Origin recognition complex subunit 1
ORC1 accumulates transiently during a synchronous cycle that takes place in third larval instar eye imaginal disc cells (Asano, 1999). The onset of accumulation appears to result from transient activation of E2F-dependent transcription in the interval from late G1 to early S phase. Due to the methods of detection in these experiments, it was not possible to determine precisely when during the cycle ORC1 disappears. Confocal microscopy has been used to examine the timing of ORC1 disappearance with respect to two cell cycle markers: cyclin B (CycB), which accumulates from S phase until its destruction by the APC during mitosis, and the phospho-histone 3 (PH3) epitope, which marks M phase (Araki, 2003).
ORC1 is first detectable in the nuclei of cells that have not yet accumulated appreciable cytoplasmic CycB, consistent with the determination that ORC1 accumulates just before S phase entry. Examination of serial optical sections reveals that both proteins persist in cells as they transit G2. As the cells approach M phase, their nuclei rise apically and accumulate PH3 upon M phase entry. Remarkably, at this stage of the cell cycle, ORC1 is distributed in a 'donut' that surrounds the PH3, presumably because it has dissociated from the chromatin. Subsequently, both PH3 and ORC1 disappear as the cells complete mitosis and enter G1 of the following cycle (Araki, 2003).
The ability to monitor endogenous ORC1 in the experiments described above is limited by the level of antigen and the sensitivity of the antibody. In part to overcome these limitations and in part to monitor the regulation of various ORC1 derivatives, a transgene was generated that encodes a fusion of ORC1 to an epitope-tagged green fluorescent protein (GFP) derivative; the resulting fusion protein was expressed under control of the native orc1 promoter. Transgenic flies were generated by standard methods, and the distributions of endogenous ORC1 and transgenic ORC1-GFP were compared (Araki, 2003).
In general, the patterns of endogenous ORC1 and transgenic ORC1-GFP are indistinguishable in ovaries, embryos and the wing imaginal disc. In addition, detailed examination of the synchronous cell cycle transition in the eye disc reveals essentially identical behavior of ORC1 and ORC1-GFP: accumulation of ORC1-GFP prior to CycB in late G1 or early S phase, persistence of both ORC1-GFP and CycB throughout G2, removal of ORC1-GFP from chromatin during M phase upon accumulation of PH3, and disappearance of all three antigens (ORC1-GFP, CycB and PH3) upon entry into the subsequent G1. Thus, addition of the GFP-myc tag does not appear to perturb the cell cycle-modulated accumulation of ORC1 significantly (Araki, 2003).
It was of interest to determine whether ORC1 accumulation is regulated in a similar fashion in the asynchronously cycling cells that make up most of the imaginal discs. This seemed likely, given the appearance of ORC1 and ORC1-GFP 'donuts' in the asynchronous mitotic cells immediately anterior to the morphogenetic furrow. However, to survey the entire population of disc cells, use was made of fluorescence-activated cell sorting (FACS). In brief, imaginal discs from transgenic animals expressing ORC1-GFP under orc1 promoter control were collected, dissociated into individual cells, and sorted according to both DNA content and green fluorescence. The wing disc, which has been characterized in great detail by this method, and the eye antennal disc complex, were examined (Araki, 2003).
The cell cycle distribution of GFP-positive cells agrees with the analysis described above: ORC1-GFP is found predominantly in cells with S and G2 DNA content, consistent with the idea that it accumulates near the G1/S boundary, persists through G2, and disappears during M phase. During this stage of development, the eye and wing discs are governed by very different developmental programs; thus, the essentially identical regulation of ORC1 in these two tissues most probably reflects regulation by intrinsic cell cycle machinery rather than by developmental cues. In summary, the ORC1 in imaginal discs persists from late G1 into M phase when most of the protein is abruptly degraded before entry into the subsequent G1 (Araki, 2003).
One possible mechanism by which E2F transcription factor 2 (E2F2) could inhibit DNA replication is to act as a transcription factor to modulate the expression of at least one crucial replication factor. For example, if E2F2 was part of a repressor complex, loss of E2f2 function could lead to increases in replication gene expression that might trigger widespread DNA synthesis. In order to test this idea, an examination was made of the abundance of several mRNAs encoded by genes either known to be (e.g. Orc1, RNR2, PCNA) or possibly (e.g. Orc2 and Orc5) regulated by E2F. RNA was extracted from total follicle cell preparations and subjected to RT-PCR. Relative to rp49 controls, more Orc5 mRNA was reproducibly (n=4) detected in Df(2L)E2f2329/Df(2L)E2f2329 or Df(2L)E2f2329/E2f1-188 mutant samples, compared with wild type. An increased Orc2 mRNA level was also detected in some experiments (two out of four). For Orc1, RNR2 and PCNA there was no substantial difference in the amount of mRNA detected between wild type and E2f2 mutants. These data suggest (1) that E2F target genes are expressed at or above wild-type levels after loss of E2f2 function and (2) that de-repression of specific target genes, such as those encoding members of the ORC complex, could contribute to the inappropriate DNA synthesis seen in E2f2 mutant follicle cells (Cayirlioglu, 2001).
