Results Probl Cell Differ (42)
P. Kaldis: Cell Cycle Regulation
DOI 10.1007/b137221/Published online: 6 July 2005
© Springer-Verlag Berlin Heidelberg 2005
Regulation of S Phase
Jamie K. Teer
1,2
· Anindya Dutta
1,2
(✉)
1
Biological and Biomedical Sciences Program, Harvard Medical School,
Boston, MA 02115, USA
[email protected]
2
Dept. Of Biochemistry, University of Virginia, Charlottesville, VA 22908, USA
[email protected]
Abstract Regulation of DNA replication is critical for accurate and timely dissemination
of genomic material to daughter cells. The cell uses a variety of mechanisms to control
this aspect of the cell cycle. There are various determinants of origin identification, as
well as a large number of proteins required to load replication complexes at these defined
genomic regions. A pre-Replication Complex (pre-RC) associates with origins in the G1
phase. This complex includes the Origin Recognition Complex (ORC), which serves to
recognize origins, the putative helicase MCM2-7, and other factors important for com-
plex assembly. Following pre-RC loading, a pre-Initiation Complex (pre-IC) builds upon
the helicase with factors required for eventual loading of replicative polymerases. The
chromatin association of these two complexes is temporally distinct, with pre-RC being
inhibited, and pre-IC being activated by cyclin-dependent kinases (Cdks). This regulation
is the basis for replication licensing, which allows replication to occur at a specific time
once, and only once, per cell cycle. By preventing extra rounds of replication within a cell
cycle, or by ensuring the cell cycle cannot progress until the environmental and intracel-

consensus seqeunce (A element) and several other B elements (Marahrens
and Stillman, 1992). These replicator sequences were used later to identify the
putative initiator proteins: the Origin Recognition Complex [ORC] (see be-
low). In Schizosaccharomyces pombe, two 30-55 base pair elements essential
for replication were discovered in origin ars3002, and similar sequences were
found in other ars regions (Dubey et al., 1996). Although the yeasts seem to
have high sequence conservation from one replicator to the next, determining
such consensus replicators in higher eukaryotes has been more difficult.
Studies in Xenopus laevis egg extracts have not identified a consensus
replicator sequence. On the contrary, early results indicate the lack of se-
quence specificity in replicating regions (Hyrien and Mechali, 1992; Hyrien
and Mechali, 1993; Mahbubani et al., 1992). Recent studies show that while
the ORC proteins may prefer AT rich DNA stretches, they show no preference
between defined origin sequences and control sequences in vitro, even with
varying ORC concentration (Vashee et al., 2003). Such random origin selec-
tion may, however, be a function of the early embryogensis system. When ori-
gin selection in the rDNA locus was studied at different times in development,
increasing origin specificity was seen as development progressed (Hyrien
et al., 1995). In early stages, origin selection was random, but when rDNA
gene expression began in late blastula and early gastrula stages, initiation
frequency decreased in the transcribed regions. This effectively limited ini-
tiation to the intergenic regions. Similar results were observed in Drosophila
embryos (Sasaki et al., 1999). Interestingly, when intact mammalian nuclei
were added to Xenopus extracts, they initiated replication at specific sites.
Disrupting the nuclei before incubation ablated this specificity (Gilbert et al.,
1995). Additionally, intact mammalian nuclei isolated before a certain time in
the G1 phase also failed to initiate specifically (Wu and Gilbert, 1996). Taken
Regulation of S Phase 33
together, these results indicate that metazoans do seem to initiate replication
at specific sites, but this specificity may be determined not by sequence, but

(Aladjem et al., 1998; Wang et al., 2004). Similar results have been observed
at the lamin B2 locus [1.2 kb] (Paixao et al., 2004), the hamster DHFR lo-
cus [5.8 kb] (Altman and Fanning, 2004), and the c-myc locus [2.4 kb] (Liu
et al., 2003): ectopically inserted sequences can confer origin activity, and
deletion of specific elements eliminates such activity. Unfortunately, the se-
quence elements do not seem to be identical, and no consensus sequences
have emerged. There does seem to be an important role of AT rich sequences,
as these are often found in critical deleted regions. Supporting this idea, an
essential AT rich element in the lamin B2 locus can substitute for the AT-
rich element in hamster ori-β locus (Altman and Fanning, 2004). One should
note that experiments showing sequence specificity generally measure origin
firing by PCR of nascent strands, while studies supporting sequence inde-
34 J.K. Teer · A. Dutta
pendent origin firing use ELFH and two dimensional gel electrophoresis. The
possibility exists that different methodologies may have different effects on
the results.
In addition to sequence effects, many studies have implicated transcrip-
tion in selection of replication origins. In the DHFR locus, transcription of
DHFR itself is required for origin firing activity, and yet origins do not fire in
the gene (Kalejta et al., 1998; Saha et al., 2004). In yeast, evidence exists for
transcriptional correlation with replication (Muller et al., 2000), but this may
be limited to few specific sites, as a genomic microarray study failed to see
a good correlation (Raghuraman et al., 2001). Many studies have shown a link
between early origin firing and transcriptional activity by looking at replica-
tion of developmentally regulated genes, as well as genes from asymmetrically
active alleles [reviewed in (Goren and Cedar, 2003)]. In the latter case, the
active alleles are replicated much earlier than the silenced alleles. Addition-
ally, replication studies using human (Jeon et al., 2005; Woodfine et al., 2004)
and Drosophila (Macalpine et al., 2004; Schubeler et al., 2002) genome tiling
microarrays show a positive correlation between early origin activity, gene

