Journal of Experimental Botany, Vol. 52, No. 355, pp. 193±202, February 2001
REVIEW ARTICLE
Origins and complexes: the initiation of DNA replication
John A. Bryant1, Karen Moore and Stephen J. Aves
School of Biological Sciences, University of Exeter, Washington Singer Laboratories, Perry Road,
Exeter EX4 4QG, UK
Received 23 August 2000; Accepted 10 October 2000
Abstract
Introduction
Eukaryotic DNA is organized for replication as multiple replicons. DNA synthesis in each replicon is
initiated at an origin of replication. In both budding
yeast, Saccharomyces cerevisiae and fission yeast,
Schizosaccharomyces pombe, origins contain specific sequences that are essential for initiation,
although these differ significantly between the two
yeasts with those of S. pombe being more complex
then those of S. cerevisiae. However, it is not yet clear
whether the replication origins of plants contain
specific essential sequences or whether origin sites
are determined by features of chromatin structure.
In all eukaryotes there are several biochemical
events that must take place before initiation can
occur. These are the marking of the origins by the
origin recognition complex (ORC), the loading onto
the origins, in a series of steps, of origin activation
factors including the MCM proteins, and the initial
denaturation of the double helix to form a replication
`bubble'. Only then can the enzymes that actually
initiate replication, primase and DNA polymerase-a,
gain access to the template. In many cells this complex series of events occurs only once per cell cycle,
ensuring that DNA is not re-replicated within one
cycle. However, regulated re-replication of DNA
within one cell cycle (DNA endoreduplication) is
relatively common in plants, indicating that the
`once-per-cycle' controls can be overridden.
Plants, in common with all eukaryotic organisms, organize their DNA for replication as multiple units known as
replicons (Van't Hof, 1985, 1988). Each replicon is
de®ned spatially by an origin of replication (ori) and
two termini: the process of replication proceeds outwards
from the origin to the termini. Whether the termini are
speci®c sites or simply zones in which replication forks
®nally stall is not clear although evidence has been
presented for speci®c termini in the replicons within the
repeated genes that encode rRNA in pea (see below)
(Hernandez et al., 1988b). However, it is clear that under
circumstances in which fewer replication origins than
usual are being utilized (see below) replication forks can
continue through sitesuzones which would in other circumstances be termini. Typically, the time taken to complete
replication within one replicon is considerably shorter
than the S-phase. For example, in pea it takes about 2 h
to complete a replicon but S-phase may last for up to 8 h
(Van't Hof and Bjerknes, 1981). The explanation for this
is that replicons are organized as time-groups or families
with each family having its particular time within S-phase
during which it is active in replication. This is most clearly
seen in the small genome of Arabidopsis thaliana with only
two replicon families, one of which completes replication
in the ®rst 2 h of S-phase whilst the second is delayed in
initiation until 35±40 min after the start of S-phase, again
taking about 2 h to complete replication (Van't Hof et al.,
1978). Clearly, the initiation of replication at the origins
of the earliest replicon family represents the ®nal stage in
the transition from G1 to S, but of course that initiation
process must also occur within each replicon family as its
time for activity arrives during S-phase. This is the setting
for this brief review which will examine what is known
about the biochemistry of initiation of replication in
plants against a background of knowledge obtained
in other eukaryotic organisms and particularly in budding
Key words: ARS, DNA polymerase, DNA replication, initiation,
MCM, ORC, origin, primase, replicon.
1
To whom correspondence should be addressed. Fax: q44 1392 264668. E-mail: [email protected]
ß Society for Experimental Biology 2001
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Bryant et al.
yeast, Saccharomyces cerevisiae
Schizosaccharomyces pombe.
and
®ssion
yeast,
Origins of replication
General features
In plants the organization of DNA as multiple replicons
can be readily visualized by ®bre autoradiography
(Van't Hof, 1975, 1985, 1988), in which replicating DNA
molecules are labelled with tritiated thymidine. Use of
increasing labelling times facilitates the determination of
`fork rate', i.e. the rate of replication. `Step down' labelling (transfer from high to low speci®c activity thymidine)
shows the bidirectional movement of the forks and facilitates the identi®cation of the origins. This in turn allows
measurement of origin-to-origin distances (i.e. replicon
lengths). In a given species, there is usually a clear mode
of replicon length with some variation either side of the
mode. Thus in pea root meristems the modal replicon size
is about 54 kb (Van't Hof and Bjerknes, 1977). However,
there are well-de®ned situations in several plant species in
which the replicon size shortens signi®cantly, implying
that more origins must be employed. This is discussed in
more detail later in the review.
