BIOLOGICAL OVERVIEW

The initiation of DNA synthesis occurs at sites bound by a heteromeric origin recognition complex (ORC). In Drosophila, the level of the large subunit, ORC1, is modulated during cell cycle progression; changes in ORC1 concentration alter origin utilization during development. The mechanisms underlying cell cycle-dependent degradation of ORC1 have been investigated. Signals in the non-conserved N-terminal domain of ORC1 mediate its degradation upon exit from mitosis and in G1 phase by the anaphase-promoting complex (APC) in vivo. Degradation appears to be the result of direct action of the APC; the N-terminal domain is ubiquitylated by purified APC in vitro. This regulated proteolysis is potent, sufficient to generate a normal temporal distribution of protein even when transcription of ORC1 is driven by strong constitutive promoters. These observations suggest that in Drosophila, ORC1 regulates origin utilization much as does Cdc6 (see Drosophila Cdc6) in budding yeast (Araki, 2003)

Control of cell cycle progression underlies the orderly proliferation of cells essential for normal development and homeostasis in adult animals. The initiation of DNA replication commits cells in organisms as diverse as yeast and mammals to progression through a complete cycle. In addition, replication is misregulated in most tumor cells, in part accounting for their phenotype. Therefore, there is a great deal of interest in understanding the molecular mechanisms of replication initiation. The initiation of DNA synthesis is perhaps best understood in the budding yeast, where discrete well-defined sequences that act as origins in vivo were defined some time ago. The foundation for mechanistic studies was the identification of a heteromeric hexamer, the origin recognition complex (ORC), that binds to yeast origins. The ORC marks origins, serving as a platform for subsequent loading of additional factors including Cdc6. In budding yeast, ORCs are monotonous: as cells progress through the cycle, they remain constitutively bound to origins and their levels do not fluctuate even when yeast enter a prolonged quiescent period that is perhaps analogous to the G₀ phase of mammalian cells. Origin utilization is governed by recruitment of Cdc6 by ORC (for reviews see Bell, 2002a and b), the abundance of which is tightly regulated during the cell cycle (Araki, 2003 and references therein).

ORC proteins are well conserved throughout metazoans, suggesting they might constitutively mark replication origins throughout the cell cycle in other organisms as well. This idea is supported by some studies of mammalian cells and of early Xenopus embryos. The abundance of ORC proteins in these cell types is also constant throughout the cell cycle, although the Xenopus ORCs are released from chromatin during M phase, unlike their budding yeast counterparts. However, other work on human cells as well as work on hamster cells and Drosophila has revealed significantly different behavior for the largest subunit, ORC1. In cycling CHO cells, ORC1, but not other ORC subunits, is released from chromatin at the end of S phase (Natale, 2000; Li, 2002). The behavior of ORC1 in cycling HeLa and Raji human cells is even more divergent from the budding yeast paradigm (Fujita, 2002; Mendez, 2002). ORC1 levels rise in G₁ and fall during S phase. Accumulation in G₁ is due, at least in part, to modulated transcription of the orc1 gene by E2F (Ohtani, 1996), a conserved transcription factor that plays a central role in orchestrating the G₁-S transition. Following S phase entry, two events lead to the disappearance of ORC1: (1) E2F activity falls so that degraded ORC1 cannot be replenished; (2)the F-box protein Skp2 mediates degradation of ORC1 either while still bound to chromatin or very quickly upon its release, since unbound protein is undetectable (Mendez, 2002). The consequences of failing to modulate either the level of ORC1 or its association with chromatin are unclear, although it has been speculated that ORC1 may be a limiting factor for pre-replication complex formation in early G₁ (Cimbora, 2001; Li, 2002; Mendez, 2002; Araki, 2003 and references therein).

