Review

The ‘ORC cycle’: a novel pathway for regulating eukaryotic DNA

replication

Melvin L. DePamphilis*

National Institute of Child Health and Human 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 mitosis is complete and a nuclear membrane is assembled. In yeast, Cdk1/Clb

phosphorylates ORC. In frog eggs, pre-RC assembly destabilizes ORC/chromatin 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, eukaryotes 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 cell 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; Cell division cycle; Ubiquitination; Nuclear structure

1. DNA replication and the cell division cycle

In normal cells, the cell division cycle is highly regulated

and consists of four easily recognized phases: G1-phase

(preparation for DNA replication), S-phase (DNA replica-

tion), G2 phase (preparation for mitosis) and M-phase

(mitosis). Following mitosis, each cell must decide whether

to enter G1-phase, which is a commitment to cell division,

or to enter a quiescent state (G0-phase). Once cells are

committed to undergoing DNA replication, they must

complete the process of cell division or die. Thus,

commitment to DNA replication is the primary point at

which cells regulate their division cycle. In contrast, cells in

G0-phase remain capable of proliferating for long periods of

time (e.g. hepatocytes, lymphocytes). Alternatively, cells

can undergo terminal differentiation (e.g. neurons, germ

cells), a state from which they do not reenter the cell

division cycle. Unregulated cell proliferation is the primary

characteristic of the pathological state known as cancer.

One critical feature of all eukaryotic cell cycles,

regardless of species or developmental stage, is that

initiation of DNA replication is limited to once per

replication origin per cell division cycle. Not all origins

are used during every cell cycle, but if they are, they cannot

be used a second time until after cell division has occurred.

Presumably, this mechanism evolved to avoid the genomic

instability that would invariably result if some sequences

were duplicated more than others and replication forks

remained during mitosis. Therefore, it is not surprising that

the proteins and sequence of events that comprise

eukaryotic DNA replication are highly conserved (Bogan

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

Gene 310 (2003) 1–15

www.elsevier.com/locate/gene

* Fax: þ1-301-480-9354.

E-mail address: depamphm@mail.nih.gov (M.L. DePamphilis).

Abbreviations: ORC, origin recognition complex; Orc1–Orc6, ORC

subunits; Mcm, 2–10, minichromosome maintenance proteins; Mcm(2–7),

heterohexameric complex of Mcm proteins 2–7; Cdc, cell division cycle

protein; Cdt1, protein encoded by Cdc10 dependent transcript 1 in

Schizosaccharomyces pombe; RLF-B, Cdt1 and replication licensing

factor B are the same protein; Cdk, cyclin dependent protein kinase; Pre-

RCs, pre-replication complexes consisting of ORC, Cdc6, Cdt1 and

Mcm(2–7); Sc, Saccharomyces cerevisiae; Sp, Schizosaccharomyces

pombe; Xl, Xenopus laevis; Dm, Drosophila melanogaster.

Eukaryotic DNA replication begins with the binding of a

six subunit ‘origin recognition complex’ (ORC) to DNA

(reviewed in Bell and Dutta, 2002; outlined in Fig. 1).

Proteins Cdc6 and Cdt1(RLF-B) are then required to load

Mcm proteins 2–7 onto the ORC/chromatin site to form a

pre-replication complex (pre-RC). Mcm2–Mcm7 exist as a

hexameric complex [Mcm(2–7)] that appears to be the

helicase responsible for unwinding parental DNA strands.

Presumably, there is at least one Mcm(2–7) complex per

replication fork, although more are possible (Edwards et al.,

2002). Pre-RCs are activated upon binding of Mcm10

protein (Wohlschlegel et al., 2002). Cdc6 is then released by

the cyclin dependent protein kinase, Cdk2/Cyclin A, and

replaced by Cdc45 with the help of the protein kinases

Cdc7/Dbf4 and Cdk2/cyclin E. Cdc45 associates with DNA

polymerase-a: DNA primase and guides it to the complex to

initiate RNA-primed DNA synthesis.

Several mechanisms have been identified that limit

initiation of eukaryotic DNA replication to once-per-origin-

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: Metaphase chromatin from cultured, somatic

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 cyclin B 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 sperm 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 somatic cell 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 enzyme 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 phosphorylation 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) 1–152

cells enter S-phase, released from pre-RCs, ubiquitinated

and degraded [(Drury et al., 1997; Weinreich et al., 2001)

and references therein]. When yeast enter M-phase, the

Cdk1/cyclin B phosphorylates any newly synthesized Cdc6,

thereby preventing it from binding to ORC until mitosis is

completed. The same mechanism exists in frogs and

mammals, except that in human cells phosphorylated

Cdc6 is transported out of the nucleus during S-phase and

remains in the cytoplasm during G2 and M-phases [(Jiang

et al., 1999; Petersen et al., 1999; Coverley et al., 2000;

Mendez and Stillman, 2000; Petersen et al., 2000;

Delmolino and Dutta, 2001) and references therein]. During

the M to G1-transition, HsCdc6 is degraded and then

resynthesized later during G1-phase (Mendez and Stillman,

2000; Petersen et al., 2000). However, the amount of

chromatin bound Cdc6 in hamster cells appears to remain

constant throughout the cell cycle, with no indication of any

degradation during G1 (Okuno et al., 2001). This difference

may reflect a larger pool of Cdc6 in human cells than

hamster cells. Nevertheless, under conditions where Cdc6

cannot be phosphorylated and remains in the nucleus during

S-phase, reinitiation of DNA replication still does not occur,

revealing the existence of other regulatory mechanisms

(Pelizon et al., 2000; Petersen et al., 2000). One of these

mechanisms is inhibition of Cdt1(RLF-B) activity by

geminin, a protein produced during the S to M transition

(Wohlschlegel et al., 2000; Tada et al., 2001). Geminin has

not been found in yeast, but in yeast, as well as in mammals,

Cdt1 is degraded during S-phase. The combined inacti-

vation of both Cdc6 and Cdt1 prevents rebinding of the

Mcm(2–7) to ORC/chromatin sites. Nevertheless, Mcm

proteins themselves are phosphorylated during S-phase and

released from chromatin. This mechanism is presumably

triggered by termination of DNA replication when two

oncoming replication forks collide. Moreover, in budding

yeast, the phosphorylated Mcm proteins are exported from

the nucleus during the G2 to M transition, thus providing an

additional barrier to reinitiation during S-phase (Labibet al.,

1999; Pasion and Forsburg, 1999; Nguyen et al., 2000). In

fission yeast as well as in the metazoa, Mcm proteins remain

in the nucleus, although their affinity for chromatin is

markedly diminished (Lei and Tye, 2001).

The question remains as to whether or not ORC activity

is also regulated.

