Molecular Cell, Vol. 12, 1067–1075, November, 2003, Copyright 2003 by Cell Press
Enigmatic Variations:
Divergent Modes of Regulating
Eukaryotic DNA Replication
Stephen E. Kearsey1,* and Sue Cotterill2,*
1
Department of Zoology
South Parks Road
Oxford OX1 3PS
United Kingdom
2
St. George’s Hospital Medical School
Cranmer Terrace
London SW16 ORE
United Kingdom
Proteins involved in DNA replication are conserved
from yeast to mammals, suggesting that the mechanism was established at an early stage of eukaryotic
evolution. In spite of this common origin, recent findings have revealed surprising variations in how replication initiation is controlled, implying that a conserved mechanism has not necessarily resulted in
regulatory conservation.
Introduction
A defining feature of eukaryotic organisms is a DNA
replication mechanism able to cope with the duplication
of large genomes. Initiation of replication from many
chromosomal origins means that the duration of S phase
is not limited by the total amount of DNA but only by
the interorigin distance, enabling the evolution of plants
and animals with genomes larger than 100 Gb. The
trade-off for this flexibility is the need for a high fidelity
control mechanism that permits origins to fire not more
than once per S phase, thus ensuring a single round of
DNA replication. Failure to execute this control leads to
activation of checkpoint responses and is potentially a
source of genome instability (Vaziri et al., 2003), so it is
not surprising that onset of replication involves a series
of steps, each of which has the potential for regulation.
Initiation involves the origin recognition complex
(ORC) which binds to DNA and provides a site on the
chromosome where additional replication factors can
bind (Figure 1 and Table 1; reviewed in Bell and Dutta,
2002). Association of Cdc6 and Cdt1 with ORC allows
the complex to load the presumptive replicative helicase
(Mcm proteins) onto chromatin. This process, called licensing or prereplicative complex (pre-RC) formation,
confers competence on the origin to fire a single time
in S phase. Elucidation of the precise physical arrangement of pre-RC components must await a detailed structural study, and encouraging progress is being made in
this area with archaeal orthologs of pre-RC factors (e.g.,
Liu et al., 2000). Onset of DNA synthesis requires the
action of the CDK and Cdc7 kinases, which trigger the
binding of additional factors such as Cdc45 and GINS
at pre-RCs. This leads eventually to the assembly of the
replisome which moves away from origins in the course
of bulk DNA synthesis. During S phase a number of
mechanisms contrive to block further pre-RC formation.
*Correspondence: [email protected] (S.E.K.) scotteri@
sghms.ac.uk (S.C.)
Review
This ensures that initiation on already replicated DNA
does not occur.
Although in general the function of individual replication proteins and the overall catalytic mechanism of
DNA replication appear to be conserved, we review here
recent findings suggesting considerable evolutionary
variation in the control of this process. Differences are
particularly apparent in the ways that the activities and
spatial distribution of components involved in pre-RC
formation are controlled (Figure 2). It is worth remembering, however, that by necessity the experimental methods used in different organisms vary widely. It is therefore possible that ultimately some supposed species
differences may turn out to be due to alternative methodologies, and this may be particularly relevant when comparing data from in vivo and in vitro experiments.
Origin Recognition Complex
ORC is a heterohexameric complex in all eukaryotic
species so far studied, but differences can be seen in
terms of its sites and modes of chromatin binding, and
the properties of individual subunits.
Site Recognition
In S. cerevisiae, interaction of ORC (ScORC) with origins
involves specific short DNA sequences, called the ACS
and B1 elements (Rao and Stillman, 1995). DNA replication starts immediately adjacent to the ORC binding site
(Bielinsky and Gerbi, 1998), and the majority of ORC
binding sites function as origins in vivo (Raghuraman et
al., 2001; Wyrick et al., 2001). Sequence-specific binding
of ScORC to DNA is ATP dependent and involves five
of the six subunits (ORC1-5) (Lee and Bell, 1997).
The situation in fission yeast is quite different. Here,
only one subunit, Orc4, is involved in the DNA binding
of the complete complex. This DNA interaction is mediated by an N-terminal domain containing AT-hook motifs, not found in other Orc4 homologs, and is ATP independent (Chuang et al., 2002; Kong and DePamphilis,
2001; Lee et al., 2001). SpORC also shows lower sequence specificity in its DNA binding than ScORC. The
complex binds to asymmetric AT-rich sequences and
associates in vitro and in vivo with multiple sites in S.
pombe origins of replication (Chuang et al., 2002; Kong
and DePamphilis, 2002; Takahashi et al., 2003). Consistent with this, S. pombe replication origins are larger
than those in S. cerevisiae and lack unique sequences
essential for initiation.
In Metazoa, ORC may also show low sequence specificity in its DNA binding. Human ORC has similar affinities for DNA fragments known to contain origins as it
does for random sequences and allows initiation on DNA
molecules in vitro with apparent disregard for their sequence (Vashee et al., 2003). This explains in vivo observations where initiation lacks sequence specificity, such
as in Xenopus embryogenesis. Strikingly, recent observations suggest that Xenopus and S. pombe ORCs compete for the same sequences, implying that they have
a similar preference for AT-rich DNA (Kong et al., 2003).
In other situations, ORC does not associate randomly
Molecular Cell
1068
Figure 1. Mechanism of DNA Replication Initiation
In the initial step of pre-RC formation or licensing, Mcm2–7 proteins bind to chromatin
in a reaction dependent on ORC, Cdt1, and
Cdc6. This step can only occur if CDK is inactive and so is restricted to late M/G1. Activation of DNA replication by CDK and Cdc7 kinases causes the binding of additional
factors as shown (for details see text). For
clarity only those proteins also present at initiation are shown at the replication fork, but
additional factors such as PCNA, DNA polymerase ␦, and RF-C are also involved in elongation. The figure does not attempt to represent accurately the specific protein-protein
interactions made during initiation on chromatin.
with chromosomal sequences, mapping instead to discrete sites that serve as origins, for instance in human
somatic cells (Ladenburger et al., 2002) and in Drosophila follicle cells at sites associated with gene amplification (Austin et al., 1999).
