Department of Genetics, University of Pennsylvania School of Medicine, Philadelphia, PA 19104-6145, USA
Author for correspondence (e-mail:
calvi{at}mail.med.upenn.edu)
Accepted 5 July 2004
![]() |
SUMMARY |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Key words: DNA replication, Cyclin E, Double-parked, Cdt1, Chorion gene amplification
![]() |
Introduction |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Although phosphorylation of pre-RC subunits appears to be important for
initiation and to block pre-RC re-assembly, the biochemical mechanisms are not
fully understood. In the yeasts Saccharomyces cerevisiae and S.
pombe, CDKs block re-replication by phosphorylating several pre-RC
targets including CDC6 and subunits of the ORC and MCM complex
(Drury et al., 2000;
Gopalakrishnan et al., 2001
;
Jallepalli etal., 1997
;
Labib et al., 1999
;
Nguyen et al., 2000
;
Nguyen et al., 2001
;
Nishitani and Nurse, 1995
;
Vas et al., 2001
). All three
of these blocks must be abrogated before even partial re-replication is
permitted in S. cerevisiae cells in G2, suggesting that
multiple reinforcing mechanisms have evolved to protect the integrity of the
genome (Nguyen et al., 2001
).
In S. pombe, however, over-expression of Cdc18 (the Cdc6 homolog)
alone, but not other pre-RC subunits, is sufficient to induce re-replication
(Nishitani and Nurse, 1995
).
Thus, whether mis-regulation of a single protein can induce re-replication may
differ among organisms. In higher eukaryotes, it also appears that CDKs block
re-replication by targeting multiple pre-RC subunits to protect genome
integrity (reviewed by Bell and Dutta,
2002
) (Delmolino et al.,
2001
; Hua et al.,
1997
; Mihaylov et al.,
2002
; Pelizon et al.,
2000
; Petersen et al.,
1999
; Saha et al.,
1998
; Yamaguchi and Newport,
2003
).
Despite the prevailing concept of redundant controls, recent evidence
suggests that regulation of Cdt1 is especially important to inhibit
re-replication. In a number of systems, over-expression of Cdt1, or
inactivation of its inhibitor Geminin, causes partial, but not full,
re-replication of the genome (Mihaylov et
al., 2002; Quinn et al.,
2001
; Tada et al.,
2001
; Vaziri et al.,
2003
; Wohlschlegel et al.,
2000
). In all organisms examined, except S. cerevisiae,
the majority of Cdt1 protein is rapidly degraded at the G1/S
transition. Evidence from several organisms suggests that Cdt1 is targeted for
degradation at the proteasome by two ubiquitin ligase complexes, an SCF (Skp1,
Cul1, F box) ubiquitin ligase that contains the specificity subunit Skp2, and
an SCF-like ubiquitin ligase that is based on Cul4
(Higa et al., 2003
;
Li et al., 2003
;
Nishitani et al., 2001
). This
degradation is probably important because over-expression of Cdt1 in p53
mutant human cells in culture can lead to partial re-replication, and
contributes to oncogenic transformation of mouse erythroid cells
(Arentson et al., 2002
;
Vaziri et al., 2003
). In
Caenorhabditis elegans, RNAi of Cul4 leads to stabilization of Cdt1
protein and polyploidization (Zhong et
al., 2003
). It is unclear, however, whether Cul4 controls
degradation of other proteins important for re-replication control. Therefore,
two important remaining questions are whether increased Cdt1 protein is
sufficient to induce genome reduplication in normal cells during development,
and what coordinates the rapid degradation of Cdt1 with the initiation of DNA
replication at the G1/S transition.
The Drosophila ortholog of Cdt1, the double-parked
(dup) gene, was initially identified as recessive embryonic lethal or
female-sterile mutants that have defects in genomic replication or
developmental amplification of eggshell (chorion) protein genes in the ovary
(Underwood et al., 1990;
Whittaker et al., 2000
). In
this report, we provide evidence that degradation of Dup is controlled in part
by cyclin E/CDK2 phosphorylation, and that additional mechanisms also ensure
Dup degradation. Control of Dup protein abundance is critical because
increased expression of Dup in diploid cells is sufficient to induce
polyploidization and cell death in developing tissues. Interestingly,
over-expression of wild-type and mutant Dup derivatives have different effects
on genomic replication than on amplification from chorion origins. These last
results provide insight into how phosphorylation regulates Dup during these
developmentally distinct replication programs, and suggest that Dup
participates in replication fork elongation during amplification.
![]() |
Materials and methods |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Construction of Dup transgenes
P{w+mC, hsp70:Myc:FL-Dup} was constructed by RT-PCR
amplification of a Dup cDNA using primers that encompass the region from the
start codon to the termination codon, subcloning into pBUM (provided by J.
Sekelsky) resulting in an in frame N-terminal fusion of a new AUG and single
Myc epitope, and finally into pP{CaSpeRhs}
(Thummel et al., 1988).
P{w+, hsp70:Myc:N-Dup} was created similarly except that it contains
a stop codon after amino acid 343. P{w+mC,
hsp70:Myc:C-Dup} was created similarly with primers that amplify from
amino acid 344 E to the stop codon of the Dup coding region. The NLS from SV40
was inserted in frame between the Myc epitope and Dup coding region.
P{w+mC, hsp70:Myc:Dup 10(A)} was created by mutating the
serines and threonines at putative phosphorylation sites to alanine using the
Stratagene multi-mutagenesis kit, and then subcloning as above. The amino acid
coordinates of the serines and threonines are: S37, S111, T158, S168, S226,
S249, T256, T264, S285, S291. Detailed information on mutagenic
oligonucleotides and methods are available upon request.
Immunofluorescent microscopy
Antibody and BrdU labeling were as previously described (Calvi and Lilly,
2003; Schwed et al., 2002).
Guinea pig polyclonal Dup antibody was used at 1:1000 dilution
(Whittaker et al., 2000
). The
affinity purified rabbit polyclonal Dup antibody (a gift from E. Beall and M.