2. Selection of initiation sites

Since ORC binding to DNA is the first step in pre-RC

assembly, ORC determines where DNA replication can

begin by binding to specific DNA sites in the genomes of

budding yeast (S. cerevisiae), fission yeast (S. pombe), flies

(Drosophila melanogaster, Sciara coprophila) and mam-

mals [(Abdurashidova et al., 2003; Bell, 2002; Keller et al.,

2002b; Kong and DePamphilis, 2002; Ladenburger et al.,

2002) and references therein]. The budding yeast S.

cerevisiae contains about two ORCs per replication origin

[600 ORC/cell (Rowley et al., 1995); 332 origins/cell

(Raghuraman et al., 2001)]. Since both ORC and Mcm

proteins are bound to 429 sites in the S. cerevisiae genome

(Wyrick et al., 2001), about 25% of these ORC/chromatin

sites do not initiate DNA replication. These pre-RCs are

silenced when DNA replication forks from neighboring,

earlier firing origins, pass through them (Santocanale et al.,

1999). Mammalian cells contain 104–105 molecules of

ORC per cell (Natale et al., 2000; Kreitz et al., 2001; Okuno

et al., 2001; Ohta et al., 2003), suggesting that they initiate

replication, on average, once every 60–600 kb (Natale et al.,

2000), consistent with the average size of replicons in

mammalian cells [200–300 kb; (Ockey and Saffhill, 1976;

Yurov and Liapunova, 1977)]. These data suggest that most,

if not all, ORCs are bound to replication origins.

Initiation sites for DNA replication are distributed

throughout all eukaryotic genomes. This insures that

genomes can be duplicated in a few minutes (e.g. rapidly

cleaving embryos of flies, frogs, and fish) to a few hours

(e.g. mammalian cells) without having to change the basic

replication fork mechanism by which DNA is replicated. In

yeast, flies and mammals, cis-acting sequences (replication

origins) determine where DNA replication will begin

[(DePamphilis, 1999; Altman and Fanning, 2001; Lu et al.,

2001; Bell and Dutta, 2002; Kong and DePamphilis, 2002)

and references therein], but with the exception of the small

(,0.1 kb) replication origins in S. cerevisiae, no consensus

sequence has been identified that is required for replication.

Replication origins in fission yeast, flies and mammals are

five to 20 times larger than those in budding yeast. They

frequently contain large AT-rich regions and genetically

identifiable sequences that are required for origin function.

The activity of these sequences is often orientation or

distance dependent. Furthermore, ORC not only binds to

replication origins in fission yeast, flies, and mammals (Bell

and Dutta, 2002), but recent studies have revealed that ORC

binds to specific sites within replication origins in S. pombe

(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;

Kong and DePamphilis, 2002).

One remarkable feature of eukaryotic DNA replication is

that selection of initiation sites varies from non-specific in

the rapidly cleaving embryos of frogs, flies, and fish, to site-

specific in yeasts, flies and mammals (DePamphilis, 1999;

Bell and Dutta, 2002). Moreover, as development of frogs

(Hyrien et al., 1995) and flies (Sasaki et al., 1999) changes

from rapid cell cleavage events prior to the onset of zygotic

gene expression to the slower cell division cycles in later

1 Yeast contain a single cyclin dependent protein kinase (Cdk) encoded

by the Cdc28 gene in Saccharomyces cerevisiae and Cdc2 gene in

Schizosaccharomyces pombe. This enzyme corresponds to Cdk1 in

metazoan cells.

M.L. DePamphilis / Gene 310 (2003) 1–15 3

embryonic stages and adult animals, initiation events

changes from non-specific to site-specific. Since only one

set of Orc genes has been discovered in frogs and flies, site-

specific initiation must be developmentally acquired

epigenetically. Epigenetic changes that can affect site

specificity include the ratio of initiation factors to DNA,

chromatin or nuclear composition and structure, DNA

methylation, and the acquisition of ORC accessory proteins

that facilitate binding of ORC to specific sequences

(DePamphilis, 1999). Recent studies have suggested that

transcription factors may also facilitate binding of ORC to

specific DNA sites (Bosco et al., 2001; Beall et al., 2002;

Saitoh et al., 2002).

3. The ‘ORC cycle” in summary

Reinitiation of DNA replication at the same replication

origins during a single cell division cycle is prevented in

eukaryotic cells by regulating at least three steps in the

assembly of pre-RCs – association of Cdc6, Cdt1 and

Mcm(2–7) proteins with chromatin (see Section 1). Recent

results have revealed a fourth regulatory step in which ORC

activity is inhibited during the G1 to S-phase transition and

not reestablished until mitosis is complete and a nuclear

membrane has reassembled. Therefore, regulation of ORC

activity becomes the premier step in determining when

DNA replication begins. Cell cycle dependent changes in

ORC activity, and in some cases the affinity of one or

more ORC subunits for chromatin, is hereafter referred to

as ‘the ORC cycle’. However, the manifestation of the ORC

cycle can vary among species and during development

(summarized in Fig. 2).

The ORC cycle was first recognized in mammalian cells

(Natale et al., 2000; Kreitz et al., 2001) where the Orc1

subunit is selectively destabilized during S-phase, ubiqui-

tinated and in some cases degraded, and then rebinds stably

to chromatin during the M to G1 transition to establish pre-

RCs at specific genomic sites (see Section 4). Since all six

ORC subunits are required for ORC activity in yeast (Bell,

2002), release of Orc1 should prevent ORC function, and in

fact, mammalian metaphase chromatin lacks functional

ORCs. Cell cycle dependent loss of ORC function is even

more obvious when mammalian somatic cell chromatin is

incubated in a Xenopus laevis (Xl) egg extract (see Section

7). In this case, XlORC binds to the cell chromatin, initiates

pre-RC assembly, and the entire XlORC is then released

upon completion of pre-RC assembly. The timing of this

release, however, appears to be dependent on chromatin

structure, because when Xenopus sperm chromatin is

incubated in the same extract, the affinity of XlORC for

sperm chromatin is reduced, but ORC remains bound to the

chromatin throughout DNA replication. In this case, XlORC

appears to be released during G2/M phase as a result of

hyperphosphorylation by Cdk1/cyclin A. In fact, phos-

phorylation has been shown to play a role in regulating ORC

activity in yeast even though all six ORC subunits remain

stably bound to chromatin throughout the cell cycle (see

Section 8). Whether or not an ORC cycle also exists in other

eukaryotes, such as flies, remains to be demonstrated,

although some evidence suggests that it does (see Section

9). Release of ORC subunits from chromatin after pre-RC

assembly, either as a programmed event during DNA

replication (Sun et al., 2002), or by selective elution using

salt or cyclin-dependent protein kinase activity (Hua and

Newport, 1998; Jares and Blow, 2000), does not interfere

with assembly of active replication forks. Therefore,

regulating ORC association with chromatin is a feasible

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

replication origins during S-phase is unknown, but in

Xenopus egg extracts (discussed below) ORC cannot rebind

to somatic cell chromatin until the next cell cycle begins.

