Cell cycle regulation of the yeast Cdc7 protein kinase by association with the Dbf4 protein
Cell cycle regulation of the yeast Cdc7 protein kinase by association with the Dbf4 protein
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DOI：
10.1128/mcb.13.5.2899-2908.1993
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发表时间：
1993-05
影响因子：
5.3
通讯作者：
A. Jackson;P. Pahl;'. K. Harrison;J. Rosamond;'. ROBERTA.SCLAFANI
中科院分区：
文献类型：
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作者：
A. Jackson;P. Pahl;'. K. Harrison;J. Rosamond;'. ROBERTA.SCLAFANI
酵母Cdc 7蛋白激酶和Dbf 4蛋白都是在有丝分裂细胞周期的G1/S期边界启动DNA复制所必需的。Cdc 7激酶功能在细胞周期中具有阶段特异性，但总Cdc 7蛋白水平保持不变。因此，Cdc 7功能的调节似乎是翻译后修饰的结果。在这项研究中，我们试图阐明负责实现这个特定的执行点Cdc 7的机制。Cdc 7激酶的活性被证明是最大的G1/S边界，通过使用与α因子或Cdc-突变体或与DNA合成或有丝分裂的抑制剂同步的培养物。因此，Cdc 7激酶受翻译后机制调节，该机制确保Cdc 7在G1/S边界的最大活性，这与Cdc 7在细胞周期中的功能一致。这种细胞周期依赖性调节可能是与Dbf 4蛋白相关的结果。在这项研究中，Dbf 4蛋白被证明是所需的Cdc 7激酶活性，Cdc 7激酶活性是热不稳定的体外提取物时，从温度敏感的dbf 4突变体生长在允许的条件下使用。在体外重建测定，除了就业的双杂交系统的蛋白质-蛋白质相互作用，已经证明，Cdc 7和Dbf 4蛋白质相互作用，在体外和体内。一个抑制突变，bob 1 -1，它可以绕过cdc 7和dbf 4的缺失突变被分离出来。然而，bob 1 -1突变不能绕过G1期的所有事件，因为它不能抑制温度敏感的cdc 4或cdc 28突变。这表明Cdc 7和Dbf 4蛋白在细胞周期中的共同点起作用。因此，由于这两种蛋白质的功能的共同点和Dbf 4蛋白对Cdc 7功能是必不可少的事实，我们建议Dbf 4可能代表特异性激活Cdc 7激酶的细胞周期蛋白样分子。
Yeast Cdc7 protein kinase and Dbf4 protein are both required for the initiation of DNA replication at the G1/S phase boundary of the mitotic cell cycle. Cdc7 kinase function is stage-specific in the cell cycle, but total Cdc7 protein levels remained unchanged. Therefore, regulation of Cdc7 function appears to be the result of posttranslational modification. In this study, we have attempted to elucidate the mechanism responsible for achieving this specific execution point of Cdc7. Cdc7 kinase activity was shown to be maximal at the G1/S boundary by using either cultures synchronized with alpha factor or Cdc- mutants or with inhibitors of DNA synthesis or mitosis. Therefore, Cdc7 kinase is regulated by a posttranslational mechanism that ensures maximal Cdc7 activity at the G1/S boundary, which is consistent with Cdc7 function in the cell cycle. This cell cycle-dependent regulation could be the result of association with the Dbf4 protein. In this study, the Dbf4 protein was shown to be required for Cdc7 kinase activity in that Cdc7 kinase activity is thermolabile in vitro when extracts prepared from a temperature-sensitive dbf4 mutant grown under permissive conditions are used. In vitro reconstitution assays, in addition to employment of the two-hybrid system for protein-protein interactions, have demonstrated that the Cdc7 and Dbf4 proteins interact both in vitro and in vivo. A suppressor mutation, bob1-1, which can bypass deletion mutations in both cdc7 and dbf4 was isolated. However, the bob1-1 mutation cannot bypass all events in G1 phase because it fails to suppress temperature-sensitive cdc4 or cdc28 mutations. This indicates that the Cdc7 and Dbf4 proteins act at a common point in the cell cycle. Therefore, because of the common point of function for the two proteins and the fact that the Dbf4 protein is essential for Cdc7 function, we propose that Dbf4 may represent a cyclin-like molecule specific for the activation of Cdc7 kinase.