How can we reconcile origins in discrete locations
with nonspecific binding of ORC to DNA? Interaction of
ORC with proteins that bind to specific DNA sequences
may be one possible explanation, as suggested by the
recruitment of HsORC to the EBV replication origin by
binding to the EBNA1 protein (Dhar et al., 2001). Perhaps
the AT-hook domain of SpOrc4 represents an extreme
situation where fusion between an ORC subunit and
another chromatin binding protein has occurred? In any
case, this comparison emphasizes that a significant difference between species is the specificity of ORC-DNA
interaction, and a variety of mechanisms for origin selection probably occur (reviewed in Gilbert, 2001).
Stability of Chromatin Association and Interactions
between Individual ORC Subunits
A clear difference between Metazoa and yeast is emerging concerning the properties of individual ORC subunits. In yeast, Orc6 is stably associated with the rest
of the ORC complex, but this is different in higher eukaryotes. In Drosophila, although Orc6 is required for
DNA replication, a pool of free protein exists that is not
chromatin bound and associates with cell membranes
(Chesnokov et al., 2001, 2003). This protein contains
a C-terminal domain that appears to be essential for
cytokinesis but not DNA replication. Related observations have been made in human cells, where Orc6 localizes to the kinetochores and other structures during
mitosis and cytokinesis (Prasanth et al., 2002). Inactivation of hsOrc6 also causes defects in cell division. Metazoan Orc6 may thus have a separate function in cytokinesis unlinked to its S phase role, but an intriguing
alternative is that this provides a mechanism for coupling DNA replication and cytokinesis pathways.
Another difference relates to the stability of chromatin
association of the ORC complex. In yeasts, the intact
ORC complex is associated with chromatin for the entire
cell cycle. In contrast, interaction of mammalian Orc1
with the ORC core changes, with the protein released
from chromatin during S phase (Kreitz et al., 2001; Li
and DePamphilis, 2002; reviewed in DePamphilis, 2003).
Moreover, in Xenopus the ORC complex shows a weakened interaction with chromatin after licensing (Rowles
et al., 1999). The transient interaction of mammalian
Orc1 may account for the disappearance of an ORClike footprint from the lamin B2 origin during mitosis and
for the fact that mitotic/early G1 phase chromatin does
not contain functional ORC (Natale et al., 2000; Yu et
al., 1998). Different fates for Orc1 have been reported,
which may reflect differences in the cell lines used. In
hamster cells, released Orc1 is monoubiquitylated and
then rebinds to chromatin during G1 after deubiquitylation, with Orc1 levels remaining constant during the cell
cycle (Li and DePamphilis, 2002). In contrast, in HeLa
cells, Orc1 is ubiquitylated on chromatin and degraded
after initiation of DNA replication (Kreitz et al., 2001;
Mendez et al., 2002). The mechanism regulating Orc1
ubiquitylation or release from chromatin in these situations is unclear, but in Xenopus, ORC phosphorylation
appears to destabiize its interaction with chromatin
(Rowles et al., 1999).
In addition to its different chromatin binding properties, Orc1 (but not other ORC subunits) is an E2F-regulatory target during the G1-S transition, suggesting that
it may have a regulatory role in ORC function (Asano
and Wharton, 1999). Orc1 also appears to be limiting
for DNA replication in at least some cell types during
Drosophila development.
Taken together, these observations suggest that
mammalian cells can control initiation using a regulatory
step involving the association of Orc1 with other ORC
subunits on chromatin. This formation of a complete
ORC complex is essential for the subsequent Mcm loading step and would thus represent the earliest step in
a
Hsk1/Dfp1(Sp),
Cdc7/ASK (Hs)
Dna43 (Sc), Cdc23(Sp)
Rad4, Cut5 (Sp), Mus101?
(Xl), TopBP1? (Hs)
Drc1 (Sp)
Sna41,Goa1(Sp)
Cdc18(Sp)
RLF-B (Xl), Tah11/Sid2
(Sc), Dup (Dm)
Alternative
Names (Species)a
⫹
⫹
⫹
⫺
⫺
⫺
ssDNA binding
protein kinase
protein kinase
synthesis of RNA primer, and
DNA
DNA synthesis
⫺
⫹
⫹
⫹
⫹
⫺
⫺
⫺
⫹
⫹
⫹ (Sp, Xl)
⫹
⫺
⫺
⫹ (Sc)
⫺
ATPase, Mcm467 subcomplex
has weak 3⬘-5⬘DNA
helicase activity
⫺
⫹
⫺
⫹
⫹
⫹
⫺
⫹
⫺
Initiation of
DNA Synthesis
Pre-RC Formation (loading of
Mcm2–7 Proteins onto DNA)
Requirement in DNA Replication
⫹
binds ATP, presumably
ATPase
ATPase, DNA binding
Biochemical Properties
Sc, S. cerevisiae; Sp, S. pombe; Dm, D. melanogaster; Xl, X. laevis; Hs, human.
DNA polymerase ⑀
DNA polymerase ␣
Sld2
GINS complex (Sld5, Psf1, Psf2,
Psf3)
Replication protein A
(RPA)
CDK
Cdc7/Dbf4
Cdc45
Sld3
Mcm10
Dpb11
Noc3
Mcm proteins (Mcm2–7)
Geminin
Cdc6
Origin recognition complex,
ORC1-6
Cdt1
Protein
Table 1. Proteins Involved in Initiation of Eukaryotic DNA Replication
⫹
⫹
⫺
⫺
⫹
?
⫹
⫹
⫹
⫹
⫺
⫺
⫹
⫺
⫺
⫺
Elongation
Reaction
yes
yes
yes
yes
yes
?
yes
yes
?
yes
?
yes
yes
no
yes
yes
yes
Identified Homologs in
Yeasts and Mammals?
Review
1069
Molecular Cell
1070
initiation of DNA replication (Biermann et al., 2002; Mendez and Stillman, 2000). Cdc6 proteolysis has been reported to occur in M-G1 by ubiquitin-mediated proteolysis dependent on APC/CDH1 (Mendez and Stillman,
2000; Petersen et al., 2000), but there seems to be no
vertebrate conservation of the S phase proteolysis
prominent in yeast.
