Munshi R, Leno G H
Department of Biochemistry, University of Mississippi Medical Center, Jackson 39216-4505, USA.
Exp Cell Res. 1998 May 1;240(2):321-32. doi: 10.1006/excr.1998.4019.
Nuclear membrane permeabilization is required for replication of quiescent (G0) cell nuclei in Xenopus egg extract. We now demonstrate that establishment of replication competence in G0 nuclei is dependent upon a positive activity present in the soluble egg extract. Our hypothesis is that G0 nuclei lose the license to replicate following growth arrest and that this positive activity is required for relicensing DNA for replication. To determine if G0 nuclei contain licensed DNA, we used the protein kinase inhibitor, 6-dimethylaminopurine (6-DMAP), to prepare egg extracts that are devoid of licensing activity. Intact nuclei, isolated from mammalian cells synchronized in G1-phase (licensed), G2-phase (unlicensed), and G0 were permeabilized and assayed for replication in 6-DMAP-treated and untreated extracts supplemented with [alpha-32P]dATP or biotinylated-dUTP. Very little radioactivity was incorporated into nascent DNA in each nuclear population; however, nearly all nuclei in each population incorporated biotin in 6-DMAP extract. The pattern of biotin incorporation within these nuclei was strikingly similar to the punctate pattern observed within nuclei incubated in aphidicolin-treated extract, suggesting that initiation events occur within most replication factories in 6-DMAP extract. However, density substitution and alkaline gel analyses indicate that the incorporated biotin within these nuclei arises from a small number of active origins which escape 6-DMAP inhibition. We conclude that 6-DMAP-treated egg extract cannot differentiate licensed from unlicensed mammalian somatic cell nuclei and, therefore, cannot be used to determine the "licensed state" of G0 nuclei using the assays described here.
非洲爪蟾卵提取物中静止（G0）期细胞核的复制需要核膜通透化。我们现在证明，G0期细胞核复制能力的建立依赖于可溶性卵提取物中存在的一种正向活性。我们的假设是，G0期细胞核在生长停滞后失去了复制许可，而这种正向活性是重新许可DNA进行复制所必需的。为了确定G0期细胞核是否含有已许可的DNA，我们使用蛋白激酶抑制剂6-二甲基氨基嘌呤（6-DMAP）制备了没有许可活性的卵提取物。从同步于G1期（已许可）、G2期（未许可）和G0期的哺乳动物细胞中分离出完整的细胞核，使其通透化，并在补充有[α-32P]dATP或生物素化-dUTP的经6-DMAP处理和未处理的提取物中检测其复制情况。每个核群体中新生DNA掺入的放射性都很少；然而，每个群体中的几乎所有细胞核在6-DMAP提取物中都掺入了生物素。这些细胞核内生物素掺入的模式与在阿非科林处理的提取物中孵育的细胞核内观察到的点状模式惊人地相似，这表明在6-DMAP提取物中，大多数复制工厂内都发生了起始事件。然而，密度置换和碱性凝胶分析表明，这些细胞核内掺入的生物素来自少数逃脱6-DMAP抑制的活性起始点。我们得出结论，经6-DMAP处理的卵提取物无法区分已许可和未许可的哺乳动物体细胞细胞核，因此，不能用于通过此处描述的检测方法来确定G0期细胞核的“许可状态”。
