Current Biology 17, 966–972, June 5, 2007 ª2007 Elsevier Ltd All rights reserved DOI 10.1016/j.cub.2007.04.055

ReportC. elegans CUL-4 Prevents Rereplicationby Promoting the Nuclear Exportof CDC-6 via a CKI-1-Dependent Pathway

Jihyun Kim,1 Hui Feng,1 and Edward T. Kipreos1,*1Department of Cellular BiologyUniversity of GeorgiaAthens, Georgia 30602-2607

Summary

Genome stability requires that genomic DNA is repli-

cated only once per cell cycle. The replication-licens-ing system ensures that the formation of prereplicative

complexes is temporally separated from the initiationof DNA replication [1–4]. The replication-licensing fac-

tors Cdc6 and Cdt1 are required for the assembly ofprereplicative complexes during G1 phase. During S

phase, metazoan Cdt1 is targeted for degradation bythe CUL4 ubiquitin ligase [5–8], and vertebrate Cdc6

is translocated from the nucleus to the cytoplasm[9, 10]. However, because residual vertebrate Cdc6

remains in the nucleus throughout S phase [10–13],it has been unclear whether Cdc6 translocation to the

cytoplasm prevents rereplication [1, 2, 14]. The inacti-vation of C. elegans CUL-4 is associated with dramatic

levels of DNA rereplication [5]. Here, we show thatC. elegans CDC-6 is exported from the nucleus during

S phase in response to the phosphorylation of multi-

ple CDK sites. CUL-4 promotes the phosphorylationand subsequent translocation of CDC-6 via negative

regulation of the CDK-inhibitor CKI-1. Rereplicationcan be induced by coexpression of nonexportable

CDC-6 with nondegradable CDT-1, indicating thatredundant regulation of CDC-6 and CDT-1 prevents re-

replication. This demonstrates that CDC-6 transloca-tion is critical for preventing rereplication and that

CUL-4 independently controls both replication-licens-ing factors.

Results

CDC-6 Is Exported from the Nucleus during S Phase

The C. elegans ortholog of the DNA-replication-licensingfactor Cdc6 is essential for DNA replication. C. eleganscdc-6(RNAi) embryos arrest with w100 cells and containonly trace amounts of DNA, indicating a total failure ofDNA replication (Figure S1 in the Supplemental Dataavailable online).

We followed CDC-6 dynamics during the first cell divi-sion of the V1–V6 hypodermal seam cells by using immu-nofluorescence with CDC-6 antibody. We determinedthe timing of S phase entry by following GFP expressedfrom the ribonucleotide reductase promoter (Prnr-1)[15]. Seam cells varied in the timing of S phase entry:105–120 min after hatch for V5, 120–135 min for V2–V4,135–150 min for V6, and 180–195 min for V1 (data notshown).

*Correspondence: ekipreos@cb.uga.edu

At 10–20 min after hatch, nuclear expression of CDC-6was first observed in the seam cells, and by 100–115 minafter hatch, nuclear levels were high (Figure 1A; data notshown). As cells entered S phase, nuclear levels of CDC-6 dropped gradually with a concomitant increase in cy-toplasmic CDC-6. Beyond 180 min after hatch, endoge-nous CDC-6 remained at high levels in the cytoplasm,although residual nuclear CDC-6 was still present(Figure 1A).

We also followed CDC-6::GFP that was expressed inseam cells under the control of the wrt-2 promoter [16].In contrast to the partial translocation of endogenousCDC-6, transgenic CDC-6::GFP appeared to localizecompletely to the cytoplasm during S phase (Figure 1B,Figure S2D). CDC-6::GFP was present throughout thecell cycle and localized to condensed chromosomesduring mitosis (Figures S2A and S2B).

To determine the time course of CDC-6 nuclear exportduring S phase, we investigated CDC-6::GFP or CDC-6::tdTomato localization at set time points (Fig-ure S2C). At 120–135 min after hatch, CDC-6::GFPremained nuclear in most of the seam cells except V5.Cytoplasmic localization was first detected at 150–165min after hatch for V2–V4, at 165–180 min for V6, andat 195–210 min for V1. The timing of CDC-6 translocationfor the seam cells correlated with the timing of S phaseentry.

To determine whether the exclusive cytoplasmic local-ization of CDC-6::GFP during S phase resulted from thedegradation ofnuclear CDC-6::GFP, wecreated aconsti-tutively nuclear-localized CDC-6::GFP by adding twoSV40 large T antigen nuclear-localization signals (NLSs)to the C terminus and inactivating the predicted nuclear-export signal (NES) through L434A and L436A amino acidsubsitutions [17, 18]. This CDC-6mNES+2NLS proteinhad stable nuclear-protein levels throughout the cell cy-cle, indicating that CDC-6 does not undergo appreciabledegradation in the nucleus during S phase (Figure S3;data not shown).

