Chromatin binding of the fission yeast replication factor mcm4 occurs during anaphase and requires ORC and cdc18
Chromatin binding of the fission yeast replication factor mcm4 occurs during anaphase and requires ORC and cdc18
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DOI：
10.1093/emboj/19.7.1681
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发表时间：
2000-04-03
期刊：
影响因子：
11.4
通讯作者：
Lindner, K
中科院分区：
文献类型：
--
作者：
Kearsey, SE;Montgomery, S;Lindner, K
我们描述了一种原位技术，用于研究裂变酵母裂糖酵母中蛋白质的染色质结合，在用绿色荧光蛋白（GFP）标记感兴趣的蛋白质后，通过细胞渗透和非离子洗涤剂洗涤，用GFP荧光检测染色质相关蛋白。细胞形态和核结构在这个过程中被保留，允许结构，如有丝分裂纺锤体被间接免疫荧光检测。因此，可以从异步培养的单个细胞中确定蛋白质染色质关联的细胞周期变化。我们将这种方法应用于DNA复制因子mcm4/cdc21，发现染色质关联发生在B后期，明显早于出芽酵母。mcm4与染色质的结合需要orc1和cdc18（在出芽酵母中与Cdc6同源）。mcm4从染色质中释放发生在S期，需要DNA复制。当过表达cdc18时，我们发现在没有有丝分裂的情况下，基因组的再复制需要mcm4，并且在进行再复制的细胞中与染色质相关。
We describe an in situ technique for studying the chromatin binding of proteins in the fission yeast Schizosaccharomyces pombe, After tagging the protein of interest with green fluorescent protein (GFP), chromatin-associated protein is detected by GFP fluorescence following cell permeabilization and washing with a non-ionic detergent. Cell morphology and nuclear structure are preserved in this procedure, allowing structures such as the mitotic spindle to be detected by indirect immunofluorescence. Cell cycle changes in the chromatin association of proteins can therefore be determined from individual cells in asynchronous cultures. We have applied this method to the DNA replication factor mcm4/cdc21, and find that chromatin association occurs during anaphase B, significantly earlier than is the case in budding yeast. Binding of mcm4 to chromatin requires orc1 and cdc18 (homologous to Cdc6 in budding yeast). Release of mcm4 from chromatin occurs during S phase and requires DNA replication, Upon overexpressing cdc18, we show that mcm4 is required for re-replication of the genome in the absence of mitosis and is associated with chromatin in cells undergoing re-replication.