Yeast
Yeast 2006; 23: 951–962.
Published online in Wiley InterScience
(www.interscience.wiley.com) DOI: 10.1002/yea.1416
Review
DNA replication in the fission yeast: robustness in the
face of uncertainty
Ioannis Legouras1 , Georgia Xouri1# , Sotiris Dimopoulos2 , John Lygeros2 and Zoi Lygerou1 *
1 School of Medicine, Laboratory of General Biology, University of Patras, Rio, Patras,
2 Department of Electrical Engineering, University of Patras, Rio, Patras, Greece
*Correspondence to:
Zoi Lygerou, Laboratory of
Gen.Biology, School of Medicine,
University of Patras, 26500 Rio,
Patras, Greece.
E-mail: [email protected]
# Present
address: European
Molecular Biology Laboratory,
Heidelberg, Germany.
Received: 16 July 2006
Accepted: 24 August 2006
Greece
Abstract
DNA replication, the process of duplication of a cell’s genetic content, must be carried
out with great precision every time the cell divides, so that genetic information
is preserved. Control mechanisms must ensure that every base of the genome is
replicated within the allocated time (S-phase) and only once per cell cycle, thereby
safeguarding genomic integrity. In eukaryotes, replication starts from many points
along the chromosome, termed origins of replication, and then proceeds continuously
bidirectionally until an opposing moving fork is encountered. In contrast to bacteria,
where a specific site on the genome serves as an origin in every cell division, in
most eukaryotes origin selection appears highly stochastic: many potential origins
exist, of which only a subset is selected to fire in any given cell, giving rise to an
apparently random distribution of initiation events across the genome. Origin states
change throughout the cell cycle, through the ordered formation and modification of
origin-associated multisubunit protein complexes. State transitions are governed by
fluctuations of cyclin-dependent kinase (CDK) activity and guards in these transitions
ensure system memory. We present here DNA replication dynamics, emphasizing
recent data from the fission yeast Schizosaccharomyces pombe, and discuss how
robustness may be ensured in spite of (or even assisted by) system randomness.
Copyright  2006 John Wiley & Sons, Ltd.
Keywords: fission yeast; Schizosaccharomyces pombe; DNA replication; origin
selection; stochasticity; licensing; random completion problem; hybrid dynamics
Contents
Introduction: uncertainty and robustness in DNA
replication
Replication initiation sites are selected
randomly
Replication forks move continuously
Discrete memory ensures once-per-cell-cycle replication
Origin sites, origin states and stochasticity
The random completion problem
Randomness as a source of robustness
Perspectives
References
Copyright  2006 John Wiley & Sons, Ltd.
Introduction: uncertainty and robustness
in DNA replication
Every time a cell divides, its entire genome must be
replicated fully and only once during S-phase of the
cell cycle, followed by chromosome segregation in
mitosis, in order to ensure that the genetic material
is transmitted to the next generation and genomic
instabilities are avoided.
In bacterial and viral genomes, replication initiates from the same genomic site in every cell,
termed the origin of replication [28]. Given the
large genomes of eukaryotes, replication would
fail to be completed in the observed time if it
952
started from a single point on every chromosome.
Thus, eukaryotes initiate replication from hundreds
of replication origins at every cell cycle [16].
These origins are able to initiate firing throughout the length of S phase. Surprisingly, accumulating evidence suggests that the location of active
origins exhibits a high degree of uncertainty in
most eukaryotic organisms [16]: although replication generally starts from defined regions, many
potential origins exist, of which only a subset will
be randomly selected to fire in any given cell in a
population. In addition, the time at which a given
origin will fire within S phase is not precisely determined.
DNA replication is therefore a complex process
which involves considerable uncertainty. It must,
however, be carried out with great precision for the
genetic information to be maintained. The foregoing features introduce some possible problems to
its successful completion:
• How are the positions of origins selected so
that DNA replication is completed within the
allocated S-phase duration?
• How does the system have memory and coordinate firing in such a way that every region is
replicated and no region is rereplicated within
the same cell cycle?