It should not be surprising that species-specific variations

exist in the regulation of ORC activity, because although

ORC proteins are highly conserved within a single

taxonomic family, conservation among all species is

modest. For example, human, hamster and mouse Orc1

proteins are ,81% identical and 84% similar in their total

amino acid sequence, and 93% identical and 95% similar in

their C-terminal portion (Bogan et al., 2000). The C-

terminal portion contains a homology with Cdc6 protein and

an ATP binding domain that appears to be required for site-

specific DNA binding (Bell, 2002). Among all species,

however, total amino acid sequence conservation drops to

24% identity and 35% similarity.

4. The ORC cycle in mammals

4.1. Selective release Of Orc1

In mammals, both the cellular concentrations of Orc

proteins 2–6 and the amount of each protein bound to

chromatin appear constant throughout the cell division cycle

[Orc2 (Ritzi et al., 1998; Saha et al., 1998; Mendez and

Stillman, 2000; Natale et al., 2000; Mendez et al., 2002;

Ohta et al., 2003), Orc3 (Mendez et al., 2002; Ohta et al.,

2003), Orc4 (Okuno et al., 2001; Ohta et al., 2002), Orc5

(Ohta et al., 2003), Orc6 (Dhar and Dutta, 2000; Mendez

et al., 2002). Nevertheless, there are changes in the

distribution of ORC subunits that suggest they have

activities independent of their role in DNA replication.

For example, both the cellular concentration (Dhar and

Dutta, 2000) and the amount of chromatin bound HsOrc6

(Mendez et al., 2002) remain constant throughout the cell

cycle, but some Orc6 is found at non-chromosomal sites in

mitotic cells (Prasanth et al., 2002). This appears to be due

to a fraction of Orc6 that is not associated with ORC.

Furthermore, the abundance of individual ORC subunits

relative to each other can vary among tissues, and some

M.L. DePamphilis / Gene 310 (2003) 1–154

subunits are expressed in non-proliferating tissues (Thome

et al., 2000). Orc1, however, is unique in that it appears to

regulate ORC activity in a cell cycle dependent manner

(Fig. 1, ‘Mammals’; Fig. 2, ‘Mammalian cells’).

In hamster cells, the cellular concentration of Orc1 is

constant throughout the cell cycle (Natale et al., 2000;

Okuno et al., 2001; Li and DePamphilis, 2002), but Orc1 is

selectively released from chromatin during the S and M-

phases while the remaining Orc proteins remain chromatin

bound when cells are lysed with a non-ionic detergent in the

presence of 0.1–0.15 M salt and ATP (Natale et al., 2000; Li

and DePamphilis, 2002). The same is true in human cells,

except that the cellular concentration of Orc1 oscillates

during the cell cycle, because Orc1 is degraded during S-

phase (Kreitz et al., 2001; Fujita et al., 2002; Mendez et al.,

2002; Ohta et al., 2003); J. Teer and A. Dutta, unpublished

data]. Previous reports that HsOrc1 levels did not decrease

during S-phase (Saha et al., 1998; Tatsumi et al., 2000)

appear to have resulted from differences in the specificity of

the antibodies used to detect HsOrc1 in various experiments

(A. Dutta; C. Obuse, personal communications).

In contrast to the reports described above, release of

chromatin bound Orc1 during S-phase in hamster cells was

not detected by Okuno et al. (2001) who concluded that both

the amount of chromatin-bound Orc1 and Orc4 as well as

their cellular concentrations remained constant throughout

the cell cycle. However, although it is not clear why they did

not observe a release of Orc1 during S-phase, it does appear

that the amount of Orc1 bound to chromatin in their

experiments was markedly diminished during M-phase,

suggesting that Orc1 is selectively destabilized sometime

during the cell cycle. The absence of Orc1 in their

chromatin-unbound fraction from M-phase cells (Okuno

et al., 2001) probably resulted from the fact that unbound

Orc1 is easily degraded, even when cell extracts are on ice,

whereas chromatin bound Orc1 is stable (Li and DePam-

philis, 2002). In addition, the amount of chromatin bound

Orc1 in M-phase arrested cells can vary depending on the

length of time cells are held in nocodazole. For example,

chromatin bound Orc1 was not detected in M-phase cells

that were not collected in nocodazole (Natale et al., 2000; Li

and DePamphilis, 2002), whereas cells arrested in nocoda-

zole could contain up to 20% of the amount of chromatin

bound Orc1 observed in G1-phase cells, depending on how

long the cells were held in nocodazole (Li and DePamphilis,

2002). Nocodazole blocks anaphase by preventing micro-

tubule assembly, which does not necessarily prevent other

cell cycle related events.

The selective release of Orc1 during S-phase accounts

for the fact that one or more ORC subunits are both

functionally and physically absent from metaphase chro-

matin. The absence of ORC activity is revealed by the fact

that hamster M-phase chromatin will replicate in a complete

Xenopus egg extract, but not in one that has been depleted of

Xenopus Orc proteins (Yu et al., 1998; Li et al., 2000; Natale

et al., 2000). In contrast, nuclei isolated in the same manner

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.

M.L. DePamphilis / Gene 310 (2003) 1–15 5

from G1-phase cells will initiate DNA replication de novo

under both conditions, revealing that hamster pre-RCs are

assembled during the M to G1 transition. Loss of ORC

activity can be accounted for by the fact that Orc1 protein is

not associated with metaphase chromatin under conditions

where other Orc proteins remain stably bound (Natale et al.,

2000; Tatsumi et al., 2000; Okuno et al., 2001; Mendez

et al., 2002) and mammalian Cdc6 protein is abundant

[(Mendez and Stillman, 2000; Petersen et al., 2000). This

observation is consistent with the cell cycle-dependent

changes observed in a footprint at the human lamin B2

origin (Abdurashidova et al., 1998) that encompasses the

start sites for leading strand DNA replication (Abdurashi-

dova et al., 2000). A large G1-phase footprint changed into a

smaller S and G2 phase footprint that disappeared during M-

phase. Recent UV-induced cross-linking experiments reveal

that the large G1-phase footprint could be accounted for by

the presence of a pre-RC containing at least Orc1, Orc2,

Cdc6, and Mcm3 proteins (Abdurashidova et al., 2003).

During S-phase, only the Orc2 protein can be cross-linked to

chromatin, consistent with the disappearance of the pre-RC

and the release of Orc1. By M-phase, not even Orc2 could

be cross-linked to DNA, suggesting additional changes

occur in the affinity of ORC for chromatin. Similarly, both

Orc1 and Orc2 can be cross-linked to specific replication

origins in vivo by treating human G1-phase cells with

formaldehyde, but only Orc2 can be cross-linked during S-

phase (Ladenburger et al., 2002). These observations in

mammalian cells are consistent with earlier reports that both

Orc1 and Orc2 are present on chromatin in Xenopus

interphase cells but not in metaphase cells (Romanowski

et al., 1996). Moreover, Orc proteins in Xenopus eggs

competent for DNA replication will bind to sperm

chromatin, whereas Orc proteins in Xenopus eggs in

metaphase II will not (Coleman et al., 1996; Hua and

Newport, 1998; Findeisen et al., 1999; Rowles et al., 1999).