Botchan) was used at 1:500. Anti-cyclin E monoclonal 8B10
(Richardson et al., 1995
) was
used at 1:5, and anti-cyclin B monoclonal
(Lehner and O'Farrell, 1990
)
was used at 1:4. Rabbit polyclonal anti-caspase 3 antibody (Cell Signaling,
Beverly, MA) was used at 1:100. For Myc/BrdU stability experiments, cells were
fixed and denatured with DNAse (Calvi and Lilly, 2003) and subsequently
labeled with polyclonal sheep anti-BrdU (Research Diagnostics, Flanders, NJ)
and monoclonal mouse anti-Myc (clone 9E10; Upstate Biotech, Lake Placid, NY),
and Toto-3 as described previously (Schwed
et al., 2002
). Slides were analyzed using a Leica SP confocal
microscope and TCS-NT software, or by using Openlab quantification software on
non-confocal images (Image Processing and Vision Co. Ltd).
Antibody production, western blots and immunoprecipitation
The anti-Geminin antibody was raised in rabbits against a synthetic peptide
(C)QQRQTLKPLQGNVNDKEN (ZYMED) corresponding to amino acid residues 24-41
predicted from the gene CG3183 in the Drosophila genomic sequence.
Antibody was affinity purified using this peptide and the Sulfolink Kit
(Pierce, Rockford, IL).
Standard methods were used for immunoprecipitation and western blot
analysis of Dup and Geminin, which were detected using the ECL kit (Amersham
Biosciences) (Harlow and Lane,
1999). For Fig. 4A,
0- to 16-hour y w embryos were homogenized in NP40 lysis buffer and
spun for 5 minutes at 4°C to remove insoluble material. Guinea pig
anti-Dup antibody was then used for standard immunoprecipitation.
Approximately 1/100 total lysate (input) and 1/3 of the pellet samples were
loaded. Dup was detected using the affinity purified polyclonal rabbit
antibody at 1:5000.
|
![]() |
Results |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
|
|
|
|
Phosphorylation of Dup depends on cyclin E/CDK2
To begin to address whether Dup is phosphorylated in vivo, we examined Dup
for post-translational modification using western blotting.
Immunoprecipitation of Dup from embryos indicated that there are at least
three isoforms of the protein, a predicted full-length 82 kDa form and a
higher molecular weight doublet of
105 kDa and
106 kDa
(Fig. 4A). To confirm that
these isoforms are indeed Dup, we raised antibodies against
Drosophila geminin protein, a tight binding inhibitor of Dup
(Quinn et al., 2001
). All
three isoforms co-immunoprecipitated with Geminin, further suggesting that
they represent Dup (Fig. 4A).
Previous reports that used independently raised antibodies against
Drosophila Geminin and Dup have also suggested the existence of these
higher molecular mass species (Higa et
al., 2003
; Quinn et al.,
2001
).
Treatment of extracts from larval brain and imaginal disc with lambda
phosphatase resulted in the disappearance of the 106 kDa band and an increase
in the intensity of the 105 kDa band, suggesting that they differ by
phosphorylation (Fig. 4B). To
ask if this phosphorylation is cyclin E dependent in vivo, we over-expressed
cyclin E or its specific inhibitor, Dacapo, using the GAL4/UAS system and a
heat inducible hsp70:GAL4 (Brand and
Perrimon, 1993; de Nooij et
al., 1996
; Lane et al.,
1996
). One hour after heat induction, cyclin E increased the
relative abundance of the 106 kDa relative to the 105 kDa isoform, whereas
over-expression of Dacapo decreased the abundance of the 106 kDa
phospho-isoform (Fig. 4B). The
increase in the 106 kDa isoform by cyclin E was due to phosphorylation because
it was completely reversed by treating the extracts with lambda phosphatase
(Fig. 4B).
To further confirm and investigate Dup phosphorylation, we transformed flies with a full-length Dup cDNA tagged on the amino terminus with the Myc epitope, and under control of the heat-inducible hsp70 promoter (hsp70:Myc:FL-Dup). One hour after heat-induced expression, western blotting of larval brain extracts with Myc antibodies gave evidence for a single band that co-migrated with the endogenous 106 kDa isoform (Fig. 4C). Treatment of these extracts with lambda phosphatase did not alter this molecular mass to 105 kDa, but over-expression of cyclin E shifted Myc:FL-Dup to a higher molecular mass, which was completely reversed by lambda phosphatase (Fig. 4C). This suggests that the abundant Myc:Dup species represents the unphosphorylated isoform that migrates at 106 kDa, because of the addition of the Myc epitope. It also confirms that Dup phosphorylation responds to cyclin E/CDK2 activity in vivo.
Cyclin E/CDK2 induces phosphorylation of the Dup N terminus which is required for normal degradation in vivo
The amino-terminal half of the Dup protein contains ten sequences that
resemble the consensus site for CDK phosphorylation ({S/T}PX{K/R}) and several
appropriately spaced RXL sites, the consensus for cyclin binding
(Fig. 5A)
(Pearson and Kemp, 1991;
Takeda et al., 2001
). To
determine if these sites are phosphorylated by cyclin E/CDK2, we transformed
flies with a Dup mutant in which all ten of the serines or threonines at these
sites were changed to alanine (hsp70:Myc:Dup 10(A))
(Fig. 5A).
Like Myc:FL-Dup, the migration of Myc:Dup 10(A) was unaffected by the addition of phosphatase to the extracts. Unlike Myc:FL-Dup, however, the migration of Myc:Dup 10(A) was not shifted upward by over-expression of cyclin E (Fig. 4C). Together with the CDK2 immunoprecipitation results, this suggests that one or more of the ten phosphorylation sites in the N terminus can be phosphorylated by cyclin E/CDK2 in dividing brain and disc cells.