ORC1 levels are also modulated in Drosophila, where some of the consequences of misaccumulation are known (Asano, 1999). As in human cells, the level of ORC1, but not ORC2 (Pak, 1997), is modulated in proliferating cells of the Drosophila embryo or imaginal disc. ORC1 accumulates at the end of G₁ as a result of transient E2F-dependent transcription, just as in human cells. Although the precise timing of ORC1 degradation was not determined, the protein disappears sometime after S phase entry but before G₁ of the subsequent cycle. Most importantly, misexpression of ORC1 causes an ensemble of phenotypes that include aberrant S phase entry, female sterility and lethality. Taken together, these suggest that at least some of the cells in embryonic, larval, imaginal and adult tissues are extremely sensitive to the level of ORC1 (Asano, 1999). Despite its importance, the pathways responsible for cell cycle-coupled degradation of ORC1 have not been described in Drosophila (Araki, 2003 and references therein).

The timing and mechanism of ORC1 degradation has been investigated. In eye imaginal disc cells, ORC1 persists from the end of G₁ until M phase, disappearing only upon completion of mitosis and entry into the subsequent G₁ phase. This finding suggests that ORC1 is degraded by the anaphase-promoting complex (APC), a key component of the cell cycle clock responsible for the cyclical degradation of mitotic cyclins and securin. In support of this idea, ORC1 degradation in vivo has been shown to be enhanced by overexpression of fizzy-related (fzr), an activating subunit of the APC. This action of Fzr apparently is direct, since Fzr-dependent ubiquitylation of ORC1 by purified APC has been demonstrated (Araki, 2003).

The experiments described in this study suggest that modulation of ORC1 levels during cell cycle progression may provide a mechanism for controlling replication origin use in metazoans. Unlike ORC1 in budding and fission yeasts, human and fly ORC1 is available during only part of the cell cycle. Thus, ORC1 recruitment to the origin may fulfill some or all of the function supplied by Cdc6 recruitment in Saccharomyces cerevisiae, transforming an inert DNA-bound apo- complex to a replication-competent complex. According to this idea, regulated degradation of ORC1 is intrinsic to cell cycle progression in human and fly cells, allowing both organisms readily to regulate origin usage during development via ORC1. While this idea is generally attractive (Cimbora, 2001; Li, 2002; Mendez, 2002), to date the only direct supporting evidence comes from flies, where origin utilization in at least two different cell types is sensitive to the level of ORC1 (Asano, 1999; Araki, 2003 and references therein).

Different aspects of cell cycle progression are driven by two proteolytic machines: the SCF ubiquitin ligase family and the APC. In principle, either proteolytic system could suffice to degrade 'spent' ORC1 after the initiation of S phase, thereby helping to reset origins and prevent re-replication before completion of the cycle. Why then do human and fly cells degrade ORC1 at different stages of the cell cycle using different mechanisms? Three possible explanations are discussed below (Araki, 2003).

One possible explanation is that degradation via the APC, as is accomplished in Drosophila, is a mechanism to restrict origin licensing to a very narrow window at the end of M or the onset of G₁. There is a brief period during the M/G₁ transition when CycB is absent but ORC1-GFP is not yet degraded. Licensing might occur only during this interval, with subsequent Fzr-dependent degradation of ORC1 limiting origin activation to the beginning of G₁. Note that the possibility that ORC1 acts late in G₁ to license origins cannot be excluded. In either case, the timing of origin activation may be different in flies and vertebrates (for a review see Bell, 2002a), where licensing is generally permitted throughout much of G₁ (Araki, 2003).

Another possible explanation is that evading degradation at the M/G₁ boundary may facilitate the switch from a canonical four-phase cell cycle to an endocycle. Throughout the life of the fly, many cells bypass M phase and re-replicate their DNA. These cells might be able to reuse ORC1 from one cycle to the next by bypassing that portion of the cell cycle when the APC is active, from M phase into G₁. In contrast, if fly ORC1 were degraded shortly after the onset of DNA synthesis, as is the case for human ORC1, the next round of DNA synthesis would require de novo synthesis of ORC1. Such a strategy would be less efficient: it might also be impossible in differentiated cells in which E2F transcription factor activity, required for ORC1 transcription in cycling cells (for a review see Dyson, 1998), is inhibited or absent (Araki, 2003).

