University of Newcastle, The Institute for Cell and Molecular Biosciences, Framlington Place, NE2 4HH, Newcastle-Upon-Tyne, UK.
Development. 2012 Jan;139(1):63-74. doi: 10.1242/dev.068676. Epub 2011 Nov 17.
Cdt1 plays a key role in licensing DNA for replication. In the somatic cells of metazoans, both Cdt1 and its natural inhibitor geminin show reciprocal fluctuations in their protein levels owing to cell cycle-dependent proteolysis. Here, we show that the protein levels of Cdt1 and geminin are persistently high during the rapid cell cycles of the early Xenopus embryo. Immunoprecipitation of Cdt1 and geminin complexes, together with their cell cycle spatiotemporal dynamics, strongly supports the hypothesis that Cdt1 licensing activity is regulated by periodic interaction with geminin rather than its proteolysis. Overexpression of ectopic geminin slows down, but neither arrests early embryonic cell cycles nor affects endogenous geminin levels; apparent embryonic lethality is observed around 3-4 hours after mid-blastula transition. However, functional knockdown of geminin by ΔCdt1_193-447, which lacks licensing activity and degradation sequences, causes cell cycle arrest and DNA damage in affected cells. This contributes to subsequent developmental defects in treated embryos. Our results clearly show that rapidly proliferating early Xenopus embryonic cells are able to regulate replication licensing in the persistent presence of high levels of licensing proteins by relying on changing interactions between Cdt1 and geminin during the cell cycle, but not their degradation.
Cdt1 在为复制授权 DNA 方面发挥着关键作用。在后生动物的体细胞中，由于细胞周期依赖性蛋白水解，Cdt1 和其天然抑制剂 geminin 的蛋白水平都呈现出相互波动。在这里，我们表明，在早期 Xenopus 胚胎快速的细胞周期中，Cdt1 和 geminin 的蛋白水平持续升高。Cdt1 和 geminin 复合物的免疫沉淀，以及它们在细胞周期中的时空动态，强烈支持了这样一种假设，即 Cdt1 的许可活性是通过与 geminin 的周期性相互作用而不是其蛋白水解来调节的。异位 geminin 的过表达虽然会减缓，但既不会阻止早期胚胎细胞周期，也不会影响内源性 geminin 水平；在中胚层转换后约 3-4 小时观察到明显的胚胎致死性。然而，缺乏许可活性和降解序列的 ΔCdt1_193-447 对 geminin 的功能敲低会导致受影响细胞的细胞周期停滞和 DNA 损伤。这导致了处理胚胎的后续发育缺陷。我们的研究结果清楚地表明，快速增殖的早期 Xenopus 胚胎细胞能够通过在细胞周期中改变 Cdt1 和 geminin 之间的相互作用来调节复制许可，而不是依赖它们的降解，从而在高水平许可蛋白的持续存在下调节复制许可。