To examine whether this phosphorylation alters Dup protein stability, we heat induced a pulse of expression of hsp70:Myc:FL-Dup and hsp70:Myc:Dup 10(A) and measured their abundance at different times thereafter. This showed that Myc:Dup 10(A) was somewhat more abundant than Myc:FL-Dup (Fig. 4D, lanes 1-3 and 7-9). Importantly, Myc:FL-Dup was more sensitive to over-expression of cyclin E than was Myc:Dup 10(A) (Fig. 4D lanes 4-6 and 10-12). Nevertheless, Dup 10(A) stability was sensitive to over-expression of cyclin E, suggesting that mutation of the ten phosphorylation sites was not sufficient to completely block cyclin E-dependent degradation (Fig. 4D,E compare lanes 7-9 with 10-12). These results suggest that phosphorylation of Dup on one or more of the ten N-terminal sites is only partially responsible for its cyclin E/CDK2-dependent degradation.
The N terminus is required for Dup degradation during S phase in different types of cell cycles in the ovary
The combined evidence suggested that both phosphorylation and degradation
of Dup at G1/S depended on cyclin E/CDK2, but that phosphorylation
of Dup protein could not fully account for its instability. To address this
question by a different method, we examined the stability of the Myc-tagged
proteins by immunofluorescence. To confirm the role of cyclin E, and expand
this analysis to different types of cell cycles in development, we focused on
follicle cells of the ovary. These cells originate from stem cells and
transition from mitotic to endocycles during precise stages of oogenesis
(Calvi et al., 1998;
Mahowald et al., 1979
;
Margolis and Spradling, 1995
).
The periodic S phases of the endocycle are controlled by cyclin E/CDK2, which
is the only known oscillating CDK activity at that time
(Lilly and Spradling, 1996
).
Previous immunolabeling had shown that Dup protein levels in the ovary
oscillate during mitotic and endocycles
(Whittaker et al., 2000
).
hsp70:Myc:FL-Dup was induced at 37°C for 30 minutes, and after 1 hour
recovery, ovaries were dissected and incubated in BrdU for 1 hour, followed by
labeling for Myc and BrdU. The number of Myc/BrdU double-labeled cells was
then counted as a measure of Dup stability during S phase. As a control for
expression, in other animals an hsp70:Myc:P transposase gene was induced,
which resulted in Myc labeling in 96% of cells (n=314) including many
that labeled with BrdU (data not shown)
(Xu and Rubin, 1993). Similar
to endogenous Dup protein, Myc:FL-Dup protein was abundant in many nuclei in
mitotically dividing and endocycling follicle cells, but there were none
double labeled for Myc and BrdU (n=213), indicating Myc:FL-Dup is
unstable in S phase (Fig. 5B
and data not shown). Myc:Dup 10(A) was also abundantly expressed in many
nuclei, and was never seen in cells in S phase (n=275)
(Fig. 5C). This is in contrast
to the western results which indicated that Myc:Dup 10(A) was at least
partially stabilized.
To further explore this, we tested two other transgenes that expressed only the N-terminal half of Dup (hsp70:Myc:N-Dup), or only the C-terminal half (hsp70:Myc:C-Dup), which contain or lack the ten phosphorylation sites, respectively (Fig. 5A). The SV40 nuclear localization signal was included in Myc:C-Dup to compensate for deletion of the natural NLS (data not shown). Similar to Myc:FL-Dup, Myc:N-Dup was also highly expressed in many cells, and in only one cell was Myc labeling seen during S phase (n=344) (Fig. 5D). In contrast, deletion of the N terminus in Myc:C-Dup led to significant stability in S phase; 22% of the follicle cells (n=309) labeled for both Myc and BrdU (Fig. 5E). This indicates that the N terminus is necessary and sufficient for normal Dup degradation in S phase. Together with the western results, the data suggest that CDK2 phosphorylation contributes to Dup instability, but there must be other mechanisms that ensure Dup degradation during S phase.
Mis-expression of Dup is sufficient to induce re-replication and cell death during cell cycles in ovary and imaginal disc
We wished to test if degradation of Dup at G1/S is essential, or
whether other mechanisms would protect genome integrity in the presence of
elevated Dup protein levels. Therefore, we heat-induced expression of Dup for
30 minutes twice a day, and examined follicle cells labeled with the
fluorescent DNA dye Toto-3 in the confocal microscope at 30 hours (three heat
pulses) and 54 hours (five heat pulses) thereafter. Heat shock alone or
induction of hsp70:Myc:P-transposase had no consistent effects
(Fig. 6A and data not shown).
For Myc:FL-Dup, however, two phenotypes were observed by 30 hours: abnormally
large nuclei, suggestive of re-replication, and small pycnotic nuclei,
suggestive of cell death (Table
1) (Fig. 6B). All
ovarioles examined (n>50) had these two phenotypes to varying
extents, with 10-80% of nuclei within mitotic stage follicle cells
appearing enlarged, and
5% appearing pycnotic. Staining with the vital
dye Acridine Orange or antibody against activated caspase-3 confirmed that
follicle cells with pycnotic nuclei were in fact undergoing cell death (data
not shown). Many stage 5 follicle cells, which are normally in the mitotic
cycle with nuclei of
3-4.5 µm in diameter, had nuclei that were
enlarged up to 15 µm in diameter, twice the diameter (eight times greater
volume) of wild-type stage 10B nuclei that have a DNA content of 16C
(Fig. 6B arrows). Follicle
cells in the endocycle also were unusually large, but cell death was less
frequently observed than in mitotically dividing cells (data not shown). Some
cells with large nuclei were actively undergoing DNA synthesis as evidenced by
incorporation of BrdU (Fig.
