Moreau J L, Marques F, Barakat A, Schatt P, Lozano J C, Peaucellier G, Picard A, Genevière A M
Laboratoire Arago, URA 2156, Banyuls-sur Mer, F66650, France.
Dev Biol. 1998 Aug 15;200(2):182-97. doi: 10.1006/dbio.1998.8961.
Earlier work reported the important role of Cdk2 as a regulator of DNA replication in somatic cells and in Xenopus extracts. In the present report we analyze in vivo the involvement of Cdk2 in DNA replication during early embryogenesis using the first mitotic cycles of sea urchin embryos. Unfertilized Sphaerechinus granularis eggs are arrested after the second meiotic cytokinesis. Fertilization resumes the block and induces DNA replication after a short lag period, making sea urchin early embryo a good model for studying in vivo the onset of DNA replication. We show that Cdk2 as well as its potential partner cyclin A are present in the nucleus in G1 and S phase and therefore available for DNA replication. In accordance with data obtained in Xenopus egg extracts we observed that Cdk2 kinase activity is low and stable during the entire cycle. However, in contrast with this in vitro system in which Cdk2 activity is required for the onset of DNA replication, the specific inhibition of Cdk2 kinase by microinjection of the catalytically inactive Cdk2-K33R or the inhibitor p21(Cip1) does not prevent DNA replication. Because olomoucine, DMAP, and emetine treatments did not preclude DNA synthesis, neither cyclin A/Cdk1 nor cyclin B/Cdk1 kinase activities are necessary to replace the absence of Cdk2 kinase in promoting DNA replication. These data suggest that during early embryogenesis Cdks activities, in particular Cdk2, are dispensable in vivo for the initiation step of DNA replication. However, the specific localization of Cdk2 in the nucleus from the beginning of M phase to the end of S phase suggests its involvement in other mechanisms regulating DNA replication such as inhibition of DNA re-replication and/or that its regulating role is achieved through a pathway independent of the kinase activity. We further demonstrate that even after inhibition of Cdk activities, the permeabilization of the nuclear membrane is required to allow a second round of DNA replication. However, in contrast to Xenopus egg extracts, re-replication can take place in the absence of DMAP-sensitive kinase.
早期研究报道了Cdk2作为体细胞和非洲爪蟾提取物中DNA复制调节因子的重要作用。在本报告中，我们利用海胆胚胎的首次有丝分裂周期，在体内分析了Cdk2在早期胚胎发育过程中参与DNA复制的情况。未受精的粒球海胆卵在第二次减数分裂胞质分裂后停滞。受精解除阻滞并在短暂延迟后诱导DNA复制，使海胆早期胚胎成为在体内研究DNA复制起始的良好模型。我们发现Cdk2及其潜在伴侣细胞周期蛋白A在G1期和S期存在于细胞核中，因此可用于DNA复制。与在非洲爪蟾卵提取物中获得的数据一致，我们观察到Cdk2激酶活性在整个周期中较低且稳定。然而，与体外系统中DNA复制起始需要Cdk2活性不同，通过显微注射催化失活的Cdk2 - K33R或抑制剂p21(Cip1)对Cdk2激酶进行特异性抑制并不能阻止DNA复制。因为olomoucine、DMAP和放线菌酮处理并不排除DNA合成，所以细胞周期蛋白A/Cdk1和细胞周期蛋白B/Cdk1激酶活性对于替代Cdk2激酶缺失以促进DNA复制并非必需。这些数据表明，在早期胚胎发育过程中，细胞周期蛋白依赖性激酶（Cdks）活性，特别是Cdk2，在体内对于DNA复制的起始步骤是可有可无的。然而，Cdk2从M期开始到S期结束在细胞核中的特异性定位表明它参与了其他调节DNA复制的机制，如抑制DNA再复制和/或其调节作用是通过独立于激酶活性的途径实现的。我们进一步证明，即使在抑制Cdk活性后，核膜通透化对于允许第二轮DNA复制也是必需的。然而，与非洲爪蟾卵提取物不同，在没有DMAP敏感激酶的情况下也可以发生再复制。
