Activation of new replication foci under conditions of replication stress

DNA damage, binding of drugs to DNA or a shortage of nucleotides can decrease the rate or completely halt the progress of replication forks. Although the global rate of replication decreases, mammalian cells can respond to replication stress by activating new replication origins. We demonstrate that a moderate level of stress induced by inhibitors of topoisomerase I, commencing in early, mid or late S-phase, induces activation of new sites of replication located within or in the immediate vicinity of the original replication factories; only in early S some of these new sites are also activated at a distance greater than 300 nm. Under high stress levels very few new replication sites are activated; such sites are located within the original replication regions. There is a large variation in cellular response to stress – while in some cells the number of replication sites increases even threefold, it decreases almost twofold in other cells. Replication stress results in a loss of PCNA from replication factories and a twofold increase in nuclear volume. These observations suggest that activation of new replication origins from the pool of dormant origins within replication cluster under conditions of mild stress is generally restricted to the original replication clusters (factories) active at a time of stress initiation, while activation of distant origins and new replication factories is suppressed.

DNA损伤、药物与DNA结合或核苷酸匮乏，均可降低复制叉的行进速率，甚至完全阻断其进程。尽管整体复制速率有所下降，但哺乳动物细胞可通过激活新的复制起点以应对复制应激。本研究证实，由拓扑异构酶I抑制剂诱导的中等强度应激，若在S早期、S中期或S晚期启动，可激活位于原始复制工厂内部或其紧邻区域内的新复制位点；仅在S早期，部分此类新位点才会在300纳米以上的远处被激活。高应激水平下，极少有新复制位点被激活，且此类位点均局限于原始复制区域内。细胞对应激的响应存在显著个体差异：部分细胞的复制位点数量甚至可增至原本的三倍，而另一些细胞则会减少近两倍。复制应激会导致增殖细胞核抗原（Proliferating Cell Nuclear Antigen, PCNA）从复制工厂中流失，并使细胞核体积增加至原本的两倍。上述观测结果表明，在轻度应激条件下，从复制簇内的休眠起源池中激活新复制起点的过程，通常仅局限于应激触发时处于活跃状态的原始复制簇（即复制工厂），而远端起点及新复制工厂的激活则受到抑制。