Directed evolution to bypass cyclin requirements for the Cdc28p cyclin-dependent kinase

To identify cyclin-dependent kinase mutants with relaxed cyclin requirement s, 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 fo r 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 highl y efficient cell cycle initiation in the complete absence of CLN genes. By gel filtration chromatography, one of the mutant Cdc28 proteins exhibited k inase 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 i nitiation.