In this overview paper we outline some of the
mechanisms that have been proposed to regulate
this random yet highly robust process, concentrating on the fission yeast Schizosaccharomyces
pombe. We survey recent findings which suggest
that randomness plays an integral role in the DNA
replication process. We also discuss the mechanisms that the cell uses to ensure that each part
of the genome replicates once, and only once, in
a normal cell cycle. Finally, we highlight potential problems associated with the random nature of
the process and survey mechanisms that have been
suggested to explain how the cell deals with these
problems.
Replication initiation sites are selected
randomly
Origin positioning is a crucial part of replication
and various organisms have evolved different ways
to define origins of replication. In bacteria, there
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I. Legouras et al.
is only a single non-redundant origin of replication. The single circular bacterial chromosome is
entirely replicated by two replication forks starting from this origin [28]. Origin positioning is
sequence-specific and origin activation is deterministic: the origin fires in every cell cycle with high
fidelity. Early work from budding yeast offered
support to the defined origin model, as S. cerevisiae origins were shown to be defined by specific
sequences and fire with high efficiency [16,22].
However, eukaryotic origins in general appear less
well defined and their firing pattern appears nondeterministic. Indeed, in early fly and frog embryos,
replication is carried out without any noticeable
sequence specificity [21,49], while in human cells,
any sequence larger than 10 kb appears suitable to
sustain replication initiation on a plasmid at random
sites inside the sequence [29,30].
Most eukaryotic cells seem to follow an intermediate route between a fully deterministic and
a fully random origin selection mechanism: replication does start from relatively specific regions
along the genome; however, these regions are not
selected in every cell cycle, thereby giving rise to a
semi-random distribution of initiation events along
the DNA. In addition, while certain origins tend to
fire early and others late, the timing of firing of
each origin is not fully determined, giving rise to
a semi-random timing pattern of initiation. Recent
work from fission yeast lends support to the notion
that stochasticity is an integral part of the DNA
replication process.
Plasmid maintenance assays have been applied
to map origins in S. pombe. The average length of
fragments capable of sustaining autonomous replicating sequence (ARS) activity was shown to be
500–1500 bp [9,14]. While two-dimensional gel
electrophoresis suggested DNA replication starts
at discrete genomic loci [14,17,58], mutational
analysis in plasmids showed lack of a consensus
sequence which could define an origin [13]. Origins
were shown to be composed of multiple redundant
elements, the sequential excision of which did not
have an all-or-none effect, but led to a gradual
decrease in origin function [9,24,42]. All identified origins were shown to reside preferentially
in intergenic locations, be above average AT rich
and contain a number of short stretches of highly
asymmetric adenine and thymine residues, which
contributed synergistically to origin activity. Characterized origins were shown to initiate replication
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DNA replication dynamics in fission yeast
at their genomic location in only a fraction of cells
in a population [14,17,25,48].
Following the completion of the S. pombe
sequencing project [53], full genome analyses
became feasible and have shed more light on the
nature and function of S. pombe origins. Segurado et al. employed a bioinformatics analysis to
find the coordinates of genomic locations of ATrich sequences (AT content >72% in a 0.5–1.0 kb
window) which could serve as origin sites [47].
This approach led to the identification of 384 ATrich islands with potential origin activity. AT-rich
islands were shown to reside in intergenic regions
and to be over-represented between divergent transcriptional units. Average origin density was one
every 33 kb, with a higher density in centromeric,
subtelomeric and mating-type loci (about four-fold
higher than the genomic average). Twenty of the
384 AT-rich islands were tested for origin function at their genomic location by two-dimensional
gel electrophoresis; 90% of the islands tested had
origin activity, predicting a total of 345 origins
associated with AT-rich islands across the fission
yeast genome.
More insight into the process was given recently
by Dai et al. [11], who showed that the properties
of known fission yeast origins were similar to
fission yeast intergenic regions in general. Of 26
intergenic regions tested on plasmids for origin
function, four functioned as monomers and 10
functioned as dimers, leading to the suggestion that
approximately half of fission yeast 4978 intergenic
regions may have potential origin function. A lack
of a consensus sequence was also noted, while
AT content and intergenic size were suggested
to constitute the major determinants of origin
activity. Based on these findings, a hypothesis
was formulated, suggesting that DNA replication
is stochastic in nature and that, from the many
potential origins across the fission yeast genome,
only a few will fire in any given cell cycle.
