Coverley D, Wilkinson H R, Downes C S
Department of Zoology, University of Cambridge, United Kingdom.
Exp Cell Res. 1996 Jun 15;225(2):294-300. doi: 10.1006/excr.1996.0179.
Eukaryotic cells normally replicate their DNA only once between mitoses. Unlike G1 nuclei, intact G2 nuclei do not replicate during incubation in Xenopus egg extract. However, artificial permeabilization of the nuclear membrane of G2 nuclei allows induction of new initiations by Xenopus egg extract. This is consistent with the action of a replication licensing factor which is believed to enter the nucleus when the nuclear membrane breaks down at mitosis. Here, we show that G2 nuclei will initiate a new round of replication in the absence of nuclear membrane permeabilization, if they are preexposed to protein kinase inhibitors in vivo. Competence to rereplicate is generated within 30 min of drug treatment, well before the scheduled onset of mitosis. This demonstrates that a protein kinase-dependent mechanism is continually active in G2 phase to actively prevent regeneration of replication capacity in mammalian cells. Kinase inhibition in G2 cells causes nuclear accumulation of replication protein A. Rereplication of kinase-inhibited G2 nuclei also depends on factors supplied by Xenopus egg extract, which are distinct from those required for replication licensing.
真核细胞通常在有丝分裂之间仅复制一次DNA。与G1期细胞核不同，完整的G2期细胞核在非洲爪蟾卵提取物中孵育时不会进行复制。然而，人工使G2期细胞核的核膜通透化可使非洲爪蟾卵提取物诱导新的起始复制。这与复制许可因子的作用一致，据信该因子在有丝分裂时核膜破裂时进入细胞核。在此，我们表明，如果G2期细胞核在体内预先暴露于蛋白激酶抑制剂，则它们在没有核膜通透化的情况下也会启动新一轮复制。重新复制的能力在药物处理后30分钟内产生，远早于预定的有丝分裂开始时间。这表明蛋白激酶依赖性机制在G2期持续活跃，以积极防止哺乳动物细胞中复制能力的再生。G2期细胞中的激酶抑制导致复制蛋白A在细胞核中积累。激酶抑制的G2期细胞核的重新复制也依赖于非洲爪蟾卵提取物提供的因子，这些因子与复制许可所需的因子不同。
