Department of Zoology, University of Cambridge, Downing Street, Cambridge CB23EJ, UK.
J Cell Sci. 2011 Jun 15;124(Pt 12):2058-69. doi: 10.1242/jcs.086561. Epub 2011 May 24.
Non-coding Y RNAs are required for the initiation of chromosomal DNA replication in mammalian cells. It is unknown how they perform this function or if they associate with a nuclear structure during DNA replication. Here, we investigate the association of Y RNAs with chromatin and their interaction with replication proteins during DNA replication in a human cell-free system. Our results show that fluorescently labelled Y RNAs associate with unreplicated euchromatin in late G1 phase cell nuclei before the initiation of DNA replication. Following initiation, Y RNAs are displaced locally from nascent and replicated DNA present in replication foci. In intact human cells, a substantial fraction of endogenous Y RNAs are associated with G1 phase nuclei, but not with G2 phase nuclei. Y RNAs interact and colocalise with the origin recognition complex (ORC), the pre-replication complex (pre-RC) protein Cdt1, and other proteins implicated in the initiation of DNA replication. These data support a molecular 'catch and release' mechanism for Y RNA function during the initiation of chromosomal DNA replication, which is consistent with Y RNAs acting as replication licensing factors.
非编码 Y RNAs 对于哺乳动物细胞中染色体 DNA 复制的起始是必需的。目前尚不清楚它们如何执行此功能，或者它们在 DNA 复制过程中是否与核结构相关联。在这里，我们在人无细胞系统中研究了 Y RNAs 与染色质的关联及其在 DNA 复制过程中与复制蛋白的相互作用。我们的结果表明，在 DNA 复制起始之前，荧光标记的 Y RNAs 在 G1 期晚期细胞核中与未复制的常染色质相关联。起始后，Y RNAs 从复制焦点中存在的新生和复制 DNA 局部位移。在完整的人细胞中，大量内源性 Y RNAs 与 G1 期核相关联，但与 G2 期核不相关联。Y RNAs 与起始识别复合物 (ORC)、复制前复合物 (pre-RC) 蛋白 Cdt1 以及其他与 DNA 复制起始相关的蛋白质相互作用并共定位。这些数据支持 Y RNA 在染色体 DNA 复制起始过程中作为复制许可因子的分子“捕获和释放”机制，这与 Y RNA 作为复制许可因子的作用一致。
