Dasso M, Smythe C, Milarski K, Kornbluth S, Newport J W
Department of Biology, University of California, San Diego, La Jolla 92093.
Ciba Found Symp. 1992;170:161-80; discussion 180-6. doi: 10.1002/9780470514320.ch11.
Somatic cells possess control mechanisms which monitor DNA replication and assure that it is complete before mitosis is initiated. We have been investigating these mechanisms in Xenopus egg extracts. Using in vitro cycling extracts, which spontaneously alternate between interphase and mitosis, we found that the onset of mitosis is inhibited by the presence of unreplicated DNA, demonstrating that the completion of DNA replication and the initiation of mitosis are coupled in these extracts. As in somatic cells, this coupling is sensitive to caffeine and to okadaic acid. In Xenopus extracts unreplicated DNA increases the tyrosine phosphorylation of p34cdc2, thereby maintaining MPF (mitosis-promoting factor) in an inactive state and preventing the onset of mitosis. The block to mitosis in the presence of unreplicated DNA can be reversed by the addition of bacterially expressed cdc25 protein. The extent of MPF activation by cdc25 protein under these conditions depends on the number of nuclei present. We have developed an assay to examine the rate of tyrosine phosphorylation on p34cdc2. It is increased by unreplicated DNA, in a manner consistent with unreplicated DNA up-regulating the kinase that phosphorylates p34cdc2. We have begun to examine how unreplicated DNA generates the signal that inhibits MPF activation by testing the ability of naked single- and double-stranded DNA templates to inhibit mitosis, and by investigating the role of RCC1, a chromatin-associated protein required for the coupling of DNA replication and mitosis.
体细胞拥有监控DNA复制的控制机制，并确保在有丝分裂开始前DNA复制完成。我们一直在非洲爪蟾卵提取物中研究这些机制。利用在体外能在间期和有丝分裂之间自发交替的循环提取物，我们发现有未复制的DNA存在时，有丝分裂的起始会受到抑制，这表明在这些提取物中DNA复制的完成与有丝分裂的起始是相互关联的。与体细胞一样，这种关联对咖啡因和冈田酸敏感。在非洲爪蟾提取物中，未复制的DNA会增加p34cdc2的酪氨酸磷酸化，从而使MPF（促有丝分裂因子）维持在无活性状态并阻止有丝分裂的起始。添加细菌表达的cdc25蛋白可逆转未复制DNA存在时对有丝分裂的阻断。在这些条件下，cdc25蛋白激活MPF的程度取决于细胞核的数量。我们开发了一种检测方法来检测p34cdc2上酪氨酸磷酸化的速率。未复制的DNA会使其增加，其方式与未复制的DNA上调磷酸化p34cdc2的激酶一致。我们已开始通过测试裸露的单链和双链DNA模板抑制有丝分裂的能力，以及通过研究RCC1（一种DNA复制与有丝分裂偶联所需的染色质相关蛋白）的作用，来探究未复制的DNA如何产生抑制MPF激活的信号。
