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The 'ORC Cycle': a Novel Pathway for Regulating Eukaryotic DNA Replication

The 'ORC Cycle': a Novel Pathway for Regulating Eukaryotic DNA Replication

310 (2003) 1–15 www.elsevier.com/locate/gene Review The ‘ORC cycle’: a novel pathway for regulating eukaryotic DNA replication

Melvin L. DePamphilis*

National Institute of Child Health and Development, Building 6/416, 9000 Rockville Pike, National Institutes of Health, Bethesda, MD 20892-2753, USA

Received 20 December 2002; received in revised form 26 February 2003; accepted 12 March 2003 Received by A.J. van Wijnen

Abstract The function of the ‘origin recognition complex’ (ORC) in eukaryotic cells is to select genomic sites where pre-replication complexes (pre-RCs) can be assembled. Subsequent activation of these pre-RCs results in bi-directional DNA replication that originates at or close to the ORC DNA binding sites. Recent results have revealed that one or more of the six ORC subunits is modified during the G1 to S-phase transition in such a way that ORC activity is inhibited until is complete and a nuclear membrane is assembled. In , Cdk1/Clb phosphorylates ORC. In frog eggs, pre-RC assembly destabilizes ORC/ sites, and ORC is eventually hyperphosphorylated and released. In mammals, the affinity of Orc1 for chromatin is selectively reduced during S-phase and restored during early G1-phase. Unbound Orc1 is ubiquitinated during S-phase and in some cases degraded. Thus, most, perhaps all, exhibit some manifestation of an ‘ORC cycle’ that restricts the ability of ORC to initiate pre-RC assembly to the early G1-phase of the cycle, making the ‘ORC cycle’ the premier step in determining when replication begins. q 2003 Published by Elsevier Science B.V.

Keywords: Origin recognition complexes; Replication origins; Orc1; cycle; Ubiquitination; Nuclear structure

1. DNA replication and the cell division cycle commitment to DNA replication is the primary point at which cells regulate their division cycle. In contrast, cells in In normal cells, the cell division cycle is highly regulated G0-phase remain capable of proliferating for long periods of and consists of four easily recognized phases: G1-phase time (e.g. hepatocytes, lymphocytes). Alternatively, cells (preparation for DNA replication), S-phase (DNA replica- can undergo terminal differentiation (e.g. , germ tion), (preparation for mitosis) and M-phase cells), a state from which they do not reenter the cell (mitosis). Following mitosis, each cell must decide whether division cycle. Unregulated is the primary to enter G1-phase, which is a commitment to cell division, characteristic of the pathological state known as . or to enter a quiescent state (G0-phase). Once cells are One critical feature of all eukaryotic cell cycles, committed to undergoing DNA replication, they must regardless of species or developmental stage, is that complete the process of cell division or die. Thus, initiation of DNA replication is limited to once per replication origin per cell division cycle. Not all origins Abbreviations: ORC, origin recognition complex; Orc1–Orc6, ORC are used during every , but if they are, they cannot subunits; Mcm, 2–10, maintenance ; Mcm(2–7), be used a second time until after cell division has occurred. heterohexameric complex of Mcm proteins 2–7; Cdc, cell division cycle ; Cdt1, protein encoded by Cdc10 dependent transcript 1 in Presumably, this mechanism evolved to avoid the genomic Schizosaccharomyces pombe; RLF-B, Cdt1 and replication licensing instability that would invariably result if some sequences factor B are the same protein; Cdk, dependent protein ; Pre- were duplicated more than others and replication forks RCs, pre-replication complexes consisting of ORC, , Cdt1 and remained during mitosis. Therefore, it is not surprising that Mcm(2–7); Sc, Saccharomyces cerevisiae;Sp,Schizosaccharomyces the proteins and sequence of events that comprise pombe; Xl, laevis; Dm, Drosophila melanogaster. * Fax: þ1-301-480-9354. eukaryotic DNA replication are highly conserved (Bogan E-mail address: [email protected] (M.L. DePamphilis). et al., 2000; Bell and Dutta, 2002).

0378-1119/03/$ - see front matter q 2003 Published by Elsevier Science B.V. doi:10.1016/S0378-1119(03)00546-8 2 M.L. DePamphilis / Gene 310 (2003) 1–15

Eukaryotic DNA replication begins with the binding of a 2002). Pre-RCs are activated upon binding of Mcm10 six subunit ‘origin recognition complex’ (ORC) to DNA protein (Wohlschlegel et al., 2002). Cdc6 is then released by (reviewed in Bell and Dutta, 2002; outlined in Fig. 1). the cyclin dependent , Cdk2/, and Proteins Cdc6 and Cdt1(RLF-B) are then required to load replaced by Cdc45 with the help of the protein Mcm proteins 2–7 onto the ORC/chromatin site to form a Cdc7/Dbf4 and Cdk2/. Cdc45 associates with DNA pre-replication complex (pre-RC). Mcm2–Mcm7 exist as a -a: DNA and guides it to the complex to hexameric complex [Mcm(2–7)] that appears to be the initiate RNA-primed DNA synthesis. responsible for unwinding parental DNA strands. Several mechanisms have been identified that limit Presumably, there is at least one Mcm(2–7) complex per initiation of eukaryotic DNA replication to once-per-origin- replication fork, although more are possible (Edwards et al., per-cell division. In yeast, Cdc6 is phosphorylated when

Fig. 1. Sequence of events during the initiation of DNA replication in mammalian chromatin. Upper panel: chromatin from cultured, mammalian cells incubated in a Xenopus egg extract. Bottom panel: DNA replication in cultured, somatic mammalian cells. In general, ORC binds to the DNA component of chromatin, but XlORC exists as a stable complex in egg extracts, whereas HsORC exists as a stable complex of Orc(2–5) to which human Orc1 and Orc6 bind only weakly. Thus, in Xenopus egg extract, the entire XlORC binds to chromatin, but in mammalian cells only the Orc1 subunit is selectively bound during the M to G1 transition (concurrent with degradation); the remaining ORC subunits remain stably bound to chromatin throughout the cell cycle. Furthermore, in Xenopus, Cdc6 facilitates binding of ORC to hamster chromatin, but not to Xenopus chromatin. In mammals, Cdc6 associates with Orc1, and both Orc1 and Cdc6 are present in mitotic cells, suggesting that an Orc1/Cdc6 complex binds either to Orc(2–5) or to Orc(2–6) complexes that are bound to chromatin. Cdt1 then brings in at least one Mcm(2–7) hexamer per replication fork. In Xenopus, ORC is released from chromatin following stable binding of Mcm proteins. In mammals, only Orc1 is released concurrent with DNA replication. In mammals, Orc1 is also associated with some component of nuclear structure during G1-phase. Following pre-replication complex (pre-RC) assembly, DNA replication is initiated by the action of Mcm10. This allows Cdk2/cyclin A to phosphorylate and release Cdc6 and for Cdc7/Dbf4 and Cdk2/cyclin E to modify one or more proteins and allow Cdc45 to bring to the replication origin DNA polymerase-a: DNA primase, the that initiates synthesis of the first RNA-primed nascent DNA strands. This event marks the beginning of S-phase. Concomitant with DNA synthesis is the inactivation of Cdt1 by geminin, and the of Mcm proteins. Cdc6 and Cdt1 are released from chromatin and eventually degraded. Mcm proteins remain in the nucleus where they are weakly associated with chromatin. M.L. DePamphilis / Gene 310 (2003) 1–15 3 cells enter S-phase, released from pre-RCs, ubiquitinated budding yeast (S. cerevisiae), fission yeast (S. pombe), flies