Optimization of the analogue-sensitive Cdc2/Cdk1 mutant by in vivo selection eliminates physiological limitations to its use in cell cycle analysis.

ABSTRACT: Analogue-sensitive (as) mutants of kinases are widely used to selectively inhibit a single kinase with few off-target effects. The analogue-sensitive mutant cdc2-as of fission yeast (Schizosaccharomyces pombe) is a powerful tool to study the cell cycle, but the strain displays meiotic defects, and is sensitive to high and low temperature even in the absence of ATP-analogue inhibitors. This has limited the use of the strain for use in these settings. Here, we used in vivo selection for intragenic suppressor mutations of cdc2-as that restore full function in the absence of ATP-analogues. The cdc2-asM17 underwent meiosis and produced viable spores to a similar degree to the wild-type strain. The suppressor mutation also rescued the sensitivity of the cdc2-as strain to high and low temperature

SUBMITTER: Aoi Y 

PROVIDER: S-EPMC4118601 | biostudies-literature | 2014 Jul

REPOSITORIES: biostudies-literature