Whether Orc1 is associated with chromatin in vivo in an

unstable state or is actually released from chromatin in vivo

as well as in cell extracts is difficult to assess. What is clear

is that Orc1 is not stably bound to chromatin in comparison

with other Orc proteins, and that metaphase cells do not

contain functional ORCs. M-phase cells permeabilized with

digitonin retain Orc1 in the chromatin pellet (Natale et al.,

2000), but this M-phase chromatin does not initiate DNA

replication in an ORC-depleted Xenopus egg extract (Yu

et al., 1998; Li et al., 2000; Natale et al., 2000). Expression

of a GFP-tagged Orc1 protein in hamster cells resulted in

GFP-labeled metaphase chromosomes (Okuno et al., 2001),

but there is no evidence that the GFP-Orc1 protein is

assembled into ORCs and if so, that the GFP-ORCs are

active.

4.2. Ubiquitination Of Orc1

Orc1 in S-phase mammalian cells is selectively ubiqui-

tinated and, under some conditions, degraded. In hamster

cells, two forms of Orc1 are released concurrently during

S-phase, one that is not ubiquitinated and one that is

mono-ubiquitinated (Li and DePamphilis, 2002). Orc1 and

mono-ubiquitinated Orc1 also were detected in the non-

chromatin bound fraction from human S-phase cells

(Mendez et al., 2002). However, in human cells, Orc1

became polyubiquitinated and was degraded via the 26S

proteasome pathway (Fujita et al., 2002; Mendez et al.,

2002), a reaction mediated by the SCFskp2 ubiquitin-ligase

complex (Mendez et al., 2002). In contrast, only trace

amounts of polyubiquitinated Orc1 were detected in

hamster cells. Hamster Orc1 was polyubiquitinated and

degraded only when it was released from nuclei into cellular

extracts. Thus, the chromatin unbound fraction of Orc1 in

non-ionic detergent extracts of hamster mitotic cells can be

lost, even when cell lysates are held in ice (Okuno et al.,

2001; Li and DePamphilis, 2002).

In contrast to Orc1, Orc2 not only remains bound to

chromatin throughout the cell cycle, but Orc2 is not a

substrate for ubiquitination, even as a soluble protein (Li

and DePamphilis, 2002). Interestingly, the half-life of Orc1

in both hamster and human exponentially proliferating cells

is ,3 h, the same as observed for Orc2 [(Li and

DePamphilis, 2002); J. Mendez, personal communication].

Therefore, it may not be necessary to deubiquitinate Ub-

Orc1 in order to reestablish functional ORC/chromatin sites

in early G1-phase, because in both hamster and human cells,

most of the Orc1 in M-phase cells will have been

synthesized during the previous 6 h.

5. Orc1 and the assembly of pre-replication complexes

Several observations suggest that the stable binding of

Orc1 to chromatin is the premier regulatory step in the

assembly of pre-RCs and thereby the commitment to cell

proliferation. First, both HsOrc1 (but not HsOrc2) and

DmOrc1 expression are regulated by E2F (Ohtani et al.,

1996; Asano and Wharton, 1999), a transcription factor that

plays an important role in driving cells from G1 into S-

phase. E2F also regulates expression of Cdc6 and Mcm

genes (Leone et al., 1998; Ohtani et al., 1998; Yan et al.,

1998), suggesting that assembly of pre-RCs depends on the

E2F/Rb pathway with ORC activity regulated by expression

of Orc1. Second, the Orc1 gene is the only ORC gene whose

expression is correlated with cell proliferation: Orc1 is

expressed only in proliferating mammalian cells whereas

Orc2–Orc5 are expressed to varying extents in many non-

proliferating cells (Thome et al., 2000). Third, stable

binding of Orc1 to chromatin immediately precedes

assembly of pre-RCs at specific chromosomal loci.

During the M to G1 transition, Orc1 rebinds stably to

chromatin with the concomitant appearance of functional

ORCs. M-phase hamster cells contain as much Orc1 as G1-

phase cells, but most, if not all, of the Orc1 in M-phase

hamster cells is not bound to chromatin and only a minor

M.L. DePamphilis / Gene 310 (2003) 1–156

fraction is ubiquitinated (Li and DePamphilis, 2002). M-

phase human cells generally contain much less Orc1 than

G1-phase human cells, as a result of Orc1 degradation

during S-phase (Mendez et al., 2002). Therefore, human

Orc1 must be resynthesized during the M to G1 transition,

whereas hamster Orc1 does not (Okuno et al., 2001). ORC

activity is restored during the first 2 h after either hamster or

human M-phase cells (arrested in nocodazole) are released

into G1-phase (Yu et al., 1998; Natale et al., 2000). This

corresponds to the time period (1–3 h post-M) when Orc1

(Natale et al., 2000; Li and DePamphilis, 2002; Mendez

et al., 2002; Ohta et al., 2003), Mcm2 and Mcm3 reassociate

stably with chromatin (Dimitrova et al., 1999; Natale et al.,

2000; Dimitrova et al., 2002; Ohta et al., 2003). Nuclei

isolated from these early G1-cells can initiate de novo DNA

replication in a Xenopus egg extract depleted either of ORC

or of Mcm proteins. The appearance of chromatin bound

Orc1 is inversely related to the lost of cyclin B (Mendez and

Stillman, 2000), which is required to drive cells into mitosis,

and directly linked to formation of an intact nuclear

envelope (Dimitrova and Gilbert, 1998; Natale et al.,

2000), which is required for initiation of DNA replication.

Thus, the bulk of Orc1 and Mcm binding occurs after

mitosis is complete and a nuclear membrane has reformed

around the genome, a time referred to either as late

telophase or early G1-phase. Consistent with this view is

the fact that HsCdc6 protein, like cyclin B, is degraded

during the M to G1 transition and then resynthesized during

G1 (Petersen et al., 2000; Mendez et al., 2002), just in time

to be incorporated into the newly reconstituted active ORC/

chromatin sites. Therefore, stable rebinding of Orc1 to

chromatin appears to be the rate-limiting step for assembly

of pre-RCs.

One experiment, however, suggests that pre-RC assem-

bly is completed about 2 h before the stable binding of

hamster Orc1, Mcm2 and Mcm3 is completed. Geminin, a

specific inhibitor of Cdt1, prevents stable binding of

Mcm(2–7) to chromatin. Once pre-RCs are assembled,

DNA replication is no longer sensitive to geminin. DNA

replication in hamster metaphase chromatin incubated in

Xenopus egg extract is sensitive to geminin, but DNA

replication in chromatin isolated 1 h after metaphase is not

(Okuno et al., 2001; Dimitrova et al., 2002). In these

experiments, Orc1 appears to bind to chromatin within the

1st h after release of cells from the nocodazole block,

consistent with the execution point for geminin. Interest-

ingly, Mcm proteins continued to bind to chromatin until

mid to late G1-phase (Dimitrova et al., 1999; Natale et al.,

2000; Dimitrova et al., 2002), suggesting that more Mcm

proteins bind to chromatin than are actually assembled into

pre-RCs. Apparent differences in the published time courses

for protein binding and the geminin execution point likely

reflect differences in experimental protocols that affect the

amount of protein recovered in different fractions and the

time required for cells to progress from M to G1 when the

synchronizing agent, nocodazole, is removed.