elements, chromatin structure itself, or a combination of effects. Thus, the
early theory of a replicator still holds true today. The increasing complexity
of higher eukaryotes simply means that the defining elements of a replicator
are themselves more multifaceted.
3
Pre-Replication Complex
3.1
ORC
The identification of replicator sequences in S. cerevisiae opened the field of
DNA replication in eukaryotes. One of the first critical discoveries stemming
from this work was the identification of the proposed initiator proteins. The
consensus A element of the ARS sequence was used to identify a six subunit
complex termed ORC, or Origin Recognition Complex (Bell and Stillman,
1992). Mutations in the A element that prevent ORC binding also prevent
replication from the mutated ARS, (Bell and Stillman, 1992; Rowley et al.,
1995) supporting the idea that budding yeast ORC is the protein initiator
responsible for recognizing specific replicator sequences. ORC is highly con-
served, with homologues identified in A.thaliana,S.pombe, D. melanogaster,
X. laevis, M. musculus, H. sapiens, and others (Carpenter et al., 1996; Dhar
and Dutta, 2000; Gavin et al., 1995; Gossen et al., 1995; Leatherwood et al.,
1996; Masuda et al., 2004; Muzi-Falconi and Kelly, 1995; Pinto et al., 1999;
Quintana et al., 1997; Quintana et al., 1998; Tugal et al., 1998). The ORC
subunits have been shown to form a functional complex in D. melanogaster
(Chesnokov et al., 1999), S. pombe (Moon et al., 1999), X. laevis (Gillespie
et al., 2001), and H. sapiens (Dhar et al., 2001a; Vashee et al., 2001).
As a replicative initiator, ORC should be able to recognize the replicator
sequences. ORC has been shown by many to bind DNA, and this binding is
dependent on ATP and the ATP binding functions of ORC (Bell and Still-
man, 1992; Chesnokov et al., 2001; Gillespie et al., 2001; Seki and Diffley,
2000). Specific replicator sequence association has been observed in S. cere-

3.4, respectively), rereplication was observed (Nguyen et al., 2001; Wilmes
et al., 2004). Similarly, S. pombe Orc2 is phosphorylated, which may be due
to its similar interaction with Cdk1/cyclin B in the G2 phase. This interaction
serves to prevent rereplication without an intervening mitosis, again ensur-
ing only one replication event per cell cycle takes place (Wuarin et al., 2002).
Phosphorylation is well studied as a molecular switch to regulate protein ac-
tivity through a variety of mechanisms and ORC phosphorylation gives the
cells a reversible way to prevent replication firing.
In higher eukaryotes, phosphorylation of ORC subunits is also observed.
In Xenopus systems, phosphorylation of ORC by cyclin A dependent kinase
activity disrupts ORC chromatin association (Findeisen et al., 1999). Simi-
larly, mammalian Orc1 is phosphorylated. In Chinese hamster ovary cells,
Orc1 interacts with cyclin A/Cdk1, which leads to the phosphorylation of
Orc1. Inhibiting this phosphorylation with drugs allows Orc1 to rebind chro-
matin, indicating that phosphorylation is important for chromatin release in
mitosis (Li et al., 2004). In human cells, Orc1 is also phosphorylated in vivo
(our unpublished results) and in vitro by cyclin A/Cdk2 (Mendez et al., 2002).
This phosphorylation seems to be required for Skp2 mediated ubiquitination
of Orc1 (Mendez et al., 2002). Similarly, hamster Orc1 is also ubiquitinated.
However, the nature and effect of these ubiquitination events is different. In
hamster cells, Orc1 seems to be mono- and di-ubiquitinated, which causes its
release from chromatin in S-phase until M-G1 (Li and DePamphilis, 2002).
Regulation of S Phase 37
In humans, several studies show that Orc1 is polyubiquitinated and then de-
graded by the proteasome during S-phase, (Fujita et al., 2002; Mendez et al.,
2002; Tatsumi et al., 2003) although this observation may result from proteol-
ysis after lysis (Ritzi et al., 2003). Although not degraded during the cell cycle,
hamster Orc1 is increasingly sensitive to proteasomal degradation when ar-
tificially released to the cytoplasm (Li and DePamphilis, 2002). In Drosophila
embryos, Orc1 is degraded in M and early G1 phases by the APC/fzr complex