and degraded [(Drury et al., 1997; Weinreich et al., 2001) (Drosophila melanogaster, Sciara coprophila) and mam- and references therein]. When yeast enter M-phase, the mals [(Abdurashidova et al., 2003; Bell, 2002; Keller et al., Cdk1/cyclin B phosphorylates any newly synthesized Cdc6, 2002b; Kong and DePamphilis, 2002; Ladenburger et al., thereby preventing it from binding to ORC until mitosis is 2002) and references therein]. The budding yeast S. completed. The same mechanism exists in frogs and cerevisiae contains about two ORCs per replication origin mammals, except that in human cells phosphorylated [600 ORC/cell (Rowley et al., 1995); 332 origins/cell Cdc6 is transported out of the nucleus during S-phase and (Raghuraman et al., 2001)]. Since both ORC and Mcm remains in the during G2 and M-phases [(Jiang proteins are bound to 429 sites in the S. cerevisiae et al., 1999; Petersen et al., 1999; Coverley et al., 2000; (Wyrick et al., 2001), about 25% of these ORC/chromatin Mendez and Stillman, 2000; Petersen et al., 2000; sites do not initiate DNA replication. These pre-RCs are Delmolino and Dutta, 2001) and references therein]. During silenced when DNA replication forks from neighboring, the M to G1-transition, HsCdc6 is degraded and then earlier firing origins, pass through them (Santocanale et al., resynthesized later during G1-phase (Mendez and Stillman, 1999). Mammalian cells contain 104–105 molecules of 2000; Petersen et al., 2000). However, the amount of ORC per cell (Natale et al., 2000; Kreitz et al., 2001; Okuno chromatin bound Cdc6 in hamster cells appears to remain et al., 2001; Ohta et al., 2003), suggesting that they initiate constant throughout the cell cycle, with no indication of any replication, on average, once every 60–600 kb (Natale et al., degradation during G1 (Okuno et al., 2001). This difference 2000), consistent with the average size of replicons in may reflect a larger pool of Cdc6 in human cells than mammalian cells [200–300 kb; (Ockey and Saffhill, 1976; hamster cells. Nevertheless, under conditions where Cdc6 Yurov and Liapunova, 1977)]. These data suggest that most, cannot be phosphorylated and remains in the nucleus during if not all, ORCs are bound to replication origins. S-phase, reinitiation of DNA replication still does not occur, Initiation sites for DNA replication are distributed revealing the existence of other regulatory mechanisms throughout all eukaryotic . This insures that (Pelizon et al., 2000; Petersen et al., 2000). One of these genomes can be duplicated in a few minutes (e.g. rapidly mechanisms is inhibition of Cdt1(RLF-B) activity by cleaving of flies, frogs, and fish) to a few hours geminin, a protein produced during the S to M transition (e.g. mammalian cells) without having to change the basic (Wohlschlegel et al., 2000; Tada et al., 2001). Geminin has replication fork mechanism by which DNA is replicated. In not been found in yeast, but in yeast, as well as in mammals, yeast, flies and mammals, cis-acting sequences (replication Cdt1 is degraded during S-phase. The combined inacti- origins) determine where DNA replication will begin vation of both Cdc6 and Cdt1 prevents rebinding of the [(DePamphilis, 1999; Altman and Fanning, 2001; Lu et al., Mcm(2–7) to ORC/chromatin sites. Nevertheless, Mcm 2001; Bell and Dutta, 2002; Kong and DePamphilis, 2002) proteins themselves are phosphorylated during S-phase and and references therein], but with the exception of the small released from chromatin. This mechanism is presumably (,0.1 kb) replication origins in S. cerevisiae, no consensus triggered by termination of DNA replication when two sequence has been identified that is required for replication. oncoming replication forks collide. Moreover, in budding Replication origins in fission yeast, flies and mammals are yeast, the phosphorylated Mcm proteins are exported from five to 20 times larger than those in budding yeast. They the nucleus during the G2 to M transition, thus providing an frequently contain large AT-rich regions and genetically additional barrier to reinitiation during S-phase (Labibet al., identifiable sequences that are required for origin function. 1999; Pasion and Forsburg, 1999; Nguyen et al., 2000). In The activity of these sequences is often orientation or fission yeast as well as in the metazoa, Mcm proteins remain distance dependent. Furthermore, ORC not only binds to in the nucleus, although their affinity for chromatin is replication origins in fission yeast, flies, and mammals (Bell markedly diminished (Lei and Tye, 2001). and Dutta, 2002), but recent studies have revealed that ORC The question remains as to whether or not ORC activity binds to specific sites within replication origins in S. pombe is also regulated. (Kong and DePamphilis, 2001; Lee et al., 2001; Kong and DePamphilis, 2002) and human cells (Abdurashidova et al., 2003), and the ORC binding sites in S. pombe ARS3001 are required for origin function (Kim and Huberman, 1998; 2. Selection of initiation sites Kong and DePamphilis, 2002). One remarkable feature of eukaryotic DNA replication is Since ORC binding to DNA is the first step in pre-RC that selection of initiation sites varies from non-specific in assembly, ORC determines where DNA replication can the rapidly cleaving embryos of frogs, flies, and fish, to site- begin by binding to specific DNA sites in the genomes of specific in , flies and mammals (DePamphilis, 1999; Bell and Dutta, 2002). Moreover, as development of frogs 1 Yeast contain a single cyclin dependent protein kinase (Cdk) encoded by the Cdc28 gene in Saccharomyces cerevisiae and Cdc2 gene in (Hyrien et al., 1995) and flies (Sasaki et al., 1999) changes Schizosaccharomyces pombe. This enzyme corresponds to Cdk1 in from rapid cell events prior to the onset of zygotic metazoan cells. to the slower cell division cycles in later 4 M.L. DePamphilis / Gene 310 (2003) 1–15 embryonic stages and adult , initiation events activity in yeast even though all six ORC subunits remain changes from non-specific to site-specific. Since only one stably bound to chromatin throughout the cell cycle (see set of Orc has been discovered in frogs and flies, site- Section 8). Whether or not an ORC cycle also exists in other specific initiation must be developmentally acquired eukaryotes, such as flies, remains to be demonstrated, epigenetically. Epigenetic changes that can affect site although some evidence suggests that it does (see Section specificity include the ratio of initiation factors to DNA, 9). Release of ORC subunits from chromatin after pre-RC chromatin or nuclear composition and structure, DNA assembly, either as a programmed event during DNA methylation, and the acquisition of ORC accessory proteins replication (Sun et al., 2002), or by selective elution using that facilitate binding of ORC to specific sequences salt or cyclin-dependent protein kinase activity (Hua and (DePamphilis, 1999). Recent studies have suggested that Newport, 1998; Jares and Blow, 2000), does not interfere factors may also facilitate binding of ORC to with assembly of active replication forks. Therefore, specific DNA sites (Bosco et al., 2001; Beall et al., 2002; regulating ORC association with chromatin is a feasible Saitoh et al., 2002). mechanism for restricting reinitiation events during S-phase without interfering with DNA replication. Whether or not ORC or an ORC subcomplex binds to newly synthesized 3. The ‘ORC cycle” in summary replication origins during S-phase is unknown, but in Xenopus egg extracts (discussed below) ORC cannot rebind