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9. Fernandez Sarabia, M. J., McInerny, C., Harris, P., Gordon, C. & Fantes, P. The cell cycle genes cdc22+
and suc22+ of the ®ssion yeast Schizosaccharomyces pombe encode the large and small subunits of
ribonucleotide reductase. Mol. Gen. Genet. 238, 241±251 (1993).
10. Kelly, T. J. et al. The ®ssion yeast cdc18+ gene product couples S phase to START and mitosis. Cell 74,
371±382 (1993).
11. Carpenter, P. B., Mueller, P. R. & Dunphy, W. Role for a Xenopus Orc2-related protein in controlling
DNA replication. Nature 379, 357±360 (1995).
12. Mechali, M. & Harland, R. M. DNA synthesis in a cell-free system from Xenopus eggs: priming and
elongation on single-stranded DNA in vitro. Cell 30, 93±101 (1982).
13. Almouzni, G. & Mechali, M. Assembly of spaced chromatin promoted by DNA synthesis in extracts
from Xenopus eggs. EMBO J. 7, 665±672 (1988).
14. Blow, J. J. Preventing re-replication of DNA in a single cell cycle: evidence for a replication licensing
factor. J. Cell Biol. 122, 993±1002 (1993).
15. Coue, M., Amariglio, F., Maiorano, D., Bocquet, S. & MeÂchali, M. Evidence for different MCM
subcomplexes with differential binding to chromatin in Xenopus. Exp. Cell Res. 245, 282±289 (1998).
16. CoueÂ, M., Kearsey, S. E. & MeÂchali, M. Chromatin binding, nuclear localization and phosphorylation
of Xenopus cdc21 are cell-cycle dependent and associated with the control of initiation of DNA
replication. EMBO J. 15, 1085±1097 (1996).
17. Rowles, A. et al. Interaction between the origin recognition complex and the replication licensing
system in Xenopus. Cell 87, 287±296 (1996).
18. Romanowski, P., Madine, M. A., Rowles, A., Blow, J. J. & Laskey, R. A. The Xenopus origin recognition
complex is essential for DNA replication and MCM binding to chromatin. Curr. Biol. 6, 1416±1425
(1996).
19. Chong, J. P., Mahbubani, H. M., Khoo, C. -Y. & Blow, J. J. Puri®cation of an MCM-containing
complex as a component of the DNA replication licensing system. Nature 375, 418±421 (1995).
20. Rowles, A., Tada, S. & Blow, J. J. Changes in association of the Xenopus origin recognition complex
with chromatin on licensing of replication origins. J. Cell Sci. 112, 2011±8 (1999).
21. Perkins, G. & Dif¯ey, J. F. Nucleotide-dependent prereplicative complex assembly by Cdc6p, a
homolog of eukaryotic and prokaryotic clamp-loaders. Mol. Cell 2, 23±32 (1998).
22. Vuillard, L., Rabilloud, T. & Goldberg, M. E. Interactions of non-detergent sulfobetaines with early
folding intermediates facilitate in vitro protein renaturation. Eur. J. Biochem. 256, 128±135 (1998).
23. Menut, S., LemaõÃtre, J. M., Hair, A. & MeÂchali, M. in Advances in Molecular Biology: A Comparative
Methods Approach to the Study of Ooocytes and Embryos (ed. Richter, J. D.) 198±226 (Oxford Univ.
Press, 1999).
24. Murray, A. W. Cell cycle extracts. Methods Cell Biol. 36, 581±605 (1991).
25. Maiorano, D., LemaõÃtre, J-M. & MeÂchali, M. Step-wise, regulated chromatin assembly of MCM2-7
proteins. J. Biol. Chem. (in the press).
Acknowledgements
We thank S. Bocquet for technical assistance in the raising of antibodies; S. Cotterill for the
cdc6 antibody; J. Blow and A. Rowles for the ORC1 antibody; and C. Jaulin, D. Fisher and
J.-M. LemaõÃtre for helpful technical tips. This work has been supported by grants from
ARC and Ligue Nationale contre le Cancer. D. M. was supported by an EC fellowship
(Biomedicine and Health) and a grant from Fondation pour la Recherche MeÂdicale.
