Lucas I, Chevrier-Miller M, Sogo J M, Hyrien O
Laboratoire de Génétique Moléculaire, Ecole Normale Supérieure, 46 rue d'Ulm, Paris Cedex 05, 75230, France.
J Mol Biol. 2000 Feb 25;296(3):769-86. doi: 10.1006/jmbi.2000.3500.
Chromosome replication initiates without sequence specificity at average intervals of approximately 10 kb during the rapid cell cycles of early Xenopus embryos. If the distribution of origins were random, some inter-origin intervals would be too long to be fully replicated before the end of S phase. To investigate what ensures rapid completion of DNA replication, we have examined the replication intermediates of plasmids of various sizes (5.3-42.2 kbp) in Xenopus egg extracts by two-dimensional gel electrophoresis and electron microscopy. We confirm that replication initiates without sequence specificity on all plasmids. We demonstrate for the first time that multiple initiation events occur on large plasmids, but not on small (<10 kb) plasmids, at average intervals of approximately 10 kb. Origin interference may prevent multiple initiation events on small plasmids. Multiple initiation events are neither synchronous nor regularly spaced. Bubble density is higher on later than on earlier replication intermediates, showing that initiation frequency increases throughout S phase, speeding up replication of late intermediates. We suggest that potential origins are abundant and randomly distributed, but that the increase of initiation frequency during S phase, and possibly origin interference, regulate origin activation to ensure rapid completion of replication.
在非洲爪蟾早期胚胎快速的细胞周期中，染色体复制以平均约10 kb的间隔无序列特异性地起始。如果起始位点的分布是随机的，那么一些起始位点间的间隔就会过长，以至于在S期结束前无法完全复制。为了研究是什么确保了DNA复制的快速完成，我们通过二维凝胶电泳和电子显微镜检查了非洲爪蟾卵提取物中各种大小（5.3 - 42.2 kbp）质粒的复制中间体。我们证实所有质粒上的复制均无序列特异性地起始。我们首次证明，大型质粒上会发生多次起始事件，而小型（<10 kb）质粒上则不会，平均间隔约为10 kb。起始位点干扰可能会阻止小型质粒上发生多次起始事件。多次起始事件既不同步也不规律分布。后期复制中间体的泡密度高于早期，这表明在整个S期起始频率增加，加速了后期中间体的复制。我们认为潜在的起始位点丰富且随机分布，但S期起始频率的增加以及可能的起始位点干扰会调节起始位点的激活，以确保复制的快速完成。