6. ORC and the ‘origin decision point’

Xenopus egg extract can initiate DNA replication de

novo in virtually any DNA, chromatin or nuclei provided,

but these initiation events are generally distributed non-

specifically throughout the DNA substrate. The only

exception is when nuclei are isolated from mammalian

cells under conditions that preserve their impermeability to

macromolecules (Gilbert et al., 1995). Under these con-

ditions, initiation events are distributed non-specifically

throughout the genome when ‘nuclei’ are isolated from M-

phase or early G1-phase cells, but at specific sites when

nuclei are taken from late G1-phase cells (Wu and Gilbert,

1996; Wu and Gilbert, 1997; Li et al., 2000; Okuno et al.,

2001). DNA replication in late G1-nuclei is independent of

XlORC proteins, begins in vitro at the same site-specific

origins of bi-directional DNA replication used by the cells

in vivo, and is sensitive to protein kinase inhibitors.

Therefore, it appears that pre-RCs in late G1-phase nuclei

are present at specific genomic sites waiting to be activated

by Cdk2/cyclin A, E and Cdc7/Dbf4 (Fig. 1). This cell cycle

dependent transition that occurs in mammalian chromatin,

from a substrate for non-specific initiation events to a

substrate for site-specific initiation events, is referred to as

the ‘origin decision point’ (ODP).

Two views of the ODP have emerged. In one view, the

ODP marks the transition from Xenopus ORC directed

initiation events to mammalian ORC directed initiation

events. Prior to the ODP in hamster cells (midpoint ,2 h

post-M), initiation of DNA replication in hamster chromatin

requires the presence of Xenopus ORC in the egg extract

(Natale et al., 2000). XlORC present in these experiments

rapidly binds to hamster metaphase chromatin within the

first few minutes of incubation (Sun et al., 2002) and is

responsible for most of the DNA replication that occurs

during the first 1–2 h post-M (Yu et al., 1998; Natale et al.,

2000) where it initiates replication non-specifically through-

out the genome (Yu et al., 1998; Dimitrova and Gilbert,

1999; Li et al., 2000; Natale et al., 2000). After the ODP in

hamster cells, Xenopus egg extract simply activates hamster

pre-RCs that were assembled in vivo. Initiation sites are

established in hamster cells upon the stable binding of Orc1

(midpoint ,1.5 h post-M) to Orc2–6/chromatin complexes

located at specific sites along the genome (Natale et al.,

2000; Li and DePamphilis, 2002). This view is supported by

the studies in human cells where Orc1 binding to chromatin

follows a similar time course to that in hamster cells

(Mendez et al., 2002) and where cross-linking studies have

revealed that Orc2 is bound to specific genomic sites, some

of which correspond to origins of bi-directional DNA

replication (Abdurashidova et al., 2003; Keller et al., 2002b;

Ladenburger et al., 2002). This view also accounts for the

observation that the normal temporal order for initiation is

absent in chromatin isolated from cells in M-phase to 1 h

post-M phase but present in chromatin isolated from late

G1-phase cells (Dimitrova and Gilbert, 1999; Li et al.,

M.L. DePamphilis / Gene 310 (2003) 1–15 7

2001). XlORC initiates replication non-specifically in

chromatin from M-phase and telophase cells.

An alternative view of the ODP is that pre-RCs are

initially assembled ‘randomly’ along the genome during the

M to G1-transition in mammalian cells, and then reorgan-

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

at the ODP, leaving those pre-RCs at specific genome sites

to initiate replication at the start of S-phase. However, this

implies the existence of a larger number of ORC/chromatin

sites in mammalian cells that the cellular concentration of

ORC would permit. This view of the ODP is based on the

conclusion that all six mammalian ORC subunits are stably

bound to chromatin throughout the cell cycle, that pre-RCs

are assembled in hamster cells within 1 h after metaphase,

and that pre-RC assembly precedes the ODP in hamster

cells (midpoint ,3 h post-M) by 2–2.5 h. Various

experimental artifacts that are innumerated in the papers

cited above may well explain some of the differences

between these two views. Nevertheless, the possibility

remains that there exists a novel, as yet undiscovered,

mechanism for determining where initiation events occur in

mammalian genomes.

6.1. Association of Orc1 with nuclear structure

The fact that assembly of pre-RCs is delayed until

mitosis is complete and a nuclear membrane is assembled

implies that nuclear structure plays a role in the initiation of

DNA replication. The nucleus appears to regulate the

concentration of proteins required to initiate DNA replica-

tion (Walter et al., 1998), to facilitate the assembly or

activity of DNA replication forks, and to determine where in

the genome initiation of DNA replication occurs (reviewed

in DePamphilis, 2000; Keller et al., 2002a). Replication

origins have been reported to be associated with ‘nuclear

matrix’ preparations (Djeliova et al., 2001), although not

with ‘nucleoskeleton’ preparations (Ortega and DePamphi-

lis, 1998), revealing that interactions between replication

origins and nuclear structure are sensitive to experimental

conditions.

Recent observations reveal that Orc1 (and to a lesser

extent the other ORC subunits) is associated with a fraction

of chromatin that is not solubilized by digestion with

endonucleases (Kreitz et al., 2001; Fujita et al., 2002), and

that this fraction accumulates during G1 phase and then

decreases during S-phase (Ohta et al., 2003). In HeLa cells,

the appearance of Orc1 following release of cells from a

metaphase block occurs concurrently with the appearance of

Orc1 and other ORC proteins in an insoluble nuclease-

resistant fraction, suggesting that newly assembled ORC/

chromatin complexes are tethered to some component of

nuclear structure and then released as the Orc1 subunit is

destabilized, ubiquitinated, and in the case of human cells,

degraded (Fig. 1, ‘Mammals’). Mcm proteins, which appear

to bind to chromatin flanking the ORC binding site, appear

in the soluble nuclease-sensitive fraction. This transition of

ORC/chromatin complexes from untethered to tethered may

contribute to the phenomenon referred to as the ‘origin

decision point’.