6B). At 54 hours (five heat pulses) there was a notable increase
in the number of dying follicle cells (data not shown). These results suggest
that over-expression of Dup is sufficient to induce re-replication and cell
death in follicle cells of the ovary.
|
|
We considered that the ability of Dup to induce re-replication may be peculiar to follicle cells because they normally undergo a developmental transition to endocycles. To test this idea, we over-expressed the various Dup transgenes in larval imaginal disc cells that have a canonical cell cycle (Fig. 7A,B). Larvae were subjected to heat induction twice a day and imaginal discs were examined after staining with the fluorescent DNA dye DAPI. As in the ovary, Myc:C-Dup and Myc:N-Dup over-expression had no obvious effects (data not shown). After 30 hours, about half of the Myc:FL-Dup imaginal discs had some nuclei that were greatly enlarged, while others were pycnotic and labeled positively for activated caspase-3 (Fig. 7C and data not shown). In some nuclei small polytene chromosomes could be seen (Fig. 7G). By 54 hours all discs displayed the enlarged and pycnotic nuclear phenotype, and disc morphology was extremely aberrant (data not shown). At 54 hours there was a decrease in the number of large nuclei with a concomitant increase in the number of dying cells, consistent with the idea that cell death is a consequence of re-replication. Similar results were obtained for Myc:Dup 10(A) over-expression except that enlarged and dying cells were more frequent (Fig. 7E).
|
Mis-expression of Dup enhances genomic replication during S phase
Although Dup mis-expression was sufficient to induce polyploidy, it was
unclear in what stage of the cell cycle this occurred. We did not see evidence
of chromosome mis-segregation after labeling with the mitotic chromosome
marker anti-phosphohistone H3, suggesting that mitotic non-disjunction was not
the cause of the 8C population (data not shown). To gain insight into the cell
cycle stage affected, we induced three pulses of Dup over 30 hours and labeled
third instar eye discs with BrdU for 1 hour. Mis-expression of Myc:FL-Dup or
Myc:Dup 10(A) resulted in a greater number of the asynchronous cycling cells
anterior to the MF labeling with BrdU (Fig.
8A-C). In contrast, Dup mis-expression did not induce an
appreciable increase in BrdU incorporation in the differentiating cells
posterior to the furrow, many of which are in G2, despite having
high levels of mis-expressed Dup protein
(Fig. 8B,C and data not shown).
Mis-expression of Dup did result in a low level of BrdU incorporation in some
cells within the morphogenetic furrow, although the majority showed a normal
G1 arrest. Most strikingly, mis-expression of Myc:FL-Dup or Myc:Dup
10(A) resulted in a wider stripe of S-phase cells incorporating BrdU just
posterior to the furrow. In wild type, nucleus-wide incorporation of BrdU
occurs for approximately four cell diameters behind the furrow, and an
additional five cell diameters have focal BrdU over heterochromatin late in S
phase (Fig. 8A). With FL-Dup
mis-expression, nucleus-wide incorporation of BrdU incorporation was seen for
approximately nine to ten cell diameters, with euchromatic replication
continuing into late S phase (Fig.
8B). The BrdU stripe was wider in Dup 10(A), indicating that
euchromatic replication was even more prolonged
(Fig. 8C). Together with the
FACS results, this enhanced genomic replication implies that Dup can induce
re-replication during S phase.
|
|
Expression of Myc:Dup 10(A) had the opposite effect; it severely inhibited
amplification with no BrdU incorporation detected at amplification foci within
3 hours (Fig. 9D). We
considered that the dominant-negative effect of the non-phosphorylatable Dup
10(A) could be due to tight binding of CDK2, thereby reducing its total
cellular activity, which is required for chorion gene amplification
(Calvi et al., 1998). To test
this, we used the MPM2 antibody, which we have previously shown detects a
subnuclear phospho-epitope whose periodic cell cycle labeling is a reporter
for cyclinE/CDK2 activity (Calvi et al.,
1998
). Although expression of Myc:Dup 10(A) completely inhibited
BrdU incorporation at chorion foci within 3 hours, it had no effect on MPM2
labeling (data not shown). Therefore, the dominant negative effect of Dup
10(A) is not due to inhibition of CDK2. Rather, the results suggest that in
the context of the full-length protein, phosphorylation of the N terminus is
required for activity during amplification. Taken together, it appears that
aspects of Dup regulation or function may differ between amplification and
genomic replication.
Previous high resolution analysis of amplification foci showed that origin
regions can be visually distinguished from migrating replication forks
(Calvi et al., 1998;
Calvi and Spradling, 2001
). Dup
antibody labeling co-localizes with BrdU and proteins at the replication fork,
suggesting that, contrary to evidence from other cell cycles, it may have a
role in elongation during amplification
(Claycomb et al., 2002
).
Myc:Dup 10(A) inhibited BrdU incorporation at amplification foci as soon as 1
hour after expression in all stages, including stages 12-13 when only
elongation of forks is occurring (data not shown)
(Claycomb et al., 2002
). This
complete and rapid inhibition of BrdU incorporation suggests that Dup is
required for fork elongation during amplification.
![]() |
Discussion |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Regulation of Dup degradation at G1/S
The results suggest that cyclin E/CDK2 phosphorylates the Dup N terminus
contributing to its instability at G1/S. Dup was degraded during
periodic endocycle S phases that are solely regulated by oscillating cyclin
E/CDK2, further supporting a link between this kinase and Dup degradation.
Although the N terminus was necessary and sufficient for degradation, mutation
of the ten N-terminal CDK sites within Dup 10(A) only partially stabilized the
protein. This suggests that there are other cyclin E/CDK2-dependent mechanisms
that trigger Dup degradation independent of these ten sites during S phase. It
has been noted that the C terminus of Dup contains a PEST sequence
(Whittaker et al., 2000), and
there are several serines and threonines in the C terminus that are potential
targets of phosphorylation (Gopalakrishnan
et al., 2001
). Although we have not directly tested the
requirement for these sites, the stability of C-Dup indicates that they are
not sufficient for degradation at G1/S. To explain our results, we
suggest a bi-phasic degradation model where cyclin E/CDK2 phosphorylation
promotes Dup degradation in late G1, whereas other fail-safe
mechanisms become operative only during S phase. This would explain why
inhibiting CDK2 and S phase entry with GMRp21 completely blocked Dup
degradation.
A number of very recent publications describe results for Cdt1 in human
cells that are similar to ours in flies
(Kondo et al., 2004;
Liu et al., 2004
;
Nishitani et al., 2004
;
Sugimoto et al., 2004
). These
results suggest that cyclin A/CDK2 phophorylates the human Cdt1 N terminus,
which enhances its binding to the Skp2 subunit of the SCF ubiquitin ligase.
