Anglana Mauro, Apiou Françoise, Bensimon Aaron, Debatisse Michelle
Institut Curie, FRE 2584, 26 rue d'Ulm, 75248 Paris, France.
Cell. 2003 Aug 8;114(3):385-94. doi: 10.1016/s0092-8674(03)00569-5.
Selection of active origins and regulation of interorigin spacing are poorly understood in mammalian cells. Using tricolor analysis of combed DNA molecules, we studied an amplified locus containing the known origin, oriGNAI3. We visualized replication firing events at this and other discrete regions and established a strict correlation between AT richness and initiation sites. We found that oriGNAI3 is the prominent origin of the domain, the firing of which correlates with silencing of neighboring sites and establishes large interorigin distances. We demonstrate that cells reversibly respond to a reduction in nucleotide availability by slowing the rate of replication fork progression; in addition, the efficiency of initiation at oriGNAI3 is lowered while other normally dormant origins in the region are activated, which results in an overall increase in the density of initiation events. Thus, nucleotide pools are involved in the specification of active origins, which in turn defines their density along chromosomes.
在哺乳动物细胞中，活跃复制起点的选择和起点间间距的调控仍知之甚少。我们通过对梳理后的DNA分子进行三色分析，研究了一个包含已知起点oriGNAI3的扩增位点。我们观察到了该位点及其他离散区域的复制起始事件，并确定了富含AT区域与起始位点之间的严格相关性。我们发现oriGNAI3是该区域的主要起点，其起始与相邻位点的沉默相关，并形成较大的起点间距离。我们证明，细胞会通过减缓复制叉前进速度来可逆地响应核苷酸可用性的降低；此外，oriGNAI3处的起始效率降低，而该区域其他通常处于休眠状态的起点被激活，这导致起始事件密度总体增加。因此，核苷酸库参与了活跃起点的指定，进而决定了它们在染色体上的密度。