7. The ORC cycle in frogs

Metaphase chromatin lacks functional ORC complexes,

and therefore when metaphase chromatin from hamster cells

is incubated in a Xenopus egg extract, XlORC rapidly binds

to the chromatin and initiates DNA replication. However, in

contrast to mammalian ORC, where only the Orc1 subunit is

destabilized upon DNA replication, the entire XlORC is

released from the somatic cell chromatin into the replication

extract (50 mM KCl) as soon as pre-RCs have been

assembled [Fig. 1, ‘Xenopus’; Fig. 2, ‘somatic cell

chromatin’; (Sun et al., 2002)]. This programmed release

of XlORC was insensitive to aphidicolin (specific inhibitor

of replicative DNA polymerases) and olomoucine (specific

inhibitor of cyclin dependent protein kinases), but sensitive

to geminin (specific inhibitor of Cdt1), and prevented by

immuno-depletion of XlMcm proteins from the egg extract.

Therefore, assembly of pre-RCs triggers release of ORC

from cell chromatin. Alternatively, ORC release may be

triggered by some event that occurs between completion of

pre-RC assembly and the action of Cdk2. For example,

Mcm10 binds to chromatin after pre-RC assembly and

before Cdk2 activity where it is required to initiate DNA

replication (Wohlschlegel et al., 2002).

Once pre-RCs were assembled in egg extract, XlORC

was no longer present on somatic cell chromatin, despite the

fact that egg extract contained a great excess of soluble

XlORC. Furthermore, XlORC could not bind to chromatin

in nuclei isolated from hamster cells that had progressed

past their ‘origin decision point’ (Sun et al., 2002). Cdk/

cyclins were not involved in these events, since neither

binding nor release of XlORC to somatic cell chromatin was

affected by olomoucine. Moreover, XlORC in meiotic eggs

(metaphase II) does not bind to sperm chromatin (Coleman

et al., 1996; Hua and Newport, 1998; Findeisen et al., 1999;

Rowles et al., 1999), and XlORC is not bound to metaphase

chromatin in Xenopus somatic cells (Romanowski et al.,

1996). This inability of XlORC in cells undergoing meiosis

or mitosis to bind to chromatin likely reflects the fact that

XlORC is hyperphosphorylated in these cells where it

associates with Cdk1/cyclin A (Romanowski et al., 2000).

These data strongly suggest that XlORC cannot rebind to

either somatic cell chromatin or sperm chromatin until cell

division is completed.

7.1. Chromatin structure and the affinity of ORC for DNA

Xenopus sperm chromatin, like mammalian metaphase

chromatin, lacks ORC proteins, and XlORC rapidly binds to

M.L. DePamphilis / Gene 310 (2003) 1–158

sperm chromatin and initiates DNA replication. However,

in this case, XlORC is not released from chromatin,

although it does undergo a demonstrable change in its

affinity for chromatin [Fig. 2, ‘sperm chromatin’; (Hua and

Newport, 1998; Rowles et al., 1999)]. Prior to pre-RC

assembly, ORC remains bound to sperm chromatin in 0.25

M salt, but after pre-RC assembly, ORC can be eluted from

sperm chromatin by exposure to high levels of Cdk2, or to

mitotic extract, or 0.1–0.15 M KCl, leaving the Mcm

proteins bound to the chromatin and capable of initiating

DNA replication. This destabilization of ORC’s association

with sperm chromatin does not require DNA replication,

protein synthesis, cyclin-dependent kinases or Cdc7. It does

require prior action of Cdc6, Mcm(2–7), and Cdt1,

suggesting that binding of the Mcm(2–7) to chromatin

weakens the association of ORC with sperm chromatin and

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

assembly. However, in the case of sperm chromatin, ORC

apparently is not released until mitosis, a step that was

blocked in these experiments by the presence of

cycloheximide.

The fact that XlORC remains bound to sperm chromatin

under conditions where it is released from somatic cell

chromatin reveals that the affinity of ORC for DNA depends

on chromatin structure. Since both chromatin substrates are

rapidly assembled into nuclei by Xenopus egg extract, these

differences likely reflect differences in their composition.

For example, somatic cell chromatin brings with it a full

complement of core and linker histones, but sperm

chromatin must undergo extensive remodeling during its

first 30 min in egg extract (Philpott and Leno, 1992).

Nucleoplasmin dependent decondensation of sperm chro-

matin is a prerequisite for XlORC binding and subsequent

assembly of pre-RCs (Gillespie et al., 2001). Remodeling

involves displacement of sperm specific protamines, uptake

of core histones H2A and H2B (histones H3 and H4 are

already present), and linker histone B4 (histone H1 is absent

from Xenopus sperm and eggs), as well as phosphorylation

of core histones and uptake of HMG-2 protein (Dimitrov

et al., 1994). Sperm chromatin is not converted into somatic

cell chromatin until after the midblastula transition when

histone H1 is expressed, the rate of cell division slows

down, and a G1-phase first appears in the cell division cycle.

7.2. Comparison of frogs and mammals

The difference in behavior between mammalian and

Xenopus ORCs is in keeping with the properties of the

purified proteins. XlOrc proteins exist as a stable complex of

XlOrc(1–6) that can be immunoprecipitated from Xenopus

egg extract (Rowles et al., 1996; Tugal et al., 1998),

although the identity of XlOrc6 has not been confirmed. In

contrast, human Orc proteins exist as a stable complex of

HsOrc(2–5) to which HsOrc1 and HsOrc6 bind only weakly

(Dhar et al., 2001; Vashee et al., 2001). Therefore, it is not

surprising that mammalian Orc1 is selectively released from

chromatin when cells enter S-phase and then rebinds when

cells enter G1-phase, while Xenopus Orc1 remains associ-

ated with the other ORC subunits, regardless of whether

they are bound or released from chromatin. Thus, XlORC is

well suited to initiating DNA replication at many sites as

cells undergo rapid cleavage events during early frog

development. Xenopus ORC appears to bind weakly to

chromatin and then release intact in order to reinitiate

quickly at other sites. Mammalian ORC, on the other hand,

is better suited to binding tightly to specific genomic sites

where it remains throughout cell proliferation and differen-

tiation. In mammals, site specificity is maintained by

releasing only one of the six subunits during S-phase,

leaving the core ORC tightly bound to a replication origin.

8. The ORC cycle in yeast

In contrast to mammalian cells, yeast ORC remains

stably bound to chromatin throughout the yeast cell cycle. In

S. cerevisiae, both DNA footprinting (Diffley et al., 1994;

Fujita et al., 1998) and immuno-precipitation (Liang and

Stillman, 1997) analyses reveal that a complete ORC binds

to yeast replication origins immediately after initiation of

replication occurs and remains there throughout the cell

division cycle (Fig. 2, ‘yeast’). Similarly, in S. pombe all six

ORC subunits remain bound to chromatin throughout the

cell cycle (Lygerou and Nurse, 1999; Kong and DePam-

philis, 2001). Nevertheless, re-initiation within a single

S-phase is prevented by a combination of ORC phosphoryl-

ation, down regulation of Cdc6 activity, and nuclear

exclusion of Mcm proteins (Nguyen et al., 2001).