Like Dup, non-phosphorylatable Cdt1 mutants were only partially stabilized,
but simultaneously inhibiting CDK2 and S phase entry with p21 completely
blocked degradation. Previous evidence in C. elegans, human and
Drosophila cells suggested that destruction of Cdt1 may be mediated
by two ubiquitin ligases, an SCF complex containing Skp2, and an SCF-like
complex based on Cul4 (Higa et al.,
2003
; Li et al.,
2003
; Nishitani et al.,
2001
; Zhong et al.,
2003
). For many substrates of the SCF, prior phosphorylation is
required for their subsequent recognition and ubiquitinylation, including
substrates phosphorylated by CDK2 at G1/S (reviewed by
Jackson et al., 2000
)
(Montagnoli et al., 1999
). It
is not known whether prior phosphorylation is required for substrate
recognition by Cul4-based ubiquitin ligases. It is tempting to speculate,
therefore, that the bi-phasic degradation of Cdt1 that we suggest may reflect
its modification by two distinct ubiquitin ligases: a
phosphorylation-dependent ubiquitinylation by the SCF complex, and a
phosphorylation-independent ubiquitinylation by a Cul4-based complex. Clearly,
more experiments are needed to sort out the complexity of this regulation.
Nonetheless, the similar results from flies and humans suggest that tight
regulation of Cdt1 abundance is a generally conserved and important mechanism
to protect genome integrity in eukaryotes.
Regulation of the pre-RC in re-replication control
CDK activity and Geminin play central roles in the block to re-replication
(reviewed by Diffley, 2001).
Our results show that Dup over-expression is sufficient to induce a full
genome reduplication in normal cells in developing tissues, transforming
diploid into polyploid cells. This phenotype is more profound than that of
Geminin mutants, suggesting that degradation of Dup protein is of highest
priority to protect genome integrity. An important caveat is that in our
experiments Dup is over-expressed and therefore not equivalent to an absence
of degradation. We have found, however, that even small, undetectable
increases in Dup protein can have profound consequences (data not shown).
Moreover, after multiple heat pulses, Dup protein was undetectable during S
phase, yet it induced extensive re-replication in most cells. The prolonged
genomic replication in the synchronized cells of the eye disc suggests that
this small increase in Dup protein may permit origins to be relicensed and
reinitiate within a single S phase. While the precise molecular mechanism for
how increased Dup promotes re-replication remains undefined, the results
indicate that even a small increase in Dup protein is sufficient to compromise
genome integrity.
The other phenotype associated with over-expression of Dup was cell death.
Dup 10(A) caused more cell death than wild-type Dup, suggesting that
phosphorylation of the Dup N terminus influences this phenotype. In human
cells re-replication due to over-expression of Cdt1 is more easily detected
when p53 is mutant, probably because they escape apoptosis triggered by
re-replication (Vaziri et al.,
2003). We therefore favor the model that Dup over-expression
induces re-replication, which in turn can lead to the activation of
checkpoints and apoptosis.
Dup function in chorion gene amplification
In recent years, the analysis of replication from the defined chorion
amplification origins has been a prominent genetic and molecular model system
for the regulation of DNA replication in metazoa. Chorion origins require
pre-RC proteins, cyclin E/CDK2 and Dbf4/Cdc7 kinases, indicating that their
regulation resembles that of genomic origins (reviewed by
Bandura and Calvi, 2002). They
clearly differ, however, in that they re-replicate at a time when no other
origins are firing, and understanding this exception should provide insight
into the rules of regulation of all origins. To our surprise, we found that
the carboxyl-terminal half of Dup, although having no effect on genomic
replication, is a hyperactive protein that causes over-amplification from
chorion origins. The Dup 10(A) mutant gave the opposite result; it was
dominant negative and strongly inhibited amplification. We propose that during
amplification phosphorylation of the Dup N terminus abrogates its inhibition
of the activity of the C terminus, explaining why deleting the N terminus
results in a hyperactive protein, whereas blocking its phosphorylation results
in an inactive protein. An important functional role for the C terminus is
consistent with its binding to MCM proteins, and the fact that among Cdt1
family members the C-terminal half is much more highly conserved than the
N-terminal half of the protein (Whittaker
et al., 2000
; Wohlschlegel et
al., 2000
; Yanagi et al.,
2002
). Most Cdt1 proteins have known or potential CDK
phosphorylation sites in their N terminus despite its poor conservation,
supporting the notion that its conserved function is to mediate regulation by
CDKs.
The different effects on amplification versus genomic replication suggest a
distinction in the regulation or function of Dup in these two processes.
Claycomb et al. had previously proposed that Dup participates in fork
elongation during amplification, based on immunolabeling at chorion foci
(Claycomb et al., 2002). We
show that in other cell cycles Dup is rapidly degraded at the onset of S phase
and not present during fork elongation, similar to results from human and
other cells. Moreover, S. cerevisiae cells experimentally depleted of
Cdt1 within S phase are able to complete genomic replication, inconsistent
with a role in elongation (Tanaka and
Diffley, 2002
). Expression of Dup 10(A), however, inhibited BrdU
incorporation within 1 hour in all stages of amplification, including late
stages when only elongation of forks is occurring. This rapid and complete
inhibition of BrdU incorporation by Dup 10(A) cannot be an indirect effect of
origin inhibition, and supports the proposed role for Dup at the replication
fork. Furthermore, this suggests that phosphorylation is important for the
function of Dup in elongation during amplification. The distinct activities of
C-Dup and Dup 10(A) in genomic replication versus amplification provide a
molecular handle on the mechanism by which these two developmental replication
programs differ, possibly resulting from the activity of Dup at the fork. The
function of Dup at the fork may be related to its known ability to load the
MCM complex helicase onto chromatin. It also raises the possibility that Cdt1
family members may act at the fork under other special circumstances.
![]() |
ACKNOWLEDGMENTS |
---|
![]() |
Footnotes |
---|
![]() |
REFERENCES |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Arentson, E., Faloon, P., Seo, J., Moon, E., Studts, J. M., Fremont, D. H. and Choi, K. (2002). Oncogenic potential of the DNA replication licensing protein CDT1. Oncogene 21,1150 -1158.[CrossRef][Medline]
Austin, R. J., Orr-Weaver, T. L. and Bell, S. P.