Proteins of the ORC complex assemble at origins of DNA replication and recruit factors (e.g. CDC45L) required to initiate bi-directional DNA synthesis. During oogenesis, localization of different ORC proteins within the follicle cell nuclei is dynamically regulated, coincident with changing patterns of DNA replication. ORC1, ORC2, ORC5, and CDC45L are distributed throughout the entire follicle cell nucleus when these cells are performing genomic replication during endocycle S phase, and after stage 10, these proteins are detected in foci that correlate with sites of chorion gene amplification. Because E2f2 mutant follicle cells fail to restrict DNA replication to gene amplification foci, the localization of replication factors was determined in follicle cells. Anti-ORC2, -ORC5 and CDC45L antibodies each label sites of chorion gene amplification in wild-type stage 10B egg chambers, presumably because these are the major sites of active DNA synthesis. This is consistent with the known role of these proteins in replication origin firing, and indeed female sterile mutants of Orc2 have reduced chorion gene amplification. In E2f2 mutant egg chambers, the distinct localization pattern of these replication proteins is lost, resulting in the detection of all three proteins throughout the entire nucleus. This phenotype is rescued by an E2f2 transgene. These data are consistent with the firing of origins in addition to those at the chorion loci causing inappropriate genomic DNA synthesis. Whether the mis-localization of ORC components and CDC45 in E2f2 mutants is a direct cause or a consequence of the ongoing ectopic replication is not known (Cayirlioglu, 2001).
Abdurashidova, G., et al. (2003). Localization of proteins bound to a replication origin of human DNA along the cell cycle. EMBO J. 22(16): 4294-4303. 12912926
Araki, M., et al. (2003). Degradation of origin recognition complex large subunit by the anaphase-promoting complex in Drosophila. EMBO J. 22: 6115-6126. 14609957
Araki, M., Yu, H. and Asano, M. (2005). A novel motif governs APC-dependent degradation of Drosophila ORC1 in vivo. Genes Dev. 19: 2458-2465. Medline abstract: 16195415
Asano, M. and Wharton, R. P. (1999). E2F mediates developmental and cell cycle regulation of ORC1 in Drosophila. EMBO J. 18(9): 2435-2448. 10228158
Badugu, R. Shareef, M. M. and Kellum, R. (2003). Novel Drosophila Heterochromatin protein 1 (HP1)/Origin recognition complex-associated protein (HOAP) repeat motif in HP1/HOAP interactions and chromocenter associations. J. Biol. Chem. 278: 34491-34498. 12826664
Beall, E. L., et al. (2002). Role for a Drosophila Myb-containing protein complex in site-specific DNA replication. Nature 420(6917): 833-837. 12490953
Bell, S. P., Mitchell, J., Leber, J., Kobayashi, R. and Stillman, B. (1995). The multidomain structure of Orc1p reveals similarity to regulators of DNA replication and transcriptional silencing. Cell 83(4): 563-8. 7585959
Bell, S. P. (2002a). The origin recognition complex: from simple origins to complex functions. Genes Dev. 16: 659-672. 11914271
Bell, S. P. and Dutta, A. (2002b). DNA replication in eukaryotic cells. Annu. Rev. Biochem. 71: 333-374. 12045100
Bosco, G., Du, W. and Orr-Weaver, T. L. (2001). DNA replication control through interaction of E2F-RB and the origin recognition complex. Nature Cell Biol. 3: 289-295. 11231579
Burke, T. W., Cook, J. G., Asano, M. and Nevins, J. R. (2001). Replication factors MCM2 and ORC1 interact with the histone acetyltransferase HBO1. J. Biol. Chem. 276(18): 15397-408. 11278932
Callebaut, I., Courvalin, J. C. and Mornon, J. P. (1999). The BAH (bromo-adjacent homology) domain: a link between DNA methylation, replication and transcriptional regulation. FEBS Lett. 446(1): 189-93. 10100640
