000202531 001__ 202531
000202531 005__ 20181203023635.0
000202531 0247_ $$2doi$$a10.1098/rsob.140063
000202531 022__ $$a2046-2441
000202531 02470 $$2ISI$$a000341627800001
000202531 037__ $$aARTICLE
000202531 245__ $$aOptimization of the analogue-sensitive Cdc2/Cdk1 mutant by in vivo selection eliminates physiological limitations to its use in cell cycle analysis
000202531 269__ $$a2014
000202531 260__ $$aLondon$$bRoyal Society, The$$c2014
000202531 300__ $$a12
000202531 336__ $$aJournal Articles
000202531 520__ $$aAnalogue-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, 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.
000202531 6531_ $$acell cycle
000202531 6531_ $$acyclin-dependent kinase
000202531 6531_ $$achemical genetics
000202531 6531_ $$aanalogue-sensitive mutant
000202531 6531_ $$afission yeast
000202531 700__ $$aAoi, Yuki$$uUniv Tokyo, Grad Sch Sci, Dept Biophys & Biochem, Tokyo 1130033, Japan
000202531 700__ $$aKawashima, Shigehiro A.$$uUniv Tokyo, Grad Sch Pharmaceut Sci, Tokyo 1130033, Japan
000202531 700__ $$0240501$$aSimanis, Viesturs$$g174228$$uEPFL SV ISREC UPSIM SV2 1830, CH-1015 Lausanne, Switzerland
000202531 700__ $$aYamamoto, Masayuki$$uUniv Tokyo, Grad Sch Sci, Dept Biophys & Biochem, Tokyo 1130033, Japan
000202531 700__ $$aSato, Masamitsu$$uUniv Tokyo, Grad Sch Sci, Dept Biophys & Biochem, Tokyo 1130033, Japan
000202531 773__ $$j4$$k7$$tOpen Biology
000202531 8564_ $$s1440277$$uhttps://infoscience.epfl.ch/record/202531/files/Open%20Biol.%202014.pdf$$zn/a
000202531 909C0 $$0252141$$pUPSIM$$xU11430
000202531 909CO $$ooai:infoscience.tind.io:202531$$pSV$$particle
000202531 917Z8 $$x181933
000202531 937__ $$aEPFL-ARTICLE-202531
000202531 973__ $$aEPFL$$rREVIEWED$$sPUBLISHED
000202531 980__ $$aARTICLE