(1999). Drosophila ORC specifically binds to ACE3, an origin of
DNA replication control element. Genes Dev.
13,2639
-2649.
Bandura, J. L. and Calvi, B. R. (2002). Duplication of the genome in normal and cancer cell cycles. Cancer Biology and Therapy 1,8 -13.[Medline]
Bell, S. and Stillman, B. (1992). ATP-dependent recognition of eukaryotic origins of DNA replication by a multiprotein complex. Nature 357,128 -134.[CrossRef][Medline]
Bell, S. P. and Dutta, A. (2002). DNA replication in eukaryotic cells. Annu. Rev. Biochem. 71,333 -374.[CrossRef][Medline]
Brand, A. and Perrimon, N. (1993). Targeted
gene expression as a means of altering cell fates and generating dominant
phenotypes. Development
118,401
-415.
Calvi, B. R. and Lilly, M. A. (2004). BrdU labeling and nuclear flow sorting of the Drosophila ovary. In Drosophila Cytogenetics Protocols (ed. D. Henderson), pp. 203-213. Totowa, USA: Humana Press.
Calvi, B. R., Lilly, M. A. and Spradling, A. C.
(1998). Cell cycle control of chorion gene amplification.
Genes Dev. 12,734
-744.
Calvi, B. R. and Spradling, A. C. (2001). The nuclear location and chromatin organization of active chorion amplification origins. Chromosoma 110,159 -172.[Medline]
Chong, J., Mahbubani, H., Khoo, C. and Blow, J. (1995). Purification of an MCM-containing complex as a component of the DNA replication licensing system. Nature 375,418 -421.[CrossRef][Medline]
Claycomb, J. M., Benasutti, M., Bosco, G., Fenger, D. D. and Orr-Weaver, T. L. (2004). Gene amplification as a developmental strategy: isolation of two developmental amplicons in Drosophila. Dev. Cell 6,145 -155.[CrossRef][Medline]
Claycomb, J. M., MacAlpine, D. M., Evans, J. G., Bell, S. P. and
Orr-Weaver, T. L. (2002). Visualization of replication
initiation and elongation in Drosophila. J. Cell Biol.
159,225
-236.
Cocker, J., Piatti, S., Santocanale, C., Nasmyth, K. and Diffley, J. (1996). An essential role for the Cdc6 protein in forming the pre-replicative complexes of budding yeast. Nature 379,180 -182.[CrossRef][Medline]
Dahmann, C., Diffley, J. F. X. and Nasmyth, K. A. (1995). S-phase-promoting cyclin-dependent kinases prevent re-replication by inhibiting the transition of replication origins to a pre-replicative state. Curr. Biol. 5,1257 -1269.[Medline]
de Nooij, J., Letendre, M. and Hariharan, I. (1996). A cyclin-dependent kinase inhibitor, Dacapo, is necessary for timely exit from the cell cycle during Drosophila embryogenesis. Cell 87,1237 -1247.[Medline]
de Nooij, J. C. and Hariharan, I. K. (1995). Uncoupling cell fate determination from patterned cell division in the Drosophila eye. Science 270,983 -985.[Abstract]
Delmolino, L. M., Saha, P. and Dutta, A.
(2001). Multiple mechanisms regulate subcellular localization of
human CDC6: NLS, NES and phosphorylation. J. Biol.
Chem. 276,26947
-26954.
Diffley, J., Cocker, J., Dowell, S. and Rowley, A. (1994). Two steps in the assembly of complexes at yeast replication origins in vivo. Cell 78,303 -316.[Medline]
Diffley, J. F. (2001). DNA replication: building the perfect switch. Curr. Biol. 11,367 -370.[CrossRef]
Dowell, S., Romanowski, P. and Diffley, J. (1994). Interaction of Dbf4, the Cdc7 protein kinase regulatory subunit, with yeast replication origins in vivo.Science 265,1243 -1246.[Medline]
Drury, L. S., Perkins, G. and Diffley, J. F. (2000). The cyclin-dependent kinase Cdc28p regulates distinct modes of Cdc6p proteolysis during the budding yeast cell cycle. Curr. Biol. 10,231 -240.[CrossRef][Medline]
Gopalakrishnan, V., Simancek, P., Houchens, C., Snaith, H. A.,
Frattini, M. G., Sazer, S. and Kelly, T. J. (2001).
Redundant control of rereplication in fission yeast. Proc. Natl.
Acad. Sci. USA 98,13114
-13119.
Harlow, E. and Lane, D. (1999). Using Antibodies: A Laboratory Manual. Cold Spring Harbor: Cold Spring Harbor Laboratory Press.
Hayles, J., Fisher, D., Woollard, A. and Nurse, P. (1994). Temporal order of S phase and mitosis in fission yeast is determined by the state of the p34cdc2-mitotic B cyclin complex. Cell 78,813 -822.[Medline]
Hengstschlager, M., Braun, K., Soucek, T., Miloloza, A. and Hengstschlager-Ottnad, E. (1999). Cyclin-dependent kinases at the G1-S transition of the mammalian cell cycle. Mutat. Res. 436,1 -9.[CrossRef][Medline]
Higa, L. A., Mihaylov, I. S., Banks, D. P., Zheng, J. and Zhang, H. (2003). Radiation-mediated proteolysis of CDT1 by CUL4-ROC1 and CSN complexes constitutes a new checkpoint. Nat. Cell Biol. 5,1008 -1015.[CrossRef][Medline]
Hua, X. H., Yan, H. and Newport, J. (1997). A
role for cdk2 kinase in negatively regulating DNA replication during S phase
of the cell cycle. J. Cell Biol.
137,183
-192.
Jackson, P. K., Eldridge, A. G., Freed, E., Furstenthal, L., Hsu, J. Y., Kaiser, B. K. and Reimann, J. D. (2000). The lore of the RINGs: substrate recognition and catalysis by ubiquitin ligases. Trends Cell Biol. 10,429 -439.[CrossRef][Medline]
Jallepalli, P., Brown, G., Muzi-Falconi, M., Tien, D. and Kelly,
T. (1997). Regulation of the replication initiator protein
p65cdc18 by CDK phosphorylation. Genes Dev.
