Section of Biochemistry and Cell Biology, Division of Life Science, Hong Kong University of Science and Technology, Clear Water Bay, Kowloon, Hong Kong, China.
J Cell Sci. 2010 Nov 15;123(Pt 22):3933-43. doi: 10.1242/jcs.075366. Epub 2010 Oct 27.
In eukaryotes, replication licensing is achieved through sequential loading of several replication-initiation proteins onto replication origins to form pre-replicative complexes (pre-RCs), and unscheduled replication licensing is prevented by cyclin-dependent kinases (CDKs) through inhibitory phosphorylations of multiple initiation proteins. It is known that CDK inactivation during mitotic exit promotes pre-RC formation for the next cell cycle. However, whether the removal of the inhibitory phosphorylations on the initiation proteins is essential and the identity of the acting phosphatase(s) remain unknown. Here, we show that cell division cycle protein 14 (Cdc14p) dephosphorylates replication-initiation proteins Orc2p, Orc6p, Cdc6p and Mcm3p to restore their competence for pre-RC assembly in the budding yeast Saccharomyces cerevisiae. Cells without functional Cdc14p fail to dephosphorylate initiation proteins and to form pre-RCs - even when CDK activities are suppressed - and cannot replicate DNA in mitotic rereplication systems, whereas pulsed ectopic expression of Cdc14p in mitotic cells results in efficient pre-RC assembly and DNA rereplication. Furthermore, Cdc14p becomes dispensable for DNA rereplication in mitotic cells with combined non-phosphorylatable and/or phosphorylation-insensitive alleles of the initiation proteins. These data unravel the essential role of Cdc14p in replication licensing, beyond its established functions in mitotic exit, providing new insight into the intricate regulation of DNA replication through the interplay of CDKs and the Cdc14p phosphatase.
在真核生物中，通过将几个起始蛋白依次加载到复制起始点上来形成预复制复合物（pre-RC），从而实现复制的起始蛋白加载，并且细胞周期蛋白依赖性激酶（CDKs）通过对多个起始蛋白的抑制性磷酸化来阻止非计划的复制起始蛋白加载。已知在有丝分裂退出过程中 CDK 的失活会促进下一个细胞周期的 pre-RC 形成。然而，起始蛋白上抑制性磷酸化的去除是否是必需的，以及起作用的磷酸酶的身份仍然未知。在这里，我们表明细胞分裂周期蛋白 14（Cdc14p）可以去磷酸化起始蛋白 Orc2p、Orc6p、Cdc6p 和 Mcm3p，以恢复它们在芽殖酵母酿酒酵母中组装 pre-RC 的能力。没有功能正常的 Cdc14p 的细胞不能去磷酸化起始蛋白，不能形成 pre-RC-即使 CDK 活性受到抑制-并且不能在有丝分裂重复复制系统中复制 DNA，而在有丝分裂细胞中脉冲异位表达 Cdc14p 会导致有效的 pre-RC 组装和 DNA 重复复制。此外，在起始蛋白的非磷酸化和/或磷酸化不敏感等位基因的组合存在的有丝分裂细胞中，Cdc14p 对于 DNA 重复复制变得可有可无。这些数据揭示了 Cdc14p 在复制起始蛋白加载中的关键作用，超出了其在有丝分裂退出中的既定功能，为通过 CDK 和 Cdc14p 磷酸酶的相互作用对 DNA 复制的复杂调控提供了新的见解。