One possible explanation for the persistence of Cdc6
in S phase in vertebrate cells is that other mechanisms
ensure that there is a block to rereplication, and Cdc6
does not have an important regulatory role. Alternatively,
Cdc6 could have a second function, distinct from Mcm
loading, which requires it to be present during S phase.
One possibility is a role in the S phase checkpoint, as
suggested by analysis of Cdc6 overexpression in human
cells (Clay-Farrace et al., 2003).
Figure 2. Nonconservation of Blocks to Pre-RC Formation/Licensing in Eukaryotes
Different mechanisms used to block Mcm2–7 chromatin association
after replication initiation in yeasts and vertebrates are shown. PreRC components are regulated by nuclear export (black arrows),
proteolysis (red X), phosphorylation (ORC), and binding of inhibitors
(geminin to Cdt1 and Crm1 to Mcms). Vertebrate cells also show
destabilization of binding of the ORC complex or Orc1 to chromatin
(green arrows). For details see text.
the sequence leading to initiation. This control step appears to be absent in yeast, although phosphorylation
of ORC is a component of the block to rereplication in
S phase, utilizing a mechanism which does not appear to
involve destabilization of the ORC subunit interactions
(Nguyen et al., 2001).
Cdc6/Cdc18
The Cdc6-ORC complex shows sequence similarities to
the RF-C clamp loader of PCNA, and it may function in
a similar way to load the Mcm complex onto DNA in an
ATP-dependent reaction (Perkins and Diffley, 1998). The
main species differences with regard to the regulation
of Cdc6 relate to how its concentration and localization
changes during the cell cycle.
In yeasts, Cdc6/Cdc18 is only present for a short window of the cell cycle. The importance of this tight regulation is dramatically demonstrated by the phenotype of
Cdc18 overexpression in fission yeast, which leads to
rereplication of DNA (Nishitani and Nurse, 1995). The
levels of Cdc6/Cdc18 are controlled by a combination
of transcriptional regulation and proteolysis. Transcription is limited to a narrow M-G1 period of the cell cycle
(Baum et al., 1998; Piatti et al., 1995), and degradation
of Cdc6/Cdc18 is triggered by CDK phosphorylation
around S phase onset in an SCF-mediated pathway
(Drury et al., 2000; Jallepalli et al., 1998). In contrast,
mammalian Cdc6 levels are fairly stable during the cell
cycle (Jiang et al., 1999; Saha et al., 1998). Although
some Cdc6 is exported to the cytoplasm during S phase,
which correlates with cyclin A-CDK2 phosphorylation
(Petersen et al., 1999), Cdc6 remains on chromatin after
Cdt1 and Geminin
Cdt1 functions with Cdc6 in promoting the chromatin
loading of Mcm proteins, but its actual biochemical
function is not understood. Both proteins independently
associate with chromatin via ORC, but unlike Cdc6, Cdt1
shows no sequence similarity with ORC subunits. Compared to most of the replication factors discussed so
far, Cdt1 homologs are rather poorly conserved. Perhaps this is related to the fact that different species
show quite distinct modes of Cdt1 regulation.
Three modes of Cdt1 regulation have been described.
The first involves ensuring that the protein is only present
during a narrow window of the cell cycle before S phase.
This type of regulation has been reported for mammalian
cells and fission yeast but is not seen in budding yeast.
In mammalian cells, this cell cycle variation appears to
be mainly due to proteolysis since Cdt1 can be stabilized
by proteasome inhibitors (Nishitani et al., 2001). In fission yeast, the levels of Cdt1 are also subject to Cdc10mediated transcriptional control (Nishitani et al., 2000).
Overriding this control has no phenotype alone, but the
rereplication caused by Cdc18 overexpression is enhanced by Cdt1. A similar effect has been reported in
checkpoint-deficient human cells (Vaziri et al., 2003).
A distinct second mode of Cdt1 control (so far only
observed in S. cerevisiae) involves cell cycle changes
in the nuclear localization of the protein. Nuclear accumulation of Cdt1 only occurs during late mitosis and
G1, and the CDK activation step necessary for replication onset effects Cdt1 exclusion from the nucleus, thus
coordinating a block to rereplication with S phase onset
(Tanaka and Diffley, 2002).
A third mechanism controlling Cdt1, seen in Metazoa
but not in plants or yeasts, involves geminin. Geminin
interacts with Cdt1 and inhibits its Mcm loading activity
(Tada et al., 2001; Wohlschlegel et al., 2000). In mammalian cells, geminin levels are high in mitosis, decline on
entry to G1 and reaccumulate on S phase entry, and
show an inverse relationship to levels of Cdt1 (Nishitani
et al., 2001). The destruction of geminin is due to APCmediated ubiquitylation (McGarry and Kirschner, 1998).
Geminin regulation in Xenopus eggs appears to be different as endogenous geminin is not all degraded on
mitotic exit but becomes incapable of inhibiting Cdt1,
perhaps due to some posttranslational mechanism
(Hodgson et al., 2002). Depleting geminin from meta-
Review
1071
phase-arrested Xenopus extracts allows some activation of licensing (Tada et al., 2001), and depletion in
vivo causes early lethality, suggesting it is an important
factor for early cell cycles (McGarry, 2002). Depletion of
Drosophila geminin also causes lethality and a rereplication phenotype (Mihaylov et al., 2002; Quinn et al., 2001).
Despite the variety of different mechanisms regulating
Cdt1, the removal of Cdt1 activity during S phase is
a unifying feature of replication control. Perhaps this
inactivation is crucial for maintaining genome stability
in eukaryotic cells?
Mcm Proteins
The six Mcm proteins (Mcm2–7) probably constitute the
replicative helicase (reviewed in Labib and Diffley, 2001).
These proteins, which share high sequence similarity
in a central domain, are required for licensing and the
elongation stage of DNA replication (Labib et al., 2000;
Prokhorova and Blow, 2000).
