Brand Normen, Faul Thomas, Grummt Friedrich
Department of Biochemistry, University of Würzburg, Biozentrum, Am Hubland, 97074 Würzburg, Germany.
Mol Genet Genomics. 2007 Dec;278(6):623-32. doi: 10.1007/s00438-007-0278-1. Epub 2007 Aug 7.
For initiation of eukaryotic DNA replication the origin recognition complex (ORC) associates with chromatin sites and constitutes a landing pad allowing Cdc6, Cdt1 and MCM proteins to accomplish the pre-replication complex (pre-RC). In S phase, the putative MCM helicase is assumed to move away from the ORC to trigger DNA unwinding. By using the fluorescence-based assays bioluminescence resonance energy transfer (BRET) and bimolecular fluorescence complementation (BiFC) we show in live mammalian cells that one key interaction in pre-RC assembly, the interaction between Orc2 and Orc3, is not restricted to the nucleus but also occurs in the cytoplasm. BRET assays also revealed a direct interaction between Orc2 and nuclear localization signal (NLS)-depleted Orc3. Further, we assessed the subcellular distribution of Orc2 and Orc3 in relation to MCM proteins Mcm3 and Mcm6 as well as to a key protein involved in elongation of DNA replication, proliferating nuclear cell antigen (PCNA). Our findings illustrate the spatial complexity of the elaborated process of DNA replication as well as that the BRET and BiFC techniques are novel tools that could contribute to our understanding of the processes at the very beginning of the duplication of the genome.
对于真核生物DNA复制的起始，起始识别复合物（ORC）与染色质位点结合，并构成一个着陆平台，使Cdc6、Cdt1和MCM蛋白能够完成预复制复合物（pre-RC）的组装。在S期，假定的MCM解旋酶被认为会从ORC上移开以触发DNA解旋。通过基于荧光的生物发光共振能量转移（BRET）和双分子荧光互补（BiFC）分析，我们在活的哺乳动物细胞中表明，pre-RC组装中的一个关键相互作用，即Orc2和Orc3之间的相互作用，不仅局限于细胞核，也发生在细胞质中。BRET分析还揭示了Orc2与核定位信号（NLS）缺失的Orc3之间的直接相互作用。此外，我们评估了Orc2和Orc3与MCM蛋白Mcm3和Mcm6以及与DNA复制延伸过程中涉及的关键蛋白增殖细胞核抗原（PCNA）相关的亚细胞分布。我们的研究结果说明了DNA复制精细过程的空间复杂性，以及BRET和BiFC技术是有助于我们理解基因组复制最初阶段过程的新工具。