Reinitiation of DNA replication at the same replication to somatic cell chromatin until the next cell cycle begins. origins during a single cell division cycle is prevented in It should not be surprising that species-specific variations eukaryotic cells by regulating at least three steps in the exist in the regulation of ORC activity, because although assembly of pre-RCs – association of Cdc6, Cdt1 and ORC proteins are highly conserved within a single Mcm(2–7) proteins with chromatin (see Section 1). Recent taxonomic family, conservation among all species is results have revealed a fourth regulatory step in which ORC modest. For example, human, hamster and mouse Orc1 activity is inhibited during the G1 to S-phase transition and proteins are ,81% identical and 84% similar in their total not reestablished until mitosis is complete and a nuclear sequence, and 93% identical and 95% similar in membrane has reassembled. Therefore, regulation of ORC their C-terminal portion (Bogan et al., 2000). The C- activity becomes the premier step in determining when terminal portion contains a homology with Cdc6 protein and DNA replication begins. Cell cycle dependent changes in an ATP binding domain that appears to be required for site- ORC activity, and in some cases the affinity of one or specific DNA binding (Bell, 2002). Among all species, more ORC subunits for chromatin, is hereafter referred to however, total amino acid sequence conservation drops to as ‘the ORC cycle’. However, the manifestation of the ORC 24% identity and 35% similarity. cycle can vary among species and during development (summarized in Fig. 2). The ORC cycle was first recognized in mammalian cells 4. The ORC cycle in mammals (Natale et al., 2000; Kreitz et al., 2001) where the Orc1 subunit is selectively destabilized during S-phase, ubiqui- 4.1. Selective release Of Orc1 tinated and in some cases degraded, and then rebinds stably to chromatin during the M to G1 transition to establish pre- In mammals, both the cellular concentrations of Orc RCs at specific genomic sites (see Section 4). Since all six proteins 2–6 and the amount of each protein bound to ORC subunits are required for ORC activity in yeast (Bell, chromatin appear constant throughout the cell division cycle 2002), release of Orc1 should prevent ORC function, and in [Orc2 (Ritzi et al., 1998; Saha et al., 1998; Mendez and fact, mammalian metaphase chromatin lacks functional Stillman, 2000; Natale et al., 2000; Mendez et al., 2002; ORCs. Cell cycle dependent loss of ORC function is even Ohta et al., 2003), Orc3 (Mendez et al., 2002; Ohta et al., more obvious when mammalian somatic cell chromatin is 2003), Orc4 (Okuno et al., 2001; Ohta et al., 2002), Orc5 incubated in a Xenopus laevis (Xl) egg extract (see Section (Ohta et al., 2003), Orc6 (Dhar and Dutta, 2000; Mendez 7). In this case, XlORC binds to the cell chromatin, initiates et al., 2002). Nevertheless, there are changes in the pre-RC assembly, and the entire XlORC is then released distribution of ORC subunits that suggest they have upon completion of pre-RC assembly. The timing of this activities independent of their role in DNA replication. release, however, appears to be dependent on chromatin For example, both the cellular concentration (Dhar and structure, because when Xenopus sperm chromatin is Dutta, 2000) and the amount of chromatin bound HsOrc6 incubated in the same extract, the affinity of XlORC for (Mendez et al., 2002) remain constant throughout the cell sperm chromatin is reduced, but ORC remains bound to the cycle, but some Orc6 is found at non-chromosomal sites in chromatin throughout DNA replication. In this case, XlORC mitotic cells (Prasanth et al., 2002). This appears to be due appears to be released during G2/M phase as a result of to a fraction of Orc6 that is not associated with ORC. by Cdk1/cyclin A. In fact, phos- Furthermore, the abundance of individual ORC subunits phorylation has been shown to play a role in regulating ORC relative to each other can vary among tissues, and some M.L. DePamphilis / Gene 310 (2003) 1–15 5

Fig. 2. Four manifestations of the ORC cycle in eukaryotes. Yeast cells: ORC (six gray cylinders) remains bound to replication origins throughout the cell cycle, but ORC is phosphorylated (-P) during the S to M periods, and this phosphorylation inhibits its ability to assemble a pre-RC. Xenopus egg extract: ORC binds to sperm chromatin, but the stability of ORC/chromatin sites is reduced (red boxes) following pre-RC assembly. ORC is phosphorylated by Cdk1/cyclin A (yellow ball) during G2/M and released from chromatin. If somatic cell chromatin instead of sperm chromatin is incubated in the extract, then ORC is released from chromatin following pre-RC assembly. Mammalian cells: The Orc1 subunit is selectively destabilized (red box) and released from chromatin when cells enter S-phase. Orc1 is then mono-ubiquitinated (Ub) and in some cases polyubiquitinated ([Ub]n) and degraded. Orc1 in mitotic cells appears hyperphosphorylated. Thus, in yeast, frogs and mammals, stable ORC/chromatin sites that can initiate assembly of a pre-RC are not present until mitosis is complete and a nuclear membrane is present. subunits are expressed in non-proliferating tissues (Thome that the amount of Orc1 bound to chromatin in their et al., 2000). Orc1, however, is unique in that it appears to experiments was markedly diminished during M-phase, regulate ORC activity in a cell cycle dependent manner suggesting that Orc1 is selectively destabilized sometime (Fig. 1, ‘Mammals’; Fig. 2, ‘Mammalian cells’). during the cell cycle. The absence of Orc1 in their In hamster cells, the cellular concentration of Orc1 is chromatin-unbound fraction from M-phase cells (Okuno constant throughout the cell cycle (Natale et al., 2000; et al., 2001) probably resulted from the fact that unbound Okuno et al., 2001; Li and DePamphilis, 2002), but Orc1 is Orc1 is easily degraded, even when cell extracts are on ice, selectively released from chromatin during the S and M- whereas chromatin bound Orc1 is stable (Li and DePam- phases while the remaining Orc proteins remain chromatin philis, 2002). In addition, the amount of chromatin bound bound when cells are lysed with a non-ionic detergent in the Orc1 in M-phase arrested cells can vary depending on the presence of 0.1–0.15 M salt and ATP (Natale et al., 2000; Li length of time cells are held in nocodazole. For example, and DePamphilis, 2002). The same is true in human cells, chromatin bound Orc1 was not detected in M-phase cells except that the cellular concentration of Orc1 oscillates that were not collected in nocodazole (Natale et al., 2000; Li during the cell cycle, because Orc1 is degraded during S- and DePamphilis, 2002), whereas cells arrested in nocoda- phase (Kreitz et al., 2001; Fujita et al., 2002; Mendez et al., zole could contain up to 20% of the amount of chromatin 2002; Ohta et al., 2003); J. Teer and A. Dutta, unpublished bound Orc1 observed in G1-phase cells, depending on how data]. Previous reports that HsOrc1 levels did not decrease long the cells were held in nocodazole (Li and DePamphilis, during S-phase (Saha et al., 1998; Tatsumi et al., 2000) 2002). Nocodazole blocks by preventing micro- appear to have resulted from differences in the specificity of tubule assembly, which does not necessarily prevent other the used to detect HsOrc1 in various experiments cell cycle related events. (A. Dutta; C. Obuse, personal communications). The selective release of Orc1 during S-phase accounts In contrast to the reports described