Correspondence and requests for materials should be addressed to M. M.
(e-mail: [email protected]).
.................................................................
The Cdt1 protein is required to license
DNA for replication in ®ssion yeast
Hideo Nishitani*²³, Zoi Lygerou*³§, Takeharu Nishimoto² & Paul Nurse*
* Imperial Cancer Research Fund, 44, Lincoln's Inn Fields, London WC2A 3PX,
UK
² Graduate School of Medical Science, Kyushu University, Fukuoka 812-8582,
Japan
³ These authors contributed equally to this work.
..............................................................................................................................................
To maintain genome stability in eukaryotic cells, DNA is licensed
for replication only after the cell has completed mitosis, ensuring
that DNA synthesis (S phase) occurs once every cell cycle1. This
licensing control is thought to require the protein Cdc6 (Cdc18 in
®ssion yeast) as a mediator for association of minichromosome
maintenance (MCM) proteins with chromatin2±10. The control is
overridden in ®ssion yeast by overexpressing Cdc18 (ref. 11)
which leads to continued DNA synthesis in the absence of
mitosis12. Other factors acting in this control have been
§ Present address: Laboratory of General Biology, School of Medicine, University of Patras, 26110, Rio,
Patras, Greece.
NATURE | VOL 404 | 6 APRIL 2000 | www.nature.com
postulated13 and we have used a re-replication assay to identify
Cdt1 (ref. 14) as one such factor. Cdt1 cooperates with Cdc18 to
promote DNA replication, interacts with Cdc18, is located in the
nucleus, and its concentration peaks as cells ®nish mitosis and
proceed to S phase. Both Cdc18 and Cdt1 are required to load the
MCM protein Cdc21 onto chromatin at the end of mitosis and this
is necessary to initiate DNA replication. Genes related to Cdt1
have been found in Metazoa and plants (A. Whitaker, I. Roysman
and T. Orr-Weaver, personal communication), suggesting that the
cooperation of Cdc6/Cdc18 with Cdt1 to load MCM proteins onto
chromatin may be a generally conserved feature of DNA licensing
in eukaryotes.
Cdc6/18 has a central role in regulating S-phase onset in a
number of eukaryotes15. We employed a re-replication assay in
®ssion yeast to identify new factors that cooperate with Cdc18 to
induce DNA synthesis. Increased expression of Cdc18 induces
®ssion yeast cells to undergo continued DNA synthesis in the
absence of mitosis, leading to re-replication12. When Cdc18 was
expressed in cells at increasing levels (low, L; medium, M; and high,
H), a corresponding increase in the extent of re-replication was
observed (Fig. 1a, b). There was hardly any re-replication in strain L,
whereas an average DNA content of 6C and 16C was observed in
strains M and H, respectively. We reasoned that co-expression of a
factor that cooperates with Cdc18 might induce the low-expressing
strain L to re-replicate more effectively. The ®ssion yeast Cdt1
protein is a good candidate for a Cdc18 partner as it is under the
same transcriptional regulation as Cdc18 (ref. 14) and, like Cdc18
(ref. 11), is required both for S-phase onset and for the DNA
replication checkpoint14. Overexpression of Cdt1 did not promote
re-replication on its own (data not shown). However, co-expression
of Cdt1 in strain L generated cells with an average DNA content of
16C, similar to the level of re-replication observed in strain H and
signi®cantly higher than the level seen in the medium-expressing
strain M (Fig. 1c, d). We saw no enhancement of DNA synthesis
when Cdc30 (SpOrc1; refs 16, 17)), Hsk1 (ref. 18; a CDC7 homologue) or Cdc22 (ref. 19; the large subunit of ribonucleotide
reductase) were co-expressed with Cdc18 (data not shown). We
conclude that Cdt1 expression enhances the effect of Cdc18 to
promote continuing DNA synthesis, indicating that Cdc18 and
Cdt1 cooperate to induce DNA replication.
