Mec1-independent activation of the Rad53 checkpoint kinase revealed by quantitative analysis of protein localization dynamics.

The replication checkpoint is essential for accurate DNA replication and repair, and maintenance of genomic integrity when a cell is challenged with genotoxic stress. Several studies have defined the complement of proteins that change subcellular location in the budding yeast Saccharomyces cerevisiae following chemically induced DNA replication stress using methyl methanesulfonate (MMS) or hydroxyurea (HU). How these protein movements are regulated remains largely unexplored. We find that the essential checkpoint kinases Mec1 and Rad53 are responsible for regulating the subcellular localization of 159 proteins during MMS-induced replication stress. Unexpectedly, Rad53 regulation of the localization of 52 proteins is independent of its known kinase activator Mec1, and in some scenarios independent of Tel1 or the mediator proteins Rad9 and Mrc1. We demonstrate that Rad53 is phosphorylated and active following MMS exposure in cells lacking Mec1 and Tel1. This noncanonical mode of Rad53 activation depends partly on the retrograde signaling transcription factor Rtg3, which also facilitates proper DNA replication dynamics. We conclude that there are biologically important modes of Rad53 protein kinase activation that respond to replication stress and operate in parallel to Mec1 and Tel1.

Errata: Functional annotation of chemical libraries across diverse biological processes.

This corrects the article DOI: 10.1038/nchembio.2436.

Errata: Functional annotation of chemical libraries across diverse biological processes.

This corrects the article DOI: 10.1038/nchembio.2436.

Functional annotation of chemical libraries across diverse biological processes.

Chemical-genetic approaches offer the potential for unbiased functional annotation of chemical libraries. Mutations can alter the response of cells in the presence of a compound, revealing chemical-genetic interactions that can elucidate a compound's mode of action. We developed a highly parallel, unbiased yeast chemical-genetic screening system involving three key components. First, in a drug-sensitive genetic background, we constructed an optimized diagnostic mutant collection that is predictive for all major yeast biological processes. Second, we implemented a multiplexed (768-plex) barcode-sequencing protocol, enabling the assembly of thousands of chemical-genetic profiles. Finally, based on comparison of the chemical-genetic profiles with a compendium of genome-wide genetic interaction profiles, we predicted compound functionality. Applying this high-throughput approach, we screened seven different compound libraries and annotated their functional diversity. We further validated biological process predictions, prioritized a diverse set of compounds, and identified compounds that appear to have dual modes of action.

Smc5/6 Mediated Sumoylation of the Sgs1-Top3-Rmi1 Complex Promotes Removal of Recombination Intermediates.

Timely removal of DNA recombination intermediates is critical for genome stability. The DNA helicase-topoisomerase complex, Sgs1-Top3-Rmi1 (STR), is the major pathway for processing these intermediates to generate conservative products. However, the mechanisms that promote STR-mediated functions remain to be defined. Here we show that Sgs1 binds to poly-SUMO chains and associates with the Smc5/6 SUMO E3 complex in yeast. Moreover, these interactions contribute to the sumoylation of Sgs1, Top3, and Rmi1 upon the generation of recombination structures. We show that reduced STR sumoylation leads to accumulation of recombination structures, and impaired growth in conditions when these structures arise frequently, highlighting the importance of STR sumoylation. Mechanistically, sumoylation promotes STR inter-subunit interactions and accumulation at DNA repair centers. These findings expand the roles of sumoylation and Smc5/6 in genome maintenance by demonstrating that they foster STR functions in the removal of recombination intermediates.

High-throughput fluorescence microscopic analysis of protein abundance and localization in budding yeast.

Proteins directly carry out and regulate cellular functions. As a result, changes in protein levels within a cell directly influence cellular processes. Similarly, it is intuitive that the intracellular localization of proteins is a key component of their functionality. Optimal activity is achieved by a combination of protein concentration, co-compartmentalization with substrates, co-factors and regulators and sequestration from deleterious locales. The proteome within a cell is highly dynamic and changes in response to different environmental conditions. High-throughput microscopic analysis in the budding yeast Saccharomyces cerevisiae has afforded proteome-wide views of protein organization in living cells, and of how protein abundance and location is regulated and remodeled in response to stress.

A high-throughput confocal fluorescence microscopy platform to study DNA replication stress in yeast cells.

High-throughput imaging of yeast cells expressing fluorescent proteins can be used to understand biological pathways in the context of spatial organization. Here we describe a method for imaging yeast cells expressing proteins tagged with green fluorescent protein (GFP) and/or red fluorescent protein (RFP), with or without drug treatment, in a 384-well format, using the PerkinElmer Opera high-content confocal imaging microscope.

Mapping the cellular response to small molecules using chemogenomic fitness signatures.

Genome-wide characterization of the in vivo cellular response to perturbation is fundamental to understanding how cells survive stress. Identifying the proteins and pathways perturbed by small molecules affects biology and medicine by revealing the mechanisms of drug action. We used a yeast chemogenomics platform that quantifies the requirement for each gene for resistance to a compound in vivo to profile 3250 small molecules in a systematic and unbiased manner. We identified 317 compounds that specifically perturb the function of 121 genes and characterized the mechanism of specific compounds. Global analysis revealed that the cellular response to small molecules is limited and described by a network of 45 major chemogenomic signatures. Our results provide a resource for the discovery of functional interactions among genes, chemicals, and biological processes.

A high-throughput yeast assay identifies synergistic drug combinations.

Drug combinations are commonly used in the treatment of a range of diseases such as cancer, AIDS, and bacterial infections. Such combinations are less likely to be thwarted by resistance, and they have the desirable potential to be synergistic. Synergistic combinations can have decreased toxicity if lower doses of the constituent agents can be used. Conversely, antagonistic combinations can lead to lower efficacy of a treatment. Unfortunately, practical limitations, including the large number of possible combinations to be tested and the importance of optimizing concentrations and order of addition, discourage systematic studies of compound combinations. To address these limitations, we present a platform to screen drug combinations at multiple concentrations with varying orders of addition in Saccharomyces cerevisiae, at high throughput. In a proof of principle, we screened all possible pairwise combinations of 11 DNA damaging agents and found that of the 66 combinations tested, six were synergistic and three were antagonistic. The strength of two-thirds of these combinations was dependent on the order in which the drugs were added to the cells. We further tested the synergistic and antagonistic combinations in two cancer cell lines and found the combination of mitomycin C and irinotecan to be synergistic in both cell lines. This pilot study demonstrates the utility of using yeast for screening large matrices of drug combinations, and it provides a means to prioritize drug combination tests in human cells. Finally, we underscore the importance of testing the order of addition for assessing drug combinations.