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 following chemically induced DNA replication stress using methyl methanesulfonate (MMS) or hydroxyurea (HU). How these protein movements are regulated remains largely unexplored.

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.

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.

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.

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.

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.