What is an origin?
Although replication origins may be `seen' in ®bre autoradiographs, it is much more dif®cult to separate a functional origin from the rest of the DNA; very few have
been isolated from the DNA of multicellular eukaryotes.
Many attempts to identify putative replication origins
have relied on assaying the ability of DNA fragments to
facilitate the so-called autonomous replication of plasmids in yeast (Saccharomyces cerevisiae) (Stinchcomb
et al., 1980). These pieces of DNA are known as ARS
elements. This was originally coined as an abbreviation
for Autonomous Replication in Saccharomyces, but now
taken as meaning autonomously replicating sequence.
Further, some authors use the term as being synonymous
with replication origin. ARSs have indeed been shown by
2-D gel electrophoresis to act as bona ®de origins in yeast
itself, both in plasmids and in chromosomal DNA
(Brewer and Fangman, 1987; Huberman et al., 1988).
The ability to isolate functional origins has also led to
their characterization in terms of sequence: ARS1 may be
taken as a typical example. It consists of four sequence
domains, A, B1, B2 and B3, all of which are needed for
function (Marahrens and Stillman, 1992). The A domain
is highly conserved between different origins and contains
the absolutely essential core sequence, AuTTTTATGu
ATTTAuT. All budding yeast replication origins have at
least a 9u11 match to this sequence and will not function
as an origin without it. The B domains, however, are
more variable from origin to origin. The functional
signi®cance of this variation is not clear, but it is tempting to speculate that it may be related to the time of
activation (often known as `®ring') of the origins within
S-phase.
DNA sequences from many other eukaryotes, including plants, have been shown to function in the budding
yeast ARS assay (Stinchcomb et al., 1980). As might be
expected from the essential nature of the ARS core
sequence, all the higher eukaryote ARS elements that
have been sequenced contain at least one essential core
sequence. Indeed, as exempli®ed by ARS elements isolated from Brassica napus (Sibson et al., 1988) some
contain more than one match. What exactly does this say
about DNA replication origins in higher eukaryotes?
Firstly, it is inevitable that an assay depending on functioning as an origin in budding yeast will select for the
sequences that yeast uses for origins. Secondly, in the
large genomes of multicellular eukaryotes a sequence of
11 base pairs is very likely to occur by chance. The presence of budding yeast-type ARS elements in plant DNA
does not therefore show that these elements are involved
in replication origins in plants. Indeed, recent studies on
origins of replication in the ®ssion yeast Schizosaccharomyces pombe have indicated that S. cerevisiae may be
a poor model for the structure of eukaryotic replication
origins. These two yeasts are evolutionarily divergent and
S. pombe ARS sequences (similarly identi®ed by plasmid
assayÐbut in S. pombe, not S. cerevisiaeÐand shown to
be bona ®de chromosomal origins) are signi®cantly
different in structure from those in S. cerevisiae. They
are much larger (500±1500 bp as opposed to -150 bp for
budding yeast) and their sequence composition is more
complex, lacking a speci®c essential ARS core sequence.
Instead they contain AT-rich 20±50 bp regions containing
clustered A or T stretches, which are required for ARS
activity (Clyne and Kelly, 1995; Dubey et al., 1996; Kim
and Huberman, 1998; Okuno et al., 1999). Unlike budding yeast, S. pombe ARSs appear to map in promoter
regions upstream from genes, suggesting that origin
function may be related to transcription (GoÂmez and
Antequera, 1999). Consistent with these differences,
S. pombe ARSs do not normally function in S. cerevisiae,
and vice versa (Clyne and Kelly, 1995). The exact extent
of similarity between S. pombe ARSs and DNA replication origins in multicellular eukaryotes remains to be
seen, but it is certainly clear that budding yeast origins
differ in signi®cant aspects from those in other eukaryotes.