above, release of for the fact that one or more ORC subunits are both chromatin bound Orc1 during S-phase in hamster cells was functionally and physically absent from metaphase chro- not detected by Okuno et al. (2001) who concluded that both matin. The absence of ORC activity is revealed by the fact the amount of chromatin-bound Orc1 and Orc4 as well as that hamster M-phase chromatin will replicate in a complete their cellular concentrations remained constant throughout Xenopus egg extract, but not in one that has been depleted of the cell cycle. However, although it is not clear why they did Xenopus Orc proteins (Yu et al., 1998; Li et al., 2000; Natale not observe a release of Orc1 during S-phase, it does appear et al., 2000). In contrast, nuclei isolated in the same manner 6 M.L. DePamphilis / Gene 310 (2003) 1–15 from G1-phase cells will initiate DNA replication de novo cells, two forms of Orc1 are released concurrently during under both conditions, revealing that hamster pre-RCs are S-phase, one that is not ubiquitinated and one that is assembled during the M to G1 transition. Loss of ORC mono-ubiquitinated (Li and DePamphilis, 2002). Orc1 and activity can be accounted for by the fact that Orc1 protein is mono-ubiquitinated Orc1 also were detected in the non- not associated with metaphase chromatin under conditions chromatin bound fraction from human S-phase cells where other Orc proteins remain stably bound (Natale et al., (Mendez et al., 2002). However, in human cells, Orc1 2000; Tatsumi et al., 2000; Okuno et al., 2001; Mendez became polyubiquitinated and was degraded via the 26S et al., 2002) and mammalian Cdc6 protein is abundant pathway (Fujita et al., 2002; Mendez et al., [(Mendez and Stillman, 2000; Petersen et al., 2000). This 2002), a reaction mediated by the SCFskp2 -ligase observation is consistent with the cell cycle-dependent complex (Mendez et al., 2002). In contrast, only trace changes observed in a footprint at the human lamin B2 amounts of polyubiquitinated Orc1 were detected in origin (Abdurashidova et al., 1998) that encompasses the hamster cells. Hamster Orc1 was polyubiquitinated and start sites for leading strand DNA replication (Abdurashi- degraded only when it was released from nuclei into cellular dova et al., 2000). A large G1-phase footprint changed into a extracts. Thus, the chromatin unbound fraction of Orc1 in smaller S and G2 phase footprint that disappeared during M- non-ionic detergent extracts of hamster mitotic cells can be phase. Recent UV-induced cross-linking experiments reveal lost, even when cell lysates are held in ice (Okuno et al., that the large G1-phase footprint could be accounted for by 2001; Li and DePamphilis, 2002). the presence of a pre-RC containing at least Orc1, Orc2, In contrast to Orc1, Orc2 not only remains bound to Cdc6, and Mcm3 proteins (Abdurashidova et al., 2003). chromatin throughout the cell cycle, but Orc2 is not a During S-phase, only the Orc2 protein can be cross-linked to substrate for ubiquitination, even as a soluble protein (Li chromatin, consistent with the disappearance of the pre-RC and DePamphilis, 2002). Interestingly, the half- of Orc1 and the release of Orc1. By M-phase, not even Orc2 could in both hamster and human exponentially proliferating cells be cross-linked to DNA, suggesting additional changes is ,3 h, the same as observed for Orc2 [(Li and occur in the affinity of ORC for chromatin. Similarly, both DePamphilis, 2002); J. Mendez, personal communication]. Orc1 and Orc2 can be cross-linked to specific replication Therefore, it may not be necessary to deubiquitinate Ub- origins in vivo by treating human G1-phase cells with Orc1 in order to reestablish functional ORC/chromatin sites formaldehyde, but only Orc2 can be cross-linked during S- in early G1-phase, because in both hamster and human cells, phase (Ladenburger et al., 2002). These observations in most of the Orc1 in M-phase cells will have been mammalian cells are consistent with earlier reports that both synthesized during the previous 6 h. Orc1 and Orc2 are present on chromatin in Xenopus cells but not in metaphase cells (Romanowski et al., 1996). Moreover, Orc proteins in Xenopus eggs 5. Orc1 and the assembly of pre-replication complexes competent for DNA replication will bind to sperm chromatin, whereas Orc proteins in Xenopus eggs in Several observations suggest that the stable binding of metaphase II will not (Coleman et al., 1996; Hua and Orc1 to chromatin is the premier regulatory step in the Newport, 1998; Findeisen et al., 1999; Rowles et al., 1999). assembly of pre-RCs and thereby the commitment to cell Whether Orc1 is associated with chromatin in vivo in an proliferation. First, both HsOrc1 (but not HsOrc2) and unstable state or is actually released from chromatin in vivo DmOrc1 expression are regulated by (Ohtani et al., as well as in cell extracts is difficult to assess. What is clear 1996; Asano and Wharton, 1999), a that is that Orc1 is not stably bound to chromatin in comparison plays an important role in driving cells from G1 into S- with other Orc proteins, and that metaphase cells do not phase. E2F also regulates expression of Cdc6 and Mcm contain functional ORCs. M-phase cells permeabilized with genes (Leone et al., 1998; Ohtani et al., 1998; Yan et al., digitonin retain Orc1 in the chromatin pellet (Natale et al., 1998), suggesting that assembly of pre-RCs depends on the 2000), but this M-phase chromatin does not initiate DNA E2F/Rb pathway with ORC activity regulated by expression replication in an ORC-depleted Xenopus egg extract (Yu of Orc1. Second, the Orc1 gene is the only ORC gene whose et al., 1998; Li et al., 2000; Natale et al., 2000). Expression expression is correlated with cell proliferation: Orc1 is of a GFP-tagged Orc1 protein in hamster cells resulted in expressed only in proliferating mammalian cells whereas GFP-labeled metaphase (Okuno et al., 2001), Orc2–Orc5 are expressed to varying extents in many non- but there is no evidence that the GFP-Orc1 protein is proliferating cells (Thome et al., 2000). Third, stable assembled into ORCs and if so, that the GFP-ORCs are binding of Orc1 to chromatin immediately precedes active. assembly of pre-RCs at specific chromosomal loci. During the M to G1 transition, Orc1 rebinds stably to 4.2. Ubiquitination Of Orc1 chromatin with the concomitant appearance of functional ORCs. M-phase hamster cells contain as much Orc1 as G1- Orc1 in S-phase mammalian cells is selectively ubiqui- phase cells, but most, if not all, of the Orc1 in M-phase tinated and, under some conditions, degraded. In hamster hamster cells is not bound to chromatin and only a minor M.L. DePamphilis / Gene 310 (2003) 1–15 7 fraction is ubiquitinated (Li and DePamphilis, 2002). M- 6. ORC and the ‘origin decision point’ phase human cells generally contain much less Orc1 than G1-phase human cells, as a result of Orc1 degradation Xenopus egg extract can initiate DNA replication de during S-phase (Mendez et al., 2002). Therefore, human novo in virtually any DNA, chromatin or nuclei provided, Orc1 must be resynthesized during the M to G1 transition, but these initiation events are generally distributed non- whereas hamster Orc1 does not (Okuno et al., 2001). ORC specifically throughout the DNA substrate. The only activity is restored during the first 2 h after either hamster or exception is when nuclei are isolated from mammalian human M-phase cells (arrested in nocodazole) are released cells under conditions that preserve their impermeability to into G1-phase (Yu et al., 1998; Natale et al., 