www.elsevier.com/locate/yexcr

Experimental Cell Research 295 (2004) 138–149

Cell cycle regulation of the licensing activity of Cdt1 in Xenopus laevis$

Domenico Maiorano, Wilfrid Rul,1 and Marcel Mechali*

Institute of Human Genetics, CNRS, 34396 Montpellier Cedex 05, France

Received 4 June 2003, revised version received 12 November 2003

Abstract

Cdt1 is a conserved replication factor required in licensing the chromosome for a single round of DNA synthesis. The activity of Cdt1 is

inhibited by geminin. The mechanism by which geminin interferes with Cdt1 activity is unknown. It is thought that geminin binds to and

sequestrate Cdt1. We show that geminin does not interfere with the chromatin association of Cdt1 and that inhibition of DNA synthesis by

geminin is observed following its accumulation on chromatin. The binding of geminin to chromatin has been investigated during S phase. We

demonstrate that loading of geminin onto chromatin requires Cdt1, suggesting that geminin is targeted at replication origins. We also show

that geminin binds chromatin at the transition from the pre-replication to pre-initiation complexes, which overlaps with the release of Cdt1.

This regulation is strikingly different from that observed in somatic cells where the chromatin binding of these proteins is mutually exclusive.

In contrast to somatic cells, we further show that geminin is stable during the early embryonic cell cycles. These results suggest a specific

regulation of origin firing adapted to the rapid cell cycles of Xenopus and indicate that periodic degradation of geminin is not relevant to

licensing during embryonic development.

D 2004 Elsevier Inc. All rights reserved.

Keywords: DNA replication; S phase; Pre-RC; Pre-IC; Geminin; Chromatin; Development

Introduction

A crucial step in initiating DNA synthesis is the opening

of the DNA double helix at replication origins, a step

necessary to form the template for the catalytical activity

of the DNA polymerases machinery [1]. Replication origins

acquire the competence to carry out this step on exit from

mitosis in a sequential reaction currently known as replica-

tion licensing, which corresponds to formation of pre-

replicative complexes [2–4]. The Origin Recognition Com-

plex (ORC) and two other proteins, Cdt1 and Cdc6 are

required, in an ORC-dependent manner, for licensing of

DNA replication origins, which corresponds to the chroma-

tin assembly of complexes of the Mini Chromosome Main-

tenance (MCM2–7) protein family, the putative replicative

0014-4827/$ - see front matter D 2004 Elsevier Inc. All rights reserved.

doi:10.1016/j.yexcr.2003.11.018

$ Supplementary data associated with this article can be found, in the

online version, at doi:10.1016/S0014-4827(03)00631-1.

* Corresponding author. Institute of Human Genetics, CNRS, 141 Rue

de la Cardonille, 34396 Montpellier Cedex 05, France. Fax: +33-4-

99619917.

E-mail address: mechali@igh.cnrs.fr (M. Mechali).1 Present address: Institute of Molecular Genetics of Montpellier

(IGMM), 1919 route de Mende 34293 Montpellier Cedex 05, France.

helicase. Cdt1 and the Cdc6 protein, both required for

licensing, appear to be differentially regulated. In Xenopus,

Cdc6 redistributes on chromatin following initiation of

DNA replication, while Cdt1 is removed from chromatin.

(Ref. [5] for review). A complex between Cdt1 and Cdc6

has been observed in fission yeast cell-free extracts [6], but

such complex has not been detected in multicellular eukar-

yotes. Following licensing, several proteins associate with

replication origins in a reaction that requires the action of S-

phase cyclin-dependent kinases (S-CDKs). One of these

proteins, Cdc45, plays a key role in this reaction allowing

the entry of DNA polymerase a at the origin [7,8], and

marks the transition from the pre-RC to the pre-initiation

complex (pre-IC).

Not only must licensing be induced to allow the G1 to S

phase transition, but it should also be repressed within S

phase to block any illegitimate re-initiation events which

would lead to chromosome imbalance and genetic instabil-

ity [9]. At least three distinct mechanisms have been shown

to prevent this phenomenon in eukaryotic cells. The first

mechanism is the repression of origins firing by high levels

of CDKs [10–12], which are thought to inactivate and/or

exclude replication proteins from the nucleus [11,13,14].

The second mechanism involves the destabilization of

D. Maiorano et al. / Experimental Cell Research 295 (2004) 138–149 139

replication proteins from chromatin during ongoing DNA

synthesis [15–18], so that cells can distinguish replicated

from unreplicated DNA. A third mechanism, which is

specific to higher eukaryotes, involves the presence of a

DNA replication inhibitor, geminin, during S phase [19–

21]. Overexpression of geminin in Xenopus inhibits DNA

replication by interfering with the assembly of MCM2–7

proteins onto chromatin [19]. Recombinant geminin can

form complexes with Cdt1 in egg extracts [20,22] and a

similar mechanism seems to operate in Drosophila [21] and

mammalian cells [20,23]. It is currently thought that gem-

inin binds to and sequestrate soluble Cdt1 thus blocking its

activity [24] for review, and a very recent study in mouse

suggests that in vitro recombinant geminin may inhibit

association of recombinant Cdt1 with naked DNA and

binding to at least one MCM subunit [25].