Direct isolation of plant DNA replication origins has
proved very dif®cult. However, multicellular eukaryotes
possess an origin of DNA replication in each non-transcribed spacer (NTS) between the repeated genes that
code for rRNA. The `bubbles' associated with initiation
at these sites are visible in electron micrographs of replicating DNA in Drosophila, Tetrahymena and Xenopus
(reviewed by Van't Hof, 1988). Van't Hof's group
The initiation of DNA replication
(Van't Hof et al., 1987a, b; HernaÂndez et al., 1988a) have
taken advantage of the high level of repetition of these
genes in pea: in cultured pea root meristems they localized
the site of initiation of replication to a 1500 bp region
within the NTS. Subsequent 2-D gel experiments con®rmed that the replication bubble that marks the initiation
of strand separation occurs within this region (Van't Hof
and Lamm, 1992). Interestingly, the region contains a very
AT-rich domain that includes four good matches to the
S. cerevisiae ARS core (HernaÂndez et al., 1988a, 1993).
On this basis, it appears that the budding yeast ARS core
sequence may also be part of plant origins, despite the
absence of this sequence in ®ssion yeast origins (as discussed above). This suggestion is supported by the ®nding
that replicative DNA isolated in very early S-phase in
the same synchronized meristem system is enriched for
similar sequences (Bryant, 1994). Further, the origins of
replication from which the chorion genes in Drosophila
are ampli®ed are also AT-rich and contain sequences very
similar to the ARS core (Austin et al., 1999; Spradling,
1999). However, some investigators have suggested that
initiation of replication in higher eukaryotes has much
`looser' sequence requirements than in budding yeast
(DePamphilis, 1993; Bogan et al., 2000), or even no
sequence requirement at all (Mechali and Kearsey, 1984;
Gillespie and Blow, 2000). In reviewing this topic, Gilbert
suggests that in animals, origins are localized to speci®c
sequences not because of the sequences per se but by
features of chromosome structure (Gilbert, 1998). This
will be considered again in the context of origin
recognition.
Understanding of the structure of plant DNA replication origins is further complicated by the ®ndings that
origin-to-origin spacing may vary in plant development,
or in response to nutrients or hormones or to experimental manipulation. Thus in Sinapis alba, the ¯oral
stimulus induces in the shoot meristem a dramatic
shortening of the S-phase that is mainly brought about
by a halving of the modal replicon length from 15 kb to
7.5 kb, i.e. twice as many origins are utilized (Jacqmard
and Houssa, 1988). Increase in the number of active
origins during the transition to ¯owering also occurs in
Silene coeli-rosa and Pharbitis nil (Durdan et al., 1998). In
Sinapis, this aspect of the ¯oral response may be mimicked by application of the hormone cytokinin (Houssa
et al., 1990). Cytokinin has the same effect on dividing
cells in the vegetative shoot apex of a grass, Lolium
temulentum and in the ovule of tomato (Lycopersicon
esculentum) (Houssa et al., 1994). In synchronized pea
root meristems, cross-linking the DNA with psoralen in
order to stall fork movement causes extra origins to be
utilized between the cross-links (Francis et al., 1985). All
these data suggest that plant DNA contains sequences
that normally do not act as origins, but can be called into
action under particular circumstances. By contrast, in
195
lettuce roots, addition of trigonelline causes a halving of
the number of active origins, with two out of every cluster
of four being silenced or switched off (Mazzuca et al.,
2000). In the Sinapis alba shoot meristem, abscisic
acid caused a doubling of replicon length (15 kb to
30 kb: Jacqmard et al., 1995) implying that only one out
of every two normally used origins was activated
(as opposed to cytokinin which causes `extra' origins to
be activated, as described above). An earlier suggestion
that is consistent with all these data was that plant DNA
contains `strong' and `weak' origins (Francis et al., 1985).
This may relate to the ®nding in S. pombe of clustered
origins of replication which demonstrate hierarchies of
initiation frequencies (Dubey et al., 1994; Okuno et al.,
1997; Kim and Huberman, 1999). However, if `strength'
or `weakness' is related to sequence, this does not explain
the selective use of only one origin in six of those located
in the non-transcribed spacers of the plant rRNA genes.