Cayirlioglu, P., Bonnette, P. C., Dickson, M. R. and Duronio, R. J. (2001). Drosophila E2f2 promotes the conversion from genomic DNA replication to gene amplification in ovarian follicle cells. Development 128: 5085-5098. 11748144
Chesnokov, I., Gossen, M., Remus, D. and Botchan, M. (1999). Assembly of functionally active Drosophila origin recognition complex from recombinant proteins. Genes Dev. 13(10): 1289-1296. 10346817
Chesnokov, I., Remus, D. and Botchan, M. (2001). Functional analysis of mutant and wild-type Drosophila origin recognition complex. Proc. Natl. Acad. Sci. 98(21): 11997-2002. 11593009
Cimbora, D. M. and Groudine, M. (2001). The control of mammalian DNA replication: a brief history of space and timing. Cell 104: 643-646. 11257218
Claycomb, J. M., et al. (2002). Visualization of replication initiation and elongation in Drosophila, Jour. Cell Biol. 159: 225-236. 12403810
Dyson, N. (1998). The regulation of E2F by pRB-family proteins. Genes Dev., 12: 2245-2262. 9694791
Fujita, M., Ishimi, Y., Nakamura, H., Kiyono, T. and Tsurumi, T. (2002) Nuclear organization of DNA replication initiation proteins in mammalian cells. J. Biol. Chem. 277: 10354-10361. 11779870
Gardner, K. A., Rine, J. and Fox, C. A. (1999). A region of the Sir1 protein dedicated to recognition of a silencer and required for interaction with the Orc1 protein in Saccharomyces cerevisiae. Genetics 151(1): 31-44. 9872946
Gavin, K. A., Hidaka, M. and Stillman, B. (1995). Conserved initiator proteins in eukaryotes. Science 270: 1667-1671. 7502077
Grallert, B. and Nurse, P. (1996). ORC1 homolog orp1 in fission yeast plays a key role in regulating onset of S phase. Genes Dev. 10(20): 2644-54. 8895665
Iizuka, M. and Stillman, B. (1999). Histone acetyltransferase HBO1 interacts with the ORC1 subunit of the human initiator protein. J. Biol. Chem. 274(33): 23027-34. 1043847
Kearsey, S. E., Montgomery, S., Labib, K. and Lindner, K. (2000). Chromatin binding of the fission yeast replication factor mcm4 occurs during anaphase and requires ORC and cdc18. EMBO J. 19(7): 1681-90. 10747035
Kong, D., Coleman, T. R. and DePamphilis, M. L. (2003). Xenopus origin recognition complex (ORC) initiates DNA replication preferentially at sequences targeted by Schizosaccharomyces pombe ORC. EMBO J. 22: 3441-3450. 12840006
Kreitz, S., et al. (2001). The human origin recognition complex protein 1 dissociates from chromatin during S Phase in HeLa cells. J. Biol. Chem. 276: 6337-6342. 11102449
Ladenburger, E.-M. Keller, C. and Knippers, R. (2002). Identification of a binding region for human origin recognition complex proteins 1 and 2 that coincides with an origin of dna replication. Mol. Cell. Biol. 22(4): 1036-1048. 11809796
Leismann, O. and Lehner, C. F. (2003) Drosophila securin destruction involves a D-box and a KEN-box and promotes anaphase in parallel with cyclin A degradation. J. Cell Sci. 116: 2453-2460. 12724352
Li, C. J. and DePamphilis, M. L. (2002). Mammalian Orc1 protein is selectively released from chromatin and ubiquitinated during the S-to-M transition in the cell division cycle. Mol. Cell Biol. 22(1): 105-16. 11739726
Loupart, M. L., Krause, S. and Heck, M. S. M. (2000). Aberrant replication timing induces defective chromosome condensation in Drosophila ORC2 mutants. Curr. Biol. 10: 1547-1556. 11137005
Lygerou, Z. and Nurse, P. (1999). The fission yeast origin recognition complex is constitutively associated with chromatin and is differentially modified through the cell cycle. J. Cell Sci. 112: 3703-12. 10523506
MacAlpine, D. M., Rodriguez, H. K. and Bell, S. P. (2004). Coordination of replication and transcription along a Drosophila chromosome. Genes Dev. 18: 3094-3105. 15601823
Mendez, J., Zou-Yang, X. H., Kim, S. Y., Hidaka, M., Tansey, W. P. and Stillman, B. (2002). Human origin recognition complex large subunit is degraded by ubiquitin-mediated proteolysis after initiation of DNA replication. Mol. Cell, 9: 481-491. 11931757