In most organisms Mcm proteins remain in the nucleus throughout the cell cycle, associating with chromatin during licensing and being displaced on replication termination. In budding yeast, however, they are
regulated by a distinct mechanism involving nuclear localization. CDK promotes Mcm nuclear export, thus coordinating initiation with a block to rereplication as seen
with Cdt1 (Labib et al., 1999). Forcing Mcm proteins to
stay in the nucleus is necessary but not sufficient to
promote rereplication (Nguyen et al., 2001). The regulated nuclear localization of these proteins, in fact, requires Cdt1. Mcms and Cdt1 form a complex, and the
nuclear accumulation of these proteins in G1 is interdependent (Tanaka and Diffley, 2002).
A novel control mechanism has recently been reported for Xenopus Mcms, reminiscent of their regulation in S. cerevisiae. During S phase, a nuclear export
receptor (Crm1) and Ran-GTP interact with soluble Mcm
in a CDK2-dependent reaction and inhibit its loading
onto chromatin (Yamaguchi and Newport, 2003). Surprisingly, this does not involve nuclear export of the
complex. Since CDK activity is low in G1, chromatin
binding of Mcm proteins can occur at this stage, and
once bound, they are refractory to Crm1 inhibition. By
preventing the rebinding of free Mcm proteins to chromatin, this provides an additional way of preventing
rereplication during early Xenopus cell cycles. Further
work will be required to determine whether this mechanism has any relationship to nuclear localization
changes of Mcm proteins seen in S. cerevisiae.
Another factor affecting Mcm chromatin association
in S. cerevisiae is Mcm10 (which is distinct in sequence
from proteins in the Mcm2–7 family). Mcm10 function
is required for stabilizing the interaction of Mcm proteins
with chromatin, suggesting it is a component of the preRC (Homesley et al., 2000), but in other organisms it
appears to function later (see below).
An additional factor related to the Mcm2–7 family,
designated Mcm8, has recently been discovered in humans (Gozuacik et al., 2003). Unlike the other Mcm proteins, Mcm8 homologs have not been identified in all
eukaryotes (e.g., it is absent in yeasts), and it is unclear
whether it is required for DNA replication.
Activating DNA Synthesis
Replication activation involves unwinding the DNA at
licensed origins and recruiting the three polymerases
(␣, ␦, and ⑀) required for DNA synthesis. During this
process a number of additional proteins associate with
the origin, and some of these have only been characterized recently from work in yeast and Xenopus. This step
is not understood in great detail in either yeast or multicellular organisms, but it is apparent that, while some
of these proteins are highly conserved, others are more
divergent, and this could reflect differences in how regulation is carried out.
Assembly of Replication Factors onto Origins in Yeast
Apart from polymerases, the proteins which have been
seen to bind to origins on replication activation are
Cdc45, Sld3, GINS, and Dpb11. Cdc45 binds to origins
on CDK and Cdc7 activation, and this association is
dependent on pre-RC formation, thus linking initiation
with earlier regulatory controls affecting pre-RC components (Zou and Stillman, 1998, 2000). This event appears
to be linked to origin unwinding, as RP-A binding is
dependent on Cdc45 chromatin association. Sld3 forms
a complex with Cdc45 (Kamimura et al., 2001), while
GINS (Go, Ichi, Nii, San; five, one, two, three in Japanese)
is a tetramer composed of Sld5, Psf1, Psf2, and Psf3
(Takayama et al., 2003). The associations of Cdc45, Sld3,
GINS, and Dpb11 with chromatin appear to be mutually
dependent, implying that they are not occurring in a
stepwise fashion. Thus, for instance, inactivation of Sld3
prevents Cdc45 and Psf1 chromatin binding, and inactivation of Psf1 prevents Dpb11 and Cdc45 chromatin
association. Assembly of this protein complex is necessary for the association of polymerases with the origin.
Cdc45 function is needed for polymerase ␣ association
(Zou and Stillman, 2000), and Dpb11 is needed for both
polymerase ␣ and ⑀ binding (Masumoto et al., 2000).
While Dpb11 only functions during initiation, Cdc45 and
GINS are needed for elongation and both behave similarly to Mcms and DNA polymerase ⑀, moving away from
origins during S phase (Kanemaki et al., 2003; Takayama
et al., 2003; Tercero et al., 2000). The biochemical functions of Cdc45 and GINS are mysterious; one idea is
that they could have a role in tethering polymerases at
the fork; alternatively, they could be needed as auxiliary
factors for the Mcm helicase.
These events in yeast are broadly consistent with what
is known in Xenopus (Kubota et al., 2003). However,
homologs of some of the S. cerevisiae factors, found
also in S. pombe, have not been found in vertebrates
(Table 1), perhaps reflecting poor sequence conservation. Another difference relates to Mcm10. Unlike the
Mcm10 involvement at the pre-RC step found in S. cerevisiae, Xenopus Mcm10 binds to chromatin after preRC formation but before activation of CDK and Cdc7
(Wohlschlegel et al., 2002). Mcm10 is subsequently required for the Cdc45 chromatin binding step, and similar
observations have also been made in fission yeast
(Gregan et al., 2003).
Roles of CDK and Cdc7 in Replication Activation
CDK and Cdc7 activation is a critical step for initiating
DNA synthesis, and this requirement is conserved in
eukaryotes. Until recently, higher eukaryotes were
thought to be different from yeasts in using CDK2 for S
phase activation and CDK1 for mitosis, but this view
Molecular Cell
1072
has had to be revised with the generation of a CDK2
knockout mouse which is largely without cell cycle or
developmental abnormalities (Ortega et al., 2003). CDK2
may still function normally for S phase activation, but
clearly other CDKs can substitute for essential functions
if CDK2 is absent.
However, it is as yet unclear whether this conserved
requirement for CDK reflects use of the same mechanism of replication activation in different eukaryotes. In
S. cerevisiae, an important substrate of CDK may be
Sld2, a protein which is capable of forming a complex
with Dpb11 (Masumoto et al., 2002). Phosphorylation
of Sld2 enhances its interaction with Dpb11, and this
appears to be necessary for onset of DNA replication.