Potential origins are 9 kb apart, but the modal replicon
length is still 54 kb although the sequences of the NTSs
are very similar. Van't Hof speculates that nucleosome
spacing may be an important factor in determining
which of these origins is active (Van't Hof, 1988). This
suggestion is clearly compatible with Gilbert's idea that
initiation of replication at speci®c sequences is dependent
on chromosome structure (as mentioned above) (Gilbert,
1998).
What happens at an origin?
Origins of replication are the places at which DNA replication starts. The general biochemical activities involved in
this are self-evident: the origins must be recognized and
activated and the synthesis of daughter strands must be
initiated. These three activities are now considered.
Recognition of origins
The presence in budding yeast of replication origin
sequences that are essential for function led, after some
false starts, to the isolation of a complex of six proteins,
the origin recognition complex (ORC) that binds to the
ars core sequence of the A-domain in the origin (Bell
and Stillman, 1992). The sequence speci®city is based on
the binding requirements of ®ve out of the six subunits of
the ORC (Lee and Bell, 1997). The role of the ORC is
to `mark' origins of replication and in budding yeast
the ORC is bound to the origin for nearly the whole of
the cell cycle (Dif¯ey and Cocker, 1992; Dutta and
Bell, 1997). However, although the ORC plays no direct
role in origin activation or in initiation, the strict
sequence requirements for ORC binding in budding
yeast that directly parallel the strict sequence requirements for origin function (as described earlier) suggest
that this marking of the origins by the ORC is essential
for the initiation of DNA replication. Since it is now clear
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Bryant et al.
that plants also possess ORC proteins (Gavin et al., 1995)
it is legitimate to ask what happens in respect of origin
recognition in those situations in which extra origins are
used, as described above. Are these extra origins already
`marked', is more ORC synthesized or recruited or do
plants have a less rigid requirement for the presence of the
ORC in the initiation of replication?
This leads back to a consideration of whether plant
and other higher eukaryotic origins are speci®c sequences.
Unfortunately, the limited characterization of plant
ORCs does not reveal anything about their sequence
requirements. However, some data from Xenopus may be
relevant here (Gillespie and Blow, 2000). A cell-free
extract of Xenopus oocytes will replicate sperm DNA,
added to the extract in the form of sperm chromatin. The
oocyte extracts `sets up' replication origins at 10 kb
intervals by the binding of the ORC. In this system at
least, the Xenopus ORC exhibits no speci®c sequence
requirements and there is no speci®c sequence associated
with the sites used as origins. This system is unusual
(indeed, Xenopus embryo cells `revert' to use of speci®c
origins at the mid-blastula transition) but, nevertheless,
the data raise the possibility that origin function in at
least some higher eukaryotes may not always depend on
speci®c sequences. By contrast, in recognizing the origin
of replication within the chorion gene in Drosophila, the
origin recognition complex shows a speci®city for the
440 bp AT-rich tract referred to earlier (Austin et al.,
1999). These data imply that origins may be at least partly
de®ned by sequence. Clearly a more detailed functional
analysis of ORCs in multi-cellular eukaryotes, including
plants, is required.