2000). This macromolecules (Gilbert et al., 1995). Under these con- corresponds to the time period (1–3 h post-M) when Orc1 ditions, initiation events are distributed non-specifically (Natale et al., 2000; Li and DePamphilis, 2002; Mendez throughout the genome when ‘nuclei’ are isolated from M- et al., 2002; Ohta et al., 2003), Mcm2 and Mcm3 reassociate phase or early G1-phase cells, but at specific sites when stably with chromatin (Dimitrova et al., 1999; Natale et al., nuclei are taken from late G1-phase cells (Wu and Gilbert, 2000; Dimitrova et al., 2002; Ohta et al., 2003). Nuclei 1996; Wu and Gilbert, 1997; Li et al., 2000; Okuno et al., isolated from these early G1-cells can initiate de novo DNA 2001). DNA replication in late G1-nuclei is independent of replication in a Xenopus egg extract depleted either of ORC XlORC proteins, begins in vitro at the same site-specific or of Mcm proteins. The appearance of chromatin bound origins of bi-directional DNA replication used by the cells Orc1 is inversely related to the lost of cyclin B (Mendez and in vivo, and is sensitive to protein kinase inhibitors. Stillman, 2000), which is required to drive cells into mitosis, Therefore, it appears that pre-RCs in late G1-phase nuclei and directly linked to formation of an intact nuclear are present at specific genomic sites waiting to be activated envelope (Dimitrova and Gilbert, 1998; Natale et al., by Cdk2/cyclin A, E and Cdc7/Dbf4 (Fig. 1). This cell cycle 2000), which is required for initiation of DNA replication. dependent transition that occurs in mammalian chromatin, Thus, the bulk of Orc1 and Mcm binding occurs after from a substrate for non-specific initiation events to a mitosis is complete and a nuclear membrane has reformed substrate for site-specific initiation events, is referred to as around the genome, a time referred to either as late the ‘origin decision point’ (ODP). or early G1-phase. Consistent with this view is Two views of the ODP have emerged. In one view, the the fact that HsCdc6 protein, like cyclin B, is degraded ODP marks the transition from Xenopus ORC directed during the M to G1 transition and then resynthesized during initiation events to mammalian ORC directed initiation G1 (Petersen et al., 2000; Mendez et al., 2002), just in time events. Prior to the ODP in hamster cells (midpoint ,2h to be incorporated into the newly reconstituted active ORC/ post-M), initiation of DNA replication in hamster chromatin chromatin sites. Therefore, stable rebinding of Orc1 to requires the presence of Xenopus ORC in the egg extract chromatin appears to be the rate-limiting step for assembly (Natale et al., 2000). XlORC present in these experiments of pre-RCs. rapidly binds to hamster metaphase chromatin within the One experiment, however, suggests that pre-RC assem- first few minutes of incubation (Sun et al., 2002) and is bly is completed about 2 h before the stable binding of responsible for most of the DNA replication that occurs hamster Orc1, Mcm2 and Mcm3 is completed. Geminin, a during the first 1–2 h post-M (Yu et al., 1998; Natale et al., specific inhibitor of Cdt1, prevents stable binding of 2000) where it initiates replication non-specifically through- Mcm(2–7) to chromatin. Once pre-RCs are assembled, out the genome (Yu et al., 1998; Dimitrova and Gilbert, DNA replication is no longer sensitive to geminin. DNA 1999; Li et al., 2000; Natale et al., 2000). After the ODP in replication in hamster metaphase chromatin incubated in hamster cells, Xenopus egg extract simply activates hamster Xenopus egg extract is sensitive to geminin, but DNA pre-RCs that were assembled in vivo. Initiation sites are replication in chromatin isolated 1 h after metaphase is not established in hamster cells upon the stable binding of Orc1 (Okuno et al., 2001; Dimitrova et al., 2002). In these (midpoint ,1.5 h post-M) to Orc2–6/chromatin complexes experiments, Orc1 appears to bind to chromatin within the located at specific sites along the genome (Natale et al., 1st h after release of cells from the nocodazole block, 2000; Li and DePamphilis, 2002). This view is supported by consistent with the execution point for geminin. Interest- the studies in human cells where Orc1 binding to chromatin ingly, Mcm proteins continued to bind to chromatin until follows a similar time course to that in hamster cells mid to late G1-phase (Dimitrova et al., 1999; Natale et al., (Mendez et al., 2002) and where cross-linking studies have 2000; Dimitrova et al., 2002), suggesting that more Mcm revealed that Orc2 is bound to specific genomic sites, some proteins bind to chromatin than are actually assembled into of which correspond to origins of bi-directional DNA pre-RCs. Apparent differences in the published time courses replication (Abdurashidova et al., 2003; Keller et al., 2002b; for protein binding and the geminin execution point likely Ladenburger et al., 2002). This view also accounts for the reflect differences in experimental protocols that affect the observation that the normal temporal order for initiation is amount of protein recovered in different fractions and the absent in chromatin isolated from cells in M-phase to 1 h time required for cells to progress from M to G1 when the post-M phase but present in chromatin isolated from late synchronizing agent, nocodazole, is removed. G1-phase cells (Dimitrova and Gilbert, 1999; Li et al., 8 M.L. DePamphilis / Gene 310 (2003) 1–15

2001). XlORC initiates replication non-specifically in to bind to chromatin flanking the ORC binding site, appear chromatin from M-phase and telophase cells. in the soluble nuclease-sensitive fraction. This transition of An alternative view of the ODP is that pre-RCs are ORC/chromatin complexes from untethered to tethered may initially assembled ‘randomly’ along the genome during the contribute to the phenomenon referred to as the ‘origin M to G1-transition in mammalian cells, and then reorgan- decision point’. ized, by some as yet unidentified mechanism, into specific sites [(Okuno et al., 2001) and references therein]. One suggested mechanism is that some pre-RCs are inactivated 7. The ORC cycle in frogs at the ODP, leaving those pre-RCs at specific genome sites to initiate replication at the start of S-phase. However, this Metaphase chromatin lacks functional ORC complexes, implies the existence of a larger number of ORC/chromatin and therefore when metaphase chromatin from hamster cells sites in mammalian cells that the cellular concentration of is incubated in a Xenopus egg extract, XlORC rapidly binds ORC would permit. This view of the ODP is based on the to the chromatin and initiates DNA replication. However, in conclusion that all six mammalian ORC subunits are stably contrast to mammalian ORC, where only the Orc1 subunit is bound to chromatin throughout the cell cycle, that pre-RCs destabilized upon DNA replication, the entire XlORC is are assembled in hamster cells within 1 h after metaphase, released from the somatic cell chromatin into the replication and that pre-RC assembly precedes the ODP in hamster extract (50 mM KCl) as soon as pre-RCs have been cells (midpoint ,3 h post-M) by 2–2.5 h. Various assembled [Fig. 1,‘Xenopus’; Fig. 2,‘somaticcell experimental artifacts that are innumerated in the papers chromatin’; (Sun et al., 2002)]. This programmed release cited above may well explain some of the differences of XlORC was insensitive to aphidicolin (specific inhibitor between these two views. Nevertheless, the possibility of replicative DNA ) and olomoucine (specific remains that there exists a