A higher eukaryotic homolog of Sld2 has not been found,
and while metazoan Cut5/Mus101/TopBP1 is distantly
related to Dpb11 (the proteins share some BRCT domains), it is unclear whether they are true homologs. In
Xenopus, Cut5/Mus101 binds to chromatin in advance
of Cdc45, rather than in a mutually dependent manner,
but is still is needed for Cdc45 and polymerase ␣ association steps (Hashimoto and Takisawa, 2003; Van Hatten
et al., 2002). The relative lack of conservation of the Sld2/
Dpb11 components, compared to many of the factors
involved in DNA synthesis during the elongation step of
replication, might reflect greater divergence of factors
involved in replication control.
As for the role of Cdc7 in replication activation, the
Mcm complex appears to be an important target in both
yeast and vertebrates (reviewed in Masai and Arai, 2002).
However, there may be species-specific differences in
the order in which Cdc7 and CDK carry out their functions. In yeast CDK must function before Cdc7 (Nougarede et al., 2000), implying that Cdc7 cannot access a
target until CDK has been activated. In contrast, in Xenopus Cdc7 can carry out its function before CDK (Jares
and Blow, 2000). Differences in the timing of chromatin
association of the Cdc7 kinase during the cell cycle have
also been reported. S. cerevisiae Cdc7 is constitutively
associated with chromatin during the cell cycle, but in
contrast, Xenopus Cdc7 only binds to chromatin after
Mcm proteins have been loaded (Jares and Blow, 2000).
This may reflect the involvement of different factors in
Cdc7 chromatin association.
A confusing aspect regarding CDK’s role in replication
is that, while CDK activity inhibits pre-RC formation in
yeasts, under some circumstances it can stimulate this
process. This has been shown for mammalian G0 cells
reentering the cell cycle (Coverley et al., 2002) and also in
certain types of cell cycle in Drosophila (Su and O’Farrell,
1998). One possible explanation for these results is that
CDK is required to inactivate an inhibitor of pre-RC formation such as geminin in some cell types.
An Evolutionary Paradox: Nonconservation
of Replication Control but Conservation
of the Replication Mechanism
In this section we discuss some speculative possibilities
that might account for the variations in DNA replication
control seen when comparing different species. The focus is on the regulation of pre-RC formation, since here
the differences are most apparent (summarized in Figure
2), but similar arguments might apply to later steps leading to DNA synthesis.
Higher Eukaryotes Need Higher Fidelity
During a single S phase in a Xenopus embryonic cell, the
number of initiation events is three orders of magnitude
greater than that in a yeast cell cycle. Even though redundancy in controls affecting pre-RC formation is
found in yeast (Nguyen et al., 2001), the increased risk of
inappropriate reinitiation, which presumably correlates
with the number of initiation events, may necessitate an
increased redundancy in control mechanisms in organisms with larger genomes. Being multicellular may provide an added incentive for initiation fidelity, as genome
instability that could result from faulty origin firing is
known to be a major factor in generating the genetic
changes needed for cancer development. Extra blocks
to rereplication such as the regulation of Cdt1 by geminin and the destabilization of Orc1 chromatin binding
after initiation may be important for maintaining the genome stability in the 1016 cell divisions that take place
in the course of a human lifetime.
Are Blocks to Pre-RC Formation Important When
Cells Exit the Cell Cycle?
Cells in multicellular organisms face different regulatory
problems compared to yeasts. The rate of yeast cell
division is primarily controlled by availability of nutrients,
whereas mammalian cells are not nutrient limited, and
whether they are proliferating or not is regulated by a
variety of complex controls involving cell-cell signaling
which ensure appropriate development and tissue maintenance. One way of controlling cell proliferation is to
block DNA replication, and many components involved
in pre-RC formation, such as Cdc6 and Mcm proteins,
are dramatically downregulated in cells that exit the cell
cycle and enter a nonproliferating state (reviewed in
Blow and Hodgson, 2002). This is also seen in G2arrested Xenopus oocytes which are replication incompetent due to the absence of Cdc6 (Lemaitre et al., 2002;
Whitmire et al., 2002). It is possible that extra controls
over pre-RC formation found in Metazoa have a special
role in effecting entry to or exit from the G0 nonproliferative state or ensuring that replication origins remain inactive in arrested cells.
Specific Cell Types Need Specific
Replication Controls?
Specialized cell types in multicellular organisms could
have a requirement for modification of a generic control
of licensing. For example, regulating the activities of
initiation factors by a combination of transcriptional control and proteolysis may be advantageous for small cells
but less suited to large embryonic cells. Here large maternal stockpiles of replication factors may favor control
mechanisms that rely on reversible protein modifications and inhibitors of pre-RC components such as geminin and Crm1, thus avoiding the need to resynthesize
replication proteins at every cell cycle. Alterations of S
phase execution to allow polyploidy, selective rereplication of parts of the genome such as in chorion gene
amplification, or suppression of S phase between meiotic nuclear divisions are likely to reflect modifications
of a basic control mechanism. Even in unicellular organisms, distinctive features of their cell biology may have
influenced regulation. Cdt1 and Mcm proteins are both
regulated by nuclear localization in budding yeast which
makes more sense for an organism with a closed mitosis
Review
1073
than for one in which nuclear membrane breakdown
occurs on entry into mitosis.
Replications Factors Function Outside of Replication?
DNA replication proteins may not be uniquely involved
in replication, and this may have led to species-specific
differences in their regulation that are difficult to account
for if only replication is considered. ORC components
have been implicated in transcriptional silencing and
chromosome structure (Foss et al., 1993) as well as in
cytokinesis. Several reports connecting the Mcm proteins with transcription components may indicate that
the roles of these proteins are wider than generally accepted (e.g., Zhang et al., 1998). Also intriguing is the
reported neuralizing property of geminin (Kroll et al.,
1998; Quinn et al., 2001). Perhaps this protein has simply
been adopted for a second function, but a more interesting possibility is that this could be related to the process
of coupling cell differentiation with exit from the cell
cycle.
Consequences of Redundancy
in Control Mechanisms?
It is worth stressing that one consequence of having
redundant controls could be to facilitate evolutionary
and developmental variations in replication regulation.
