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

Yuki Aoi, Shigehiro A. Kawashima, Viesturs Simanis, Masayuki Yamamoto, Masamitsu Sato

Research output: Contribution to journalArticle

11 Citations (Scopus)

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 ATPanalogues. 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, genotoxins and an anti-microtubule drug. We have used cdc2-asM17 to show that Cdc2 activity is required to maintain the activity of the spindle assembly checkpoint. Furthermore, we also demonstrate that maintenance of the Shugoshin Sgo1 at meiotic centromeres does not require Cdc2 activity, whereas localization of the kinase aurora does. The modified cdc2-asM17 allele can be thus used to analyse many aspects of cell-cycle-related events in fission yeast.

Original languageEnglish
Article number140063
JournalOpen Biology
Volume4
Issue numberJULY
DOIs
Publication statusPublished - 2014 Jul 2
Externally publishedYes

Fingerprint

Schizosaccharomyces
Genetic Suppression
Cell Cycle
Cells
Phosphotransferases
Aurora Kinases
Yeast
M Phase Cell Cycle Checkpoints
Temperature
Centromere
Mutagens
Meiosis
Spores
Microtubules
Adenosine Triphosphate
Alleles
Maintenance
Pharmaceutical Preparations
Defects

Keywords

  • Analogue-sensitive mutant
  • Cell cycle
  • Chemical genetics
  • Cyclin-dependent kinase
  • Fission yeast

ASJC Scopus subject areas

  • Biochemistry, Genetics and Molecular Biology(all)
  • Neuroscience(all)
  • Immunology
  • Medicine(all)

Cite this

Optimization of the analogue-sensitive Cdc2/Cdk1 mutant by in vivo selection eliminates physiological limitations to its use in cell cycle analysis. / Aoi, Yuki; Kawashima, Shigehiro A.; Simanis, Viesturs; Yamamoto, Masayuki; Sato, Masamitsu.

In: Open Biology, Vol. 4, No. JULY, 140063, 02.07.2014.

Research output: Contribution to journalArticle

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