In S. cerevisiae, ScOrc1, ScOrc2 and ScOrc6 contain

consensus phosphorylation sites, and these proteins are

phosphorylated by the cyclin dependent protein kinase

Cdk1(Cdc28)/Clb1 during the G1 to S transition (Nguyen

et al., 2001). ScORC proteins are hyperphosphorylated

during the S to M transition, and then hypophosphorylated

during early G1-phase when pre-RC assembly occurs.

Genetic alterations of ScOrc proteins that prevent their

phosphorylation do not affect either DNA replication or cell

division. However, when this alteration is accompanied by

alterations in both Cdc6 and Mcm proteins that prevent their

export from the nucleus, then DNA replication is reinitiated

prior to cell division, resulting in polyploid cells. In other

words, all three regulatory pathways must be inactivated in

order to induce reinitiation of DNA replication within a

single cell division cycle.

A similar phenomenon exists in S. pombe. SpOrc2

undergoes dephosphorylation during the M to G1 transition

(Lygerou and Nurse, 1999; Kong and DePamphilis, 2001),

and is hyperphosphorylated when cells enter S-phase and

Mcm proteins are released from chromatin (Vas et al., 2001;

Wuarin et al., 2002). Furthermore, the mitotic B type cyclin

M.L. DePamphilis / Gene 310 (2003) 1–15 9

Cdc13 complexed with the Cdk1(Cdc2)1 protein kinase

associates with replication origins during S-phase and

remains there during G2 and early M-phases (Wuarin

et al., 2002). This association is ORC-dependent, apparently

by association of Cdk1(Cdc2) with the Orc2 subunit, and

prevents reinitiation of DNA replication before mitosis has

been completed. These data strongly suggest that the

phosphorylated state of SpOrc2 determines ORC activity.

8.1. Comparison of yeast and frogs

The ORC cycle in yeast resembles that in Xenopus egg

extract when sperm chromatin in the substrate (Fig. 2). ORC

proteins in both species are hyperphosphorylated during

metaphase by Cdk1, and phosphorylation inhibits its ability

to initiate pre-RC assembly. Phosphorylated XlORC binds

poorly to chromatin (Romanowski et al., 2000), while pre-

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

phosphorylation of XlORC subunits, like those in yeast, also

occurs during DNA replication is not clear. Whether or not

yeast ORC, like XlORC, can be selectively eluted at lower

salt concentrations following pre-RC assembly or DNA

replication also is not clear. Dephosphorylation of the Orc2

subunit occurs during the M to G1 transition in Xenopus

(Sun et al., 2002) and in yeast (Nguyen et al., 2001; Vas

et al., 2001), presumably as an activation step in pre-RC

assembly. Phosphorylation of ORC may alter its affinity

either for chromatin or for other pre-RC proteins during the

G1 to S transition, while chromatin composition may

determine whether the modified ORC subunits are remain

bound or are released. Interestingly, yeast chromatin, like

Xenopus sperm chromatin, lacks a classical histone H1

linker (Landsman, 1996), suggesting that reduced chromatin

condensation may facilitate binding of ORC during S-phase.

9. The ORC cycle in flies

Direct analyses of cell cycle dependent changes in the

activity, post-translational modifications or chromatin

affinity of DmORC are not yet available. However, several

observations are consistent with a cell cycle dependent,

differential association of DmOrc proteins with chromatin.

In Drosophila, Orc2 is distributed fairly homogeneously in

interphase nuclei, while it only remains bound to the

heterochromatic region of chromosomes through mitosis in

embryos and larval neuroblasts (Pak et al., 1997; Loupart

et al., 2000). Mutations in Orc2 exhibited delayed entry into

S-phase as well as delayed exit from metaphase with some

euchromatic regions replicating even later than heterochro-

matin (Loupart et al., 2000). In addition, mitotic chromo-

somes were irregularly condensed, suggesting a novel role

for ORC in chromosome architecture as well as DNA

replication. In contrast to Orc2, Orc1 levels change

dramatically throughout Drosophila development, and its

accumulation is regulated by E2F-dependent transcription

(Asano and Wharton, 1999). In embryos, Orc1 accumulates

preferentially in proliferating cells, and in the eye imaginal

disc, Orc1 accumulation is cell cycle regulated, with high

levels in late G1 and S phase. Moreover, overexpression of

Orc1 altered the pattern of DNA synthesis, implicating Orc1

in regulating initiation of DNA replication.

The DNA binding activity of purified DmORC is largely

non-specific and ATP-independent, although DmORC, like

ScORC, requires only the ORC1 component of the complex

to bind ATP for tight DNA interactions (Chesnokov et al.,

2001). In vivo, DmORC does bind to specific genomic sites

(Austin et al., 1999; Bielinsky et al., 2001). Such site-

specific DNA binding may require association with other

proteins (Bosco et al., 2001; Beall et al., 2002), and these

associations may regulate ORC activity as well.

10. Mechanisms that regulate ORC activity

The results described above reveal at least five

mechanisms by which ORC activity is regulated during

cell proliferation.

10.1. Ubiquitination

Since both Orc1 and Ub-Orc1 are rapidly released from

chromatin when hamster cells enter S-phase and Ub-Orc1 is

largely absent from mitotic cells, ubiquitination per se is not

likely the cause of Orc1 release, but a mechanism to prevent

its reassociation with ORC/chromatin sites during S-phase.

Two phenomena suggest that Ub-Orc1 may become

sequestered in the nuclear membrane. First, several

examples have been reported where monoubiquitination is

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

ubiquitin conjugates proteins with a single adduct and this

modification generally results in the protein’s relocation to

the nucleus (Wilson and Rangasamy, 2001). Some cells,

such as HeLa and other cancer cells, may contain larger

pools of Orc1 that cause it to be exported to the cytoplasm

where it is polyubiquitinated and degraded. In fact,

differences in pool sizes could account for the observation

that Cdc6 is exported from the nucleus and degraded in

human cells but not in hamster cells (Okuno et al., 2001).

10.2. Phosphorylation

Phosphorylation of Orc proteins can affect both their

affinity for chromatin and their activity. In yeast, Cdk1/cy-

clin B prevents pre-RC assembly until mitosis has been

completed by phosphorylating one or more of the ORC

subunits and Cdc6. In frogs, both Orc1 and Orc2 are

hyperphosphorylated in metaphase-arrested eggs relative to

M.L. DePamphilis / Gene 310 (2003) 1–1510

activated eggs (Carpenter and Dunphy, 1998; Tugal et al.,

1998). Moreover, XlORC can be selectively released from

chromatin by incubating chromatin either in a metaphase

extract (Rowles et al., 1999) or with Cdk1(Cdc2)/cyclin A

(Hua and Newport, 1998; Findeisen et al., 1999). In

mammals, hyperphosphorylation of Orc1 has been detected

in metaphase cells (Tatsumi et al., 2000; Thome et al.,

2000), and over-expression of cyclin A results in export of

Orc1 to the cytoplasm, a process that is dependent on the

phosphorylation status of Cdk target sites in Orc1 (Laman

et al., 2001). Given the fact that Cdk2/cyclin A interacts

with HsOrc1 (Mendez et al., 2002) and Cdk1/cyclin A

interacts with XlORC (Romanowski et al., 2000), cyclin

dependent phosphorylation may be the mechanism that

releases ORC proteins from chromatin during the G1 to S

transition, and the released protein then may be exported to

the cytoplasm where it undergoes polyubiquitination and

degradation.