To determine whether Cdt1 and Cdc18 can physically interact,
Cdt1 was tagged with glutathione S-transferase (GST±Cdt1) and
Cdc18 with the myc epitope (myc±Cdc18), and both fusion
proteins were co-expressed in ®ssion yeast. As shown in Fig. 2a,
precipitation of GST±Cdt1 (and not the control GST protein)
speci®cally co-precipitated myc±Cdc18 (compare lanes 3 and 4,
lower panel). The reciprocal experiment is shown in Fig. 2b;
immunoprecipitation of myc±Cdc18 speci®cally co-precipitates
GST±Cdt1. The association between Cdt1 and Cdc18 was con®rmed using cells expressing untagged Cdt1 and Cdc18 (Fig. 2c);
immunoprecipitation with anti-Cdt1 antibodies speci®cally brings
down Cdc18 (lane 4). To map the domain of Cdc18 required for
Cdt1 interaction, the amino-terminal and carboxy-terminal regions
of Cdc18 were tagged with the haemagglutinin (HA) epitope and
independently expressed together with either GST±Cdt1 or GST
alone as a control. As shown in Fig. 2d, the C terminus but not the N
terminus of Cdc18 is speci®cally co-precipitated with GST±Cdt1
(lane 6). We therefore conclude that Cdc18 can form in vivo
complexes with Cdt1, primarily through its C terminus. We have
previously shown that it is the C terminus of Cdc18 which induces
re-replication and activates the DNA replication checkpoint,
whereas the N terminus associates with the Cdc2 protein kinase20.
The re-replication induced by the C terminus of Cdc18 is also
enhanced by co-expression of Cdt1, to a level similar to that seen
with the full-length protein (Fig. 2e).
Cdc18 is located in the nucleus, and its protein levels vary
periodically through the cell cycle12,21. To determine whether Cdt1
© 2000 Macmillan Magazines Ltd
625
letters to nature
behaves similarly, we investigated the levels and subcellular localization of Cdt1 through the cell cycle. We found that Cdt1 protein
levels ¯uctuated as cells progressed through a synchronous cell
cycle, peaking at the end of mitosis, in parallel with Cdc18 protein
levels (Fig. 2f). Both Cdc18 and Cdt1 localized to the nuclei of
binucleate cells that have just completed mitosis and are in G1 or
early S phase (Fig. 2g). Either no, or very weak, nuclear staining was
observed in septated cells which were further on in S phase, or in G2
cells. These observations were con®rmed using synchronous cultures (data not shown). Cdt1 and Cdc18 are therefore present in the
cell nucleus after mitosis when DNA licensing takes place.
DNA licensing is believed to require chromatin association of the
MCM proteins2±5, and in budding yeast and Xenopus this association depends on the Cdc6 protein6±10, the homologue of Cdc18 in
these organisms. To con®rm MCM protein association with chromatin in ®ssion yeast, and establish the requirement for Cdc18 and
a
Total
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Figure 1 Cdt1 enhances the ability of Cdc18 to induce continuing DNA synthesis. a, b,
Three strains were constructed which express Cdc18, under the inducible nmt promoter,
at different levels (L, low; M, medium; H, high). The promoter was derepressed and cells
collected at the indicated time points (in h). The DNA content was analysed by
¯uorescence-activated cell sorting (FACS) analysis (a) and the level of Cdc18 produced
was monitored by western blotting (b). Cdc2 protein levels are shown as loading control.
c, d, Strain L was transformed with plasmid pREP4±Cdt1, in which Cdt1 is under the nmt
promoter, and expression of both Cdc18 and Cdt1 was induced. As controls, strains M
and H transformed with vector alone were used. DNA content was measured by FACS (c)
and levels of both Cdc18 and Cdt1 were assessed by western blotting (d). Cdt1
overexpression led to a small increase in Cdc18 protein levels, perhaps due to stabilization
of the Cdc18 protein in rereplicating cells. The band marked with an asterisk in d is a nonspeci®c band that serves as loading control.
