Abstract
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.
This is a preview of subscription content, log in via an institution to check access.
Similar content being viewed by others
References
Angers S, Salahpour A, Joly E, Hilairet S, Chelsky D, Dennis M, Bouvier M (2000) Detection of beta 2-adrenergic receptor dimerization in living cells using bioluminescence resonance energy transfer (BRET). Proc Natl Acad Sci USA 97:3684–3689
Ayoub MA, Levoye A, Delagrange P, Jockers R (2004) Preferential formation of MT1/MT2 melatonin receptor heterodimers with distinct ligand interaction properties compared with MT2 homodimers. Mol Pharmacol 66:312–321
Bell SP, Dutta A (2002) DNA replication in eukaryotic cells. Annu Rev Biochem 71:333–374
Boute N, Boubekeur S, Lacasa D, Issad T (2003) Dynamics of the interaction between the insulin receptor and protein tyrosine-phosphatase 1B in living cells. EMBO Rep 4:313–319
Boyer PD (1993) The binding change mechanism for ATP synthase-some probabilities and possibilities. Biochim Biophys Acta 1140:215–250
Dhar SK, Delmolino L, Dutta A (2001) Architecture of the human origin recognition complex. J Biol Chem 276:29067–29071
Frouin I, Montecucco A, Spadari S, Maga G (2003) DNA replication: a complex matter. EMBO Rep 4:666–670
Germain-Desprez D, Bazinet M, Bouvier M, Aubry M (2003) Oligomerization of TIF1 transcriptional regulators and interaction with ZNF74 nuclear matrix protein revealed by BRET in living cells. J Biol Chem 278:22367–22373
Grinberg AV, Hu CD, Kerppola TK (2004) Visualization of Myc/Max/Mad family dimers and the competition for dimerization in living cells. Mol Cell Biol 24:4294–4308
Hozak P, Hassan AB, Jackson DA, Cook PR (1993) Visualization of replication factories attached to nucleoskeleton. Cell 73:361–373
Hu CD, Kerppola TK (2003) Simultaneous visualization of multiple protein interactions in living cells using multicolor fluorescence complementation analysis. Nat Biotechnol 21:539–545
Hu CD, Chinenov Y, Kerppola TK (2002) Visualization of interactions among bZIP and Rel family proteins in living cells using bimolecular fluorescence complementation. Mol Cell 9:789–798
Jackson DA (1995) Nuclear organization: uniting replication foci, chromatin domains and chromosome structure. Bioessays 17:587–591
Kelman Z, Hurwitz J (1998) Protein-PCNA interactions: a DNA-scanning mechanism? Trends Biochem Sci 23:236–238
Kneissl M, Putter V, Szalay AA, Grummt F (2003) Interaction and assembly of murine pre-replicative complex proteins in yeast and mouse cells. J Mol Biol 327:111–128
Labib K, Tercero JA, Diffley JF (2000) Uninterrupted MCM2-7 function required for DNA replication fork progression. Science 289:1643–1647
Laskey RA, Madine MA (2003) A rotary pumping model for helicase function of MCM proteins at a distance from replication forks. EMBO Rep 4:26–30
Laskey RA, Goerlich D, Madine MA, Makkerh JPS, Romanowski P (1996) Regulatory roles of the nuclear envelope. Exp Cell Res 229:204–211
Leonhardt H, Rahn HP, Weinzierl P, Sporbert A, Cremer T, Zink D, Cardoso MC (2000) Dynamics of DNA replication factories in living cells. J Cell Biol 149:271–280
Mercier JF, Salahpour F, Angers S, Breit A, Bouvier M (2002) Quantitative assessment of beta 1- and beta 2-adrenergic receptor homo- and heterodimerization by bioluminescence resonance energy transfer. J Biol Chem 277:44925–44931
Nakayasu H, Berezney R (1989) Mapping replication sites in the eukaryotic cell nucleus. J Cell Biol 108:1–11
O´Keefe RT, Henderson SC, Spector DL (1992) Dynamic organization of DNA replication in mammalian cell nuclei: spatially and temporally defined replication of chromosome-specific α-satellite DNA sequences. J Cell Biol 116:1095–1110
Pasion SG, Forsburg SL (1999) Nuclear localization of Schizosaccharomyces pombe Mcm2/Cdc19p requires MCM complex assembly. Mol Biol Cell 10:4043–4057
Prasanth SG, Mendez J, Prasanth KV, Stillman B (2004a) Dynamics of pre-replication complex proteins during the cell division cycle. Philos Trans R Soc Lond B Biol Sci 359:7–16
Prasanth SG, Prasanth KV, Siddiqui K, Spector DL, Stillman B (2004b) Human Orc2 localizes to centrosomes, centromeres and heterochromatin during chromosome inheritance. EMBO J 23:2651–2663
Radichev I, Kwon SW, Zhao Y, DePamphilis ML, Vassilev A (2006) Genetic analysis of human Orc2 reveals specific domains that are required in vivo for assembly and nuclear localization of the origin recognition complex. J Biol Chem 281:23264–23273
Schwacha A, Bell SP (2001) Interactions between two catalytically distinct MCM subgroups are essential for coordinated ATP hydrolysis and DNA replication. Mol Cell 8:1093–1104
Springer J, Nanda I, Hoehn K, Schmid M, Grummt F (1999) Identification and chromosomal localization of murine ORC3, a new member of the mouse origin recognition complex. Cytogenet Cell Genet 87:245–251
Takahara K, Bong M, Brevard R, Eddy RL, Haley LL, Sait SJ, Shows TB, Hoffman GG, Greenspan DS (1996) Mouse and human homologues of the yeast origin of replication recognition complex subunit ORC2 and chromosomal localization of the cognate human gene ORC2L. Genomics 31:119–122
Vashee S, Simancek P, Challberg MD, Kelly TJ (2001) Assembly of the human origin recognition complex. J Biol Chem 276:26666–26673
Wang Y, Wang G, O’Kane DJ, Szalay AA (2001) A study of protein–protein interaction in living cells using luminescence resonance energy transfer (LRET) from Renilla luciferase to Aequorea GFP. Mol Gen Genet 264:578–587
Wyman C, Botchan M (1995) A familiar ring to DNA polymerase processivity. Curr Biol 5:334–337
Xu Y, Piston DW, Johnson CH (1999) A bioluminescence resonance energy transfer (BRET) system: application to interacting circadian clock proteins. Proc Natl Acad Sci USA 96:151–156
You Z, Ishimi Y, Masai H, Hanaoka F (2002) Roles of Mcm7 and Mcm4 subunits in the DNA helicase activity of the mouse Mcm4/6/7 complex. J Biol Chem 277:42471–42479
Acknowledgments
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.
Author information
Authors and Affiliations
Corresponding author
Additional information
Communicated by A. Aguilera.
Rights and permissions
About this article
Cite this article
Brand, N., Faul, T. & Grummt, F. Interactions and subcellular distribution of DNA replication initiation proteins in eukaryotic cells. Mol Genet Genomics 278, 623–632 (2007). https://doi.org/10.1007/s00438-007-0278-1
Received:
Accepted:
Published:
Issue Date:
DOI: https://doi.org/10.1007/s00438-007-0278-1