with this, overexpression of Cdt1 alone leads to extensive re-replication in
human cells (Vaziri et al., 2003).
In addition to its function as a replication licensing factor, Cdt1 has re-
cently been implicated in preventing replication initiation after DNA damage
in human cells. Cdt1 levels were found to be profoundly decreased after UV
irradiation, and an E3 ligase, Cul4A-Roc1-Ddb1, was responsible for signaling
38 J.K. Teer · A. Dutta
this degradation via the proteasome (Higa et al., 2003; Hu et al., 2004). In-
terestingly, a separate study implicated the SCF
Skp2
complex in the radiation
induced degradation of Cdt1 (Kondo et al., 2004). It remains to be resolved
which ubiquitin ligase is primarily responsible for both the cell cycle depen-
dent modifications and the DNA damage induced modifications.
3.3
Cdc6
Cdc6 was originally identified in S. cerevisiae as a protein essential for cell
cycle progression (Hartwell et al., 1974), and was thereafter shown to have an
early DNA synthesis defect (Hartwell, 1976). Cdc6 interacts with ORC, form-
ing a complex with an extended nuclease protected DNA footprint (Cocker
et al., 1996; Liang et al., 1995). Furthermore, Cdc6 expression is required for
MCM loading in budding yeast. Interestingly, phosphorylation of Cdc6 by B-
type cyclin/Cdk complexes prevented the loading of Cdc6, illustrating a pow-
erful way for the cells to regulate pre-RC formation (Donovan et al., 1997;
Tanaka et al., 1997). By phosphorylating Cdc6 in S and G2/M, its activity was
limited, preventing inappropriate origin firing in mitosis. ScCdc6 was also
found to be marked for degradation at the G1/S transition by Clb/Cdc28 and
the Cdc4/Cdc34/Cdc53 ubiquitination machinery, adding a further layer of
regulation (Drury et al., 1997; Elsasser et al., 1999). A similar gene, Cdc18, was
identified in S. pombe, and is also required for S-phase. Indeed, its overex-

This observation may reconcile earlier observed differences, allowing Cdc6 to
bind chromatin in S-phase in a tenuous manner so that rapid regulation can
be achieved when needed.
Cdc6 is a AAA+ ATPase as is its homolog Orc1 (Neuwald et al., 1999), and
is therefore also regulated in cis. The recently solved structure of an archaeal
Cdc6 ortholog confirms the presence of a AAA+ ATPase domain, contain-
ing Walker A and B motifs, and well as several sensor regions thought to
detect nucleotide binding status (Liu et al., 2000). Several studies have been
carried out to characterize the importance of its ATP binding and hydrolysis
activities. From these studies, it appears that the Walker A motif (nucleotide
binding) may be important for Cdc6 binding to chromatin, and Walker B mo-
tif (nucleotide hydrolysis) is involved in MCM2-7 loading (Herbig et al., 1999;
Perkins and Diffley, 1998; Weinreich et al., 1999). In addition to replication
defects caused by mutation in the Walker A and B regions, certain mutations
in the sensor regions are also detrimental to replication, often by failing to
recruit MCM (Schepers and Diffley, 2001). These studies indicate the ATP
binding and hydrolysis of Cdc6 are critical to its function in many different
organisms.
In addition to its direct regulation, which serves to limit replication to
once, and only once per cell cycle by controlling MCM loading, Cdc6 also
has several other secondary roles. Interestingly, Cdc6 seems to regulate ORC
by inhibiting its non-specific DNA binding (Harvey and Newport, 2003;
Mizushima et al., 2000). By increasing sequence specificity of ORC, Cdc6 may
be playing an indirect role in origin selection, especially in higher eukary-
otes where consensus initiators have been elusive. This function may also help
prevent inefficient fork firing by ensuring ORCs are directed to specific sites,
presumably spaced evenly along the chromosome.
Cdc6 is not only regulated by the cell-cycle; it is also cleaved or degraded
during apoptosis (Blanchard et al., 2002; Pelizon et al., 2002). It is not entirely
clear why Cdc6 would be a target for apoptotic machinery; the cell no longer