Inactivation of three controls affecting Cdc6, Mcms, and
ORC is necessary to get moderate rereplication in budding yeast (Nguyen et al., 2001). During evolution, this
redundancy might allow loss of one control mechanism
without lethal consequences, and subsequently a different one could be added to satisfy the regulatory requirements of a particular organism. The relative importance
of particular regulatory steps in licensing could also vary
in different cell types during development, which might
affect S phase execution in subtle ways. A cell cycle
whereby, for instance, Orc1 is rate limiting for pre-RC
formation could have distinct characteristics compared
to one in which Cdt1 activity is limiting, such as in the
distribution of replication origins.
specifically binds to ACE3, an origin of DNA replication control element. Genes Dev. 13, 2639–2649.
Concluding Remarks
Comparative analysis of DNA replication mechanisms in
different organisms has highlighted variations in control
mechanisms, and it will be of interest to determine
whether replication controls comparing neoplastic or
different types of normal cells in the same organism
diverge. DNA replication is an important target for antiproliferative drugs, and the selectivity of such agents
would be improved if DNA replication can be selectively
inhibited in target cells owing to an idiosyncrasy in their
initiation control.
Foss, M., McNally, F.J., Laurenson, P., and Rine, J. (1993). Origin
recognition complex (ORC) in transcriptional silencing and DNA replication in S. cerevisiae. Science 262, 1838–1844.
Acknowledgments
We apologize to authors whose work has not been cited owing to
space limitations. We are grateful to Erik Boye and John Diffley for
comments. Work in the authors’ laboratories is supported by grants
from AICR, BBSRC, Cancer Research UK, and LRF.
References
Asano, M., and Wharton, R.P. (1999). E2F mediates developmental
and cell cycle regulation of ORC1 in Drosophila. EMBO J. 18, 2435–
2448.
Austin, R.J., Orr Weaver, T.L., and Bell, S.P. (1999). Drosophila ORC
Baum, B., Nishitani, H., Yanow, S., and Nurse, P. (1998). Cdc18
transcription and proteolysis couple S phase to passage through
mitosis. EMBO J. 17, 5689–5698.
Bell, S., and Dutta, A. (2002). DNA replication in eukaryotic cells.
Annu. Rev. Biochem. 71, 333–374.
Bielinsky, A., and Gerbi, S. (1998). Mapping initiation sites for DNA
replication: the transition point from continuous to discontinuous
DNA synthesis in yeast ARS1. Science 279, 95–98.
Biermann, E., Baack, M., Kreitz, S., and Knippers, R. (2002). Synthesis and turn-over of the replicative Cdc6 protein during the HeLa
cell cycle. Eur. J. Biochem. 269, 1040–1046.
Blow, J., and Hodgson, B. (2002). Replication licensing—defining
the proliferative state. Trends Cell Biol. 12, 72–78.
Chesnokov, I., Remus, D., and Botchan, M. (2001). Functional analysis of mutant and wild-type Drosophila origin recognition complex.
Proc. Natl. Acad. Sci. USA 98, 11997–12002.
Chesnokov, I.N., Chesnokova, O.N., and Botchan, M. (2003). A cytokinetic function of Drosophila ORC6 protein resides in a domain
distinct from its replication activity. Proc. Natl. Acad. Sci. USA
100, 9150–9155.
Chuang, R.Y., Chretien, L., Dai, J., and Kelly, T.J. (2002). Purification
and characterization of the Schizosaccharomyces pombe origin recognition complex: interaction with origin DNA and Cdc18 protein.
J. Biol. Chem. 277, 16920–16927.
Clay-Farrace, L., Pelizon, C., Santamaria, D., Pines, J., and Laskey,
R.A. (2003). Human replication protein Cdc6 prevents mitosis
through a checkpoint mechanism that implicates Chk1. EMBO J.
22, 704–712.
Coverley, D., Laman, H., and Laskey, R.A. (2002). Distinct roles for
cyclins E and A during DNA replication complex assembly and activation. Nat. Cell Biol. 4, 523–528.
DePamphilis, M. (2003). The ‘ORC cycle’: a novel pathway for regulating eukaryotic DNA replication. Gene 310, 1–15.
Dhar, S.K., Yoshida, K., Machida, Y., Khaira, P., Chaudhuri, B.,
Wohlschlegel, J.A., Leffak, M., Yates, J., and Dutta, A. (2001). Replication from oriP of Epstein-Barr virus requires human ORC and is
inhibited by geminin. Cell 106, 287–296.
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.
Gilbert, D. (2001). Making sense of eukaryotic DNA replication origins. Science 294, 96–100.
Gozuacik, D., Chami, M., Lagorce, D., Faivre, J., Murakami, Y., Poch,
O., Biermann, E., Knippers, R., Brechot, C., and Paterlini Brechot,
P. (2003). Identification and functional characterization of a new
member of the human Mcm protein family: hMcm8. Nucleic Acids
Res. 31, 570–579.
Gregan, J., Lindner, K., Brimage, L., Franklin, R., Namdar, M., Hart,
E.A., Aves, S.J., and Kearsey, S.E. (2003). Fission yeast Cdc23/
Mcm10 functions after pre-replicative complex formation to promote Cdc45 chromatin binding. Mol. Biol. Cell 14, 3876–3887.
Hashimoto, Y., and Takisawa, H. (2003). Xenopus Cut5 is essential
for a CDK-dependent process in the initiation of DNA replication.
EMBO J. 22, 2526–2535.
Hodgson, B., Li, A., Tada, S., and Blow, J.J. (2002). Geminin becomes
activated as an inhibitor of Cdt1/RLF-B following nuclear import.
Curr. Biol. 12, 678–683.
Homesley, L., Lei, M., Kawasaki, Y., Sawyer, S., Christensen, T., and
Tye, B.K. (2000). Mcm10 and the MCM2-7 complex interact to initiate
DNA synthesis and to release replication factors from origins. Genes
Dev. 14, 913–926.
Jallepalli, P.V., Tien, D., and Kelly, T.J. (1998). sud1(⫹) targets cyclindependent kinase-phosphorylated Cdc18 and Rum1 proteins for
Molecular Cell
1074
degradation and stops unwanted diploidization in fission yeast.