11,2767
-2779.
Kondo, T., Kobayashi, M., Tanaka, J., Yokoyama, A., Suzuki, S.,
Kato, N., Onozawa, M., Chiba, K., Hashino, S., Imamura, M. et al.
(2004). Rapid degradation of Cdt1 upon UV-induced DNA damage is
mediated by SCFSkp2 complex. J. Biol. Chem.279
, 27315-27319.
Labib, K., Diffley, J. F. and Kearsey, S. E. (1999). G1-phase and B-type cyclins exclude the DNA-replication factor Mcm4 from the nucleus. Nat. Cell Biol. 1, 415-422.[CrossRef][Medline]
Landis, G., Kelley, R., Spradling, A. and Tower, J.
(1997). The k43 gene, required for chorion gene amplification and
diploid cell chromosome replication, encodes the Drosphila homolog of yeast
origin recognition complex subunit 2. Proc. Natl. Acad. Sci.
USA 94,3888
-3892.
Lane, M., Sauer, K., Wallace, K., Jan, Y., Lehner, C. and Vaessin, H. (1996). Dacapo, a cyclin-dependent kinase inhibitor, stops cell proliferation during Drosophila development. Cell 87,1225 -1235.[Medline]
Lehner, C. F. and O'Farrell, P. H. (1990). The roles of Drosophila Cyclins A and B in mitotic control. Cell 61,535 -547.[Medline]
Lei, M., Kawasaki, Y., Young, M. R., Kihara, M., Sugino, A. and
Tye, B. K. (1997). Mcm2 is a target of regulation by
Cdc7-Dbf4 during the initiation of DNA synthesis. Genes
Dev. 11,3365
-3374.
Li, X., Zhao, Q., Liao, R., Sun, P. and Wu, X.
(2003). The SCF(Skp2) ubiquitin ligase complex interacts with the
human replication licensing factor Cdt1 and regulates Cdt1 degradation.
J. Biol. Chem. 278,30854
-30858.
Liang, C., Weinreich, M. and Stillman, B. (1995). ORC and Cdc6p interact and determine the frequency of initiation of DNA replication in the genome. Cell 81,667 -676.[Medline]
Lilly, M. and Spradling, A. (1996). The Drosophila endocycle is controlled by Cyclin E and lacks a checkpoint ensuring S-phase completion. Genes Dev. 10,2514 -2526.[Abstract]
Liu, E., Li, X., Yan, F., Zhao, Q. and Wu, X.
(2004). Cyclin-dependent kinases phosphorylate human Cdt1 and
induce its degradation. J. Biol. Chem.
279,17283
-17288.
Mahowald, A., Caulton, J., Edwards, M. and Floyd, A. (1979). Loss of centrioles and polyploidization in follicle cells of Drosophila melanogaster. Exp. Cell Res. 118,404 -410.[Medline]
Maiorano, D., Moreau, J. and Mechali, M. (2000). XCDT1 is required for the assembly of pre-replicative complexes in Xenopus laevis. Nature 404,622 -625.[CrossRef][Medline]
Margolis, J. and Spradling, A. (1995).
Identification and behavior of epithelial stem cells in the Drosophila ovary.
Development 121,3797
-3807.
Masumoto, H., Muramatsu, S., Kamimura, Y. and Araki, H. (2002). S-Cdk-dependent phosphorylation of Sld2 essential for chromosomal DNA replication in budding yeast. Nature 415,651 -655.[CrossRef][Medline]
McGarry, T. J. and Kirschner, M. W. (1998). Geminin, an inhibitor of DNA replication, is degraded during mitosis. Cell 93,1043 -1053.[Medline]
Meyer, C. A., Jacobs, H. W., Datar, S. A., Du, W., Edgar, B. A.
and Lehner, C. F. (2000). Drosophila Cdk4 is required
for normal growth and is dispensable for cell cycle progression.
EMBO J. 19,4533
-4542.
Mihaylov, I. S., Kondo, T., Jones, L., Ryzhikov, S., Tanaka, J.,
Zheng, J., Higa, L. A., Minamino, N., Cooley, L. and Zhang, H.
(2002). Control of DNA replication and chromosome ploidy by
geminin and cyclin A. Mol. Cell Biol.
22,1868
-1880.
Montagnoli, A., Fiore, F., Eytan, E., Carrano, A. C., Draetta,
G. F., Hershko, A. and Pagano, M. (1999).
Ubiquitination of p27 is regulated by Cdk-dependent phosphorylation and
trimeric complex formation. Genes Dev.
13,1181
-1189.
Neufeld, T. P., de la Cruz, A. F., Johnston, L. A. and Edgar, B. A. (1998). Coordination of growth and cell division in the Drosophila wing. Cell 93,1183 -1193.[Medline]
Nguyen, V. Q., Co, C., Irie, K. and Li, J. J. (2000). Clb/Cdc28 kinases promote nuclear export of the replication initiator proteins Mcm2-7. Curr. Biol. 10,195 -205.[CrossRef][Medline]
Nguyen, V. Q., Co, C. and Li, J. J. (2001). Cyclin-dependent kinases prevent DNA re-replication through multiple mechanisms. Nature 411,1068 -1073.[CrossRef][Medline]
Nishitani, H., Lygerou, Z. and Nishimoto, T.
(2004). Proteolysis of DNA replication licensing factor Cdt1 in
S-phase is performed independently of Geminin through its N-terminal region.
J. Biol. Chem. 279,30807
-30816.
Nishitani, H., Lygerou, Z., Nishimoto, T. and Nurse, P. (2000). The Cdt1 protein is required to license DNA for replication in fission yeast. Nature 404,625 -628.[CrossRef][Medline]
Nishitani, H. and Nurse, P. (1995). p65cdc18 plays a major role controlling the initiation of DNA replication in fission yeast. Cell 83,397 -405.[Medline]
Nishitani, H., Taraviras, S., Lygerou, Z. and Nishimoto, T.
(2001). The human licensing factor for DNA replication Cdt1
accumulates in G1 and is destabilized after initiation of S-phase.