We have analyzed the molecular basis for Cdt1 inhibi-

tion by geminin. Recombinant geminin added to egg

extracts binds to chromatin, but chromatin binding per se

is not sufficient to block Cdt1 activity, as inhibition of pre-

replication complexes (pre-RC) formation is observed

when geminin accumulates on chromatin. In addition, we

show that geminin associates with chromatin in a Cdt1-

dependent manner precisely at the transition from the pre-

RC to the pre-initiation complexes (pre-IC), and at the

same time as the entry of the Cdc45 protein. We also show

that geminin is a relatively stable protein during early

Xenopus development. Our results suggest that geminin

might have two main functions in regulating licensing, one

is to prevent re-entry into S phase before mitosis exit, and

second, in preventing re-firing of replication origins within

one cell cycle.

Materials and methods

Xenopus egg extracts

Xenopus egg extracts and demembranated sperm nuclei

were prepared as described [43]. Metaphase-arrested egg

extracts were released in interphase by addition of 0.4 mM

CaCl2 in the presence or absence of 250 Ag/ml of cyclo-

heximide. Cycling extracts were prepared as described [38].

Replication reaction was carried out by supplementing egg

extracts with cycloheximide (250 Ag/ml), demembranated

sperm nuclei (3 ng/Al), an energy regeneration system (10

mM creatine phosphate; 10 Ag/ml creatine kinase; 1 mM

ATP; 1 mM MgCl2) and 20 ACi/ml of [a-32P]dATP. Incor-

poration of the radiolabeled nucleotide was monitored by

trichloric acid precipitation on GF/C filters (Whatmann) on

ice.

Analysis of Cdt1 and geminin protein complexes

Fractionation of Xenopus egg cytosol on sucrose gra-

dients was performed essentially as previously described

[28]. Briefly, metaphase-arrested extracts were diluted 5-fold

in buffer A containing 5 mM EGTA pH 7.7 and a mixture of

both phosphatases and kinases inhibitors (Buffer B: 0.5 AMokadaic acid, 25 mM NaF, 1 mM sodium pyrophosphate, 1

mM phenyl phosphate, 0.2 mg/ml a naphthyl phosphate, 10

mM sodium orthovanate) to keep intact MPF activity

(Doree, personal communication). Interphasic egg extracts

were obtained by activation of metaphase-arrested eggs

extracts with calcium as described above following incuba-

tion at room temperature for 15 min. Extracts were then

transferred on ice and diluted 5-fold with buffer A (100 mM

KCl, 0.1 mM CaCl2, 1 mMMgCl2, 10 mM K-Hepes pH 7.7,

5 Ag/ml leupeptin, pepstatin, and aprotinin). The diluted

extracts (0.2 ml) were then centrifuged for 10 min at 12 000� g and the supernatants were layered onto a 5-ml linear 5%

to 20% sucrose gradient in buffer A (interphasic extract) or

buffer B (metaphase extract) containing 0.1 AM instead of

0.5 AM okadaic acid. All gradients were centrifuged at 26

000 rpm, 20 h, at 2jC in a SW 55Ti rotor for 22 h. Fractions

of 0.180 ml were collected and aliquots of each fraction

were analyzed by immunoblotting. Marker proteins were run

in parallel. Replication reactions were performed as de-

scribed [43]. For replication of chromatin templates, chro-

matin purification was as described [29]. Isolated chromatin

fractions were resuspended in XB (10mM Hepes KOH pH

7.7; 100mM KC1; 1mM MgC12; 0.1 mM CaCl2; 5%

sucrose) + protease inhibitors and added to mock-depleted

or Cdt1-depleted extracts.

Extracts treated as described above, were also fraction-

ated by gel filtration chromatography on a 25-ml superose 6

column (Pharmacia) run at a constant rate of 0.4 ml per

minute in a FPLC system (Pharmacia). Before chromatog-

raphy, the column was equilibrated in XB + 5 mM EGTA

for mitotic extract and XB for interphasic egg extracts.

Thirty-six fractions of 0.2 ml each were collected and

analyzed by SDS-PAGE followed by Western blot.

Proteins

Recombinant geminin-Del was produced as described

[19]. Purified protein was dialyzed against 10 mM Tris–

HCl pH 8.0, 300 mM NaCl at 4jC and stored at �80jC.Recombinant XCdc6 was produced and purified at more

than 90% homogeneity as described [31]. Immunopurifica-

tion of the MCM2–7 complex from Xenopus eggs was done

as described [29]. Recombinant Cdt1 was produced and

purified as described [26]. The activity of the purified

proteins was determined by complementation of eggs

extracts depleted with antibodies specific for the cor-

responding proteins. For replication assays, purified pro-

teins were diluted in XB and added to Xenopus egg extracts

at 1:20 ratio. For geminin-treated extracts, recombinant

geminin was pre-incubated in egg extracts for 10 min on

ice. We have found that this pre-incubation step greatly

increases the efficiency of geminin activity (data not

shown).

l Cell Research 295 (2004) 138–149

Antibodies

A polyclonal antibody specific for the geminin protein

was raised in rabbits by injection of the recombinant protein

expressed in Escherichia coli BL21(EDE3). Cdc6 antibod-

ies were raised as described [31]. The Cdc6 antibody

recognizes a single polypeptide of 61 kDa in egg extracts

[44]. Antibodies specific for Cdt1 and MCMs have been

previously described [26,34]. ORC1 antibodies were a kind

gift from Dr. J. Blow [45]. The Cdc45 antibody was a gift

from Dr. J. Walter [8]. Cyclin B2 antibody was obtained

from D. Fisher (IGH, Montpellier). Depletion experiments

were performed as described [29]. For depletion of meta-

phase-arrested eggs extracts, protein-A sepharose beads

coupled to antibodies were pre-equilibrated in XB + 5

mM EGTA before depletion. Mock-depleted metaphasic

egg extracts replicated efficiently only upon activation by

addition of 0.4 mM CaCl2 as expected, while Cdt1-depleted

extracts did not replicate with or without calcium addition

(data not shown) which demonstrates that the depletion

procedure did not cause a spontaneous release of the extract

in interphase. Reconstitution experiments were carried out

as described [26,29,31].