acting together, namely Cdc6ucdc18 and cdt1 (Maiorano
et al., 2000; Nishitani et al., 2000) (Fig. 1). In budding
yeast, only Cdc6 appears to be involved at this point
(Cocker et al., 1996). Both Cdc6ucdc18 and cdt1 are
regulated by transcription during G1 (Drury et al., 1997;
Lopez-Girona et al., 1998). The dissociation of the
Cdc6ucdc18 protein from the pre-replicative complex (in
®ssion yeast this is driven by a cdc2-kinase-mediated
phosphorylation: Lopez-Girona et al., 1998) and its
subsequent degradation (Drury et al., 1997) all contribute
to the control system ensuring that in a normal cell cycle,
each origin is only ®red once. Further, in budding yeast,
the activity of the MCM complex appears to be
dependent on the dissociation of the Cdc6 protein from
chromatin (Hua and Newport, 1998). The MCM complex
also interacts with Mcm10ucdc23 (S. cerevisiaeuS. pombe)
which is essential for correct DNA replication (Merchant
et al., 1997; Aves et al., 1998; Homesley et al., 2000). The
MCM complex is then phosphorylated by the Cdc7-Dbf4
protein kinase, without which the complex cannot
Activation of origins
In order to set the scene for this topic it is necessary to
discuss data from budding yeast and from ®ssion yeast. In
budding yeast, the region of the origin protected by the
ORC (as de®ned by DNAse protection footprinting)
is extended, from late anaphase and throughout the
G1 phase, by the binding of additional components
(Dif¯ey et al., 1994; Cocker et al., 1996). The increase in
the footprint is due to the binding of an `origin
loading factor' (see below) followed by the binding of
an additional protein complex known as the MCM
complex, consisting of six different MCM proteins
(MCMs 2-7), (so named because they were discovered
as being the products of genes essential for minichromosome maintenance in yeast). The binding of the
MCM complex (or, more probably, two complete MCM
complexes: see below) actually requires the prior recognition of an ORC-marked origin by the loading factor
(Cocker et al., 1996; Kearsey et al., 2000). In ®ssion yeast
and vertebrates the loading factor consists of two proteins
Fig. 1. Diagram illustrating the steps involved in activation of a
replication origin. (A) The origin is `marked' by the origin recognition
complex (ORC). (B) The `loading factors', Cdc6ucdc18 and cdt1 bind to
the DNA adjacent to the ORC. (C) Two MCM complexes load onto the
origin. (D) The loading factors have been displaced from the DNA; the
two MCM complexes have moved apart along the template, generating a
replication `bubble' and displacing the ORC; at each `fork' the Cdc45
protein binds. This will lead to the loading of the initiation complex at
each fork (see Fig. 2). Note: The exact spatial relationships between
several of these proteins are unknown and should not be deduced from
their positions in the diagram.
The initiation of DNA replication
activate the origin. Following this, the MCM proteins are
joined by the protein encoded in budding yeast by
CDC45, the role of which is to facilitate the loading of
the initiation complex (Mimura and Takisawa, 1998; Tye,
1999; Walter and Newport, 2000). This sequence of events
is summarized diagramatically in Fig. 1.
So, what is the role of the MCM complex? MCM
proteins contain a sequence domain that indicates the
possession of DNA helicaseuDNA-dependent ATPase
activity (Koonin, 1993). Direct biochemical demonstration of this helicase activity in individual MCM proteins
is dif®cult and current evidence suggests that the activity
is associated with a sub-complex consisting of MCM4, 6
and 7 (Ishimi, 1997). Rather confusingly, MCM2 appears
to inhibit this helicase activity (Ishimi, 1997). Interestingly, evidence from the archaeon, Methanobacterium
thermoautotrophicum, in which a single MCM gene is
present, shows that the protein exists both as a monomer
and as a double hexamer complex (Kelman et al., 1999)
with both a DNA-dependent ATPase activity and 39 to 59
helicase activity suf®cient to unwind 500 base pairs of
DNA (Kelman et al., 1999; Chong et al., 2000; Shechter
et al., 2000). Although the archaeal system is considerably
simpler than that of eukaryotes, these results suggest that
the MCM complex may act as the replicative DNA
helicase in eukaryotes and archaea (but see below).
Features that are clear are that the activity of the
MCM complex ®rstly displaces, at least temporarily, the
ORC from the origin and secondly allows Cdc45mediated access to the template of the initiation complex,
including primase and DNA polymerase-a. This implies
that extensive enough strand separation has occurred to
allow the formation of a replication bubble. Further
evidence that it is the helicase activity of the MCM
complex that separates the two strands comes from
®ndings that in budding yeast the two MCM complexes
remain associated with or even generate the two moving
replication forks, consistent with their DNA helicase
activity referred to above (Aparicio et al., 1997; Tye,
1999). However, this may not be true of all eukaryotes: in
Xenopus, for example, MCMs are displaced from chromatin after replication is initiated (Coue et al., 1996).
Further, in animals (Wang, 1996) and plants (JA Bryant,
unpublished data) a separate helicase enzyme is part of
the multi-protein complex associated with DNA polymerase-a-primase (see below). The presence of both this
helicase and at least one of the MCM proteins makes it
unclear as to which of these is involved in separating the
DNA strands to generate the replication forks. It may of
course be both.