J. Biol. Chem. 276,44905
-44911.
Noton, E. and Diffley, J. F. (2000). CDK inactivation is the only essential function of the APC/C and the mitotic exit network proteins for origin resetting during mitosis. Mol. Cell 5,85 -95.[Medline]
Pearson, R. B. and Kemp, B. E. (1991). Protein kinase phosphorylation site sequences and consensus specificity motifs: tabulations. Methods Enzymol. 200, 62-81.[Medline]
Pelizon, C., Madine, M. A., Romanowski, P. and Laskey, R. A.
(2000). Unphosphorylatable mutants of Cdc6 disrupt its nuclear
export but still support DNA replication once per cell cycle. Genes
Dev. 14,2526
-2533.
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.
Piatti, S., Bohm, T., Cocker, J., Diffley, J. and Nasmyth, K. (1996). Activation of S-phase-promoting CDKs in late G1 defines a "point of no return" after which Cdc6 synthesis cannot promote DNA replication in yeast. Genes Dev. 10,1516 -1531.[Abstract]
Quinn, L. M., Herr, A., McGarry, T. J. and Richardson, H.
(2001). The Drosophila Geminin homolog: roles for Geminin in
limiting DNA replication, in anaphase and in neurogenesis. Genes
Dev. 15,2741
-2754.
Rialland, M., Sola, F. and Santocanale, C.
(2002). Essential role of human CDT1 in DNA replication and
chromatin licensing. J. Cell Sci.
115,1435
-1440.
Richardson, H., O'Keefe, L., Reed, S. and Saint, R.
(1995). Ectopic cyclin E expression induces premature entry into
S phase and disrupts pattern formation in the Drosophila eye imaginal disc.
Development 121,3371
-3379.
Saha, P., Chen, J., Thome, K. C., Lawlis, S. J., Hou, Z. H.,
Hendricks, M., Parvin, J. D. and Dutta, A. (1998).
Human CDC6/Cdc18 associates with Orc1 and cyclin-cdk and is selectively
eliminated from the nucleus at the onset of S phase. Mol. Cell
Biol. 18,2758
-2767.
Schwed, G., May, N., Pechersky, Y. and Calvi, B. R.
(2002). Drosophila minichromosome maintenance 6 is required for
chorion gene amplification and genomic replication. Mol. Biol.
Cell 13,607
-620.
Sclafani, R. A. (2000). Cdc7p-Dbf4p becomes
famous in the cell cycle. J. Cell Sci.
113,2111
-2117.
Spradling, A. C. and Rubin, G. M. (1982). Transposition of cloned P elements into Drosophila germ line chromosomes. Science 218,341 -347.[Medline]
Sugimoto, N., Tatsumi, Y., Tsurumi, T., Matsukage, A., Kiyono,
T., Nishitani, H. and Fujita, M. (2004). Cdt1
phosphorylation by cyclin A-dependent kinases negatively regulates its
function without affecting geminin binding. J. Biol.
Chem. 279,19691
-19697.
Tada, S., Li, A., Maiorano, D., Mechali, M. and Blow, J. J. (2001). Repression of origin assembly in metaphase depends on inhibition of RLF-B/Cdt1 by geminin. Nat. Cell Biol. 3, 107-113.[CrossRef][Medline]
Takeda, D. Y., Wohlschlegel, J. A. and Dutta, A.
(2001). A bipartite substrate recognition motif for
cyclin-dependent kinases. J. Biol. Chem.
276,1993
-1997.
Tanaka, S. and Diffley, J. F. (2002). Interdependent nuclear accumulation of budding yeast Cdt1 and Mcm2-7 during G1 phase. Nat. Cell Biol. 4, 198-207.[CrossRef][Medline]
Thomas, B. J., Gunning, D. A., Cho, J. and Zipursky, L. (1994). Cell cycle progression in the developing Drosophila eye: roughex encodes a novel protein required for the establishment of G1. Cell 77,1003 -1014.[Medline]
Thummel, C. S., Boulet, A. M. and Lipshitz, H. D. (1988). Vectors for Drosophila P-element-mediated transformation and tissue culture transfection. Gene 74,445 -456.[CrossRef][Medline]
Underwood, E., Briot, A., Doll, K., Ludwiczak, R., Otteson, D.,
Tower, J., Vessey, K. and Yu, K. (1990). Genetics of
51D-52A, a region containing several maternal-effect genes and two
maternal-specific transcripts in Drosophila. Genetics
126,639
-650.
Vas, A., Mok, W. and Leatherwood, J. (2001).
Control of DNA rereplication via Cdc2 phosphorylation sites in the origin
recognition complex. Mol. Cell Biol.
21,5767
-5777.
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.[Medline]
Whittaker, A. J., Royzman, I. and Orr-Weaver, T. L.
(2000). Drosophila double parked: a conserved, essential
replication protein that colocalizes with the origin recognition complex and
links DNA replication with mitosis and the down-regulation of S phase
transcripts. Genes Dev.
14,1765
-1776.
Wohlschlegel, J. A., Dwyer, B. T., Dhar, S. K., Cvetic, C.,
Walter, J. C. and Dutta, A. (2000). Inhibition of
eukaryotic DNA replication by geminin binding to Cdt1.
Science 290,2309
-2312.
Xu, T. and Rubin, G. M. (1993). Analysis of
genetic mosaics in developing and adult Drosophila tissues.
Development 117,1223
-1237.
Yamaguchi, R. and Newport, J. (2003). A role for Ran-GTP and Crm1 in blocking re-replication. Cell 113,115 -125.[Medline]
Yanagi, K., Mizuno, T., You, Z. and Hanaoka, F.
(2002). Mouse geminin inhibits not only Cdt1-MCM6 interactions
but also a novel intrinsic Cdt1 DNA binding activity. J. Biol.
Chem. 277,40871
-40880.
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.[CrossRef][Medline]
Zou, L. and Stillman, B. (1998). Formation of a
preinitiation complex by S-phase cyclin CDK-dependent loading of Cdc45p onto
chromatin. Science 280,593
-596.
Related articles in Development: