Directed evolution to bypass cyclin requirements for the Cdc28p cyclin-dependent kinase
Directed evolution to bypass cyclin requirements for the Cdc28p cyclin-dependent kinase
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DOI：
10.1016/s1097-2765(00)80337-8
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发表时间：
1999-09-01
期刊：
影响因子：
16
通讯作者：
Cross, FR
中科院分区：
文献类型：
--
作者：
Levine, K;Kiang, L;Cross, FR
为了鉴定具有宽松细胞周期蛋白要求的细胞周期蛋白依赖性激酶突变体，选择了CDC 28等位基因，这些等位基因可以拯救酵母菌株，该酵母菌株表达作为其唯一CLN G1细胞周期蛋白的突变体Cln 2 p（K129 A、E183 A），该突变体缺乏Cdc 28 p结合。突变型CDC 28对该菌株的拯救依赖于突变型cln 2-KAEA，但额外的诱变和DNA改组产生了多重突变型CDC 28-BYC等位基因（CLN旁路），其可以在完全不存在CLN基因的情况下支持高效的细胞周期启动。通过凝胶过滤层析，突变Cdc 28蛋白表现出激酶活性与细胞周期蛋白游离单体。因此，突变体的CLN旁路活性可能是由组成性的，细胞周期蛋白非依赖性的活性，表明细胞周期蛋白的Cdk靶向是不需要细胞周期启动。
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.