Maiorano Domenico, Rul Wilfrid, Méchali Marcel
Institute of Human Genetics, CNRS, 34396 Montpellier Cedex 05, France.
Exp Cell Res. 2004 Apr 15;295(1):138-49. doi: 10.1016/j.yexcr.2003.11.018.
Cdt1 is a conserved replication factor required in licensing the chromosome for a single round of DNA synthesis. The activity of Cdt1 is inhibited by geminin. The mechanism by which geminin interferes with Cdt1 activity is unknown. It is thought that geminin binds to and sequestrate Cdt1. We show that geminin does not interfere with the chromatin association of Cdt1 and that inhibition of DNA synthesis by geminin is observed following its accumulation on chromatin. The binding of geminin to chromatin has been investigated during S phase. We demonstrate that loading of geminin onto chromatin requires Cdt1, suggesting that geminin is targeted at replication origins. We also show that geminin binds chromatin at the transition from the pre-replication to pre-initiation complexes, which overlaps with the release of Cdt1. This regulation is strikingly different from that observed in somatic cells where the chromatin binding of these proteins is mutually exclusive. In contrast to somatic cells, we further show that geminin is stable during the early embryonic cell cycles. These results suggest a specific regulation of origin firing adapted to the rapid cell cycles of Xenopus and indicate that periodic degradation of geminin is not relevant to licensing during embryonic development.
Cdt1是一种保守的复制因子，在许可染色体进行一轮DNA合成中发挥作用。Geminin可抑制Cdt1的活性。Geminin干扰Cdt1活性的机制尚不清楚。人们认为Geminin会结合并隔离Cdt1。我们发现Geminin并不干扰Cdt1与染色质的结合，且在Geminin在染色质上积累后才观察到其对DNA合成的抑制作用。在S期对Geminin与染色质的结合进行了研究。我们证明Geminin加载到染色质上需要Cdt1，这表明Geminin靶向复制起点。我们还表明，Geminin在从复制前复合体到起始前复合体的转变过程中与染色质结合，这与Cdt1的释放时间重叠。这种调控与体细胞中观察到的情况显著不同，在体细胞中这些蛋白质与染色质的结合是相互排斥的。与体细胞不同，我们进一步表明Geminin在早期胚胎细胞周期中是稳定的。这些结果表明，针对非洲爪蟾快速细胞周期存在一种特定的复制起点激活调控机制，并且表明Geminin的周期性降解与胚胎发育过程中的许可无关。