novel, as yet undiscovered, inhibitor of cyclin dependent protein kinases), but sensitive mechanism for determining where initiation events occur in to geminin (specific inhibitor of Cdt1), and prevented by mammalian genomes. immuno-depletion of XlMcm proteins from the egg extract. Therefore, assembly of pre-RCs triggers release of ORC 6.1. Association of Orc1 with nuclear structure from cell chromatin. Alternatively, ORC release may be triggered by some event that occurs between completion of The fact that assembly of pre-RCs is delayed until pre-RC assembly and the action of Cdk2. For example, mitosis is complete and a nuclear membrane is assembled Mcm10 binds to chromatin after pre-RC assembly and implies that nuclear structure plays a role in the initiation of before Cdk2 activity where it is required to initiate DNA DNA replication. The nucleus appears to regulate the replication (Wohlschlegel et al., 2002). concentration of proteins required to initiate DNA replica- Once pre-RCs were assembled in egg extract, XlORC tion (Walter et al., 1998), to facilitate the assembly or was no longer present on somatic cell chromatin, despite the activity of DNA replication forks, and to determine where in fact that egg extract contained a great excess of soluble the genome initiation of DNA replication occurs (reviewed XlORC. Furthermore, XlORC could not bind to chromatin in DePamphilis, 2000; Keller et al., 2002a). Replication in nuclei isolated from hamster cells that had progressed origins have been reported to be associated with ‘nuclear past their ‘origin decision point’ (Sun et al., 2002). Cdk/ matrix’ preparations (Djeliova et al., 2001), although not were not involved in these events, since neither with ‘nucleoskeleton’ preparations (Ortega and DePamphi- binding nor release of XlORC to somatic cell chromatin was lis, 1998), revealing that interactions between replication affected by olomoucine. Moreover, XlORC in meiotic eggs origins and nuclear structure are sensitive to experimental (metaphase II) does not bind to sperm chromatin (Coleman conditions. et al., 1996; Hua and Newport, 1998; Findeisen et al., 1999; Recent observations reveal that Orc1 (and to a lesser Rowles et al., 1999), and XlORC is not bound to metaphase extent the other ORC subunits) is associated with a fraction chromatin in Xenopus somatic cells (Romanowski et al., of chromatin that is not solubilized by digestion with 1996). This inability of XlORC in cells undergoing endonucleases (Kreitz et al., 2001; Fujita et al., 2002), and or mitosis to bind to chromatin likely reflects the fact that that this fraction accumulates during and then XlORC is hyperphosphorylated in these cells where it decreases during S-phase (Ohta et al., 2003). In HeLa cells, associates with Cdk1/cyclin A (Romanowski et al., 2000). the appearance of Orc1 following release of cells from a These data strongly suggest that XlORC cannot rebind to metaphase block occurs concurrently with the appearance of either somatic cell chromatin or sperm chromatin until cell Orc1 and other ORC proteins in an insoluble nuclease- division is completed. resistant fraction, suggesting that newly assembled ORC/ chromatin complexes are tethered to some component of 7.1. Chromatin structure and the affinity of ORC for DNA nuclear structure and then released as the Orc1 subunit is destabilized, ubiquitinated, and in the case of human cells, Xenopus sperm chromatin, like mammalian metaphase degraded (Fig. 1, ‘Mammals’). Mcm proteins, which appear chromatin, lacks ORC proteins, and XlORC rapidly binds to M.L. DePamphilis / Gene 310 (2003) 1–15 9 sperm chromatin and initiates DNA replication. However, (Dhar et al., 2001; Vashee et al., 2001). Therefore, it is not in this case, XlORC is not released from chromatin, surprising that mammalian Orc1 is selectively released from although it does undergo a demonstrable change in its chromatin when cells enter S-phase and then rebinds when affinity for chromatin [Fig. 2, ‘sperm chromatin’; (Hua and cells enter G1-phase, while Xenopus Orc1 remains associ- Newport, 1998; Rowles et al., 1999)]. Prior to pre-RC ated with the other ORC subunits, regardless of whether assembly, ORC remains bound to sperm chromatin in 0.25 they are bound or released from chromatin. Thus, XlORC is M salt, but after pre-RC assembly, ORC can be eluted from well suited to initiating DNA replication at many sites as sperm chromatin by exposure to high levels of Cdk2, or to cells undergo rapid cleavage events during early frog mitotic extract, or 0.1–0.15 M KCl, leaving the Mcm development. Xenopus ORC appears to bind weakly to proteins bound to the chromatin and capable of initiating chromatin and then release intact in order to reinitiate DNA replication. This destabilization of ORC’s association quickly at other sites. Mammalian ORC, on the other hand, with sperm chromatin does not require DNA replication, is better suited to binding tightly to specific genomic sites protein synthesis, cyclin-dependent kinases or Cdc7. It does where it remains throughout cell proliferation and differen- require prior action of Cdc6, Mcm(2–7), and Cdt1, tiation. In mammals, site specificity is maintained by suggesting that binding of the Mcm(2–7) to chromatin releasing only one of the six subunits during S-phase, weakens the association of ORC with sperm chromatin and leaving the core ORC tightly bound to a replication origin. thus increases its sensitivity both to salt and to phosphoryl- ation. Thus, XlORC undergoes a programmed change in its affinity for chromatin immediately following pre-RC 8. The ORC cycle in yeast assembly. However, in the case of sperm chromatin, ORC apparently is not released until mitosis, a step that was In contrast to mammalian cells, yeast ORC remains blocked in these experiments by the presence of stably bound to chromatin throughout the yeast cell cycle. In cycloheximide. S. cerevisiae, both DNA footprinting (Diffley et al., 1994; The fact that XlORC remains bound to sperm chromatin Fujita et al., 1998) and immuno-precipitation (Liang and under conditions where it is released from somatic cell Stillman, 1997) analyses reveal that a complete ORC binds chromatin reveals that the affinity of ORC for DNA depends to yeast replication origins immediately after initiation of on chromatin structure. Since both chromatin substrates are replication occurs and remains there throughout the cell rapidly assembled into nuclei by Xenopus egg extract, these division cycle (Fig. 2, ‘yeast’). Similarly, in S. pombe all six differences likely reflect differences in their composition. ORC subunits remain bound to chromatin throughout the For example, somatic cell chromatin brings with it a full cell cycle (Lygerou and Nurse, 1999; Kong and DePam- complement of core and linker , but sperm philis, 2001). Nevertheless, re-initiation within a single chromatin must undergo extensive remodeling during its S-phase is prevented by a combination of ORC phosphoryl- first 30 min in egg extract (Philpott and Leno, 1992). ation, down regulation of Cdc6 activity, and nuclear Nucleoplasmin dependent decondensation of sperm chro- exclusion of Mcm proteins (Nguyen et al., 2001). matin is a prerequisite for XlORC binding and subsequent In S. cerevisiae, ScOrc1, ScOrc2 and ScOrc6 contain assembly of pre-RCs (Gillespie et al., 2001). Remodeling consensus phosphorylation sites, and these proteins are involves displacement of sperm specific protamines, uptake phosphorylated by the cyclin dependent protein kinase of core histones H2A and H2B (histones H3 and H4 