CUL-4 Is Required for CDC-6 Phosphorylationand Nuclear Export in S Phase

In cul-4(RNAi) larvae, blast cells arrest in S phase and un-dergo extensive rereplication, and this is associated witha failure to degrade CDT-1 [5]. We sought to addresswhether CUL-4 also regulates the licensing factor CDC-6 during S phase. Endogenous CDC-6 localization incul-4(RNAi) larvae was observed by immunofluores-cence. Prior to S phase, nuclear-localized CDC-6 accu-mulated in cul-4(RNAi) seam cells with a time course sim-ilar to that of the wild-type (Figure 1A; data not shown).However, during S phase, CDC-6 in cul-4(RNAi) seamcells remained nuclear with no appreciable increase incytoplasmic staining (Figure 1A). CUL-4 was similarlyrequired for the nuclear export of exogenous CDC-6::GFP during S phase (Figure 1B). These results indicatethat CDC-6 nuclear export requires the CUL-4 ubiquitinligase.

CUL-4 Promotes CDC-6 Nuclear Export967

Figure 1. CDC-6 Is Exported from the Nu-

cleus during S Phase in a CUL-4-Dependent

Manner

(A) Endogenous CDC-6 is exported to the

cytoplasm during S phase in wild-type larvae

but not in cul-4(RNAi) larvae. Wild-type and

cul-4(RNAi) larvae were observed at 100–

115 min after hatch (G1 phase, upper panels)

and at 180–195 min after hatch (S phase,

lower panels). Animals were stained with

anti-CDC-6 (green), DAPI (blue), and anti-

AJM-1 (red overlay, staining adhesion junc-

tions to indicate seam-cell boundaries [35]).

(B) CDC-6::GFP translocates from the nu-

cleus to the cytoplasm during S phase in

wild-type larvae (top), but remains nuclear

in cul-4(RNAi) larvae (bottom). CDC-6::GFP

that was expressed from the wrt-2 promoter

was observed at 90–105 min after hatch (G1

phase, upper left panels) and at 195–210

min after hatch (S or G2 phase, lower left

panels). Prnr-1::tdTomato was used as an S

phase marker (right panels).

Scale bars represent 10 mm.

In humans, Cdc6 nuclear export is triggered by thephosphorylation of three CDK phosphorylation sites thatare located near two N-terminal NLSs [9, 10] (Figure 2A).C. elegans CDC-6 has nine sites similar to the CDKphosphorylation consensus, of which six are locatednear three N-terminal NLSs (Figure 2A).

To examine whether phosphorylation near the NLSspromotes CDC-6 nuclear export, we generated a phos-pho-specific antibody against the threonine-131 CDKphosphorylation site (Figures 2A and 2B). An anti-phos-pho-T131-CDC-6 signal was not detected in wild-typeV1–V6 seam cells before S phase, but it was observedin both the nucleoli and cytoplasm of S phase seam cells(Figure 2C). This indicates that at least a subset of CDC-6 that undergoes nuclear export is phosphorylated onposition T131. Within the nucleus, the obvious nucleolaranti-phospho-T131-CDC-6 signal contrasted with themore uniform nuclear signal observed with the CDC-6antibody, suggesting either that a higher percentage ofnucleolar-localized CDC-6 is phosphorylated on theT131 site or that the phosphorylation of T131 induces nu-cleolar localization. In contrast to wild-type larvae, ananti-phospho-T131-CDC-6 signal was not detected incul-4(RNAi) larvae during S phase, indicating that CUL-4 is required for the phosphorylation of T131 (Figure 2C).

Phosphorylation of Multiple CDK Sites Is Requiredfor CDC-6 Nuclear Export

To determine the functional relevance of CDC-6 phos-phorylation, we expressed CDC-6::GFP with potentialN-terminal CDK phosphorylation sites replaced by un-phosphorylatable alanines. All of the CDC-6::GFP CDK-site mutants had normal nuclear localization in G1 phase(Figure S4; data not shown).

Single mutations of T48, S93, T131, and T145 eachcaused the partial retention of CDC-6::GFP in the

nucleus during S phase, with the T145 mutation havingthe greatest effect (Figure 2E). Combining T48, S109,T131, and T145 mutations gave approximately the sametranslocation rate as the T145 mutation alone (Figure 2E).However, mutation of all six N-terminal CDK phosphor-ylation sites (CDC-6mCDK) completely blocked translo-cation during S phase (Figure 2E, Figure S4). This impliesthat the phosphorylation of multiple sites is requiredfor CDC-6 nuclear export. No obvious defects in DNAreplication or cell-cycle progression resulted from ex-pression of the constitutively nuclear-localized CDC-6mCDK or CDC-6mNES+2NLS proteins, suggestingthat continuous CDC-6 nuclear localization does not, byitself, deregulate DNA replication.