626
Figure 2 Cdt1 can form a complex with Cdc18, and accumulates in the cell nucleus in G1.
a, Cell extracts were prepared from cells co-expressing myc±Cdc18 (in pRMH41) and GST
(lanes 1 and 3) or GST±Cdt1 (lanes 2 and 4) (both in pREP4) and the GST proteins
precipitated with glutathione beads. Cell extracts (lanes 1 and 2) and precipitates (lanes 3
and 4) were subjected to western blotting with anti-Cdt1 (upper panel) or anti-Cdc18
antibodies (lower panel). 5 mg cell extract and the precipitate from 25 mg extract, and 1 mg
extract and the precipitate from 125 mg extract, were loaded for the Cdt1 and Cdc18 blots,
respectively. b, Myc±Cdc18 in the cell extract used in a lane 2, was precipitated with antimyc (lane 1) or anti-HA antibodies (lane 2), and the precipitates blotted with anti-Cdt1 (upper
panel) or anti-Cdc18 antibodies (lower panel). Precipitates from 125 mg and 25 mg of
extract were loaded for the Cdt1 blot and the Cdc18 blot (lower panel), respectively.
c, Untagged Cdc18 and Cdt1 were co-expressed using pREP41±Cdc18 and pREP4±Cdt1.
Immunoprecipitation was performed using anti-Cdc18 (lane 2) or anti-Cdt1 antibodies (lane
4) or pre-immune serum for Cdc18 (lane 3) or Cdt1 (lane 5). 25 mg of total cell extract (lane
1) and precipitates from 125 mg extract (lanes 2±5) were western blotted using anti-Cdc18
antibodies. d, N terminus of Cdc18 (residues 1±141 in pRHA41) tagged with HA was coexpressed with GST±Cdt1 (lanes 1 and 5) or GST alone (lanes 3 and 7), both in pREP4X.
Similarly, the C terminus of Cdc18 (residues 150 to end in pRHA41) tagged with HA was coexpressed with GST±Cdt1 (lanes 2, 6 and 9) or GST alone (lanes 4 and 8). 2.5 mg cell
extract (lanes 1±4) and precipitates from 250 mg extract using glutathione beads (lanes 5±
8) or BSA beads as control (lane 9) were western blotted with anti-HA antibodies. e, Cdc18C (in pREP81) was expressed alone (upper panel) or with Cdt1(in pREP42) (lower panel) and
the DNA content measured after 20 h expression. f, cdc25-22 cells were synchronized at
the G2±M transition by incubating for 4 h at the non-permissive temperature and released
into a synchronous cell cycle. Samples were taken every 20 min and the levels of Cdc18
(top), Cdt1 (middle) and a-tubulin (bottom, loading control) were assessed by western
blotting. Per cent septation is indicated at the bottom. Initiation of DNA replication
immediately precedes the peak of septation. g, Cells expressing Cdc18±myc (upper panel)
or Cdt1±myc (lower panel), both under their endogenous promoters, were ®xed with
methanol and stained with anti-myc antibody (left), propidium iodide (middle) or visualized by
Nomarski optics (right). Only binucleate cells show strong nuclear staining for either protein.
© 2000 Macmillan Magazines Ltd
NATURE | VOL 404 | 6 APRIL 2000 | www.nature.com
letters to nature
Cdt1 for this association, we raised antibodies against Cdc21 (ref.
22; an MCM4 homologue) and developed a fractionation protocol
(Fig. 3a) which enriches for chromatin-associated components7,8,23.
In the total cell extracts and detergent-soluble protein fractions,
Cdc21 protein levels remained constant through the cell cycle.
