A dual system to manipulate protein levels for DNA replication- and cell cycle-related studies.

Author

Néstor García-Rodríguez

Affiliation

Centro Andaluz de Biología Molecular y Medicina Regenerativa (CABIMER), Universidad de Sevilla-CSIC-Universidad Pablo de Olavide, Sevilla, Spain; Departamento de Genética, Universidad de Sevilla, Sevilla, Spain. Electronic address: [Email]

Abstract

Investigation of cell cycle-regulated processes often necessitates the rapid manipulation of individual protein levels in synchronized populations over the course of a cell cycle. In the budding yeast, the two major orthogonal approaches by which this is accomplished are conditional gene expression via inducible or repressible promoters and regulated metabolic destabilization of a protein of interest via ubiquitin-mediated degradation signals. Here, we describe an application of these principles to the investigation of DNA damage signaling during replication. Using a combination of conditional gene expression via a tetracycline-repressible promoter and inducible protein degradation via an auxin-regulated degron system, we have analyzed the cross talk between factors controlling the bypass of lesions during replication and the activation of the DNA damage checkpoint. Here, we describe the basic principles underlying our experimental system and provide a detailed protocol to analyze the cross talk between damage bypass and checkpoint signaling. Finally, we discuss the advantages and disadvantages of using this approach and point out possible applications to other regulatory events and other organisms.

Keywords

AID*-tag,Budding yeast,Cell cycle,Checkpoint,DNA damage bypass,DNA repair,DNA replication,Degron,Tet-promoter,Ubiquitin,

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