are Cdk1(Cdc28)/Clb1 during the G1 to S transition (Nguyen already present), and linker B4 (histone H1 is absent et al., 2001). ScORC proteins are hyperphosphorylated from Xenopus sperm and eggs), as well as phosphorylation during the S to M transition, and then hypophosphorylated of core histones and uptake of HMG-2 protein (Dimitrov during early G1-phase when pre-RC assembly occurs. et al., 1994). Sperm chromatin is not converted into somatic Genetic alterations of ScOrc proteins that prevent their cell chromatin until after the midblastula transition when phosphorylation do not affect either DNA replication or cell histone H1 is expressed, the rate of cell division slows division. However, when this alteration is accompanied by down, and a G1-phase first appears in the cell division cycle. alterations in both Cdc6 and Mcm proteins that prevent their export from the nucleus, then DNA replication is reinitiated 7.2. Comparison of frogs and mammals prior to cell division, resulting in polyploid cells. In other words, all three regulatory pathways must be inactivated in The difference in behavior between mammalian and order to induce reinitiation of DNA replication within a Xenopus ORCs is in keeping with the properties of the single cell division cycle. purified proteins. XlOrc proteins exist as a stable complex of A similar phenomenon exists in S. pombe. SpOrc2 XlOrc(1–6) that can be immunoprecipitated from Xenopus undergoes dephosphorylation during the M to G1 transition egg extract (Rowles et al., 1996; Tugal et al., 1998), (Lygerou and Nurse, 1999; Kong and DePamphilis, 2001), although the identity of XlOrc6 has not been confirmed. In and is hyperphosphorylated when cells enter S-phase and contrast, human Orc proteins exist as a stable complex of Mcm proteins are released from chromatin (Vas et al., 2001; HsOrc(2–5) to which HsOrc1 and HsOrc6 bind only weakly Wuarin et al., 2002). Furthermore, the mitotic B type cyclin 10 M.L. DePamphilis / Gene 310 (2003) 1–15

Cdc13 complexed with the Cdk1(Cdc2)1 protein kinase dramatically throughout Drosophila development, and its associates with replication origins during S-phase and accumulation is regulated by E2F-dependent transcription remains there during G2 and early M-phases (Wuarin (Asano and Wharton, 1999). In embryos, Orc1 accumulates et al., 2002). This association is ORC-dependent, apparently preferentially in proliferating cells, and in the eye imaginal by association of Cdk1(Cdc2) with the Orc2 subunit, and disc, Orc1 accumulation is cell cycle regulated, with high prevents reinitiation of DNA replication before mitosis has levels in late G1 and . Moreover, overexpression of been completed. These data strongly suggest that the Orc1 altered the pattern of DNA synthesis, implicating Orc1 phosphorylated state of SpOrc2 determines ORC activity. in regulating initiation of DNA replication. The DNA binding activity of purified DmORC is largely 8.1. Comparison of yeast and frogs non-specific and ATP-independent, although DmORC, like ScORC, requires only the ORC1 component of the complex The ORC cycle in yeast resembles that in Xenopus egg to bind ATP for tight DNA interactions (Chesnokov et al., extract when sperm chromatin in the substrate (Fig. 2). ORC 2001). In vivo, DmORC does bind to specific genomic sites proteins in both species are hyperphosphorylated during (Austin et al., 1999; Bielinsky et al., 2001). Such site- metaphase by Cdk1, and phosphorylation inhibits its ability specific DNA binding may require association with other to initiate pre-RC assembly. Phosphorylated XlORC binds proteins (Bosco et al., 2001; Beall et al., 2002), and these poorly to chromatin (Romanowski et al., 2000), while pre- associations may regulate ORC activity as well. RC assembly is inhibited by Cdk1 dependent phosphoryl- ation of ScORC (Nguyen et al., 2001) or by association of SpORC with Cdk1 (Wuarin et al., 2002). Whether or not 10. Mechanisms that regulate ORC activity phosphorylation of XlORC subunits, like those in yeast, also occurs during DNA replication is not clear. Whether or not The results described above reveal at least five yeast ORC, like XlORC, can be selectively eluted at lower mechanisms by which ORC activity is regulated during salt concentrations following pre-RC assembly or DNA cell proliferation. replication also is not clear. Dephosphorylation of the Orc2 subunit occurs during the M to G1 transition in Xenopus 10.1. Ubiquitination (Sun et al., 2002) and in yeast (Nguyen et al., 2001; Vas et al., 2001), presumably as an activation step in pre-RC Since both Orc1 and Ub-Orc1 are rapidly released from assembly. Phosphorylation of ORC may alter its affinity chromatin when hamster cells enter S-phase and Ub-Orc1 is either for chromatin or for other pre-RC proteins during the largely absent from mitotic cells, ubiquitination per se is not G1 to S transition, while chromatin composition may likely the cause of Orc1 release, but a mechanism to prevent determine whether the modified ORC subunits are remain its reassociation with ORC/chromatin sites during S-phase. bound or are released. Interestingly, yeast chromatin, like Two phenomena suggest that Ub-Orc1 may become Xenopus sperm chromatin, lacks a classical histone H1 sequestered in the nuclear membrane. First, several linker (Landsman, 1996), suggesting that reduced chromatin examples have been reported where monoubiquitination is condensation may facilitate binding of ORC during S-phase. required for endosomal sorting of proteins into membrane vesicles (Hicke, 2001; Haglund et al., 2002). Second, a protein called SUMO that is very similar in structure to 9. The ORC cycle in flies ubiquitin conjugates proteins with a single adduct and this modification generally results in the protein’s relocation to Direct analyses of cell cycle dependent changes in the the nucleus (Wilson and Rangasamy, 2001). Some cells, activity, post-translational modifications or chromatin such as HeLa and other cancer cells, may contain larger affinity of DmORC are not yet available. However, several pools of Orc1 that cause it to be exported to the cytoplasm observations are consistent with a cell cycle dependent, where it is polyubiquitinated and degraded. In fact, differential association of DmOrc proteins with chromatin. differences in pool sizes could account for the observation In Drosophila, Orc2 is distributed fairly homogeneously in that Cdc6 is exported from the nucleus and degraded in interphase nuclei, while it only remains bound to the human cells but not in hamster cells (Okuno et al., 2001). heterochromatic region of chromosomes through mitosis in embryos and larval (Pak et al., 1997; Loupart 10.2. Phosphorylation et al., 2000). in Orc2 exhibited delayed entry into S-phase as well as delayed exit from metaphase with some Phosphorylation of Orc proteins can affect both their euchromatic regions replicating even later than heterochro- affinity for chromatin and their activity. In yeast, Cdk1/cy- matin (Loupart et al., 2000). In addition, mitotic chromo- clin B prevents pre-RC assembly until mitosis has been somes were irregularly condensed, suggesting a novel role completed by phosphorylating one or more of the ORC for ORC in architecture as well as DNA subunits and Cdc6. In frogs, both Orc1 and Orc2 are replication. In contrast to Orc2, Orc1 levels change hyperphosphorylated in metaphase-arrested eggs relative to M.L. DePamphilis / Gene 310 (2003) 1–15 11 activated eggs (Carpenter and Dunphy, 1998; Tugal et al., is released under DNA replication conditions. In the case of 