Further evidence for the stochastic nature of
origin selection was given by Patel et al. [43]
Labelling of newly synthesized DNA with thymidine analogues in the presence of hydroxyurea (to
arrest fork progression), as well as pulse-labelling
of DNA in early and late S-phase, was used
to identify replication intermediates. These intermediates were analysed at the single-cell level
with DNA combing. In combination, fluorescence
in situ hybridization (FISH) was used to map the
Copyright  2006 John Wiley & Sons, Ltd.
953
replicating areas on specific genomic locations.
After measurement of more than 1000 interbubble distances, the distribution of interorigin gaps
was found to be exponential, clearly indicative of
a random initiation pattern across the genome. The
stochasticity of origin firing was further solidified
through the analysis of origins at the ura4 and nmt1
regions. The firing probabilities of origins in these
regions were in the approximate range 10–70%. In
addition, this analysis showed no evidence for coordinated regulation of neighbouring origins, while
labelling of DNA intermediates in two sequential
S-phases showed that origin selection is random
between successive cell cycles.
From these analyses, key characteristics of fission yeast origins have emerged (Figure 1A): localization in intergenic locations and preferential association with promoter regions; absence of a specific
sequence-level definition; high AT content and AT
asymmetry; correlation with large intergenic size.
Why are origins located exclusively in intergenic
regions? An obvious explanation is that ongoing
transcription and origin specification are mutually
exclusive; origin specification may not be possible in transcribed regions which are constantly
scanned by the transcriptional machinery. In addition, elements in promoter regions which enhance
transcriptional activity could have a concomitant
positive effect on origin specification.
The most striking emerging feature of origin
specification in fission yeast, however, is its highly
probabilistic nature. Figure 1B shows the spatial
stochasticity in origin selection. From a large pool
of potential origins, a different collection is selected
in every cell cycle, which leads to a semi-random
distribution of selected origins. This distribution is
different in every cell cycle of every cell. Figure 1C
shows the temporal stochasticity in origins firing.
It is known that origins do not fire concurrently at
the G1 –S transition but rather have the potential to
fire throughout S-phase. While some origins tend
to fire early and others late, there is a continuous
distribution of firing events throughout S phase and
it is highly unlikely that timing is deterministically
defined in a precise time point, giving rise to
temporal stochasticity.
The two aspects of stochasticity are in fact
linked, as the probability of firing of a given
origin will affect the average time within S at
which it will fire, while an intrinsically late firing origin will tend to appear inefficient, as it will
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954
I. Legouras et al.
Figure 1. (A) Origin characteristics. Fission yeast origins are located in intergenic regions and are characterized by high
AT content and regions of AT asymmetry. The length of the intergenic region is also a determinant in origin specification.
(B) Spatial stochasticity in origin selection. Many putative origins exist along the fission yeast genome which will fire in
only a certain fraction of cells in a population (percentage efficiencies are shown above origin locations). Every cell in
the population will therefore be characterized by a different subset of active origins. (C) Temporal stochasticity in origin
selection. Origins fire throughout S phase and, while some tend on average to fire early and others late, there is uncertainty
as to when each origin will fire in any given cell in the population
have a high probability of being replicated passively before it fires. The early or late character
of a given origin may be deterministically defined
independently of its firing efficiency. It is, however, conceivable that the observed timing of firing represents a read-out of firing efficiency for
at least some origins, with highly efficient origins tending by probability to replicate early in
S phase, in contrast to inefficient origins appearing, on average, late. Kim and Huberman [25]
analysed the replication timing of several fission
yeast ARS elements, using two-dimensional gel
electrophoresis and various synchronization methods, and found that origins tend to be replicated in
a given order. In cells arrested in early S phase
by hydroxyurea treatment, early-replicating ARS
elements contained replication intermediates while
late-replicating ones did not, whereas in cells lacking the Rad3 or Cds1 checkpoint kinases, almost
all regions tested contained replication intermediates, suggesting active repression of late-replicating
regions, a result which was further supported by
subsequent full-genome analysis [15]. Yompakdee
Copyright  2006 John Wiley & Sons, Ltd.