Chromatin isolation

Chromatin formed in egg extracts was isolated as previ-

ously described [29]. Briefly, reactions were diluted 5-fold

in ice-cold CPB (50 mM KCl; 20 mM Hepes-KOH, pH 7.7;

5 mM MgCl2; 2% sucrose; 0.1% NP-40; leupeptin, aproti-

nin, and pepstatin, 5 Ag/ml each) and centrifuged through a

0.7-M sucrose cushion made in XB. For experiments

involving transfer of salt-washed chromatin in egg extracts,

reactions were diluted 5-fold in CPB without NP-40, sup-

plemented with the required amount of KCl.

D. Maiorano et al. / Experimenta140

Results

Recombinant geminin inhibits DNA replication following its

accumulation on chromatin

Addition of recombinant geminin to Xenopus egg extracts

results in inhibition of DNA replication, which may mimic

failure to degrade geminin on mitotic exit. Inhibition of DNA

replication is efficiently rescued by addition of recombinant

Cdt1, which demonstrates that the block is reversible [22]. It

is thought that geminin blocks DNA replication by a mech-

anism that appears to involve its binding to Cdt1, thereby

preventing the access of Cdt1 to chromatin. Nevertheless, the

exact mechanism of Cdt1 inhibition by geminin is not

known.

To address this point in detail, we have investigated the

interactions of Cdt1 and geminin with chromatin during the

cell cycle. Recombinant Xenopus geminin was purified to

homogeneity (Fig. 1A) and polyclonal antibodies were

raised against the purified recombinant protein (Fig. 1B

and Materials and methods). Recombinant geminin inhi-

bited DNA replication in a concentration range similar to

that previously described when added to egg extracts in

vitro (See Refs. [19,20,22] and Supplementary data S1). It

has been shown that failure to initiate DNA synthesis in

geminin-treated extracts is due to the absence of at least

three subunit of the MCM2–7 complex onto chromatin,

MCM3 [19], MCM4 [20], and MCM7 [22], but it is not

known whether geminin also inhibits the binding to chro-

matin of all MCM2–7 subunits. As shown in Fig. 1C,

inhibition of licensing by geminin resulted in a complete

inhibition of binding of all subunits of the MCM2–7

complex, while the Cdc6 protein accumulated onto chro-

matin as expected [19,22]. This mode of inhibition is very

similar to inhibition of licensing obtained by removal of

Cdt1 [22,26] and is consistent with the model of inhibition

of geminin preventing Cdt1 from binding to chromatin.

However, further analysis of these chromatin fractions

revealed that both recombinant and endogenous geminin

are bound to chromatin at the stage of formation of pre-

initiation complexes (Fig. 1D), even at concentrations that

do not interfere with DNA replication (e.g., 10–20 nM, see

supplementary data S1). Addition of increasing amounts of

geminin to the egg extract resulted in a corresponding

accumulation of geminin on chromatin, but the level of

chromatin-bound Cdt1 remained fairly stable. The slight

increase in Cdt1 bound to chromatin in the presence of a

concentration of 80 nM of geminin is not significant as it

was not observed in an independent experiment (see sup-

plementary data S1).

We conclude that during formation of the pre-initiation

complex (a) geminin does not interfere with the chromatin

association of Cdt1; (b) accumulation of geminin on chro-

matin correlates with the inhibition of DNA replication, but

Cdt1 binding is unaffected.

Geminin binds to chromatin at the pre-RC to pre-IC

transition

It has been previously shown that in an in vitro recon-

stituted system geminin requires Cdt1 to bind to chromatin,

which suggests that geminin may be targeted at DNA

replication origins [27]. However, the timing of chromatin

binding of geminin during S phase has not been previously

investigated. We have analyzed the dynamics of nuclear

translocation and chromatin association of endogenous

geminin and Cdt1 in synchronized egg extracts (Fig. 2).

Demembranated sperm nuclei were incubated in interphasic

Xenopus egg extract and detergent-resistant chromatin frac-

tions were isolated and analyzed by Western blot with

specific antibodies. Cdt1, like Cdc6, binds very rapidly to

chromatin (1 min), while MCM3 started to accumulate

about 5 min later, consistent with previous results [28–

30], and with the notion that both Cdt1 and Cdc6 are

required for MCM2–7 chromatin loading to form mature

Fig. 2. Dynamics of geminin binding to chromatin during S phase. Xenopus demembranated sperm nuclei were incubated in interphasic egg extracts and

detergent-resistant chromatin fractions were isolated at the indicated time points as described in Materials and methods. Eluted proteins were analysed by

Western blot with the indicated antibodies. DNA replication was monitored in parallel. Arrow indicates the Cdc45 protein.

Fig. 1. Geminin inhibits licensing on chromatin. (A) Coomassie blue staining of protein fractions eluted from a Nickel-NTA column (Qiagen) loaded with an

Escherichia coli lysate expressing recombinant His-tagged Xenopus geminin. M: molecular weight markers; Tot: total cell lysate; FT: column flow-through;

Wash: column wash with 20 mM imidazole; El: eluate with 250 mM imidazole. (B) Specificity of the anti-geminin polyclonal antibody. Western blot of

Xenopus egg extract released in interphase (25 Ag each lane) with either pre-immune serum (PI) or serum from rabbits injected with recombinant geminin (I).

(C) Western blot of proteins bound to chromatin fractions in the presence (+) or absence (�) of recombinant geminin. No: fraction obtained by centrifugation of

egg extracts in the absence of sperm nuclei. MCM2-7 proteins were revealed with an antibody that cross-react with all six MCM subunits (see Materials and

methods). (D) Western blot of proteins bound to chromatin fractions obtained in the presence of the indicated amounts of recombinant geminin (Rec) or BSA

(160 nM). The antibody also reveals the presence of endogenous geminin on chromatin (Endo). Chromatin fractions were isolated after a 20-min incubation of

demembranated sperm nuclei in egg extracts.

D. Maiorano et al. / Experimental Cell Research 295 (2004) 138–149 141

Fig. 3. Cdt1 is required for the loading of geminin onto chromatin and is not

required for processive DNA synthesis. (A) Cdt1 is required for the

association of geminin with chromatin. Western blot of chromatin formed in

either Mock-depleted or Cdt1-depleted egg extracts and isolated after 60

min incubation at 23jC in the corresponding egg extracts. Indicated

proteins were revealed with specific antibodies. (B) Western blot of proteins

bound to chromatin isolated in the presence of either 50 or 150 mM KCl.

MCM proteins were revealed with an anti MCM2-7 antibody (see Materials

and methods). Numbers on the right hand side of the panel hybridized with

an anti-MCM2-7 antibody indicate MCM subunits. (C) Kinetics of DNA

replication (expressed as incorporation of a-32P dATP, cpm) of either salt-

washed chromatin or demembranated sperm nuclei (circles), in Xenopus

egg extract depleted with a Cdt1-specific antibody.

D. Maiorano et al. / Experimental Cell Research 295 (2004) 138–149142

pre-Replication Complexes (pre-RCs; [26,31]). Geminin

was not present during the formation of the pre-RCs but it

bound to chromatin abruptly only at the onset of DNA

replication (15 min in this experiment). The time of asso-

ciation of geminin with chromatin strikingly coincided with

the binding of Cdc45, a protein required for the formation of

pre-initiation complexes (pre-ICs), which accumulates on

chromatin only following nuclear membrane formation [7].

Fig. 2 also shows that Cdt1 was displaced from chromatin

shortly after loading of geminin, whereas geminin remained

bound to chromatin during the whole S phase and was

slowly and partially removed afterwards. Cdc6, which is

also required for the loading of the MCM2–7 helicase, did

not show similar dynamics of chromatin binding. Its binding

to chromatin during S phase is consistent with previous

reports [31,32], while its accumulation occurred at the same

time as geminin. As expected, MCM3 was displaced from

chromatin as soon as DNA was replicated [33,34], while

Cdc45 showed dynamics similar to geminin.

Altogether, these results show that the binding to chro-

matin of endogenous geminin and Cdt1 is different, al-

though they are both bound to chromatin in a narrow

window of time corresponding to the transition from pre-

RCs to pre-ICs, suggesting a role for geminin immediately

after the licensing step of DNA replication.

To determine whether the association of geminin with

chromatin during S phase depends on the previous binding

of the Cdt1 protein, we have analyzed the binding of

geminin to chromatin formed in egg extracts depleted of

the Cdt1 protein. As shown in Fig. 3A, geminin associated

to chromatin in mock-depleted egg extracts, but it did not in

Cdt1-depleted egg extracts, while the ORC1 protein was

bound to chromatin in both types of extracts. These results

demonstrate that association of geminin with chromatin

during S phase requires the presence of the Cdt1 protein

arguing that geminin is targeted at DNA replication origins.

To determine whether Xenopus Cdt1 may also have some

role in elongation of chromatin templates, as suggested in

Drosophila [35], the Cdt1 protein was removed from

licensed chromatin by a salt wash (Fig. 3B) and the isolated

chromatin was tested for its ability to replicate in Cdt1-

depleted egg extracts (Fig. 3C). We have previously shown

that Cdt1 is not required for the retention of the MCM2

protein onto chromatin [26]. Fig. 3B shows that this is also

true for the whole MCM2–7 complex, which remains

bound to chromatin after removal of Cdt1 by a salt wash.

Moreover, licensed chromatin, with no Cdt1 (Fig. 3C,

diamonds), replicated efficiently in a Cdt1-depleted egg

extract, at a rate similar to chromatin containing Cdt1

(squares), indicating that all origins were fully licensed.

Conversely, replication of sperm chromatin (unlicensed

chromatin, circles) was abolished in Cdt1-depleted extracts,

as expected. This is in agreement with previous observations

showing that Cdt1 is not required for complementary strand

DNA synthesis in vitro [26] and demonstrates that once

licensing has occurred, Cdt1 is not required for DNA

synthesis in Xenopus egg extracts. These results are also

consistent with the data presented in Fig. 2 showing that

Cdt1 leaves chromatin upon initiation of DNA synthesis.

Cdt1 and geminin protein complexes in the cell cycle

A complex between geminin and Cdt1 has been observed

in Drosophila [21] Xenopus [36] and mammalian cells [20].

In Xenopus, Cdt1 and geminin have been shown to co-

fractionate by gel filtration and form very large complexes

Fig. 4. Cell cycle-dependent complex formation between Cdt1 and geminin. (A) Xenopus egg extracts arrested in mitosis (Metaphase) or released into interphase

(Interphase) were fractionated on a linear 5% to 20% sucrose gradient (see Materials and methods). Fractions were collected and the sedimentation profile of the

indicated proteins was determined byWestern blot. Numbers indicate fractions of the gradient. The size and the sedimentation position (arrows) of globular protein

used as standards is indicated on top of the panel. (B) Extracts were fractionated on a Superose 6 gel filtration column. Fractions were analyzed for the presence of

Cdt1 and geminin by Western blot. The size and the position (arrows) of globular protein markers used to calibrate the column are indicated on top of the panel.

Table 1

Cdt1 and geminin complexes in mitosis and interphase egg extracts

Protein Metaphase Interphase

Method Method

Sucrose

gradient

Gel

filtration

(kDa)

Sucrose

gradient

Gel

filtration

(kDa)

Cdt1 5.5s

(90 kDa)

400 4.4s

(66 kDa)

160, 600

9s

(200 kDa)

Geminin 3.5s

(50 kDa)

440, 380 9s

(90 kDa)

160, 250, 480

Note. Sedimentation coefficients (s) and molecular weights (kDa) for Cdt1

and geminin complexes observed following either sedimentation on a linear

5 to 20% sucrose gradient or chromatography on a superose 6 column (see

Fig. 4).

D. Maiorano et al. / Experimental Cell Research 295 (2004) 138–149 143

at metaphase, while in interphase most of geminin is

released from the Cdt1-containing complex. The stoichiom-

etry and the composition of these proteins complexes have

not been analyzed. To determine the size of Cdt1-geminin

complexes with more accuracy, we have analyzed complex

formation by another method, that is, sedimentation in

sucrose gradients (Fig. 4). Unlike what was observed by

gel filtration, at metaphase, Cdt1 and geminin co-sediment

in a low molecular weight range (60–90 kDa), which

corresponds to a sedimentation coefficient between 4.4s

and 5.5s, respectively. The size of these complexes suggests

formation of a heterodimer of geminin and Cdt1 (whose

molecular weight is of 25 and 70 kDa, respectively). MCM4

sediments as a complex of 200–230 kDa as previously

shown [28], but does not co-sediment with geminin nor with

Cdt1. When this same metaphasic egg extract was activated

to enter into interphase, by addition of calcium, MCM4

sedimented with an apparent molecular mass of 350 kDa,

consistent with previous results [28], and readily demon-

strates that the extract had successfully been released in

interphase. Cdt1 exhibited a significant change in its sedi-

mentation profile, as a new peak of sedimentation in a

higher molecular weight range (200 kDa, 9s) was observed

that did not extensively overlap with that of geminin that

sedimented with a sedimentation coefficient of 5.5s. Immu-

noprecipitation experiments with specific antibodies from

both metaphase and interphase egg extracts demonstrate

complex formation between Cdt1 and geminin (see supple-

mentary data S2). Immunoprecipitation of Cdt1 from frac-

tions 15–17 in interphase reveals the presence of a protein

of ca. 130 kDa in complex with Cdt1 (data not shown),

suggesting that in interphase, a large fraction of Cdt1 may

form a heterodimer. Again, no co-sedimentation with MCM4

was detected even in interphase.

Given the discrepancy observed between the sedimenta-

tion of Cdt1 and geminin in sucrose gradients compared to

that reported by gel filtration [36], we have run a gel

filtration on a superose 6 column in parallel (Fig. 4B and

Table 1). Surprisingly, in metaphase, Cdt1 eluted with an

apparent molecular weight of 400 kDa and overlapped with

geminin which showed a larger elution profile ranging from

D. Maiorano et al. / Experimental Cell Research 295 (2004) 138–149144

230 to 670 kDa, which is similar to what has been

previously reported [36]. In interphase, Cdt1 showed two

distinct elution peaks, similar to what was observed by

sedimentation in sucrose gradients (Fig. 4A), although the

apparent mass of the proteins significantly differs. One

elution peak was observed at 160 kDa overlapping with

geminin and a second peak was observed at 600 kDa that

did not significantly overlap with geminin. From these

experiments, we conclude that Cdt1 and geminin form

different complexes in both metaphase and interphase,

which do not exhibit similar values by gel filtration and

sucrose gradients sedimentation, suggesting that these com-

plexes are asymmetric.

Geminin is a stable protein during Xenopus early

embryogenesis

Unlike somatic cells [19,20,23] in Xenopus egg extracts,

geminin is only partially degraded at mitotic exit so that a

Fig. 5. Protein synthesis-dependent degradation of geminin upon mitotic exit and

(time 0) were released into interphase by addition of calcium in the presence or

indicated time after release in interphase and the stability of the indicated proteins w

as described in Materials and methods, were incubated at room temperature with or

points. Arrows indicate the onset of mitosis. (C) The stability of geminin in metaph

0) and released in interphase (Ca++) was determined by Western blot.

fraction of geminin persists in very early interphase [36].

However, it is not known if this feature is specific of

geminin or whether it is a general feature of APC substrates

in egg extracts.

To address this point, we have compared the stability of

geminin with that of another substrate of APC, cyclinB [37].

Mitotic exit and entry into S phase was reproduced in vitro

by addition of calcium to egg extract arrested in metaphase

of meiosis II (Ref. [38] and Materials and methods). Fig. 5A

(� cycloheximide) shows that a significant fraction (40%)

of geminin is resistant to degradation, confirming previous

results [36]. This regulation is specific of geminin as cyclin

B2, a substrate of the APC, was completely degraded during

the time course of the experiment. The MCM4 protein, a

substrate of the Cdc2/cyclin B mitotic kinase, is rapidly

dephosphorylated upon calcium addition (30 min), indicated

by its faster migration during electrophoresis compared to

mitotic egg extracts (time 0) [34,39], demonstrating the

synchronous and complete release of the mitotic extract

cell cycle stability in vitro. (A) Xenopus egg extracts blocked in metaphase

absence of cycloheximide (250 Ag/ml). Protein samples were taken at the

ere determined by Western blot. (B) Xenopus cycling egg extracts, prepared

without cycloheximide and protein samples were taken at the indicated time

ase-arrested egg extract Mock-depleted or depleted of the Cdt1 protein (time

D. Maiorano et al. / Experimental Cell Research 295 (2004) 138–149 145

into interphase. Cdt1 is also dephosphorylated upon mitotic

exit, as previously shown [26,31], but its level did not

change throughout interphase.

The degradation-resistant geminin pool present in inter-

phase could be due to de novo synthesis of geminin

replacing the degraded maternal store. Hence, the same

experiment was performed in presence of cycloheximide

to inhibit protein synthesis. Surprisingly, we observed that

addition of cycloheximide stabilized geminin in the extract

for at least 40 min compared to the untreated extract (Fig.

5A, + cycloheximide), while this treatment did not interfere

with the degradation of cyclin B2 nor did alter the level of

other proteins. This result suggests that the residual geminin

that persists in interphase is likely to be of maternal origin,

and that the mechanism responsible for its partial degrada-

tion is under translational control.

To confirm that geminin is not completely degraded

following fertilization, we have prepared egg extracts re-

leased in interphase that could support up to two cell cycles

in vitro, the so-called cycling extracts, in which periodic

synthesis and degradation of mitotic cyclins drive the cell

cycle [38]. These extracts (Fig. 5B, � cycloheximide)

supported up to two successive mitosis in vitro, judged

upon phosphorylation and dephosphorylation of the MCM4

protein (� cycloheximide, arrows). Geminin was detectable

in these extracts, confirming that in interphase, a fraction of

geminin is resistant to degradation; however, the levels of

geminin did not vary significantly during the two successive

cell cycles. Cdt1 remained stable throughout the experiment,

Fig. 6. Cdt1 does not associate with Cdc6 or MCM2-7 proteins and plays a uniqu

depleted with non-specific antibodies (Mock) or antibodies specific for the indicat

Depletion of egg extracts with MCM3 also removes associated MCM2-7 proteins

depleted or Cdt1-depleted egg extracts supplemented with buffer (XB, Materials a

determined after 90 min incubation at room temperature.

and showed a slower electrophoretic mobility at the time of

phosphorylation of MCM4 (arrows), which we have previ-

ously reported to be due to mitotic phosphorylation [26].

Inhibition of protein synthesis stabilized the levels of

geminin (Fig. 5B, + cycloheximide) as observed in Fig.

5A, while this treatment had no effect on the level of Cdt1

and inhibited the phosphorylation of the MCM4 protein, as

expected if the mitotic kinase was inactivated due to

inhibition of cyclins synthesis.

Partial degradation of geminin was also observed in vivo,

following fertilization of Xenopus eggs and no degradation

of geminin was observed during mitotic exit of the first

cleavage (data not shown). We conclude that geminin is a

rather stable protein during the early embryonic cell cycles

of Xenopus.

Only Cdt1-bound geminin is degraded upon mitotic exit

As a large part of Cdt1 and geminin form a complex in

mitosis, we further considered the possibility that complex

formation with Cdt1 may protect geminin from degradation.

To address this point, we have immunodepleted Cdt1 from

metaphase-arrested egg extracts and determined the stability

of the remaining Cdt1-free geminin following entry into

interphase by calcium addition. Fig. 5C shows that upon

Cdt1 depletion, MCM4 was fully phosphorylated (time 0)

and its dephosphorylation occurred normally upon entry

into S phase as in untreated extracts (compare with Fig. 5A,

and data not shown). Cdt1-free geminin (time 0) was

e role in licensing. (A, B) Egg extracts released in interphase were double-

ed proteins. Protein samples of supernatants were analysed by Western blot.

[29]. (C, D) Replication of Xenopus demembranated sperm nuclei in Mock-

nd methods) or the indicated purified proteins. Rate of DNA synthesis was

D. Maiorano et al. / Experimental Cell Research 295 (2004) 138–149146

entirely resistant to degradation upon mitotic exit (10 min)

and early interphase (30 min) indicating that only the

geminin that is engaged in a complex with Cdt1 is degraded

upon mitotic exit, thus producing geminin-free Cdt1.

Cdt1, Cdc6, and MCM2–7 proteins reside in distinct

complexes in Xenopus eggs

Cdt1 by itself is not sufficient to allow licensing, since

the Cdc6 protein is also required [6,14,26,40]. Consistent

with these observations, a complex between Cdt1 and Cdc6

has been observed in fission yeast [6], however, such

complex has not been observed in higher eukaryotes.

A series of observations predict that Cdt1 and Cdc6 may

not form complexes, although this point has not yet been

formally demonstrated. For instance, in Xenopus, Cdt1 and

Cdc6 co-purify on several chromatographic columns, al-

though they are eluted in different fractions during the last

step of purification [22,40]. Fig. 6A shows that removal of

Cdt1 from Xenopus eggs extracts does not remove the Cdc6

protein and that removal of Cdc6 from egg extracts does not

remove Cdt1. No complex with MCM2–7 proteins could be

detected using a similar approach (Fig. 6B) and no com-

plexes could be detected in immunoprecipitates (data not

shown). These results are consistent with the finding that

depletion of Cdt1 from Xenopus egg extracts does not

remove the MCM2–7 proteins [26] and that Cdt1 and

MCM4 show distinct sedimentation profiles in sucrose

gradients (Fig. 4). We conclude that although Cdc6,

MCM2–7, and Cdt1 are involved in the same biochemical

pathway, they do not form stable complexes in Xenopus egg

extracts.

We have further asked whether the activities of these

proteins may be redundant in Xenopus and if overexpression

of Cdc6 may compensate for the loss of Cdt1 (Fig. 6C).

Interphasic Xenopus eggs extracts that were depleted of

Cdt1 could not initiate DNA replication and neither Cdc6

nor the MCM2–7 complex could rescue DNA replication in

these extracts, while these proteins complemented egg

extracts depleted with the corresponding antibodies (Fig.

6D and Materials and methods). These results are consistent

with genetic data obtained in yeast showing that both Cdc6

and Cdt1 are essential proteins and demonstrate that Cdt1,

Cdc6, and the MCM2–7 have distinct, non-interchangeable

functions in the licensing reaction.

Discussion

Molecular basis of Cdt1 inhibition by geminin

In this report, we have provided evidence that inhibition

of Cdt1 by geminin does not occur simply by complex

formation. We have shown that when present at concen-

trations that do not interfere with DNA synthesis, geminin

binds chromatin, demonstrating that its presence on chro-

matin is not sufficient to inhibit DNA synthesis. Moreover,

we have shown that accumulation of geminin on chromatin

does result in inhibition of DNA synthesis, but geminin does

not interfere with the chromatin association of Cdt1, which

remains bound. We have shown that the binding of geminin

to chromatin requires the Cdt1 protein. A similar result has

been obtained with purified proteins in vitro [27]. These

results suggest that in a first step, geminin is very likely

targeted at DNA replication origins via association with

Cdt1, and perhaps in a second Cdt1-independent step,

geminin remains on chromatin, possibly blocking Cdt1

binding sites and locking origins in an unlicensed state.

This model is consistent with the observation that in

interphase, most geminin does not form stable complexes

with Cdt1 (Refs. [22,36] and this paper), so that Cdt1-free

geminin may accumulate on chromatin independently of

Cdt1, which is released thereafter. We speculate that the

accumulation of geminin on chromatin may take place by

oligomerization. The divergence between the sedimentation

values of geminin and its molecular weight values obtained

by gel filtration are consistent with this hypothesis.

These results are in contrast with very recent data

obtained in mouse showing that in vitro recombinant

geminin interferes with a DNA binding activity associated

with recombinant Cdt1 [25], which may point to important

differences in reconstitution experiments using naked DNA

or chromatin. Our data suggest that geminin is not solely a

soluble inhibitor of Cdt1, but that inhibition of DNA

replication correlates with the accumulation of geminin on

chromatin.

Alternative association of Cdt1 and geminin with

chromatin: a molecular switch from a competent to a

licensing-incompetent state of replication origins?

We have demonstrated that Cdt1 has no role in DNA

synthesis once licensing has occurred, as for Cdc6 and the

ORC complex [16]. These results explain why recombinant

geminin does not inhibit DNA synthesis if added after the

licensing reaction has occurred [19] and suggest that a fine

balance between the levels of geminin and Cdt1 is crucial in

regulating licensing.

The residual amount of geminin that persists in inter-

phase binds to chromatin only after the licensing reaction

has occurred, but before the onset of processive DNA

synthesis. Very recent data have suggested that geminin is

activated in the nucleus, but from these experiments, it was

not clear at what stage of S phase geminin binds to

chromatin [36]. In this paper, we have analyzed in detail

the dynamics of chromatin binding of Cdt1 and geminin and

shown a clear difference in their regulation compared to

somatic cells. First, we have shown that geminin binds to

chromatin much earlier than in somatic cells, where its

binding occurs during mid-S phase, well after the activation

of processive DNA synthesis and Cdt1 degradation [23].

Moreover, we have defined a very narrow window of time,

D. Maiorano et al. / Experimental Cell Research 295 (2004) 138–149 147

which corresponds to the formation of the pre-initiation

complex, when both Cdt1 and geminin are bound to

chromatin. Interestingly, the dynamics of chromatin release

of Cdt1 are different from those of Cdc6, which persists on

chromatin after initiation of DNA synthesis. Although it has

been reported that Cdc6 transiently dissociates from chro-

matin after licensing [12,32], we find that it remains

associated with chromatin, a result similar to what was

originally reported [31]. This difference might reflect the

protocols used to purify chromatin as well as the use of

detergents at high concentration.

Once DNA synthesis has started, Cdt1 is removed from

chromatin while geminin still persists. This regulation may

be specific to Xenopus early development, as there are no

G1 and G2 phases and DNA replication origins occur at

short intervals in the genome, allowing an accelerated rate

of DNA synthesis. In this specific context, the persistence of

a stable pool of geminin in the egg may provide enough

inhibitor to immediately prevent illegitimate re-initiation

during the same S phase, and acting as a molecular switch

for the control of MCM helicase loading. In Xenopus early

embryonic cell cycles, Cdt1 is stable throughout the cell

cycle while in somatic cells, Cdt1 is entirely degraded upon

entry into S phase [23]. Thus, in Xenopus, control of origin

re-firing by geminin may require an intermediate stage when

chromatin binding of geminin overlaps with the binding of

Cdt1, as opposed to somatic cells in which Cdt1 is unstable

and the binding to chromatin of these two proteins is

exclusive.

Degradation of geminin at each mitotic exit is not relevant

to regulation of licensing during Xenopus early

development

Unlike somatic cells in egg extracts, geminin is not

completely degraded upon mitotic exit [36]. In this paper,

we have shown that upon mitotic exit, only the geminin

pool, which is bound to Cdt1, is degraded. Hodgson et al.

[36] have reached a similar conclusion based on the obser-

vation that a large fraction of geminin (75%) is unable to

associate with Cdt1 in interphase, which persists throughout

interphase and early development (this paper). These obser-

vations are in agreement with recent data obtained in

Drosophila [41] showing that geminin is present at high

levels irrespective of the cell cycle stage during early

development. We have shown that partial degradation of

geminin occurs in in vitro egg extracts obtained from

unfertilized eggs, which are blocked at metaphase of mei-

osis II. However, degradation of geminin was not observed

in cycling egg extracts through two consecutive mitoses, nor

in vivo, except at fertilization when part of the geminin pool

is degraded (data not shown) similar to what we observe in

vitro. We conclude that geminin degradation does not occur

during the early embryonic cell cycles of Xenopus. Degra-

dation of geminin depends on the activity of the APC

complex [19], as for cyclin B. However, we have shown

that geminin behaves differently from cyclin B, which is

entirely degraded upon entry into interphase. Moreover,

only recombinant geminin, but not endogenous geminin

[36], can be completely degraded at the metaphase to

anaphase transition, indicating that part of the maternal

geminin pool may be modified to prevent its degradation.

Very recent data suggest that geminin can form dimers in

solution [42]. Analysis of protein complexes by sedimenta-

tion through sucrose gradients is consistent with the pres-

ence of a heterodimer of geminin and Cdt1 in metaphase.

These data differ from those very recently obtained in

Xenopus using superose 6 gel filtration [36]. These experi-

ments revealed one Cdt1 complex of 500 kDa and two

geminin complexes of 250–300 and 500 kDa in metaphase,

whereas in interphase Cdt1 appeared in a single complex of

450 kDa, which is separated from the 250–300 kDa

geminin complex. These discrepancies may be due to the

differences in the two methods used (gel filtration and

sucrose gradient sedimentation), which are influenced by

the shape of the proteins. We have confirmed this hypothesis

by analysis of Cdt1 and geminin complexes by both

methods, suggesting that the proteins may have an extended

shape.

Given that Cdt1 does form complexes with geminin both

in metaphase and interphase, and that Cdt1 is phosphory-

lated in mitosis [26], phosphorylation of Cdt1 may not be

necessary for its interaction with geminin. Previous obser-

vations have suggested that geminin is responsible for about

50% of the inhibition of licensing in metaphase-arrested

eggs extracts, and that CDKs may account for the remaining

inhibition observed [22]. Our results suggest that CDKs

may regulate the licensing activity of Cdt1 independently of

its interaction with geminin.

In interphase, Cdt1 is dephosphorylated and forms an

additional complex, which had not been previously ob-

served [36]. In this complex, Cdt1 is mainly geminin-free,

and sediments with a molecular mass consistent with either

a trimer of Cdt1, or Cdt1 in a complex with additional(s)

protein(s). Interestingly, in human HeLa cells it has been

shown that Cdt1 forms complexes with geminin and an

unidentified protein of 130 kDa [20] that we have also

detected in Cdt1 immunoprecipitated from egg extracts

(data not shown).

While Cdt1 forms complexes with geminin, we could not

observe complex formation between Cdt1, Cdc6, and the

MCM2–7 proteins, extending our previous data [26], which

is different from yeast [6,14]. No in vivo interactions

between Cdt1 and the MCM2–7 complex nor with Cdc6

have been reported in somatic cells, which suggests that

these results are not due to differences between eggs and

somatic cells. Our results demonstrate that these proteins

provide non-redundant activities in replication licensing.

Furthermore, removal of Cdt1 from licensed chromatin did

not affect the rate of DNA synthesis showing that, unlike the

case in Drosophila [35] in Xenopus, Cdt1 is dispensable for

the elongation step of DNA synthesis. This difference may

D. Maiorano et al. / Experimental Cell Research 295 (2004) 138–149148

be due to a peculiar regulation of Cdt1 in Drosophila tissues

undergoing a strong level of DNA amplification or to the

slow progression of replication forks during amplification.

Acknowledgments

We thank Dr. J. Blow (University of Dundee, UK) for the

anti-ORC1 antibody, Dr. J. Walter (Harvard Medical School,

Boston. USA) for the anti-Cdc45 antibody, Dr. D. Fisher

(IGH, Montpellier) for the anti-cyclin B2 antibody and Dr. T.

McGarry (BID Medical Center, Boston. USA) for geminin

clones. We thank S. Bocquet for purification of baculovirus

Cdc6 and antibody production. We thank D. Gregoire and D.

Fisher for critical reading of the manuscript. We thank C.

Franckhauser for help and suggestions in gel filtration

chromatography.

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