The situation in plants is, in general, less well-de®ned
than that described above for budding yeast and for ®ssion yeast. Homologues of the CDC6 and cdt1genes have
been detected in plants (Lin et al., 1999; Nishitani et al.,
2000; Whittaker et al., 2000) and their sequences are
197
similar enough to those of budding yeast anduor ®ssion
yeast to indicate a similar function. It is also clear that
plants possess all the members of the MCM complex, as
detected by immunological screening, from cDNA or gene
sequences and by protein puri®cation (Ivanova et al.,
1994; Springer et al., 1995; Sabelli et al., 1996; Moore et al.,
1998; Munns et al., 1998). The gene sequences are again
similar to those of their homologues in yeast, and, for
each member of the MCM complex, are very similar
between different plants. For example, the sequence of
MCM3 from pea is, at the amino acid level, 69% identical
to that from maize, 67% identical to Arabidopsis, and
51% identical to the human MCM3 (Moore et al., 1998).
This is a very high level of sequence conservation and
implies an essential function right across the eukaryotes.
One slight puzzle, however, is that although the sequences
of plant MCM genes may be translated to give protein
sequences of similar lengths to those of other eukaryotes,
the MCM proteins that have been detected all appear to
be smaller on the basis of their mobility in denaturing
polyacrylamide gels. For example, the pea MCM3
appears to have an Mr of c. 66 kDa (Munns et al.,
1998) whereas the size and sequence of the mRNA
suggests a protein of about 100 kDa. The reason for this
discrepancy is not known at present although it is possible either that the plant MCMs are subject to
partial proteolysis or that they behave anomalously in
polyacrylamide gels.
Although the `behaviour' of plant MCM proteins on
the DNA template has not been established, it is clear
that they are nuclear-located (Ivanova et al., 1994; Sabelli
et al., 1999). Further, a recent immunological study with
the maize MCM3 indicates that the intra-nuclear location
of the protein changes during the cell cycle in a manner
that is at least consistent with an association with chromatin for part of the cycle (Sabelli et al., 1999). However,
any dependence of such a binding on the ORC or on
a Cdc6 homologue has yet to be demonstrated. One
further point about plant MCMs is that an MCM3-like
protein has been detected in the primase±polymerase-a
complex (Munns et al., 1998), i.e. the replication initiation
complex (see below). For mammals, the ®rst puri®cation
of an MCM protein was that of MCM3 from the human
primase±polymerase-a (ThoÈmmes et al., 1992) and it
raises the possibility that MCM3 may have a direct role in
loading the initiation complex on to the activated origin.
Initiation of DNA synthesis
Of the multiple DNA polymerases that occur in
eukaryotes, only one is able to initiate the synthesis of
new strands. This is DNA polymerase-a, the initiating
ability of which resides in its close association with
DNA primase (Foiani et al., 1997). The latter is in effect
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Bryant et al.
a very specialized RNA polymerase that lays down
oligo-ribonucleotide primers of about 11 bases on the
single-stranded DNA template generated by the activation of the replication origins and the subsequent outward
movement of the replication forks. The details of this
process have been elucidated with a model system for
eukaryotic DNA replication, namely the replication of a
viral DNA, SV40 (Waga and Stillman, 1994, 1998). In
this system, the activation of the viral origin of replication
and at least the initial separation of the parental DNA
strands is brought about by a virus-encoded protein, the
T-antigen. However, all subsequent steps in replication
are mediated by the host cell (i.e. monkey or human)
enzymes. The ®rst step is the laying down of the primers
for the leading strands; the primers are then extended
a short distance by polymerase-a. However, the polymerase-a very quickly `hands over' to the much more
processive DNA polymerase-d that mediates the bulk of
daughter strand synthesis (details of the latter lie outside
the scope of this review which is focused on initiation). At
this point, the primase±polymerase-a complex switches
strands and becomes responsible for the initiation of
every Okazaki fragment on the lagging strand (Fig. 2).