and Huberman [57] identified a 200 bp sequence
containing GC-rich elements which could confer
a late-firing character to an origin on a plasmid,
and similar sequences were identified close to latereplicating regions in the genome. The above data
suggest that origin timing patterns may be determined, at least for some origins. Patel et al. [43], on
the other hand, found no evidence for exclusively
late-firing origins in the fission yeast genome. Further experiments will be required to clarify whether
deterministic or probabilistic firing timing patterns
prevail for the majority of fission yeast origins.
Replication forks move continuously
Starting at origins, replication forks move bidirectionally along the chromosome in a continuous fashion. Forks are generally considered to be
moving stably on DNA until they meet another
fork moving in the opposite direction. In that case
every part between the two origins from which
the forks initiated is replicated. Many origins are
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DNA replication dynamics in fission yeast
955
actually replicated by passing replication forks that
originated from nearby origins, so a single fork traverses large parts of the chromosomes. Fork speed
may vary considerably along the genome and may
be dependent on primary sequence as well as higher
order chromatin structure. Replication pause sites
or fork barriers exist along the genome, such as
in the ribosomal RNA repeats [45]. Such barriers
may be required to ensure that head-on collisions
between RNA and DNA polymerases are avoided.
In order to safeguard the continuous movement of
the replication forks, control mechanisms ensure
that polymerases remain on the DNA and that the
forks do not collapse even if replication is impeded.
Ablation of certain control proteins results in polymerase fall off and can lead to severe chromosomal
instabilities through recombination [1,3].
Discrete memory ensures once per cell
cycle replication
Origins can be in two discrete states: pre-replicative, when they are ready to fire as soon as conditions permit; and post-replicative, when they cannot
fire. The transition from the post-replicative state
to the pre-replicative state takes place in M/G1
through the process of licensing. The transition
from the pre-replicative state to the post-replicate
state takes place during S phase, through origin
activation or passive replication. The two transitions are temporally separated and involve the formation and modification of multi-subunit protein
complexes on origin DNA. While origin characteristics are not conserved during evolution, the
factors which bind to origins to define origin states
function in very similar ways in all organisms,
and our current understanding of the process stems
from experiments in several model systems.
In this section we outline the process with which
the cell ‘stores information’ in this discrete memory
and the molecular mechanism behind this process,
concentrating on data from fission yeast.
Figure 2. Formation of multisubunit protein complexes
dictates origin state transitions. Origin sites are marked by
the origin recognition complex (ORC), which is present
on chromatin throughout the cell cycle. Following mitosis,
loading of Cdc18 and Cdt1 onto chromatin recruits the
MCM complex, thereby forming the prereplicative complex
(PreRC) and licensing DNA for replication. At the G1 –S
transition, further factors are recruited to origin-bound
complexes, leading to activation of the PreRC to PreIC
and subsequent initiation of DNA replication. Origin
firing is linked to inhibition of licensing and conversion
of origin-bound complexes to the post-replicative state
(PostRC)
Licensing: transition to the pre-replicative state
Licensing occurs from late mitosis and through G1
phase and is a prerequisite for DNA replication
[4,38]. It involves the sequential recruitment of
licensing factors on origin sequences forming a
multisubunit complex, termed the pre-replicative
Copyright  2006 John Wiley & Sons, Ltd.
complex (PreRC, Figure 2). Only origins at the
pre-replicative state possess the licence to replicate
during the following S phase.