CDC-6 Nuclear Export Is Independent of CDT-1

DegradationCdt1 and Cdc6 physically interact in fission yeast andmammals [19, 20]. This raises the possibility that CDT-1degradation functions as a necessary precedent forCDC-6 nuclear export, perhaps by allowing kinases togain access to CDC-6 phosphorylation sites. To addresswhether the nuclear retention of CDC-6 in cul-4(RNAi)animals is a secondary consequence of CDT-1 perdur-ance, we asked whether the expression of a nondegrad-able CDT-1 prevents CDC-6 nuclear export in a wild-typebackground.

Human Cdt1 can be stabilized during S phase by themutation of CDK phosphorylation sites and the pro-liferating cell nuclear antigen (PCNA)-binding PCNA-interaction protein motif (PIP)-box sequence [8, 11]. Tostabilize C. elegans CDT-1, we substituted alanines forthree conserved PIP-box residues and five N-terminalCDK phosphorylation residues to create the CDT-1mCDK+PIP3A mutant (Figure S5A). CDT-1mCDK+PIP3A::GFP was present in 83% of S phase seam cells

Current Biology968

Figure 2. CDC-6 Is Phosphorylated in a CUL-4-Dependent Manner on CDK Sites during S Phase

(A) Schematic representation of human and C. elegans CDC-6 orthologs. Potential CDK phosphorylation sites are shown as black bars labeled S

(serine) or T (threonine); NLSs are shown as yellow boxes (the first C. elegans NLS is bipartite, and the next two are simple NLSs [36]); and Walker

A and B domains are shown as green boxes (these are required for the loading of Mcm2-7 onto chromatin [37]). In humans, the phosphorylation

of S54, S74, and S106 (red) near the two N-terminal NLSs is required for Cdc6 nuclear export [9, 10]. C. elegans CDC-6 has two consensus CDK

sites (S/T-P-X-K/R; purple lettering) and seven minimum CDK phosphorylation sites (S/T-P; blue) [38]. Six of these potential CDK phosphoryla-

tion sites are located near the NLSs. The scale bar represents 100 amino acids.

(B) Characterization of affinity-purified phospho-T131-CDC-6 antibody. The wild-type C. elegans lysate, either untreated (left lane) or treated with

l phosphatase (right lane), was analyzed by western blot. Note that phospho-T131-CDC-6 antibody recognized the endogenous CDC-6 protein

but could not recognize CDC-6 after treatment with phosphatase, thereby demonstrating specificity.

(C) CDC-6 T131 phosphorylation in wild-type and cul-4(RNAi) seam cells in G1 and S or G2 phases. Wild-type (top) and cul-4(RNAi) (bottom) larvae

were observed in G1 phase (upper panels) and in S phase (lower panels). Animals were stained with anti-phospho-T131-CDC-6 (green), DAPI

(blue), and anti-AJM-1 (red overlay).

(D) RNAi depletion of cki-1 in cul-4(gk434) restores the phosphorylation of CDC-6 in S phase. The staining is as in (C) for cul-4(gk434) (top) and

cul-4(gk434), cki-1(RNAi) (bottom) seam cells in S phase (180–195 min after hatch).

(E) Subcellular localization of wild-type and CDK-site-mutant CDC-6::GFP proteins expressed from the wrt-2 promoter. Serine and thre-

onine residues in the predicted N-terminal CDK phosphorylation sites were replaced by alanines, making the sites unphosphorylatable.

CDC-6::GFP localization in V1–V6 seam cells during S or G2 phase (180–225 min after hatch) is plotted on a continuum from nuclear lo-

calization to cytoplasmic localization. Numbers on the left of the graph indicate the serine or threonine residues replaced with alanine in

the mutant proteins. The panels to the right show epifluorescence images of CDK-site-mutant CDC-6::GFP proteins in V1–V6 seam cells

at 195–225 min after hatch. Numbers in the upper left corner indicate the serine or threonine residues replaced with alanine. In the

CUL-4 Promotes CDC-6 Nuclear Export969

Figure 3. CUL-4 Regulates CDC-6 Nuclear

Export through a CKI-1-Dependent Pathway

(A) CDT-1 perdurance does not disrupt CDC-6

nuclear export. CDC-6::tdTomato is exported

in the presence of stabilized CDT-1::GFP at

195–210 min after hatch (S or G2 phase).

Pwrt-2::CDC-6::tdTomato (red) and Pnhr-

168::CDT-1mCDK+PIP3A::GFP (green) were

expressed in a strain that has ajm-1::GFP

(green) marking seam-cell boundaries.

(B) CKI-1 accumulates in enlarged cul-

4(RNAi) seam cells. L2-stage wild-type and

arrested L2-stage cul-4(RNAi) larvae were

stained with anti-CKI-1 (green) and DAPI

(blue). Arrows indicate seam cells.