Although the details of initiation were worked out with
the SV40 model system, there is extensive biochemical
and genetic evidence from a range of eukaryotes to
con®rm the role of primase±polymerase-a as the initiating
enzyme complex. Focusing speci®cally on plants, DNA
polymerases of the a-type have been extensively puri®ed
from several plant species including pea (Stevens and
Bryant, 1976; Bryant et al., 1992), wheat (Litvak and
Castroviejo, 1985) maize (Coello and Vazquez Ramos,
1995), rice (Sala et al., 1981), and spinach (Misumi and
Weissbach, 1982). The pea polymerase-a has an associated primase activity that is able to use ribonucleotides
to synthesize primers on single-stranded M13 bacteriophage DNA; these may then be extended by the polymerase
itself (Bryant et al., 1992). Puri®cation of the pea primase
has so far proved impossible because the enzyme loses
activity on separation from the polymerase. However,
the tobacco (Garcia Maya and Buck, 1998) and wheat
(Laquel et al., 1994) primases have been puri®ed and the
speci®c primase activity of the latter has been demonstrated both on its own and in add-back experiments with
DNA polymerase-a.
Primase and DNA polymerase-a are obviously closely
associated. Nevertheless, there is some evidence that the
polymerase needs assistance in locating the primers laid
down by its associated primase activity. In pea, a DNAbinding protein that initially co-puri®es with polymerasea (Al Rashdi and Bryant, 1994) has been shown to bind
strongly to ds-ss junctions such as would be generated
at the 39-OH terminus of a primer (Burton et al., 1997).
In add-back experiments with primase±polymerase-a,
the DNA-binding protein stimulates signi®cantly the
Fig. 2. Diagram illustrating the initiation of DNA replication at one
replication fork. (A) The primase component of DNA polymerasea-primase synthesizes an oligoribonucleotide primer on the leading
(continuous) strand. (B) DNA polymerase-a synthesizes a short DNA
extension of the primer. (C) DNA polymerase-a-primase switches to the
lagging (discontinous) strand where the primase initiates the ®rst
Okazaki fragment by synthesizing an oligoribonucleotide primer. (D)
DNA polymerase-a continues the synthesis of the ®rst Okazaki
fragment. In the meantime, DNA polymerase-d has taken over the
synthesis of DNA on the leading strand. Polymerase-d is held in place on
the template by PCNA (proliferating cell nuclear antigen). Note: For the
sake of clarity the sub-unit structures of the polymerases, primase and
PCNA have been omitted from the diagram, as has the presence of a
number of accessory proteins.
polymerase activity when the polymerase is working on
M13 DNA templates that have been primed by the
associated primase activity or by the addition of a single
sequencing primer. It does not stimulate polymerase-a
when the latter is working on a template with very closely
spaced primers, such as `gapped' DNA (Bryant et al., 2000).
A very similar primer-recognition protein has also been
puri®ed from human cells (Jindal and Vishwanatha, 1990)
The initiation of DNA replication
where its role has been described as ensuring `productive'
binding of the polymerase to the template adjacent to the
39-OH primer terminus. However, such an activity does
not feature in the details of initiation worked out for the
SV40 model system (Waga and Stillman, 1994, 1998) and
it remains to be seen whether this is a universal feature of
the initiation of DNA synthesis in eukaryotes.
