p34cdc2 kinase activity is maintained upon activation of the replication checkpoint in Schizosaccharomyces pombe.

All eukaryotes use feedback controls to order and coordinate cell cycle events. In Schizosaccharomyces pombe, several classes of checkpoint genes serve to ensure that DNA replication is complete and free of error before the onset of mitosis. Wild-type cells normally arrest upon inhibition of DNA synthesis or in response to DNA damage, although the exact mechanisms controlling this arrest are unclear. Genetic evidence in fission yeast suggests that the dependence of mitosis upon completion of DNA replication is linked to the regulation of the p34cdc2 cyclin-dependent kinase. It has been hypothesized that inhibition of DNA synthesis triggers down-regulation of p34cdc2 kinase activity, although this has never been shown biochemically. We analyzed the activity of p34cdc2 in wild-type and checkpoint-defective cells treated with a DNA synthesis inhibitor. Using standard in vitro assays we demonstrate that p34cdc2 kinase activity is maintained in wild-type cells arrested at the replication checkpoint. We also used a novel in vivo assay for p34cdc2 kinase activity, in which we expressed a fragment of the human retinoblastoma tumor suppressor protein in fission yeast. Phosphorylation of this fragment of the human retinoblastoma tumor suppressor protein is dependent on p34cdc2 kinase activity, and this activity is also maintained in cells arrested at the replication checkpoint. These data suggest that the mechanism for cell-cycle arrest in response to incomplete DNA synthesis is not dependent on the attenuation of p34cdc2 activity. IMAGES:

所有真核生物均通过反馈调控机制，有序协调细胞周期各事件的进程。在粟酒裂殖酵母（Schizosaccharomyces pombe）中，多类检验点基因（checkpoint genes）的功能是确保有丝分裂启动前，DNA复制已完成且未出现差错。野生型细胞通常会在DNA合成被抑制或遭遇DNA损伤时发生细胞周期阻滞，尽管目前尚不明确调控该阻滞的确切分子机制。粟酒裂殖酵母中的遗传学证据表明，有丝分裂对DNA复制完成的依赖性，与p34cdc2细胞周期蛋白依赖性激酶（cyclin-dependent kinase）的调控密切相关。已有假说提出，DNA合成抑制会触发p34cdc2激酶活性的下调，但该假说尚未通过生化实验得到证实。本研究针对经DNA合成抑制剂处理的野生型细胞与检验点缺陷型细胞，分析了p34cdc2的激酶活性。通过标准体外实验（in vitro assays），我们证实：在复制检验点发生阻滞的野生型细胞中，p34cdc2激酶活性得以维持。此外，我们还采用了一种针对p34cdc2激酶活性的新型体内检测方法：在粟酒裂殖酵母中表达人类视网膜母细胞瘤抑癌蛋白（retinoblastoma tumor suppressor protein）的一段片段。该片段的磷酸化依赖于p34cdc2激酶活性，且在复制检验点阻滞的细胞中，该活性同样得以维持。上述实验结果表明，针对DNA合成不完全所引发的细胞周期阻滞机制，并非依赖于p34cdc2活性的减弱。图像：