1998). Moreover, XlORC can be selectively released from sperm chromatin, the entire ORC becomes salt-sensitive. chromatin by incubating chromatin either in a metaphase extract (Rowles et al., 1999) or with Cdk1(Cdc2)/cyclin A 10.5. Auxiliary protein interactions (Hua and Newport, 1998; Findeisen et al., 1999). In mammals, hyperphosphorylation of Orc1 has been detected Interactions between ORC and other proteins can in metaphase cells (Tatsumi et al., 2000; Thome et al., facilitate pre-RC assembly (Fig. 1). For example, HsOrc1 2000), and over-expression of cyclin A results in export of binds Cdc6 (Saha et al., 1998), and Cdc6 facilitates binding Orc1 to the cytoplasm, a process that is dependent on the of XlORC to somatic cell chromatin (Sun et al., 2002) and phosphorylation status of Cdk target sites in Orc1 (Laman ScORC to its cognate replication origin (Mizushima et al., et al., 2001). Given the fact that Cdk2/cyclin A interacts 2000). Moreover, ScCdc6 specifically recognizes the ATP- with HsOrc1 (Mendez et al., 2002) and Cdk1/cyclin A bound state of ScOrc1 (Klemm and Bell, 2001). Therefore, interacts with XlORC (Romanowski et al., 2000), cyclin Orc1 may recruit Cdc6, which is present during M-phase, dependent phosphorylation may be the mechanism that and this Orc1/Cdc6 complex may then target specifically releases ORC proteins from chromatin during the G1 to S one of the many Orc2–6 complexes that are bound to transition, and the released protein then may be exported to various sites along the genome (the ‘Jesuit Model’ for site the cytoplasm where it undergoes polyubiquitination and selection, (DePamphilis, 1993, 1996, 1999). degradation. Interactions between ORC and other proteins can also prevent pre-RC assembly. For example, Skp2, a component 10.3. DNA replication of the mammalian ubiquitination system, binds to Orc1 and is required for Orc1 ubiquitination during S-phase (Mendez et al., 2002). Association of ORC/chromatin sites with DNA replication appears to be required to destabilize Cdk1/cyclin B during mitosis in S. pombe prevents Orc1 in mammalian cells, because significant amounts Orc1 assembly of pre-RCs (Wuarin et al., 2002). Sic1, a specific are not released from chromatin in cells arrested at their inhibitor of Cdks, prevents the S-phase-specific Cdk1/Clb5 G1/S interphase (Li and DePamphilis, 2002). Therefore, protein kinase in S. cerevisiae from interacting with ORC, Orc1 release in mammals may be triggered by DNA but does not prevent the G1–phase specific Cdk1/Cln2 synthesis through the origin region. This hypothesis is kinase from binding to ORC (Weinreich et al., 2001). supported by data from yeast and flies. Therefore, Sic1 can prevent Cdk1 activation of pre-RCs Orc1, Orc4, and Orc5 from yeast and flies can each bind during the G1 to S-phase transition, without also preventing ATP, and the same ORC subunits in all other eukaryotes subsequent inactivation of pre-RC assembly by the same contain potential ATP binding sites [reviewed in (Bell, protein kinase during the S to M-phase transition. 2002)]. Moreover, the affinity of both DmORC (Chesnokov et al., 2001) and ScORC (Klemm et al., 1997) for DNA increases by binding of ATP to the Orc1 subunit. ATP 11. Regulating initiation of DNA replication by binding to ScOrc1 is required for site-specific binding of regulating ORC activity ScORC to origin DNA. The rate of ATP hydrolysis is then reduced in response to origin binding, suggesting that ATP One can envisage at least four advantages to regulating is not hydrolyzed until a subsequent step in replication. That ORC activity. The first is to prevent assembly of pre-RCs step appears to be the generation of single stranded DNA at during S-phase by inactivating the premier step in their replication origins. ScORC binds to ssDNA, but its affinity assembly. By applying this strategy to newly assembled for ssDNA is dependent only on ssDNA length, not on either ORC/chromatin sites as well as to those sites where DNA sequence or ATP binding. In fact, association of initiation has already occurred, initiation of DNA replica- ScORC with ssDNA stimulates ATP hydrolysis, and tion is restricted to once-per-origin-per-cell division cycle. stabilizes an altered ORC structure (Lee et al., 2000). In Moreover, the assembly of pre-RCs cannot begin again until yeast, this altered ORC structure may be locked in place by mitosis is complete and a nuclear membrane has been phosphorylation of ORC subunits and thereby prevent reassembled. To this end, mammals have taken the more subsequent pre-RC assembly. In mammals, this altered sophisticated approach. Since Cdc6 binds specifically to ORC structure may cause selective release of Orc1. Orc1, selective inactivation of Orc1 would directly prevent recruitment of Cdc6 to ORC/chromatin sites without 10.4. Pre-replication complex assembly disturbing the genomic locations of the remaining chroma- tin bound Orc proteins. In Xenopus egg extract, assembly of pre-RCs triggers a A second advantage is to provide the metazoa with a change in the affinity of ORC for chromatin that is mechanism for selecting which of the many potential manifested differently, depending on the chromatin sub- initiation sites along the genome will be activated [‘Jesuit strate. In the case of somatic cell chromatin, the entire ORC Model’, (DePamphilis, 1993, 1996)]. A core complex 12 M.L. DePamphilis / Gene 310 (2003) 1–15 consisting of Orc(2–5) or Orc(2–6) could be assembled at Giacca, M., Falaschi, A., 2000. Start sites of bidirectional DNA many potential initiation sites, but which of these sites is synthesis at the human lamin B2 origin. Science 287, 2023–2026. Abdurashidova, G., Danailov, M.B., Ochem, A., Triolo, G., Vindigni, A., activated during each round of cell proliferation would be Riva, S., Falaschi, A., 2003. Localization of proteins bound to a determined by the binding either of Orc1 or of an Orc1/Cdc6 replication origin of human DNA along the cell cycle, submitted. complex. This would help to resolve the problem of Altman, A.L., Fanning, E., 2001. The Chinese hamster dihydrofolate preventing DNA replication from interfering with DNA reductase replication origin beta is active at multiple ectopic transcription as the pattern of gene expression and chromosomal locations and requires specific DNA sequence elements for activity. Mol. Cell. Biol. 21, 1098–1110. chromatin organization changes during develop- Asano, M., Wharton, R.P., 1999. E2F mediates developmental and cell ment. 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For example, cells that Bell, S.P., Dutta, A., 2002. DNA replication in eukaryotic cells. Annu. Rev. Biochem. 71, 333–374. have sustained DNA damage, or that have entered a Bielinsky, A.K., Blitzblau, H., Beall, E.L., Ezrokhi, M., Smith, H.S., quiescent or terminally differentiated state could prevent Botchan, M.R., Gerbi, S.A., 2001. Origin recognition complex binding activation of the pathway leading to S-phase by inactivating to a metazoan replication origin. Curr. Biol. 11, 1427–1431. one or more ORC subunits. Human cells treated with Bogan, J.A., Natale, D.A., DePamphilis, M.L., 2000. Initiation of eukaryotic DNA replication: conservative or liberal? J. Cell. Physiol. adriamycin, a DNA damage-inducing agent, do, in fact, 184, 139–150. selectively degrade Orc1 (Mendez et al., 2002). Bosco, G., Du, W., Orr-Weaver, T.L., 2001. DNA replication control Finally, the existence of multiple mechanisms for through interaction of E2F-RB and the origin recognition complex. 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