Once and once only
In a `normal' cell cycle, the processes described above
occur just once for each replicon; DNA replication is
completed, but is not re-initiated. Detailed discussion of
this lies outside the scope of this review. It is, however,
interesting to note that several different levels of control
may be envisaged. At the level of origin activation,
Laskey and his associates have proposed that at least in
vertebrates, origins must be licensed by a speci®c factor
that either leaves the nucleus or is degraded after initiation and that a new population of licensing factor may
only gain access to the origins after breakdown of the
nuclear envelope during mitosis. There is in fact some
good evidence for such a factor in vertebrates (Coverley
and Laskey, 1994; ThoÈmmes and Blow, 1997; Donaldson
and Blow, 1999), but not yet in other eukaryotes. Indeed,
for yeasts, which undergo a closed mitosis, this hypothesis
is clearly not applicable in detail. In respect of the yeasts,
the inactivation and turnover of the MCM loading
factors (Cdc6 in budding yeast and cdc18 plus cdt1 in
®ssion yeast) have already been noted as has the cellcycle-phase-dependent transcription of the corresponding
genes. A control based on the availability of these loading
factors may therefore be envisaged. Some support for this
view comes from the ®nding that over-expression of cdc18
in ®ssion yeast does indeed lead to extra rounds of DNA
replication in the absence of mitosis (Lopez-Girona et al.,
1998) (although over-expression of Cdc6 in budding yeast
does not: Drury et al., 1997). However, the most widely
applicable hypothesis is that the normal ¯uctuations of
the cyclin-dependent kinases, determining entry into S
and into M, coupled with the operation of checkpoints, is
enough to ensure that DNA is replicated only once per
cell cycle (Larkins et al., 2001). For example, the ability of
MCMs to bind to chromatin may be modi®ed by phosphorylation by cdks. The G1 and s cdk±cyclin complexes
(cdk±cyclin-A and cdk±cyclin-E) phosphorylate MCMs
at a low level and this permits the MCMs to bind to
chromatin. However, chromatin binding is very much
reduced or even abolished when the MCMs are phosphorylated by cdk±cyclin-B, the kinase that drives the cell
through G2uM. Destruction of cyclin-B in mitosis is
therefore required to permit MCM binding in order to
allow DNA replication in the next cell cycle. None of this
of course precludes the possibility of other levels of
199
regulation such as other licensing factors that may associate with the MCMs, although recent data from plants
and mammals certainly point to the central role of the
cyclin-dependent kinases even in complex multi-cellular
eukaryotes (Larkins et al., 2001; Hattori et al., 2000).
In plants, there are many cell types in which DNA
endoreduplication (re-replication of DNA without an
intervening mitosis) occurs in a highly regulated way as
part of cellular differentiation. Indeed, Larkins and his
colleagues (Larkins et al., 2001) suggest that endoreduplication is so common in plants that it should not
be thought of as an abnormal cell cycle, but simply
a commonly occurring variant of the cell cycle. The same
authors conclude from their own work and those of
others that endoreduplication may be achieved by a loss
of the cyclins and cyclin-dependent kinases that drive the
cell into M-phase. If a background of cdk±cyclin D
expression (Riou-Khamlichi et al., 1999) is maintained in
order to keep the cells potentiated for division (Frank and
SchmuÈlling, 1999), then the normal cell-cycle-based
¯uctuations in the activity of the A and the E cyclins
(plus their corresponding kinases) will be adequate to
drive the cell into repeated S-phases, provided that the
relevant checkpoints may be bypassed (Larkins et al.,
2001). Some evidence that is consistent with this view
comes from the results of Jacqmard et al. who showed
that cyclin-B was present in Arabidopsis cells undergoing
the `normal' mitotic cell cycle, but not in cells that were
endoreduplicating their DNA (Jacqmard et al., 1999).
Concluding comments
The ability to replicate the genetic material is the most
fundamental aspect of living cells. The essential physicochemical features of the process were deduced from DNA
structure nearly 50 years ago (Watson and Crick, 1953)
and con®rmed by the elegant experiments of Meselson
and Stahl ®ve years later (Meselson and Stahl, 1958). Yet
the detailed biochemistry of replication is still being
worked out, especially in eukaryotes. It remains a major
and exciting challenge in this age of genomics to reach
a fuller understanding of the mechanisms and of their
control and then, perhaps even more challenging, to
understand how these processes are integrated within
plant development. There is still much to do.
Note added in proof
The essential role of the MCM complex helicase activity
in strand separation throughout the S-phase has recently
been demonstrated in Saccharomyces cerevisiae. Labib K,
Tercero JA, Dif¯ey JFX. 2000. Uninterrupted MCM 2±7
function is required for DNA replication fork progress.
Science 288, 1643±1647.
200
Bryant et al.
Acknowledgements
KM thanks the BBSRC for a studentship; JAB and SJA
acknowledge grant support from the BBSRC, NATO and the
EU. We are grateful for fruitful discussions with many
colleagues over several years, and especially wish to thank
Dennis Francis, Annie Jacqmard and Jack Van't Hof.
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