A central component believed to provide spatial
regulation of origin positioning is the origin
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956
recognition complex (ORC). ORC is a six-subunit
complex originally identified in budding yeast,
based on its ability to bind in a sequence-specific
manner to origins of replication [2]. Subsequent
analyses permitted characterization of fission yeast
ORCs and both in vivo and in vitro experiments
revealed specific association of ORC subunits with
origins of replication [7,27,34,41,51]. One of the
fission yeast ORC subunits, Orc4, possesses multiple AT-hook motifs at its amino-terminal part
which were shown to interact with the AT-rich S.
pombe origins [8,32]. These data suggest that the
ORCs may define which regions in the genome
have the potential to act as origins of replication.
The ORC is found on chromatin throughout the
cell cycle, albeit differentially modified [34], and
is therefore unlikely to be the major determinant
of when licensing takes place. The temporal regulation of licensing appears to be mainly provided
by two other licensing factors, Cdc18 and Cdt1.
Cdc18 was originally discovered through genetic
screening [37]. It is tightly regulated during the cell
cycle at both the transcriptional and the proteolytic
level and is present only in late M and G1 phase,
when it is recruited to origin DNA. Cdt1 (cdc10dependent transcript 1) was initially identified as
a target of the G1 transcription factor Cdc10
[19]. Further analysis showed that it is absolutely
required for origin licensing in this organism [39].
It is expressed similarly to Cdc18, peaking close to
the M–G1 transition.
Dependent on the presence of ORC, Cdc18
and Cdt1, the MCM complex is recruited onto
chromatin, and this loading confers to DNA the
licence to replicate. The six subunit MCM complex
associates with chromatin in a cell cycle-specific
manner and is believed to act as the replicative
helicase during the following S phase [31].
Apart from regulating once per cell-cycle replication, licensing also appears important for blocking mitosis until replication has taken place. Cells
depleted of Cdc18 or Cdt1 are not only unable to
initiate DNA replication, but also do not sense the
presence of unreplicated DNA and enter abortive
mitosis, resulting in a ‘cut’ phenotype [19,23].
Activation and passive replication: transition to
the post-replicative state
At the onset of S phase, several factors, pivotal
amongst which is Cdc45, associate with the already
Copyright  2006 John Wiley & Sons, Ltd.
I. Legouras et al.
formed pre-RC to assemble the pre-initiation
complex (PreIC, Figure 2; reviewed in Refs 12,38).
This complex is able to promote replisome formation at origin sites and facilitate DNA unwinding,
thereby leading to initiation of replication. Several
factors such as Sld3, Cut5/Rad4 (a Dpb11 homologue), Cdc23 (a homologue of MCM10) and the
GINS complex, as well as the S phase cyclindependent kinase (S-CDK) and Cdc7-Dbf4 kinase
(DDK, Hsk1-Dfp1 in fission yeast), are required for
stable Cdc45 association with origins, activation of
the PreIC complex and DNA replication initiation.
It is clear that origin specification and firing
is a two-step process: licensing takes place at
late M/early G1 while activation of licensed origins takes place at the G1 –S transition. In order
to ensure once-per-cell-cycle replication, licensing
must be coupled to passage through mitosis, while
activation of origins and initiation of replication
must be coupled to the inactivation of de novo
licensing.
What triggers the transitions and sets the
guards?
Early research in fission yeast received a major
boost from the observation that mutants undergoing
over-replication could be isolated and studied,
thereby allowing the identification of factors which
are essential for once per cell cycle replication.
Genetic screens to identify over-replicating mutant
strains were carried out and identified two classes
of mutants (Figure 3; reviewed in Ref. 35).
In the first class, ectopic activation of key licensing factors by overexpression of Cdc18 [40], Cdc18
together with Cdt1 [39,56], or mutations in Orc2
[52,55] results in repeated rounds of DNA replication in the absence of mitosis. Strikingly, another
class of mutants had defects due to inactivation
of the mitotic cyclin-dependent kinase (M-CDK,
cdc2/cdc13 [10,18]), suggesting that the mitotic
CDK has a dual role: to promote mitosis at the end
of the cell cycle and to inhibit DNA replication
until mitosis has been completed.