However, the appearance of Cdc21 in the chromatin-enriched
preparation was periodic through the cell cycle. When synchronized
cells completed mitosis, Cdc21 became associated with chromatin
and this association persisted through G1 into S phase (Fig. 3b, left
bottom panel, Fig. 3c). It decreased to a low level during G2 and
reappeared after the second mitosis. SpOrc1 protein levels in total,
T
soluble and chromatin fractions were constant and served as loading
controls. A signi®cant fraction of the chromatin-associated Cdc21
migrated slower than the detergent-soluble protein (Fig. 3d, lanes 1
and 2). This was due to phosphorylation of the chromatin-associated Cdc21 (Fig. 3d, lanes 3 and 4). We used a strain in which
Cdc18 was under the control of the thiamine-repressible low
strength nmt promoter to test the effect of Cdc18 depletion on
Cdc21 chromatin association. Cells depleted of Cdc18 at the G2±M
transition completed mitosis normally but failed to initiate DNA
replication (data not shown). In the absence of Cdc18, there was no
increase in Cdc21 chromatin association (Fig. 3e) or phosphorylation (data not shown) as cells completed mitosis and entered G1.
This establishes that Cdc18 is required for the chromatin association and phosphorylation of Cdc21 at the G1±S transition. Cdt1
was also found to be present in the chromatin-enriched fraction
during the G1 and S phases of the cell cycle, but in contrast to Cdc21
there was no requirement for Cdc18 to bring about this association
(Fig. 3e).
We next investigated the effects of Cdt1 depletion on DNA
replication and MCM association with chromatin. To ®nd out
whether Cdt1 is primarily required for early or late S phase, cells
in which Cdt1 is under the control of the nmt promoter were
arrested with hydroxyurea at the beginning of S phase. Thiamine
E
a
a
Spheroplasts
Triton lysis
Total cell lysate (T)
Centrifugation through sucrose
Supernatant
Triton extractable (E)
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DNase I digestion
Centrifugation
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insoluble
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chromatin associated (C)
b
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SpOrc1
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+Thiamine
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1C 2C
1C 2C
Cdc21
c
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Total
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control
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Cdc18
Cdc18
Figure 3 Cdc21 associates with chromatin as cells prepare for S phase in a Cdc18dependent manner. a, Flowchart of chromatin association assay. b, c, cdc25-22 HA-Orc1
cells were blocked at the G2±M transition and released into a synchronous cell cycle.
Samples collected every 15 min were fractionated as shown in a. The total cell lysate (T),
Triton extractable (E) and chromatin±associated fractions (C) were analysed by western
blotting using anti-Cdc21 (left) or anti-HA antibodies (right). A higher exposure is shown
for Cdc21 (C). We estimate that around 20% of the total Cdc21 is found in the chromatinenriched fraction in G1. Per cent septated cells are indicated at the bottom. FACS analysis
(c) shows when initiation of DNA replication occurs. d, The Triton extractable (lane 1) and
chromatin-associated fraction (lanes 2±4) from the 60 min time point of b were western
blotted for Cdc21 without prior treatment (lane 2) or after incubation with calf intestinal
alkaline phosphatase in the absence (lane 3) or presence (lane 4) of phosphatase
inhibitors. e, cdc25-22 cells expressing Cdc18 under the control of the thiaminerepressible nmt promoter as their only functional copy were blocked at G2±M for 4 h at
36 8C in the absence (-T) or presence (+T) of thiamine and then released to 25 8C (always
- or +T). Samples were taken every 25 min and fractionated as in a. Total cell lysate and
chromatin-associated fractions were western blotted using anti-Cdc21, anti-Cdt1, and
anti-Cdc18 antibodies as shown. A higher exposure is shown for the chromatinassociated fraction of Cdc21. As loading controls, an anti-a-tubulin western blot is shown
for the total cell lysate, and a Coomassie-stained gel of the chromatin-associated
fractions.
NATURE | VOL 404 | 6 APRIL 2000 | www.nature.com
+T
0 25 50 75 100 125 150
Chromatin associated
+T
-T
0 25 50 75 100 125 150
0 25 50 75 100 125 150
Loading
control
Cdt1
Figure 4 Cdt1 depletion blocks the initiation of DNA replication by inhibiting the
association of Cdc21 with chromatin. a, b, Cells expressing Cdt1 under the control of the
thiamine repressible nmt promoter as their only functional copy (nmt-cdt1) were arrested
at early S phase with hydroxyurea (HU), in the absence or presence of thiamine (-T, +T).
