Goldar Arach, Labit Hélène, Marheineke Kathrin, Hyrien Olivier
Service de Biologie Intégrative et de Génétique Moléculaire, Commissariat à l'Energie Atomique, Gif-sur-Yvette, France.
PLoS One. 2008 Aug 6;3(8):e2919. doi: 10.1371/journal.pone.0002919.
Eukaryotic cells seem unable to monitor replication completion during normal S phase, yet must ensure a reliable replication completion time. This is an acute problem in early Xenopus embryos since DNA replication origins are located and activated stochastically, leading to the random completion problem. DNA combing, kinetic modelling and other studies using Xenopus egg extracts have suggested that potential origins are much more abundant than actual initiation events and that the time-dependent rate of initiation, I(t), markedly increases through S phase to ensure the rapid completion of unreplicated gaps and a narrow distribution of completion times. However, the molecular mechanism that underlies this increase has remained obscure.
METHODOLOGY/PRINCIPAL FINDINGS: Using both previous and novel DNA combing data we have confirmed that I(t) increases through S phase but have also established that it progressively decreases before the end of S phase. To explore plausible biochemical scenarios that might explain these features, we have performed comparisons between numerical simulations and DNA combing data. Several simple models were tested: i) recycling of a limiting replication fork component from completed replicons; ii) time-dependent increase in origin efficiency; iii) time-dependent increase in availability of an initially limiting factor, e.g. by nuclear import. None of these potential mechanisms could on its own account for the data. We propose a model that combines time-dependent changes in availability of a replication factor and a fork-density dependent affinity of this factor for potential origins. This novel model quantitatively and robustly accounted for the observed changes in initiation rate and fork density.
CONCLUSIONS/SIGNIFICANCE: This work provides a refined temporal profile of replication initiation rates and a robust, dynamic model that quantitatively explains replication origin usage during early embryonic S phase. These results have significant implications for the organisation of replication origins in higher eukaryotes.
真核细胞似乎无法在正常S期监测复制完成情况，但必须确保可靠的复制完成时间。这在非洲爪蟾早期胚胎中是一个尖锐的问题，因为DNA复制起点是随机定位和激活的，导致了随机完成问题。DNA梳理、动力学建模以及其他使用非洲爪蟾卵提取物的研究表明，潜在起点比实际起始事件丰富得多，并且起始的时间依赖性速率I(t)在整个S期显著增加，以确保未复制间隙的快速完成以及完成时间的窄分布。然而，这种增加背后的分子机制仍然不清楚。
方法/主要发现：利用先前和新的DNA梳理数据，我们证实了I(t)在S期增加，但也确定它在S期末之前逐渐下降。为了探索可能解释这些特征的合理生化情况，我们对数值模拟和DNA梳理数据进行了比较。测试了几个简单模型：i) 从已完成的复制子中回收有限的复制叉成分；ii) 起点效率的时间依赖性增加；iii) 最初有限因子可用性的时间依赖性增加，例如通过核输入。这些潜在机制都无法单独解释这些数据。我们提出了一个模型，该模型结合了复制因子可用性的时间依赖性变化以及该因子对潜在起点的叉密度依赖性亲和力。这个新模型定量且稳健地解释了观察到的起始速率和叉密度的变化。
结论/意义：这项工作提供了复制起始速率的精细时间分布以及一个稳健的动态模型，该模型定量解释了早期胚胎S期复制起点的使用情况。这些结果对高等真核生物中复制起点的组织具有重要意义。