It is interesting to note that the two classes of
mutants are believed to give rise to different types
of genome over-replication (Figure 3): misregulation of licensing factors is believed to give rise
to repeated firing of origins within the same Sphase (rereplication), while M-CDK inactivation
gives rise to complete rounds of S phase without
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957
Figure 4. The continuous fluctuation in CDK activity
coordinates cell cycle progression (see text)
Figure 3. Defects in system memory lead to
over-replication. Two classes of system perturbations can
lead to loss of controls ensuring once-per-cell-cycle replication. Ectopic expression of key licensing factors (Cdc18,
the prototype of this category, is depicted) leads to refiring
of origins within the same S-phase (rereplication). Defects
in mitotic CDK activity lead to complete cycles of S phase
in the absence of mitosis (endoreduplication)
an intervening mitosis (endoreduplication). These
two types of over-replication are reminiscent of
physiologically occurring over-replication phenomena, such as chorion gene amplification (rereplication) or polyploidization (endoreduplication), and
may be related to certain chromosomal abnormalities observed in cancer cells.
These experiments showed that accurate regulation of licensing factors, as well as CDK activity, is essential for the cell to remember which
genomic regions have been replicated. We now
know that this memory is achieved through the
cross-talk between CDK and licensing factors.
Continuous changes in CDK activity dictate which
specific function should take place, while the discrete states of origins provide memory. In Figure 4,
the so-called quantitative model of cell cycle
regulation is illustrated [50]. Cell cycle events
are regulated by the periodic fluctuations in the
activity of CDKs, the master regulators of the
cell cycle. There are two identified thresholds in
CDK activity. Threshold 1 defines entry into S
phase and threshold 2 defines entry into mitosis.
Copyright  2006 John Wiley & Sons, Ltd.
To avoid incomplete DNA replication, regulatory
mechanisms have evolved which prevent CDK
activity from reaching threshold 2 if any region of
the genome remains unreplicated. Before replication, and while CDK activity is low, origins are
present in the pre-replicative state. Only in this
state can they support initiation of DNA replication when CDK activity passes threshold 1. When
an origin fires, or when it is passively replicated by
a passing replication fork from a nearby origin, it
automatically switches to the post-replicative state
and can no longer support initiation of replication.
CDK activities over threshold 1 inhibit conversion
of the post-replicative state to the pre-replicative
state. Only when cell division is completed and
CDK activity resets to zero can origins reacquire
the pre-replicative state. With this simple mechanism, rereplication is inhibited.
Origin sites, origin states and
stochasticity
How does the cross-talk between putative origin
sites along the genome and trans-acting factors
imposing origin states gives rise to the observed
stochasticity in origin usage in fission yeast?
The fact that only a subset of putative origins
fire in any given cycle suggests that there is a
rate-limiting step in the process of origin licensing
and/or activation:
1. At the level of licensing. Dai et al. [11] suggested that every intergenic region in the
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958
genome is a potential ORC binding site. Orc4
contains multiple AT-hook motifs at its Nterminus and it could bind with varying affinities to multiple asymmetric AT regions present
in different numbers in every intergenic region
along the genome. The number of potential Orc4
binding sites greatly exceeds the number of
ORC molecules present in the cell and therefore ORC would associate with only a subset of
potential origins in any given cell.
2. At the level of origin activation. A factor(s)
required for origin activation may be limiting and therefore only a subset of licensed
origins will initiate replication. Candidates for
such a limiting factor include Cdc45, cyclindependent kinase activity and DDB kinase activity, and differences in efficiencies between origins could reflect the accessibility of licensed
origins to these factors, e.g. due to chromatin
structure.
It should be noted, however, that the observed
efficiency of firing of an origin in a cell population
is not only affected by the intrinsic propensity of
this origin to fire in any given cell cycle but also by
the probability that it is passively replicated from
an adjacent origin. In an extreme case scenario,
randomness may be solely generated by passive
replication coupled to a degree of uncertainty in
timing of origin firing, even if most potential
origins are able to fire in every cell cycle (see
Redundancy model, below).
I. Legouras et al.
Several models could explain how the random
completion problem may be overcome [16,20].
Properties of fission yeast replication render some
of the models suitable, while others are inconsistent
with recent research findings.
