Interactions and subcellular distribution of DNA replication initiation proteins in eukaryotic cells
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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.
KeywordsDNA replication Pre-replicative complex (pre-RC) Nuclear import Bioluminescence resonance energy transfer (BRET) Bimolecular fluorescence complementation (BiFC)
We thank Dr. T. Kerppola (University of Michigan, Ann Arbor, MI) for kindly providing the plasmids for the BiFC assays, M. Lepke for the construction of ORC2-Rluc, Dr. Christoph Lambert (University of Würzburg) for making available the spectrofluorometer and V. Kriegisch, R. Stahl and E. Gärtner for expert technical assistance. This work was financially supported by the Deutsche Forschungsgemeinschaft.
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