(C) cki-1(RNAi) restores CDC-6 nuclear export

in cul-4(gk434) mutant seam cells. Pwrt-

2::CDC-6::GFP was observed in cul-4(gk434)

and cul-4(gk434), cki-1(RNAi) larvae at 195–

210 min after hatch (S or G2 phase). For both

genetic backgrounds, the expression of

Pwrt-2::CDC-6::GFP was nuclear during G1

phase (data not shown).

Scale bars represent 10 mm.

(n = 18), indicating that the mutant protein is partially sta-bilized during S phase (Figure S5B).

We observed that despite the presence of stabilizedCDT-1mCDK+PIP3A::GFP, CDC-6::tdTomato was stillexported to the cytoplasm during S phase, indicatingthat CDC-6 nuclear export occurs independently ofCDT-1 degradation (Figure 3A). It should be noted thatthe stabilized CDT-1 protein is functional and can pro-mote DNA replication (see below).

cki-1 Inactivation Rescues CDC-6 Nuclear

Export in cul-4 MutantsWe have found that CUL-4 negatively regulates the levelof the CDK inhibitor CKI-1, with CKI-1 accumulatingin cul-4(RNAi) rereplicating cells (Figure 3B) [7]. cki-1RNAi reduces the size of nuclei and DNA levels incul-4(gk434) seam cells, indicating suppression of thecul-4 rereplication phenotype (Figure S6A) [7]. cki-1RNAi does not affect the accumulation of CDT-1 incul-4(gk434) mutants, indicating that the preventionof rereplication is independent of CDT-1 accumulation[7].

We hypothesized that CUL-4 promotes CDC-6 nuclearexport by negatively regulating CKI-1 levels and thatCKI-1 accumulation in cul-4 mutants prevents CDK(s)from phosphorylating CDC-6 to induce translocation.To test this model, we investigated the localization ofPwrt-2::CDC-6::GFP in cki-1(RNAi), cul-4(gk434) mu-tants. CDC-6 is predominantly nuclear localized duringS phase in 73% of cul-4(gk434) mutant V2–V6 seam cells(n = 30) (the failure to observe full penetrance for nuclearlocalization is likely the effect of cul-4 maternal product[5]). The RNAi depletion of cki-1 in the cul-4(gk434)mutant significantly increases the percentage of seamcells with cytoplasmically localized CDC-6::GFP (83%[n = 29], versus 27% with no RNAi [n = 30]), indicating

that CKI-1 is required for CDC-6 nuclear retention incul-4 mutants (Figure 3C).

A prediction of the model is that cki-1(RNAi) willrestore the phosphorylation of CDC-6 in cul-4(gk434)mutants during S phase. The status of CDC-6 phosphor-ylation was assessed by immunofluorescence withphospho-T131-CDC-6 antibody. In S phase, an anti-phospho-T131-CDC-6 signal was detected in a relativelysmall percentage of cul-4(gk434) seam cells (33% [n =30], versus 80% for the wild-type [n = 41]) (Figures 2Cand 2D). However, cki-1 RNAi in cul-4(gk434) mutants in-creased the percentage of seam cells with phospho-T131

signal to the wild-type level (82% [n = 45]) (Figure 2D). Ifcki-1 RNAi induces CDC-6 nuclear export in cul-4 mu-tants by permitting CDC-6 phosphorylation, then non-phosphorylatable CDC-6mCDK should not undergo nu-clear export in cki-1(RNAi), cul-4(gk434) seam cells, andthis lack of nuclear export was observed (47/47 nuclearlocalized) (Figure S6B). These results indicate that CKI-1is essential for the prevention of CDC-6 phosphorylationin cul-4 mutants.

Deregulation of Both CDT-1 and CDC-6Can Induce Rereplication

We investigated the biological significance of CDT-1degradation and CDC-6 nuclear export by overexpress-ing wild-type and deregulated proteins with the heat-shock promoters hsp16-2 and hsp16-41 [21]. Wild-typeCDT-1 and CDC-6, or stabilized CDT-1 (CDT-1mCDK+PIP6A) and nonexportable CDC-6 (CDC-6m5CDK), wereexpressed individually or in combination (Figure 4A).Heat-shock expression of individual deregulated CDT-1 or CDC-6 produced higher levels of lethality (embry-onic- or L1-stage arrest) than did wild-type proteins (Fig-ure 4A). Combinations of CDT-1 and CDC-6 producedmore lethality than individually expressed proteins.

CDC-6m48.131.145 image, an inset shows a longer exposure of the V6 seam cell, whose expression was too weak to view with the nor-

mal exposure.

Error bars represent the standard error of the mean (SEM). See Experimental Procedures for the number of cells analyzed. Scale bars represent

10 mm.