Origin redundancy model
One solution to the random completion problem
would be a high redundancy of putative origins
possessing high intrinsic firing efficiencies. In this
model (Figure 5A), putative origins are present in
high excess, spaced closely together, and can all
fire within the length of S phase. A degree of
stochasticity in timing of activation within S phase
will result in early firing of some origins and passive replication of origins that have not yet fired,
giving rise to a random distribution of active origins in any given cycle and a low observed firing
efficiency for any given origin in a population of
cells. However, since every non-replicated origin
The random completion problem
In contrast to deterministic origin selection, where
origins fire in most cells and therefore inter-origin
distances and S phase length are precisely defined,
random origin selection may result in the occasional formation of large inter-origin gaps and a
possible inability to replicate those gaps within the
time allocated for S phase (the random completion
problem). It should be noted, however, that DNA
replication dynamics at the whole-genome level are
the result of many parameters, such as replication
speed at any given location, origin number and
spacing, intrinsic origin efficiencies and S phase
duration, which will influence S-phase completion.
Accurate genome-wide analysis of DNA is therefore required to reveal the extent of the random
completion problem for a given organism.
Copyright  2006 John Wiley & Sons, Ltd.
Figure 5. Possible solutions to the random completion
problem (see text). Origin redundancy. (A) Defined spacing.
(B) Redistribution. (C) ‘G2 ’ replication. Positions of putative
origins are marked with vertical red lines. Unreplicated
DNA is marked in blue, replicated DNA in green. In (B),
inter-origin distances in individual cells in a population are
marked. In (D), the maximum inter-origin region in each
cell is marked. Regions unable to become fully replicated
during what we define as S-phase are shown in red
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DNA replication dynamics in fission yeast
can initiate replication and will do so if not passively replicated soon enough, the random completion problem will not ensue. This explanation
is attractive for the early divisions of Xenopus
embryos where transcription is not yet active, replication can initiate at any sequence and there is a
large excess of licensing and initiation factors. It is,
however, unclear whether it could explain timely
replication in organisms where restrictions to origin positioning exist and factors are limiting, such
as in fission yeast, where origins are located exclusively in intergenic regions, or in mammalian cells,
where ongoing transcription and chromatin structure are unlikely to permit origins to be located
anywhere along the genome.
Defined spacing model
The defined spacing model [5,21] postulates that
though origins can be selected from a pool of putative origins, there is a defined spacing between origins that fire in any given cell, which ensures that
large inter-origins gaps do not appear (Figure 5B).
Using yeast, bacterial and viral plasmids, it has
been observed that replication never starts from a
second origin if their spacing is less than a specific
length [6,36,46] while larger plasmids are able to
initiate replication from multiple points in Xenopus
extracts [33].
The mechanism that measures and defines interorigin distances could rely on chromatin structure
or lateral inhibition. Higher-order chromatin structure could induce spatial constraints to origin selection, while lateral inhibition is an active mechanism
which could explain origin interference. Sequential
addition of a factor on DNA which is recruited
at origins, extends bidirectionally and covers DNA
sequences flanking the origin could provide lateral
inhibition, and MCM proteins have been proposed
as such a factor [20,54].
This model, although attractive, nevertheless
seems highly unlikely for fission yeast. Recent findings revealed an exponential distribution of interorigin distances analysed at the single-cell level
by DNA combing, which clearly contradicts the
defined spacing model [43].
Redistribution model
The redistribution model posits an increase in firing efficiency of origins remaining unreplicated as
Copyright  2006 John Wiley & Sons, Ltd.
959
S phase proceeds [33]. In contrast to the redundancy model postulating an excess of factors for
initiation, the redistribution model assumes a ratelimiting factor, which enables only a specific number of potential origins to be selected for firing at
any time (Figure 5C). This factor is not consumed
by the firing process but is released and retains ability to activate further origins. As S phase proceeds
and more origins convert to the post-replicative
state, the pool of the available limiting factor is
constantly increasing, thus facilitating activation
of the remaining origins. Unreplicated regions can
thus replicate fast in late S, through efficient firing
of late origins induced by redistribution of the limiting factor. Note that for this model to hold true,
the limiting factor must function at the level of origin activation, rather than origin licensing, since de
novo licensing is believed to be globally inhibited
as soon as S-phase starts. Cdc45 offers a likely
candidate for such a rate-limiting factor.