After 4 h, HU was washed off and cells were released, always -T or +T. A western blot
with anti-Cdt1 antibodies is shown in a. Coomassie staining serves as loading control.
Cdt1 levels decrease after 4 h in HU even in the absence of thiamine, indicating that Cdt1
is unstable in HU blocked cells. DNA content at the indicated times (h) is shown in b.
c, cdc25-22 nmt-cdt1 cells were arrested with HU for 6 h at 25 8C without or with
thiamine (-T or +T). After removal of the HU, cells were shifted to 36 8C for 3 h to arrest
them at the G2±M transition and then released into a synchronous cell cycle, always in
the presence or absence of thiamine. Samples were collected every 25 min and
processed as in Fig. 3a. The total cell lysate and the chromatin-associated fraction were
western blotted and visualized with anti-Cdc21, anti-Cdc18 and anti-Cdt1 antibodies as
indicated while an a-tubulin western blot and Coomassie staining serve as loading
controls for the total lysate and chromatin-extractable fractions, respectively.
© 2000 Macmillan Magazines Ltd
627
letters to nature
was added to half the culture at the same time as the hydroxyurea to
repress Cdt1 expression, and a western blot showed that Cdt1 levels
dropped by over 90% within two hours (Fig. 4a). After washing off
the hydroxyurea, both the cells expressing Cdt1 (Fig. 4b, left panels)
and the Cdt1 depleted cells (Fig. 4b, right panels) completed DNA
replication within one hour. Thus, Cdt1 depletion does not prevent
the later stages of DNA replication. However, cells lacking Cdt1 were
unable to undergo a second round of DNA replication and
accumulated in G1. We conclude that Cdt1 is required for the
initial stages of DNA replication. Cells lacking Cdt1 subsequently
proceeded inappropriately into mitosis, generating a `cut' phenotype (data not shown), verifying that Cdt1 is required for the
checkpoint control inhibits mitosis when S phase has not taken
place14. To determine whether Cdt1 depleted cells were defective in
the association of Cdc21 with chromatin, cells were released from a
hydroxyurea block, synchronized at the G2/M transition, and
samples taken as cells completed mitosis and progressed to S
phase. Like Cdc18 depleted cells, Cdt1 depleted cells were defective
in both the chromatin association and phosphorylation of Cdc21
(Fig. 4c). In contrast, Cdc18 could still associate with chromatin in
the absence of Cdt1, although possibly at a reduced level. We
conclude that Cdt1 is required to promote the association of
Cdc21 with chromatin in preparation for S phase.
We have identi®ed Cdt1 as being required to license DNA for
replication after exit from mitosis. It can associate with Cdc18 in
cells, it accumulates within the nucleus, associates with chromatin
and is expressed periodically during the cell cycle, peaking from
mitotic exit to the onset of S phase. Elevated Cdt1 expression
promotes the ability of Cdc18 to bring about continued DNA
synthesis, and the absence of either Cdt1 or Cdc18 blocks the
association of Cdc21 (MCM4) with chromatin and prevents the
initiation of DNA replication. We propose that Cdt1 acts cooperatively with Cdc18 during the G1 phase of the cell cycle, bringing
about the construction of pre-initiation complexes on replication
origins by loading MCM proteins onto chromatin associated with
the origin recognition complex (ORC). Genes related to Cdt1 have
been found in Drosophila, human, mouse, zebra®sh, Caenorhabditis
elegans and Arabidopsis, although not yet in budding yeast, and
mutant analysis of the Cdt1 related gene in Drosophila indicates that
it is required for DNA replication (A. Whitaker, I. Roysman and
T. Orr-Weaver, personal communication). This suggests that the
licensing function of Cdt1 for S-phase onset may be conserved in
multicellular eukaryotes. Its apparent absence from budding yeast
may be due to a greater divergence of the primary sequence of Cdt1
in this organism, or it may re¯ect differences in origin organization
or chromatin structure.
