and is a hallmark of cancer cells. The regulation of the kinetochore is therefore of critical importance in maintaining genome integrity. Since a number of cancer cells have over-active kinetochore genes, it has been proposed that an excess of kinetochore proteins can disrupt the normal assembly or maintenance of kinetochores. We tested this idea in yeast by increasing the amount of a specific kinetochore protein, but found no effect upon the normal loading of kinetochore proteins. Instead, we find that two ubiquitin ligases play a role in maintaining the normal balance of the different kinetochore proteins and that this correlates with correct segregation of the chromosomes.

Re-localization of proteins is a hallmark of the DNA damage response. We use high-throughput microscopic screening of the yeast GFP fusion collection to develop a systems-level view of protein re-organization following drug-induced DNA replication stress. Changes in protein localization and abundance reveal drug-specific patterns of functional enrichments. Classification of proteins by sub-cellular destination allows the identification of pathways that respond to replication stress. We analyzed pairwise combinations of GFP fusions and gene deletion mutants to define and order two novel DNA damage responses. In the first, Cmr1 forms subnuclear foci that are regulated by the histone deacetylase Hos2 and are distinct from the typical Rad52 repair foci. In a second example, we find that the checkpoint kinases Mec1/Tel1 and the translation regulator Asc1 regulate P-body formation. This method identifies response pathways that were not detected in genetic and protein interaction screens, and can be readily applied to any form of chemical or genetic stress to reveal cellular response pathways.

Systematic biological screens typically identify many genes or proteins that are implicated in a specific phenotype. However, deriving mechanistic insight from these screens typically involves focusing upon one or a few genes within the set in order to elucidate their precise role in producing the phenotype. To find these critical genes, researchers use a variety of tools to query the set of genes to uncover underlying common genetic or physical interactions or common functional annotations (e.g. gene ontology terms). Not only it is necessary to find previous screens containing genes in common with the new set, but also useful to easily access the individual manuscript or study that classified those genes. Unfortunately, no tool currently exists to facilitate this task. We have developed a web-based tool (ScreenTroll) that queries one or more genes against a database of systematic yeast screens. The software determines which genome-wide yeast screens also identified the queried gene(s) and the resulting screens are listed in an order based on the extent of the overlap between the queried gene(s) and the open reading frames (ORFs) characterized in each individual yeast screen. In a separate list, the corresponding ORFs that are found in both the queried set of genes and each individual genome-wide screen are displayed along with links to the relevant manuscript via NIH's PubMed database. ScreenTroll is useful for comparing a list of ORFs with genes identified in a wide array of published genome-wide screens. This comparison informs users whether any of their queried ORFs overlaps a previous study in the ScreenTroll database. By listing the manuscript of the published screen, users can read more about the phenotype associated with that study. Together, this information provides insight into the function of the queried genes and helps the user focus on a subset of them.

For adult stem cells to both self-renew and give rise to differentiating progenitors, they must undergo an inherently asymmetric division. This defining model of asymmetric cell division requires either that stem cells preferentially distribute internal factors, thereby maintaining a stem cell phenotype in one lineage, or that extrinsic signals determine the fate of daughter cells, allowing the maintenance of one stem cell lineage. Although microbial systems are often used to model asymmetry, lineage-specific asymmetry has not been characterized in these organisms. Recently, we identified a stem-cell-like lineage-specific pattern of kinetochore asymmetry in postmeiotic yeast spores. Because the function of the kinetochore is to segregate chromosomes, this asymmetry has the potential to segregate sister chromatids nonrandomly. This may be relevant to stem cells because more than 30 years ago, it was proposed that stem cells selectively segregate one strand of their chromosomes into the self-renewing stem cell lineage (Cairns 1975). Although advanced labeling methods have provided evidence to both support and refute this hypothesis, it remains unclear how nonrandom sister-chromatid segregation might be achieved in a stem cell lineage. We have identified a kinetochore-specific mechanism in yeast that could support lineage-specific nonrandom sister-chromatid segregation and we discuss the implications of this observation.

Polyploid cells are a characteristic feature of certain human tissues, and notably many cancers. In a systematic genomic screen in yeast, Storchova and co-workers identified the genetic requirements of tetraploidy. Surprisingly, they showed that only three connected pathways are essential for the viability of tetraploid yeast cells. These data provide exciting new targets that might be essential specifically in polyploid cancer cells.

A number of cellular proteins localize to discrete foci within cells, for example DNA repair proteins, microtubule organizing centers, P bodies or kinetochores. It is often possible to measure the fluorescence emission from tagged proteins within these foci as a surrogate for the concentration of that specific protein. We wished to develop tools that would allow quantitation of fluorescence foci intensities in high-throughput studies. As proof of principle we have examined the kinetochore, a large multi-subunit complex that is critical for the accurate segregation of chromosomes during cell division. Kinetochore perturbations lead to aneuploidy, which is a hallmark of cancer cells. Hence, understanding kinetochore homeostasis and regulation are important for a global understanding of cell division and genome integrity. The 16 budding yeast kinetochores colocalize within the nucleus to form a single focus. Here we have created a set of freely-available tools to allow high-throughput quantitation of kinetochore foci fluorescence. We use this ‘FociQuant’ tool to compare methods of kinetochore quantitation and we show proof of principle that FociQuant can be used to identify changes in kinetochore protein levels in a mutant that affects kinetochore function. This analysis can be applied to any protein that forms discrete foci in cells.

The RAD52 gene is essential for homologous recombination in the yeast Saccharomyces cerevisiae. RAD52 is the archetype in an epistasis group of genes essential for DNA damage repair. By catalyzing the replacement of replication protein A with Rad51 on single-stranded DNA, Rad52 likely promotes strand invasion of a double-stranded DNA molecule by single-stranded DNA. Although the sequence and in vitro functions of mammalian RAD52 are conserved with those of yeast, one difference is the presence of introns and consequent splicing of the mammalian RAD52 pre-mRNA. We identified two novel splice variants from the RAD52 gene that are expressed in adult mouse tissues. Expression of these splice variants in tissue culture cells elevates the frequency of recombination that uses a sister chromatid template. To characterize this dominant phenotype further, the RAD52 gene from the yeast Saccharomyces cerevisiae was truncated to model the mammalian splice variants. The same dominant sister chromatid recombination phenotype seen in mammalian cells was also observed in yeast. Furthermore, repair from a homologous chromatid is reduced in yeast, implying that the choice of alternative repair pathways may be controlled by these variants. In addition, a dominant DNA repair defect induced by one of the variants in yeast is suppressed by overexpression of RAD51, suggesting that the Rad51-Rad52 interaction is impaired.

Type I DNA restriction/modification enzymes protect the bacterial cell from viral infection by cleaving foreign DNA which lacks N6-adenine methylation within a target sequence and maintaining the methylation of the targets on the host chromosome. It has been noted that the genes specifying type I systems can be transferred to a new host lacking the appropriate, protective methylation without any adverse effect. The modification phenotype apparently appears before the restriction phenotype, but no evidence for transcriptional or translational control of the genes and the resultant phenotypes has been found. Type I enzymes contain three types of subunit, S for sequence recognition, M for DNA modification (methylation), and R for DNA restriction(cleavage), and can function solely as a M2S1 methylase or as a R2M2S1 bifunctional methylase/nuclease. We show that the methylase is not stable at the concentrations expected to exist in vivo, dissociating into free M subunit and M1S1, whereas the complete nuclease is a stable structure. The M1S1 form can bind the R subunit as effectively as the M2S1 methylase but possesses no activity; therefore, upon establishment of the system in a new host, we propose that most of the R subunit will initially be trapped in an inactive complex until the methylase has been able to modify and protect the host chromosome. We believe that the in vitro assembly pathway will reflect the in vivo situation, thus allowing the assembly process to at least partially explain the observations that the modification phenotype appears before the restriction phenotype upon establishment of a type I system in a new host cell.

High-throughput approaches are beginning to have an impact on many areas of yeast biology. Two recent studies, using different experimental platforms, provide insight into new pathways involved in the response of yeast to DNA damage.