Replication in ‘G2 ’?
A possible, although somewhat heretical, solution
would be that S phase occasionally does run into
what we term the ‘G2 phase’ (Figure 5D). S phase
is defined based on our ability to detect ongoing
DNA replication; however, a small amount of late
replication in certain cells in a population could go
undetected and proceed during what we define as
the G2 phase. Given a functional S–M checkpoint
to sense the presence of unreplicated DNA and
arrest M phase entry until replication has been
completed, variations in the length of S phase
between individual cells in a population could be
inconsequential. This solution appears particularly
attractive for fission yeast, given its unusually long
G2 phase. G2 is approximately four times longer
than an average S phase, providing ample time
for duplication of late-replicating regions of the
genome corresponding to randomly arising large
inter-origin gaps. In the very rare cases in which
unreplicated parts still persist after G2 completion,
the S–M checkpoint will ensure that mitosis is
arrested until the remaining genomic regions are
replicated.
Although this explanation appears unlikely for
the early rapid division of amphibian and insect
embryos, which lack a G2 phase and an efficient
S–M checkpoint [26,44], it could hold true for
several other organisms, provided that they have
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DOI: 10.1002/yea
960
a checkpoint to ensure complete genome replication before mitosis. In this scenario, the time
required for DNA replication would show heterogeneity within a population, with no clear distinction between S and G2 , the sum of S and G2 for
a given cell being the total time required for DNA
synthesis and cellular growth to take place in order
to prepare the cell for the next mitosis.
Randomness as a source of robustness
Since randomness in origin selection can lead to
problems, why have organisms not opted for a
deterministic mode of origin specification? In fact
it appears that a degree of randomness in initiation of replication is the rule for most organisms,
rather than the exception. Despite making the process more unpredictable, randomness may also be
a source of robustness. Randomness is intricately
linked to redundancy: many potential origins exist,
of which only a subset will be chosen in any given
cycle. Redundancy is a clear source of robustness by providing system versatility and plasticity: in a changing environment, some origins may
be unable to fire and having a large number of
potential origins offers a clear benefit. In a deterministic model, inability of a given origin to fire
would have an immediate effect on the overall process. In a redundant, random model, such a failure
would be inconsequential. For example, if transcription through a given region and initiation of
replication from that region are indeed mutually
exclusive, higher eukaryotes must contain a large
number of potential origins to accommodate different transcriptional programmes in different cell
types.
Perspectives
We have described the process of DNA replication, highlighting the control mechanisms that
ensure robustness in the face of randomness. However, several key questions require further investigation. Given random origin selection, is there
a random completion problem in fission yeast, or
is the number and spacing of origins sufficient to
ensure replication within the allocated time? If a
random completion problem exists, are there control mechanisms in place to avoid the consequences
Copyright  2006 John Wiley & Sons, Ltd.
I. Legouras et al.
of prolonged S phase duration, or is there S-phase
length heterogeneity and ‘G2 ’ replication? Are
there intrinsically early firing and late firing origins in fission yeast, or is the tendency of certain
origins to appear earlier than others a probabilistic
event reflecting their firing efficiencies?
Recent genome-wide experiments based on microarrays provide hope for answering these questions. In addition, experiments at the single-cell
level will be required to address open questions and
the development of methodologies permitting fullgenome single cell analyses will become increasingly needed. Given the complexity and probabilistic nature of the process, system behaviour will,
however, be difficult to capture and accurately analyse at the whole-genome level. Analytical methods,
modelling and in silico experiments will be needed
to analyse the data and gain insight into potential
mechanisms.
Acknowledgements
We are most grateful to Julian Blow, Jacky Hales and
Hideo Nishitani for critical reading of the manuscript and
helpful suggestions. Our work on fission yeast replication
was supported by the European Commission, under the
project HYGEIA (NEST-4995), and by the HFSPO.
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