Yamaguchi R, Dutta A
Division of Molecular Oncology, Brigham and Women's Hospital, Harvard Medical School, Boston, Massachusetts 02115, USA.
Exp Cell Res. 2000 Nov 25;261(1):271-83. doi: 10.1006/excr.2000.5053.
Replication of the mammalian genome occurs only once per cell cycle and is under strict spatiotemporal control. DNA synthesis first takes place in the inner nucleus and moves gradually to the area subjacent to the nuclear membrane as S-phase progresses. We found that proteasome inhibitors specifically reduce DNA synthesis from later replicating origins but not that from earlier replicating origins. When MG132 was added in mid S-phase and washed off in late S-phase, however, DNA synthesis resumed not at the nuclear periphery, where it was last seen, but back in the inner nucleus. Analysis of DNA from these cells showed that mid to late replicating genes were rereplicated resulting in the overreplication of DNA. Our results suggest the existence of proteasome-dependent mechanisms regulating the orderly progression of S-phase. The transient treatment of mid S-phase cells with MG132 resulted in overreplication of DNA providing an easy experimental method to perturb the "once per cell cycle" control of genome replication in mammalian cells.
哺乳动物基因组的复制在每个细胞周期中仅发生一次，且受到严格的时空控制。随着S期的进展，DNA合成首先在内核中进行，并逐渐向核膜下方的区域移动。我们发现蛋白酶体抑制剂特异性地减少了来自后期复制起点的DNA合成，但不影响早期复制起点的DNA合成。然而，当在S期中期加入MG132并在S期末期洗脱时，DNA合成不是在最后观察到的核周边恢复，而是回到内核中。对这些细胞的DNA分析表明，中期到后期复制的基因被重新复制，导致DNA过度复制。我们的结果表明存在蛋白酶体依赖性机制来调节S期的有序进展。用MG132对S期中期细胞进行短暂处理导致DNA过度复制，为扰乱哺乳动物细胞基因组复制的“每个细胞周期一次”控制提供了一种简单的实验方法。