Proc. Natl. Acad. Sci. USA 95, 8159–8164.
autonomously replicating sequence region of budding yeast. Mol.
Cell. Biol. 20, 2809–2817.
Jares, P., and Blow, J.J. (2000). Xenopus cdc7 function is dependent
on licensing but not on XORC, XCdc6, or CDK activity and is required
for XCdc45 loading. Genes Dev. 14, 1528–1540.
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.
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.
McGarry, T.J. (2002). Geminin deficiency causes a Chk1-dependent
G2 arrest in Xenopus. Mol. Biol. Cell 13, 3662–3671.
Kamimura, Y., Tak, Y.S., Sugino, A., and Araki, H. (2001). Sld3, which
interacts with Cdc45 (Sld4), functions for chromosomal DNA replication in Saccharomyces cerevisiae. EMBO J. 20, 2097–2107.
Kanemaki, M., Sanchez Diaz, A., Gambus, A., and Labib, K. (2003).
Functional proteomic identification of DNA replication proteins by
induced proteolysis in vivo. Nature 423, 720–724.
Kong, D., and DePamphilis, M.L. (2001). Site-specific DNA binding
of the Schizosaccharomyces pombe origin recognition complex is
determined by the Orc4 subunit. Mol. Cell. Biol. 21, 8095–8103.
Kong, D., and DePamphilis, M. (2002). Site-specific ORC binding,
pre-replication complex assembly and DNA synthesis at Schizosaccharomyces pombe replication origins. EMBO J. 21, 5567–5576.
Kong, D., Coleman, T., and DePamphilis, M. (2003). Xenopus origin
recognition complex (ORC) initiates DNA replication preferentially
at sequences targeted by Schizosaccharomyces pombe ORC.
EMBO J. 22, 3441–3450.
Kreitz, S., Ritzi, M., Baack, M., and Knippers, R. (2001). The human
origin recognition complex protein 1 dissociates from chromatin
during S phase in HeLa cells. J. Biol. Chem. 276, 6337–6342.
Kroll, K.L., Salic, A.N., Evans, L.M., and Kirschner, M.W. (1998).
Geminin, a neuralizing molecule that demarcates the future neural
plate at the onset of gastrulation. Development 125, 3247–3258.
Kubota, Y., Takase, Y., Komori, Y., Hashimoto, Y., Arata, T., Kamimura, Y., Araki, H., and Takisawa, H. (2003). A novel ring-like complex of Xenopus proteins essential for the initiation of DNA replication. Genes Dev. 17, 1141–1152.
Labib, K., and Diffley, J. (2001). Is the MCM2-7 complex the eukaryotic DNA replication fork helicase? Curr. Opin. Genet. Dev. 11, 64–70.
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.
Labib, K., Tercero, J.A., and Diffley, J.F. (2000). Uninterrupted
MCM2-7 function required for DNA replication fork progression.
Science 288, 1643–1647.
Ladenburger, E.M., Keller, C., and Knippers, R. (2002). Identification
of a binding region for human origin recognition complex proteins
1 and 2 that coincides with an origin of DNA replication. Mol. Cell.
Biol. 22, 1036–1048.
Lee, D.G., and Bell, S.P. (1997). Architecture of the yeast origin
recognition complex bound to origins of DNA replication. Mol. Cell.
Biol. 17, 7159–7168.
Lee, J.K., Moon, K.Y., Jiang, Y., and Hurwitz, J. (2001). The Schizosaccharomyces pombe origin recognition complex interacts with
multiple AT-rich regions of the replication origin DNA by means of
the AT-hook domains of the spOrc4 protein. Proc. Natl. Acad. Sci.
USA 98, 13589–13594.
Lemaitre, J.M., Bocquet, S., and Mechali, M. (2002). Competence to
replicate in the unfertilized egg is conferred by Cdc6 during meiotic
maturation. Nature 419, 718–722.
Li, C.J., and DePamphilis, M.L. (2002). Mammalian Orc1 protein is
selectively released from chromatin and ubiquitinated during the
S-to-M transition in the cell division cycle. Mol. Cell. Biol. 22,
105–116.
Liu, J., Smith, C.L., DeRyckere, D., DeAngelis, K., Martin, G.S., and
Berger, J.M. (2000). Structure and function of Cdc6/Cdc18: implications for origin recognition and checkpoint control. Mol. Cell 6,
637–648.
Masai, H., and Arai, K. (2002). Cdc7 kinase complex: a key regulator
in the initiation of DNA replication. J. Cell. Physiol. 190, 287–296.
Masumoto, H., Sugino, A., and Araki, H. (2000). Dpb11 controls the
association between DNA polymerases alpha and epsilon and the
McGarry, T.J., and Kirschner, M.W. (1998). Geminin, an inhibitor of
DNA replication, is degraded during mitosis. Cell 93, 1043–1053.
Mendez, J., and Stillman, B. (2000). Chromatin association of human
origin recognition complex, cdc6, and minichromosome maintenance proteins during the cell cycle: assembly of prereplication
complexes in late mitosis. Mol. Cell. Biol. 20, 8602–8612.
Mendez, J., Zou Yang, X.H., Kim, S.Y., Hidaka, M., Tansey, W.P.,
and Stillman, B. (2002). Human origin recognition complex large
subunit is degraded by ubiquitin-mediated proteolysis after initiation
of DNA replication. Mol. Cell 9, 481–491.
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.
Natale, D.A., Li, C.J., Sun, W.H., and DePamphilis, M.L. (2000). Selective instability of Orc1 protein accounts for the absence of functional
origin recognition complexes during the M-G(1) transition in mammals. EMBO J. 19, 2728–2738.
Nguyen, V.Q., Co, C., and Li, J.J. (2001). Cyclin-dependent kinases
prevent DNA re-replication through multiple mechanisms. Nature
411, 1068–1073.
Nishitani, H., and Nurse, P. (1995). p65cdc18 plays a major role
controlling the initiation of DNA replication in fission yeast. Cell
83, 397–405.
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.