Murakami, H., Yanow, S. K., Griffiths, D., Nakanishi, M. and Nurse P. (2002). Maintenance of replication forks and the S-phase checkpoint by Cdc18p and Orp1p. Nat. Cell Biol. 4(5): 384-8. 11988741
Muzi-Falconi, M. and Kelly, T. J. (1995). Orp1, a member of the Cdc18/Cdc6 family of S-phase regulators, is homologous to a component of the origin recognition complex. Proc. Natl. Acad. Sci. 92: 12475-12479. 8618924
Natale, D. A., Li, C. J., Sun, W. H. and DePamphilis, M. L. (2000). Selective instability of Orc1 protein accounts for the absence of functional origin recognition complexes during the M-G(1) transition in mammals. EMBO J. 19: 2728-2738. 10835370
Noguchi, K., Vassilev, A., Ghosh, S., Yates, J. L. and Depamphilis, M. L. (2006). The BAH domain facilitates the ability of human Orc1 protein to activate replication origins in vivo. EMBO J. 25(22): 5372-82. 17066079
Ogawa, Y., Takahashi, T. and Masukata, H. (1999). Association of fission yeast Orp1 and Mcm6 proteins with chromosomal replication origins. Mol. Cell. Biol. 19(10): 7228-36. 1049065
Ohta, S., et al., (2003). The ORC1 cycle in human cells: II. Dynamic changes in the human ORC complex during the cell cycle. J Biol Chem 278,41535-41540. 12909626
Ohtani, K., et al. (1996). Expression of the HsOrc1 gene, a human ORC1 homolog, is regulated by cell proliferation via the E2F transcription factor. Mol. Cell. Biol. 16: 6977-6984. 8943353
Okudaira, K., et al. (2005). Transcriptional regulation of the Drosophila orc2 gene by the DREF pathway. Biochim. Biophys. Acta 1732(1-3): 23-30. 16343659
Pak, D.T., et al. (1997). Association of the origin recognition complex with heterochromatin and HP1 in higher eukaryotes. Cell 91(3): 311-323.
Pinto, S. et al. (1999). latheo encodes a subunit of the origin recognition complex and disrupts neuronal proliferation and adult olfactory memory when mutant. Neuron 23: 45-54.
Rohrbough, J., Pinto, S., Mihalek, R. M., Tully, T. and Broadie, K. (1999). latheo, a Drosophila gene involved in learning, regulates functional synaptic plasticity. Neuron 23: 55-70.
Saha, P., Chen, J., Thome, K. C., Lawlis, S. J., Hou, Z. H., Hendricks, M., Parvin, J. D. and Dutta, A. (1998). Human CDC6/Cdc18 associates with Orc1 and cyclin-cdk and is selectively eliminated from the nucleus at the onset of S phase. Mol. Cell. Biol. 18: 2758-2767. 9566895
Saha, T., Ghosh, S., Vassilev, A. and DePamphilis, M. L. (2006). Ubiquitylation, phosphorylation and Orc2 modulate the subcellular location of Orc1 and prevent it from inducing apoptosis. J. Cell Sci. 119(Pt 7): 1371-82. 16537645
Saitoh, Y., et al. (2002). Functional domains involved in the interaction between Orc1 and transcriptional repressor AlF-C that bind to an origin/promoter of the rat aldolase B gene. Nucleic Acids Res. 30: 5205-5212. 12466545
Takeda, D. Y., Shibata, Y., Parvin, J. D. and Dutta, A. (2005). Recruitment of ORC or CDC6 to DNA is sufficient to create an artificial origin of replication in mammalian cells. Genes Dev. 19(23): 2827-36. 16322558
Tatsumi, Y., et al. (2003). The ORC1 Cycle in Human Cells I. Cell cycle-regulated oscillation of human ORC1. J. Biol. Chem. 278: 41528-41534. 12909627
Thome, K. C., et al., (2000). Subsets of human origin recognition complex (ORC) subunits are expressed in non-proliferating cells and associate with non-ORC proteins. J. Biol. Chem. 275: 35233-35241. 10954718
Vashee, S., Simancek, P., Challberg, M. D. and Kelly, T. J. (2001). Assembly of the human origin recognition complex. J. Biol. Chem. 276(28): 26666-73. 11323433
Wang, B., et al. (1999). The essential role of Saccharomyces cerevisiae CDC6 nucleotide-binding site in cell growth, DNA synthesis, and Orc1 association. J. Biol. Chem. 274(12): 8291-8. 10075735
date revised: 25 August 2008
Home page: The Interactive Fly © 2003 Thomas B. Brody, Ph.D.
The Interactive Fly resides on the
Society for Developmental Biology's Web server.