erate helicase (DNA unwinding) activity (Ishimi, 1997; You et al., 1999). The
fission yeast MCM4,6,7 complex also has helicase activity (Lee and Hurwitz,
2000). Interestingly, when MCM2 or MCM3,5 were present in the complex,
helicase activity was lost (Lee and Hurwitz, 2000; Sato et al., 2000; You et al.,
1999). An archaeal MCM protein has been identified, and also has a helicase
activity (Chong et al., 2000; Kelman et al., 1999; Shechter et al., 2000). Electron
microscopy studies of the MCM complex indicate that they form a heterohex-
amer ring structure (Adachi et al., 1997; Sato et al., 2000). This is supported by
a recent report which proposes double head-to-head hexamers from a crys-
tal structure of an archaeal MCM (Fletcher et al., 2003). This structure shares
similar features with that of T-antigen, suggesting a common function to un-
wind double strand DNA (Li et al., 2003a) Despite the inhibitory nature of
MCM2, 3, and 5, they, like MCM4,6,7, are essential in yeasts [for review see
(Dutta and Bell, 1997; Kelly and Brown, 2000)]. This puzzling result seems
to indicate that MCM4,6,7 serves as the catalytic helicase domain, while the
other subunits act to modulate MCM activity. The exact mechanism of such
regulation is still unknown.
Like certain ORC subunits and Cdc6, each member of the MCM2-7 com-
plex has ATPase activity. This ATPase activity is critical for viability; mutants
Regulation of S Phase 41
in the Walker A domains show S-phase defects and cell cycle arrest (Schwacha
and Bell, 2001). Biochemical analysis of MCM helicase activity showed that
the ATPase activity of these proteins is required for the helicase activity
(Ishimi, 1997; Lee and Hurwitz, 2000; You et al., 1999). These studies indicated
that the helicase activity of these proteins is most likely the in vivo func-
tion required for replication. Interestingly, Walker A mutations in MCM4,6,7
are much more toxic to yeast than similar mutations in MCM2,3,5. Although
MCM2,3,5 are actually inhibitory for helicase activity (as described above),
they are required for optimal ATPase activity of the complex (Schwacha and
Bell, 2001). This further supports the role of the MCM4,6,7 complex as the

ruption of MCM6 binding may be preventing the recruitment of the MCM2-7
complex. Additionally, the same study suggested that Cdt1 can bind DNA,
42 J.K. Teer · A. Dutta
and this interaction was also disrupted by geminin inhibition. A more re-
cent study suggests that a Cdt1 binding activity to MCM2 and to Cdc6 is also
inhibited by geminin binding (Cook et al., 2004). Structural studies indicate
that geminin forms a coiled-coil dimer which, together with an N-terminal
flexible portion, executes a bipartite interaction with Cdt1 (Lee et al., 2004;
Saxena et al., 2004). Initiation inhibition by geminin is now thought to be
a part of the replication licensing system that prevents multiple rounds of
replication in Xenopus egg extracts (Arias and Walter, 2004; Li and Blow,
2004). Although Cdt1 is the primary target of regulation, geminin provides
an additional pathway to inactivate Cdt1, and thus preventing inappropri-
ate replication. In mammalian systems, overexpression of Cdt1 or depletion
of geminin is sufficient to cause massive rereplication and checkpoint acti-
vation (Melixetian et al., 2004; Zhu et al., 2004). Abrogating this checkpoint
causes mitotic distress and eventual cell death, demonstrating the importance
of limiting replication to a single event per cell cycle.
3.6
Summary
Thepre-RCiscomposedofavarietyofproteinsthatservetorecruitMCM2-7
to selected genomic regions, establishing a potential site for replication ini-
tiation (Fig. 1). The chromatin loading of MCM2-7 depends on the loading
of Cdt1 and Cdc6, which in turn depend on the loading of ORC. This step-
Fig. 1 Regulation of the pre-RC. The pre-RC functions to load MCM2-7 complex, the
potential replicative helicase. ORC binds chromatin initially at origins. Its binding is re-
quired for the chromatin association of Cdt1 and Cdc6, which are themselves required
to load the MCM2-7 complex. This loading occurs in late mitosis until late G1. Upon
entryintoS-phase,increasingcyclin/Cdk activity inhibits the loading of MCM2-7. This
increased cyclin/Cdk activity promotes Orc1 and Cdc6 dissociation from chromatin,

Mcm10 (also named DNA43) was originally identified in two independent
screens; it was identified in a screen for DNA synthesis errors (Solomon et al.,
1992) as well as in the mini-chromosome maintenance screen that identi-
fied the members of MCM2-7 complex (Maine et al., 1984). Mcm10 mutants
showed defects in S-phase, specifically with origin firing and in fork elonga-
tion, and were shown to interact with the MCM2-7 complex (Aves et al., 1998;
Merchant et al., 1997). The interaction with MCM7 in particular is critical for
proper replication (Homesley et al., 2000). These results point to a vital role
for yeast Mcm10 in replication.
Several recent studies have helped determine a likely function for Mcm10.
In Xenopus eggextractsandfissionyeast,Mcm10isloadedontochromatin
in an MCM2-7 dependent manner, and is itself required to load Cdc45 in
G1/early S [discussed below] (Gregan et al., 2003; Wohlschlegel et al., 2002b).