Hendrickson M, Madine M, Dalton S, Gautier J
Roche Institute of Molecular Biology, Nutley, NJ 07110, USA.
Proc Natl Acad Sci U S A. 1996 Oct 29;93(22):12223-8. doi: 10.1073/pnas.93.22.12223.
In eukaryotes, tight regulatory mechanisms ensure the ordered progression through the cell cycle phases. The mechanisms that prevent chromosomal DNA replication from taking place more than once each cell cycle are thought to involve the function of proteins of the minichromosome maintenance (MCM) family. Here, we demonstrate that Xenopus MCM4, a member of the MCM protein family related to Spcdc21/ ScCDC54, is part of a large protein complex comprising several other MCM proteins. MCM4 undergoes cell cycle-dependent phosphorylation both in cleaving embryos and in cell-free extracts. MCM4 phosphorylation starts concomitantly with the clearing of the MCM complex from the chromatin during S phase. Phosphorylation is carried out by cdc2/cyclinB protein kinase, which phosphorylates MCM4 in vitro at identical sites as the ones phosphorylated in vivo. Phosphorylation is specific for cdc2 protein kinase since MCM4 is not a substrate for other members of the cdk family. Furthermore, phosphorylation of MCM4 dramatically reduces its affinity for the chromatin. We propose that the cell cycle-dependent phosphorylation of MCM4 is a mechanism which inactivates the MCM complex from late S phase through mitosis, thus preventing illegitimate DNA replication during that period of the cell cycle.
在真核生物中，严格的调控机制确保细胞周期各阶段有序进行。人们认为，防止染色体DNA在每个细胞周期中复制不止一次的机制涉及微小染色体维持（MCM）家族蛋白质的功能。在此，我们证明非洲爪蟾MCM4（一种与裂殖酵母Spcdc21/酿酒酵母ScCDC54相关的MCM蛋白家族成员）是一个包含其他几种MCM蛋白的大型蛋白质复合物的一部分。在分裂胚胎和无细胞提取物中，MCM4都会经历细胞周期依赖性磷酸化。MCM4磷酸化在S期与MCM复合物从染色质上清除同时开始。磷酸化由cdc2/细胞周期蛋白B蛋白激酶进行，该激酶在体外对MCM4的磷酸化位点与体内磷酸化位点相同。磷酸化对cdc2蛋白激酶具有特异性，因为MCM4不是细胞周期蛋白依赖性激酶（cdk）家族其他成员的底物。此外，MCM4的磷酸化显著降低了其对染色质的亲和力。我们提出，MCM4的细胞周期依赖性磷酸化是一种机制，该机制从S期后期到有丝分裂期间使MCM复合物失活，从而防止在细胞周期的该时期发生非法DNA复制。