M
Methods
Strains and cell culture conditions
All strains were derived from the wild-type 972 h- or 975 h+and standard media and
growth conditions were used24,25. To construct the nmt cdc18 integrants for re-replication
experiments, plasmid pJK-nmt-cdc18 (ref. 12) was used to transform a leu1-32 ura4D-18
strain, and three integrants expressing Cdc18 to different levels were isolated. The Cdt1
deletion strain was constructed as described14. The strains used for switch-off experiments
were: cdc25-22 cdc18::ura4+ ura4D-18 leu1-32 [pREP81±cdc18] and cdc25-22 cdt1:: ura4+
ura4D-18 leu1-32 [pREP81±cdt1]. Myc-tagged Cdc18 or Cdt1 were constructed by
inserting the myc epitope at the C terminus of the corresponding endogenous gene.
Plasmids
pREP41±myc±Cdc18 was constructed by inserting the Sal1±BamH1 cdc18+cDNA to
plasmid pRMH41. Plasmids expressing either the C terminus (150 to end) or N terminus
(1±141) of Cdc18 tagged with HA were constructed by inserting the polymerase chain
reaction (PCR) products into pRHA41. The cdt1+ gene was ampli®ed by PCR using
genomic DNA as a template, cloned and sequenced. We found one base difference to the
published sequence (from T to C at position 53). This difference was con®rmed by
sequencing the cdt1+ gene isolated from a genomic library.
Antibodies and immunodetection
The anti-Cdc18 antibody has been described12. For cdt1 antibody production, His-tagged
Cdt1 (residue 201±end in vector pQE) puri®ed from Escherichia coli was used for rabbit
628
immunizations. Anti-Cdc21 antibodies were produced against the C terminus of Cdc21 as
described22.
Total cell extract used for western blotting was prepared by glass beads24. For pull-down
experiments, cells were spheroplasted, lysed in HB buffer17 and spun down at 100,000g for
30 min. The supernatant was mixed with antibodies or glutathione beads. Immuno¯uorescence analysis was done with methanol ®xed cells processed as described24.
Chromatin association assay
Spheroplast preparation and lysis was performed as described23. The total cell extract was
overlaid on a 30% sucrose cushion, and spun at 16,000g for 15 min. The material
remaining soluble was removed (Triton extractable fraction) while the chromatinassociated proteins were released from the washed and repelleted chromatin by incubation
with 277 U DNase I (Sigma) for 10 min at 25 8C in a buffer containing 20 mM Hepes
pH 7.9, 1.5 mM magnesium acetate, 50 mM potassium acetate, 10% glycerol, 0.5 mM
DTT, 150 mM NaCl, protease and phosphatase inhibitors, and were then analysed by
SDS±polyacrylamide gel electrophoresis.
Received 8 November 1999; accepted 3 February 2000.
1. Blow, J. J. & Laskey, R. A. A role for the nuclear envelope in controlling DNA replication within the cell
cycle. Nature 332, 546±548 (1988).
2. Kubota, Y., Mimura, S., Nishimoto, S., Takisawa, H. & Nojima, H. Identi®cation of the yeast MCM3related protein as a component of Xenopus DNA replication licensing factor. Cell 81, 601±609 (1995).
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Acknowledgements
We thank J. Bahler, J. Dif¯ey, D. Grif®ths, J. Hayles and H. Murakami for critical reading of
the manuscript, T. Orr-Weaver for communicating results before publication, Y. Adachi,
T. Carr, S. Kearsey, K. Nasmyth and T. Tanaka for clones and antibodies, and N. Peat for
help with ®gure preparation. This work was supported by the ICRF, an EEC Marie Curie
fellowship (Z.L.), and HFSPO and Uehara Foundation fellowships (H.N.).
Correspondence and requests for materials should be addressed to Z.L.
(e-mail: [email protected]).
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