Current Biology970

Figure 4. Expression of Deregulated CDT-1 and CDC-6 Produces Rereplication

(A) Graph of viability upon overexpression of wild-type or deregulated CDT-1 and CDC-6 transgenes. ‘‘CDT-1 WT’’ and ‘‘CDC-6 WT’’ are wild-type

genes, ‘‘CDT-1 mut’’ is CDT-1mCDK+PIP6A, and ‘‘CDC-6 mut’’ is CDC-6m5CDK. Each gene was expressed under the control of both the

hsp16-41 and hsp16-21 heat-shock promoters. Embryos from injected hermaphrodites were incubated at 25�C (a semipermissive temperature

for hsp expression [39]) and heat shocked for 30 min at 33�C 12 hr prior to harvest. The percentages of viable progeny are listed to the right of

each bar in the graph.

(B) Expression of nondegradable CDT-1 and nonexportable CDC-6 induces DNA rereplication. Epifluorescence images of a wild-type embryo

and transgenic embryos expressing either CDT-1mCDK+PIP6A plus wild-type CDC-6 or CDT-1mCDK+PIP6A plus CDC-6m5CDK. Animals

were stained with DAPI (blue) and anti-SPD-2 (red), the latter of which highlights centrosomes [23]. Arrows indicate pairs of centrosomes in cells

with increased DNA content (the cell on the left has 13.9C DNA content, and the cell on the right has 11.1C). The scale bar represents 10 mm.

However, w100% lethality was obtained only when de-regulated CDC-6 was combined with either wild-typeCDT-1 or deregulated CDT-1 (Figure 4A).

Increased nuclear DNA levels were observed in 47%(n = 36) of embryos expressing nonexportable CDC-6with stabilized CDT-1 (21.5 6 2.4C DNA content [n =63], versus 2.4 6 0.3C [n = 13] for wild-type embryonicinterphase cells) (Figure 4B). In contrast, other combi-nations of wild-type and/or deregulated CDT-1 andCDC-6 did not produce noticeably increased DNAlevels (Figure 4B; data not shown). Increased DNA con-tent can arise either from rereplication, in which DNAreplication initiates continuously during S phase, orfrom failed mitosis, in which cells with duplicated ge-nomes re-enter the cell cycle without DNA segregation.These two mechanisms can be distinguished by analy-sis of centrosome numbers. Cells that undergo failedmitosis have extra centrosomes because centrosomeswill duplicate in the subsequent cell cycle, whereasrereplicating cells that undergo S phase arrest haveonly two centrosomes [22]. We analyzed centrosomenumbers with SPD-2 antibody, which stains centrioles[23]. We observed that cells with excessive DNA levelshad only two centrosomes, implying that the increasedploidy arises from rereplication (Figure 4B). Therefore,the stabilized CDT-1 and nonexportable CDC-6 arefunctional and can act in concert to induce DNArereplication.

Discussion

CDC-6 Phosphorylation-Dependent Nuclear Export

Prevents DNA RereplicationWe found that C. elegans CDC-6 is exported from thenucleus during S phase, similar to vertebrate Cdc6[1, 2, 14], suggesting that this is an ancient regulatorymechanism. Further, our study indicates that the strat-egy to trigger Cdc6 nuclear export is also conserved,with the phosphorylation of multiple CDK sites used toinactivate N-terminal NLSs in both C. elegans and verte-brates. In C. elegans, all six N-terminal CDK sites must bephosphorylated to promote CDC-6 nuclear export. Inter-estingly, the phosphorylation of T131 is associated withboth nuclear export and nucleoli localization. This sug-gests the possibility that the phosphorylation of a subsetof sites, although not sufficient to induce nuclear export,can direct CDC-6 to specific subnuclear locations.

In humans and Xenopus, ectopically expressed Cdc6is completely exported from the nucleus during S phase;in contrast, a substantial fraction of endogenous Cdc6remains nuclear localized during S phase [9–13]. Strik-ingly, we observed a similar result in C. elegans, witha substantial fraction of endogenous CDC-6 remainingin the nucleus during S phase, whereas ectopicallyexpressed CDC-6 appears exclusively cytoplasmic.The reason(s) for these differential localizations are notunderstood.

CUL-4 Promotes CDC-6 Nuclear Export971

The presence of nuclear-localized Cdc6 during Sphase in mammalian cells has led to the proposal thatCdc6 translocation is not important for restrainingDNA-replication licensing [1, 2, 14]. Further, there is cur-rently no evidence for a functional role of Cdc6 translo-cation in preventing rereplication [1, 2, 14]. In this study,we observed that nonexportable CDC-6 can synergizewith deregulated CDT-1 to induce rereplication. This im-plies that CDC-6 translocation is a redundant safeguardto prevent the reinitiation of DNA replication. This pro-vides the first evidence in any organism of a functionalrole for phosphorylation-dependent CDC-6 nuclearexport.

Deregulation of Both CDC-6 and CDT-1 Is Required

for RereplicationIn S. pombe, the overexpression of the Cdc6 ortholog(Cdc18) is sufficient to induce significant rereplication[24]. In contrast, overexpression of Cdc6 does notinduce rereplication in S. cerevisiae, Drosophila, orhumans [25–27]. In humans, co-overexpression of wild-type Cdt1 and Cdc6 in cells that lack a cell-cycle check-point produces only modest rereplication in a subset ofcells [27].

We observed that coexpression of nondegradableCDT-1 and nonexportable CDC-6 produced significantrereplication in a subset of early-stage C. elegans em-bryos. In contrast, overexpression of combinations ofderegulated and wild-type CDT-1 or CDC-6 did not in-duce rereplication. This indicates that redundant regula-tion of CDT-1 and CDC-6 prevents rereplication. We didnot observe rereplication in every embryonic cell ex-pressing deregulated CDT-1 and CDC-6. This suggeststhat there might be additional mechanisms that act inthe early embryo to limit rereplication.

The expression of combinations of wild-type and de-regulated CDT-1 and CDC-6 produced an embryonic le-thality that was not associated with increased DNAlevels. The cause of this lethality is unclear, but it mightarise from changes in DNA-replication timing, which isknown to produce embryonic arrest [28].

CUL-4 Regulates Both CDC-6 and CDT-1Replication-Licensing Factors

Inactivation of CUL-4 produces dramatic levels of rere-plication that are associated with a failure to degradeCDT-1 [5]. However, overexpressing Cdt1 in fissionyeast does not induce rereplication, and overexpressinghuman Cdt1 several-log-fold higher than the endoge-nous protein produces only modest rereplication ina subset of cells [27, 29, 30]. Given the negligible or lim-ited effects of greatly overexpressing Cdt1 in other or-ganisms, it was hard to reconcile the substantial rerepli-cation associated with merely failing to degrade CDT-1during S phase in cul-4(RNAi) animals.

Our work reveals that the CDC-6 replication-licensingfactor is also deregulated in cul-4(RNAi) animals. CDC-6remains nuclear throughout S phase in cul-4(RNAi) ani-mals, and this is correlated with a failure to phosphory-late CDC-6 on CDK sites. CUL-4 negatively regulates thelevels of the CDK inhibitor CKI-1 [7]. The negative regu-lation of CKIs of the CIP/KIP family by CUL-4 is con-served in Drosophila and humans [31–33]. cki-1 RNAisuppresses rereplication in cul-4 mutants without

affecting CDT-1 accumulation [7], indicating that CKI-1is independently required for the induction of rereplica-tion. Significantly, the presence of CKI-1 is required forthe block on CDC-6 phosphorylation and nuclear exportin cul-4(gk434) cells. Our results suggest that CUL-4promotes CDC-6 nuclear export by negatively regulat-ing CKI-1 levels, thereby allowing CDK(s) to phosphory-late CDC-6 and induce its nuclear export. The evidencethat CDK(s) are the relevant kinases is that CDC-6is phosphorylated on CDK consensus sites and thephosphorylation is blocked by a CDK inhibitor. In yeastand mammals, CDK activity prevents rereplication, andsiRNA codepletion of CDK1 and CDK2 in human cells in-duces limited rereplication [1, 3, 34]. Our results suggestthat in metazoa, Cdc6 is one of the critical targets ofCDKs for preventing rereplication. Our work further indi-cates that CUL-4 is a master regulator that restrainsDNA replication through two independent pathways:mediating CDT-1 degradation and promoting CDC-6nuclear export via the negative regulation of CKI-1.

Supplemental Data

Experimental Procedures and six figures are available at http://

www.current-biology.com/cgi/content/full/17/11/966/DC1/.

Acknowledgments

We are grateful to Yuji Kohara, Roger Tsien, Richard Roy, Andrew

Fire, Kevin O’Connell, and Sudhir Nayak for clones, strains or re-

agents; Christopher Dowd for technical assistance; members of

the Kipreos laboratory for critical reading of the manuscript; and

the Caenorhabditis Genetics Center for strains. This work was sup-

ported by grant R01GM055297 from the National Institute of General

Medical Sciences (NIGMS) (National Institutes of Health [NIH]) to

E.T.K.

Received: August 21, 2006

Revised: April 23, 2007

Accepted: April 23, 2007

Published online: May 17, 2007

References

1. Arias, E.E., and Walter, J.C. (2007). Strength in numbers: Pre-

venting rereplication via multiple mechanisms in eukaryotic

cells. Genes Dev. 21, 497–518.

2. Blow, J.J., and Dutta, A. (2005). Preventing re-replication of

chromosomal DNA. Nat. Rev. Mol. Cell Biol. 6, 476–486.

3. Machida, Y.J., Hamlin, J.L., and Dutta, A. (2005). Right place,

right time, and only once: Replication initiation in metazoans.

Cell 123, 13–24.

4. Takeda, D.Y., and Dutta, A. (2005). DNA replication and progres-

sion through S phase. Oncogene 24, 2827–2843.

5. Zhong, W., Feng, H., Santiago, F.E., and Kipreos, E.T. (2003).

CUL-4 ubiquitin ligase maintains genome stability by restraining

DNA-replication licensing. Nature 423, 885–889.

6. Arias, E.E., and Walter, J.C. (2006). PCNA functions as a molec-

ular platform to trigger Cdt1 destruction and prevent re-replica-

tion. Nat. Cell Biol. 8, 84–90.

7. Kim, Y., and Kipreos, E.T. (2007). The Caenorhabditis elegans

replication licensing factor CDT-1 is targeted for degradation

by the CUL-4/DDB-1 complex. Mol. Cell. Biol. 27, 1394–1406.

8. Senga, T., Sivaprasad, U., Zhu, W., Park, J.H., Arias, E.E., Walter,

J.C., and Dutta, A. (2006). PCNA is a co-factor for Cdt1 degrada-

tion by CUL4/DDB1 mediated N-terminal ubiquitination. J. Biol.

Chem. 281, 6246–6252.

9. Jiang, W., Wells, N.J., and Hunter, T. (1999). Multistep regulation

of DNA replication by Cdk phosphorylation of HsCdc6. Proc.

Natl. Acad. Sci. USA 96, 6193–6198.

Current Biology972

10. Petersen, B.O., Lukas, J., Sorensen, C.S., Bartek, J., and Helin, K.

(1999). Phosphorylation of mammalian CDC6 by cyclin A/CDK2

regulates its subcellular localization. EMBO J. 18, 396–410.

11. Coverley, D., Pelizon, C., Trewick, S., and Laskey, R.A. (2000).

Chromatin-bound Cdc6 persists in S and G2 phases in human

cells, while soluble Cdc6 is destroyed in a cyclin A-cdk2 depen-

dent process. J. Cell Sci. 113, 1929–1938.

12. Fujita, M., Yamada, C., Goto, H., Yokoyama, N., Kuzushima, K.,

Inagaki, M., and Tsurumi, T. (1999). Cell cycle regulation of hu-

man CDC6 protein. Intracellular localization, interaction with

the human mcm complex, and CDC2 kinase-mediated hyper-

phosphorylation. J. Biol. Chem. 274, 25927–25932.

13. Alexandrow, M.G., and Hamlin, J.L. (2004). Cdc6 chromatin af-

finity is unaffected by serine-54 phosphorylation, S-phase pro-

gression, and overexpression of cyclin A. Mol. Cell. Biol. 24,

1614–1627.

14. DePamphilis, M.L., Blow, J.J., Ghosh, S., Saha, T., Noguchi, K.,

and Vassilev, A. (2006). Regulating the licensing of DNA replica-

tion origins in metazoa. Curr. Opin. Cell Biol. 18, 231–239.

15. Hong, Y., Roy, R., and Ambros, V. (1998). Developmental regula-

tion of a cyclin-dependent kinase inhibitor controls postembry-

onic cell cycle progression in Caenorhabditis elegans. Develop-

ment 125, 3585–3597.

16. Aspock, G., Kagoshima, H., Niklaus, G., and Burglin, T.R. (1999).

Caenorhabditis elegans has scores of hedgehog-related genes:

Sequence and expression analysis. Genome Res. 9, 909–923.

17. Bogerd, H.P., Fridell, R.A., Benson, R.E., Hua, J., and Cullen,

B.R. (1996). Protein sequence requirements for function of the

human T-cell leukemia virus type 1 Rex nuclear export signal

delineated by a novel in vivo randomization-selection assay.

Mol. Cell. Biol. 16, 4207–4214.

18. la Cour, T., Kiemer, L., Molgaard, A., Gupta, R., Skriver, K., and

Brunak, S. (2004). Analysis and prediction of leucine-rich nuclear

export signals. Protein Eng. Des. Sel. 17, 527–536.

19. Cook, J.G., Chasse, D.A., and Nevins, J.R. (2004). The regulated

association of Cdt1 with minichromosome maintenance pro-

teins and Cdc6 in mammalian cells. J. Biol. Chem. 279, 9625–

9633.

20. Nishitani, H., Lygerou, Z., Nishimoto, T., and Nurse, P. (2000).

The Cdt1 protein is required to license DNA for replication in fis-

sion yeast. Nature 404, 625–628.

21. Stringham, E.G., Dixon, D.K., Jones, D., and Candido, E.P.M.

(1992). Temporal and spatial expression patterns of the small

heat shock (hsp16) genes in transgenic Caenorhabditis elegans.

Mol. Biol. Cell 3, 221–223.

22. Feng, H., Zhong, W., Punkosdy, G., Gu, S., Zhou, L., Seabolt,

E.K., and Kipreos, E.T. (1999). CUL-2 is required for the G1-to-

S-phase transition and mitotic chromosome condensation in

Caenorhabditis elegans. Nat. Cell Biol. 1, 486–492.

23. Kemp, C.A., Kopish, K.R., Zipperlen, P., Ahringer, J., and

O’Connell, K.F. (2004). Centrosome maturation and duplication

in C. elegans require the coiled-coil protein SPD-2. Dev. Cell 6,

511–523.

24. Nishitani, H., and Nurse, P. (1995). p65cdc18 plays a major role

controlling the initiation of DNA replication in fission yeast. Cell

83, 397–405.

25. Crevel, G., Mathe, E., and Cotterill, S. (2005). The Drosophila

Cdc6/18 protein has functions in both early and late S phase in

S2 cells. J. Cell Sci. 118, 2451–2459.

26. Nguyen, V.Q., Co, C., and Li, J.J. (2001). Cyclin-dependent ki-

nases prevent DNA re-replication through multiple mechanisms.

Nature 411, 1068–1073.

27. Vaziri, C., Saxena, S., Jeon, Y., Lee, C., Murata, K., Machida, Y.,

Wagle, N., Hwang, D.S., and Dutta, A. (2003). A p53-dependent

checkpoint pathway prevents rereplication. Mol. Cell 11, 997–

1008.

28. Encalada, S.E., Martin, P.R., Phillips, J.B., Lyczak, R., Hamill,

D.R., Swan, K.A., and Bowerman, B. (2000). DNA replication de-

fects delay cell division and disrupt cell polarity in early Caeno-

rhabditis elegans embryos. Dev. Biol. 228, 225–238.

29. Yanow, S.K., Lygerou, Z., and Nurse, P. (2001). Expression of

Cdc18/Cdc6 and Cdt1 during G2 phase induces initiation of

DNA replication. EMBO J. 20, 4648–4656.

30. Gopalakrishnan, V., Simancek, P., Houchens, C., Snaith, H.A.,

Frattini, M.G., Sazer, S., and Kelly, T.J. (2001). Redundant con-

trol of rereplication in fission yeast. Proc. Natl. Acad. Sci. USA

98, 13114–13119.

31. Banks, D., Wu, M., Higa, L.A., Gavrilova, N., Quan, J., Ye, T.,

Kobayashi, R., Sun, H., and Zhang, H. (2006). L2DTL/CDT2 and

PCNA interact with p53 and regulate p53 polyubiquitination

and protein stability through MDM2 and CUL4A/DDB1 com-

plexes. Cell Cycle 5, 1719–1729.

32. Bondar, T., Kalinina, A., Khair, L., Kopanja, D., Nag, A., Bagchi,

S., and Raychaudhuri, P. (2006). Cul4A and DDB1 associate

with Skp2 to target p27Kip1 for proteolysis involving the COP9

signalosome. Mol. Cell. Biol. 26, 2531–2539.

33. Higa, L.A., Yang, X., Zheng, J., Banks, D., Wu, M., Ghosh, P.,

Sun, H., and Zhang, H. (2006). Involvement of CUL4 ubiquitin

E3 ligases in regulating CDK inhibitors Dacapo/p27Kip1 and

cyclin E degradation. Cell Cycle 5, 71–77.

34. Machida, Y.J., and Dutta, A. (2007). The APC/C inhibitor, Emi1, is

essential for prevention of rereplication. Genes Dev. 21, 184–

194.

35. Koppen, M., Simske, J.S., Sims, P.A., Firestein, B.L., Hall, D.H.,

Radice, A.D., Rongo, C., and Hardin, J.D. (2001). Cooperative

regulation of AJM-1 controls junctional integrity in Caenorhabdi-

tis elegans epithelia. Nat. Cell Biol. 3, 983–991.

36. Jans, D.A., Xiao, C.Y., and Lam, M.H. (2000). Nuclear targeting

signal recognition: A key control point in nuclear transport? Bio-

essays 22, 532–544.

37. Cook, J.G., Park, C.H., Burke, T.W., Leone, G., DeGregori, J.,

Engel, A., and Nevins, J.R. (2002). Analysis of Cdc6 function in

the assembly of mammalian prereplication complexes. Proc.

Natl. Acad. Sci. USA 99, 1347–1352.

38. Pearson, R.B., and Kemp, B.E. (1991). Protein kinase phosphor-

ylation site sequences and consensus specificity motifs: Tabu-

lations. Methods Enzymol. 200, 62–81.

39. Dawe, A.S., Smith, B., Thomas, D.W., Greedy, S., Vasic, N.,

Gregory, A., Loader, B., and de Pomerai, D.I. (2006). A small tem-

perature rise may contribute towards the apparent induction by

microwaves of heat-shock gene expression in the nematode

Caenorhabditis elegans. Bioelectromagnetics 27, 88–97.