10.3. DNA replication

DNA replication appears to be required to destabilize

Orc1 in mammalian cells, because significant amounts Orc1

are not released from chromatin in cells arrested at their

G1/S interphase (Li and DePamphilis, 2002). Therefore,

Orc1 release in mammals may be triggered by DNA

synthesis through the origin region. This hypothesis is

supported by data from yeast and flies.

Orc1, Orc4, and Orc5 from yeast and flies can each bind

ATP, and the same ORC subunits in all other eukaryotes

contain potential ATP binding sites [reviewed in (Bell,

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

binding to ScOrc1 is required for site-specific binding of

ScORC to origin DNA. The rate of ATP hydrolysis is then

reduced in response to origin binding, suggesting that ATP

is not hydrolyzed until a subsequent step in replication. That

step appears to be the generation of single stranded DNA at

replication origins. ScORC binds to ssDNA, but its affinity

for ssDNA is dependent only on ssDNA length, not on either

DNA sequence or ATP binding. In fact, association of

ScORC with ssDNA stimulates ATP hydrolysis, and

stabilizes an altered ORC structure (Lee et al., 2000). In

yeast, this altered ORC structure may be locked in place by

phosphorylation of ORC subunits and thereby prevent

subsequent pre-RC assembly. In mammals, this altered

ORC structure may cause selective release of Orc1.

10.4. Pre-replication complex assembly

In Xenopus egg extract, assembly of pre-RCs triggers a

change in the affinity of ORC for chromatin that is

manifested differently, depending on the chromatin sub-

strate. In the case of somatic cell chromatin, the entire ORC

is released under DNA replication conditions. In the case of

sperm chromatin, the entire ORC becomes salt-sensitive.

10.5. Auxiliary protein interactions

Interactions between ORC and other proteins can

facilitate pre-RC assembly (Fig. 1). For example, HsOrc1

binds Cdc6 (Saha et al., 1998), and Cdc6 facilitates binding

of XlORC to somatic cell chromatin (Sun et al., 2002) and

ScORC to its cognate replication origin (Mizushima et al.,

2000). Moreover, ScCdc6 specifically recognizes the ATP-

bound state of ScOrc1 (Klemm and Bell, 2001). Therefore,

Orc1 may recruit Cdc6, which is present during M-phase,

and this Orc1/Cdc6 complex may then target specifically

one of the many Orc2–6 complexes that are bound to

various sites along the genome (the ‘Jesuit Model’ for site

selection, (DePamphilis, 1993, 1996, 1999).

Interactions between ORC and other proteins can also

prevent pre-RC assembly. For example, Skp2, a component

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

Cdk1/cyclin B during mitosis in S. pombe prevents

assembly of pre-RCs (Wuarin et al., 2002). Sic1, a specific

inhibitor of Cdks, prevents the S-phase-specific Cdk1/Clb5

protein kinase in S. cerevisiae from interacting with ORC,

but does not prevent the G1–phase specific Cdk1/Cln2

kinase from binding to ORC (Weinreich et al., 2001).

Therefore, Sic1 can prevent Cdk1 activation of pre-RCs

during the G1 to S-phase transition, without also preventing

subsequent inactivation of pre-RC assembly by the same

protein kinase during the S to M-phase transition.

11. Regulating initiation of DNA replication by

regulating ORC activity

One can envisage at least four advantages to regulating

ORC activity. The first is to prevent assembly of pre-RCs

during S-phase by inactivating the premier step in their

assembly. By applying this strategy to newly assembled

ORC/chromatin sites as well as to those sites where

initiation has already occurred, initiation of DNA replica-

tion is restricted to once-per-origin-per-cell division cycle.

Moreover, the assembly of pre-RCs cannot begin again until

mitosis is complete and a nuclear membrane has been

reassembled. To this end, mammals have taken the more

sophisticated approach. Since Cdc6 binds specifically to

Orc1, selective inactivation of Orc1 would directly prevent

recruitment of Cdc6 to ORC/chromatin sites without

disturbing the genomic locations of the remaining chroma-

tin bound Orc proteins.

A second advantage is to provide the metazoa with a

mechanism for selecting which of the many potential

initiation sites along the genome will be activated [‘Jesuit

Model’, (DePamphilis, 1993, 1996)]. A core complex

M.L. DePamphilis / Gene 310 (2003) 1–15 11

consisting of Orc(2–5) or Orc(2–6) could be assembled at

many potential initiation sites, but which of these sites is

activated during each round of cell proliferation would be

determined by the binding either of Orc1 or of an Orc1/Cdc6

complex. This would help to resolve the problem of

preventing DNA replication from interfering with DNA

transcription as the pattern of gene expression and

chromatin organization changes during animal develop-

ment. The ORC cycle would allow some replication origins

to be inactivated while activating others. A dramatic

example of this is the transition from non-specific selection

of initiation sites in embryos undergoing rapid cell

cleavages to site-specific initiation in cells of the same

animal undergoing a normal mitotic cell cycle (Hyrien et al.,

1995; Sasaki et al., 1999).

Third, the ORC cycle offers an excellent opportunity for

checkpoint control mechanisms. For example, cells that

have sustained DNA damage, or that have entered a

quiescent or terminally differentiated state could prevent

activation of the pathway leading to S-phase by inactivating

one or more ORC subunits. Human cells treated with

adriamycin, a DNA damage-inducing agent, do, in fact,

selectively degrade Orc1 (Mendez et al., 2002).

Finally, the existence of multiple mechanisms for

regulating initiation of DNA replication serves to prevent

terminally differentiated cells from reentering their cell

proliferation cycle. Cells entering a quiescent state have

been shown to lose their ability to establish pre-replication

complexes at specific genomic sites (Wu and Gilbert, 1997)

and their ability to bind Cdc6 and Mcm proteins to their

chromatin (Madine et al., 2000; Sun et al., 2001). Moreover,

terminally differentiated cells lack critical proteins required

for assembly of pre-replication complexes (Lu et al., 1999).

While the mechanisms involved have not yet been

elucidated, destabilization of one or more ORC subunits,

followed by the sequestration of Ub-Orc or the destruction

of [Ub]n-Orc1 may well trigger a cascade of events that

shuts down cell proliferation pathways.

Acknowledgements

The author wishes to thank Anindya Dutta, Arturo

Falaschi, Juan Mendez and Chikashi Obuse for sharing

unpublished results with him. In addition, the author is

grateful to Anindya Dutta, Margarete Heck and the three

reviewers for their thoughtful suggestions.

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