Another possible explanation is that the ORC may have important biological roles outside S phase, in which case prolonging the life of ORC1, the least stable component, through a greater portion of the cell cycle might be important. Three lines of evidence support the idea that the Drosophila ORC is involved in events outside S phase. (1) Genetic studies reveal that the ORC appears to play a role in regulating chromosome condensation and position effect variegation (Pak, 1997; Loupart, 2000). ORC1 may play a central role in mediating such events, since it interacts with the heterochromatin-associated protein HP-1 (Pak, 1997). (2) The terminal phenotype of cells in ORC2 and ORC5 mutant flies is not S phase arrest, as might be expected, but rather M phase arrest with irregular chromosome condensation (although these phenotypes might be secondary to defects in the rate of DNA synthesis) (Loupart, 2000; Chesnokov, 2001). (3) The phenotype of ORC3 mutant flies, as well as the distribution of ORC3 protein, suggest a novel role in controlling synaptic plasticity that is independent of the cell cycle (Pinto, 1999; Rohrbough, 1999). Further definition of the role of Drosophila ORC1 will require characterization of an appropriate mutant (Araki, 2003).

The N-terminal domain of ORC1 contains a number of signals that mediate APC-dependent degradation of other proteins, including a KEN box, several canonical D boxes (RxxLxxxxN/Q/E/D), as well as a variant D box (KxxLxxxxN) that has been shown to mediate APC-dependent degradation of Drosophila securin (Leismann, 2003). A preliminary analysis suggests that none of these is responsible for the regulated degradation of ORC1. This observation is not terribly surprising, given the recent identification of a number of other signals that mediate APC-dependent degradation of substrates in other systems. The Drosophila ORC1 signal apparently is recognized by vertebrate factors, and thus it will be of interest in future to define the signal and determine whether it mediates destruction of other fly and mammalian proteins (Araki, 2003).

Although transcriptional and post-translational mechanisms appear to contribute to setting the ORC1 level in Drosophila, cell cycle-regulated degradation plays the dominant role. E2F-dependent transcription of the ORC1 gene is narrowly confined to late G₁ and early S phase, yielding a relatively brief window for the generation of protein (Asano, 1999). Although constitutive proteolysis might suffice to degrade this protein within a relatively brief window, in fact ORC1 degradation is itself cell cycle regulated to allow catastrophic disappearance of ORC1 abruptly during exit from mitosis. Moreover, persistent APC activity into G₁ is sufficient to generate an essentially normal temporal distribution of ORC1 protein even if transcription of ORC1 is uncoupled from its normal, E2F-dependent signals and is driven constitutively. It is imagined that, in the absence of such potent proteolysis, transcriptional overexpression of ORC1 would cause much more striking organismal and cellular phenotypes than have been reported (Araki, 2003).

The work reported in this study extends the structural and functional analogy between ORC1 and Cdc6 described previously. Both ORC1 (in flies) and Cdc6 (in the yeasts) can govern origin utilization. Also, both ORC1 (in flies) and Cdc6 (in mammalian cells) are degraded by the APC. Thus, ORC1 appears to serve dual functions at origins of replication, acting as both a regulatory factor and a structural component of the origin-binding complex (Araki, 2003).

GENE STRUCTURE

cDNA clone length - 3155

Bases in 5' UTR - 111

Exons - 3

Bases in 3' UTR - 269

PROTEIN STRUCTURE

Amino Acids - 924

Structural Domains

Using sensitive methods of sequence analysis including hydrophobic cluster analysis, a hitherto undescribed family of modules, the BAH (bromo-adjacent homology) family, is described which includes proteins such as eukaryotic DNA (cytosine-5) methyltransferases, the origin recognition complex 1 (Orc1) proteins, as well as several proteins involved in transcriptional regulation. The BAH domain appears to act as a protein-protein interaction module specialized in gene silencing, as suggested for example by its interaction within yeast Orc1p with the silent information regulator Sir1p. The BAH module might therefore play an important role by linking DNA methylation, replication and transcriptional regulation (Callebaut, 1999).

date revised: 11 June 2024

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