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.
Nougarede, R., Della Seta, F., Zarzov, P., and Schwob, E. (2000).
Hierarchy of S-phase-promoting factors: yeast Dbf4-Cdc7 kinase
requires prior S-phase cyclin-dependent kinase activation. Mol. Cell.
Biol. 20, 3795–3806.
Ortega, S., Prieto, I., Odajima, J., Martin, A., Dubus, P., Sotillo, R.,
Barbero, J.L., Malumbres, M., and Barbacid, M. (2003). Cyclindependent kinase 2 is essential for meiosis but not for mitotic cell
division in mice. Nat. Genet. 35, 25–31.
Perkins, G., and Diffley, J.F. (1998). Nucleotide-dependent prereplicative complex assembly by Cdc6p, a homolog of eukaryotic and
prokaryotic clamp-loaders. Mol. Cell 2, 23–32.
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.
Petersen, B.O., Wagener, C., Marinoni, F., Kramer, E.R., Melixetian,
M., Denchi, E.L., Gieffers, C., Matteucci, C., Peters, J.M., and Helin,
K. (2000). Cell cycle- and cell growth-regulated proteolysis of mammalian CDC6 is dependent on APC-CDH1. Genes Dev. 14, 2330–
2343.
Piatti, S., Lengauer, C., and Nasmyth, K. (1995). Cdc6 is an unstable
protein whose de novo synthesis in G1 is important for the onset
of S phase and for preventing a ‘reductional’ anaphase in the budding yeast Saccharomyces cerevisiae. EMBO J. 14, 3788–3799.
Prasanth, S.G., Prasanth, K.V., and Stillman, B. (2002). Orc6 involved
in DNA replication, chromosome segregation, and cytokinesis. Science 297, 1026–1031.
Prokhorova, T.A., and Blow, J.J. (2000). Sequential MCM/P1 subcomplex assembly is required to form a heterohexamer with replication licensing activity. J. Biol. Chem. 275, 2491–2498.
Quinn, L.M., Herr, A., McGarry, T.J., and Richardson, H. (2001). The
Review
1075
Drosophila Geminin homolog: roles for Geminin in limiting DNA replication, in anaphase and in neurogenesis. Genes Dev. 15, 2741–2754.
Raghuraman, M.K., Winzeler, E.A., Collingwood, D., Hunt, S., Wodicka, L., Conway, A., Lockhart, D.J., Davis, R.W., Brewer, B.J., and
Fangman, W.L. (2001). Replication dynamics of the yeast genome.
Science 294, 115–121.
Rao, H., and Stillman, B. (1995). The origin recognition complex
interacts with a bipartite DNA binding site within yeast replicators.
Proc. Natl. Acad. Sci. USA 92, 2224–2228.
Rowles, A., Tada, S., and Blow, J.J. (1999). Changes in association
of the Xenopus origin recognition complex with chromatin on licensing of replication origins. J. Cell Sci. 112, 2011–2018.
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.
Su, T.T., and O’Farrell, P.H. (1998). Chromosome association of
minichromosome maintenance proteins in Drosophila endoreplication cycles. J. Cell Biol. 140, 451–460.
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.
Takahashi, T., Ohara, E., Nishitani, H., and Masukata, H. (2003).
Multiple ORC-binding sites are required for efficient MCM loading
and origin firing in fission yeast. EMBO J. 4, 964–974.
Takayama, Y., Kamimura, Y., Okawa, M., Muramatsu, S., Sugino,
A., and Araki, H. (2003). GINS, a novel multiprotein complex required
for chromosomal DNA replication in budding yeast. Genes Dev.
17, 1153–1165.
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.
Tercero, J.A., Labib, K., and Diffley, J.F. (2000). DNA synthesis at
individual replication forks requires the essential initiation factor
Cdc45p. EMBO J. 19, 2082–2093.
Van Hatten, R.A., Tutter, A.V., Holway, A.H., Khederian, A.M., Walter,
J.C., and Michael, W.M. (2002). The Xenopus Xmus101 protein is
required for the recruitment of Cdc45 to origins of DNA replication.
J. Cell Biol. 159, 541–547.
Vashee, S., Cvetic, C., Lu, W., Simancek, P., Kelly, T.J., and Walter,
J.C. (2003). Sequence-independent DNA binding and replication initiation by the human origin recognition complex. Genes Dev. 17,
1894–1908.
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.
Whitmire, E., Khan, B., and Coue, M. (2002). Cdc6 synthesis regulates replication competence in Xenopus oocytes. Nature 419,
722–725.
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.
Wohlschlegel, J.A., Dhar, S.K., Prokhorova, T.A., Dutta, A., and Walter, J.C. (2002). Xenopus Mcm10 binds to origins of DNA replication
after Mcm2-7 and stimulates origin binding of Cdc45. Mol. Cell
9, 233–240.
Wyrick, J.J., Aparicio, J.G., Chen, T., Barnett, J.D., Jennings, E.G.,
Young, R.A., Bell, S.P., and Aparicio, O.M. (2001). Genome-wide
distribution of ORC and MCM proteins in S. cerevisiae: high-resolution mapping of replication origins. Science 294, 2357–2360.
Yamaguchi, R., and Newport, J. (2003). A role for Ran-GTP and
Crm1 in blocking re-replication. Cell 113, 115–125.
Yu, G., Wu, J.R., and Gilbert, D.M. (1998). Analysis of mammalian
origin specification in ORC-depleted Xenopus egg extracts. Genes
Cells 3, 709–720.
Zhang, J.J., Zhao, Y., Chait, B.T., Lathem, W.W., Ritzi, M., Knippers,
R., and Darnell, J.E., Jr. (1998). Ser727-dependent recruitment of
MCM5 by Stat1alpha in IFN-gamma-induced transcriptional activation. EMBO J. 17, 6963–6971.
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.
Zou, L., and Stillman, B. (2000). Assembly of a complex containing
Cdc45p, replication protein A, and Mcm2p at replication origins
controlled by S-phase cyclin-dependent kinases and Cdc7p-Dbf4p
kinase. Mol. Cell. Biol. 20, 3086–3096.