Directed evolution to bypass cyclin requirements for the Cdc28p cyclin-dependent kinase.
To identify cyclin-dependent kinase mutants with relaxed cyclin requirements, CDC28 alleles were selected that could rescue a yeast strain expressing as its only CLN G1 cyclin a mutant Cln2p (K129A,E183A) that is defective for Cdc28p binding. Rescue of this strain by mutant CDC28 was dependent upon the mutant cln2-KAEA, but additional mutagenesis and DNA shuffling yielded multiply mutant CDC28-BYC alleles (bypass of CLNs) that could support highly efficient cell cycle initiation in the complete absence of CLN genes. By gel filtration chromatography, one of the mutant Cdc28 proteins exhibited kinase activity associated with cyclin-free monomer. Thus, the mutants' CLN bypass activity might result from constitutive, cyclin-independent activity, suggesting that Cdk targeting by cyclins is not required for cell cycle initiation.
Levine
K
K
Rockefeller University, New York, New York, USA.
Kiang
L
L
Jacobson
M D
MD
Fisher
R P
RP
Cross
F R
FR
D000483:Alleles; D017489:CDC28 Protein Kinase, S cerevisiae / Q000235:genetics* / Q000378:metabolism*; D050756:Cyclin-Dependent Kinase Inhibitor Proteins; D016213:Cyclins / Q000378:metabolism*; D019020:Directed Molecular Evolution / Q000379:methods; D004789:Enzyme Activation / Q000235:genetics; D019143:Evolution, Molecular; D005656:Fungal Proteins / Q000378:metabolism; D005816:Genetic Complementation Test; D016296:Mutagenesis; D011487:Protein Conformation; D011494:Protein Kinases / Q000378:metabolism; D011995:Recombination, Genetic; D029701:Saccharomyces cerevisiae Proteins; D012641:Selection, Genetic; D015003:Yeasts / Q000235:genetics
C082721:CLN2 protein, S cerevisiae; D050756:Cyclin-Dependent Kinase Inhibitor Proteins; D016213:Cyclins; D005656:Fungal Proteins; C086926:SIC1 protein, S cerevisiae; D029701:Saccharomyces cerevisiae Proteins; D011494:Protein Kinases; C102333:histone H1 kinase; D017489:CDC28 Protein Kinase, S cerevisiae