Independent Meeting held at the University of Nottingham, 30–31 January 2003. Edited by T. Allers and E. Bolt (Nottingham).
Initiation of archaeal DNA replication
E.R. Jenkinson and J.P.J. Chong1
Centre for Extremophile Research, Department of Biology and Biochemistry, University of Bath, Claverton Down, Bath BA2 7AY, U.K.
Abstract
The identification of DNA as the genetic material and the elucidation of its structure by Watson and Crick
[Watson and Crick, (1953) Nature (London) 171, 737–738], which has its 50th anniversary this year, first
suggested the simple elegance with which the problem of passing on precise genetic information from
one generation to the next could be solved. Semi-conservative replication is perhaps one of the simplest
biological concepts to explain and understand. However, despite an enormous amount of effort in the
intervening years, details of the way in which this process is regulated and performed are still unclear in
many organisms. Recent work suggests that, due to their simplicity, the Archaea may make a good model
for understanding some of the aspects of eukaryotic replication that still elude us.
An overview of replication
Replication is an essential process in both prokaryotic and
eukaryotic organisms that must occur in order for cells to
divide and proliferate. To ensure the integrity of the genome
for future generations, the duplication of the genetic material
has to be tightly controlled, as alterations in chromosome
number result for the most part in decreased cell viability. In
all organisms, replication must occur from a defined point
on the chromosome: the origin sequence. In Escherichia
coli the circular chromosome consists of a single replicon
(a region of DNA replicated by the action of a single pair
of replication forks), and bi-directional replication proceeds
from a single origin (oriC). In eukaryotes, many origins of
replication (10 000–100 000) are required in order to make
each replicon a manageable size. This is due to the sheer
quantity of DNA to be copied and further necessitated
by the packaging of the DNA into multiple chromosomes.
Having multiple origins on multiple chromosomes massively
increases the complexity of the DNA-replication task, and
so eukaryotic cells ensure each replicon is replicated only
once by a process of replication licensing, which functions to
maintain the correct gene copy number in daughter cells.
All organisms use initiator proteins to recognize and
begin replication at origins of replication. In E. coli, the
Key words: DNA replication, initiation, mini-chromosome maintenance (MCM).
Abbreviations used: pre-RC, pre-replicative complex: ORC, origin-recognition complex; MCM,
mini-chromosome maintenance; SSB, single-stranded DNA-binding protein.
1
To whom correspondence should be addressed (e-mail [email protected]).
widely studied DnaA protein is responsible for origin recognition [1–3]. The subsequent loading of the DnaB helicase by interaction with DnaC [4], and activation of the
helicase to initiate unwinding of the duplex, allows entry
of the DNA polymerase machinery so that replication can
proceed. Initiator proteins are also required in eukaryotes
but the formation of a pre-replicative complex (pre-RC)
is essential before the firing of origins can occur [5].
The origin-recognition complex (ORC) is a multisubunit
protein complex responsible for binding to and marking
the origin sequences in all eukaryotes [6–8]. During the
G1 phase of the cell cycle, ORC also functions to
recruit Cdc6 and Cdt1 proteins. Controlling the location
and availability of these proteins has a significant role
in determining the once-per-cell-cycle rule of replication
[9–13]. The most important function of these proteins is the
recruitment of the mini-chromosome maintenance (MCM)
complex to the origin [14], suggesting that they play a
role equivalent to bacterial DnaC. Loading of the MCM
proteins is the final step in establishing the pre-RC and
subsequent signals from cyclin-dependent kinases and Dbfdependent kinases activate the licensed origin for initiation
of replication. DNA replication systems are illustrated in
Figure 1.
Molecular Mechanisms and Manipulation in Archaea Independent Meeting
Molecular Mechanisms and
Manipulation in Archaea
Archaea as a model system
The Archaea have been identified as the ‘third domain
of life’ [15]. They are divided into three main phyla, the
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Figure 1 DNA replication in the three domains of life
In each example, replication proceeds bi-directionally from single (prokaryotes, Archaea) or multiple (eukaryotes)
origin sequences. Origins are recognized by specific sequences in prokaryotes. Less is known about the eukaryal origin
sequences although consensus elements are found in Saccharomyces cerevisiae and to a certain extent in Schizosaccharomyces pombe. In each case, initiation requires loading of a helicase protein to unwind the double helix: DnaB in
prokaryotes, MCM complexes in eukaryotes and Archaea. CDK, cyclin-dependent kinase; DDK, Dbf-dependent
kinase.
crenarchaeota, the euryarchaeota and the korarchaeota
(with the recent addition of a fourth group named the
nanoarchaeota on the basis of their size [16]). The Archaea
have a prokaryotic cell morphology showing an absence
of nuclear structures, cytoskeletons and organelles [17,18].
Evidence from studied species points to a prokaryote-like
genome arrangement and it has recently become evident
for some species that a single bi-directional origin of
replication is employed [19]. Sequence information from
several archaeal genomes suggests a degree of homology
between eukaryotic transcription, translation and replication
proteins while most metabolic processes are comparable
with prokaryotes [17]. Many euryarchaeal species also
contain homologues to the eukaryotic histone proteins,
present in eukaryotes to organize chromatin into higherorder structures but normally absent from prokaryotic cells
[20,21]. The Archaea thus pose an interesting evolutionary
question as regards their phylogenetic relationship with
the other two domains of life [15,17]. Archaea provide us
with a simplified model containing a more primitive protein
network that may aid in unravelling the complex replication
mechanisms observed in eukaryotes. This situation can
be exploited to try to understand the processes of DNA
replication in eukaryotes. In particular, studies on two
sequenced Archaea, the euryarchaeon Methanothermobacter
thermautotrophicus and the crenarchaeon Sulfolobus solfataricus, are helping to shed light on the involvement of
the MCM complex in pre-RC formation and replication
initiation.
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Pre-RC formation and replication licensing
in Archaea
The process of replication appears to be conserved between
Archaea and eukaryotes with some significant differences.
The lack of an obvious ORC is the first major difference
in Archaea. In every sequenced archaeal species to date,
homologues to the ORC proteins are absent. However, most
Archaea (except Methanococcus jannaschii and Methanopyrus
kandleri) have one or more Cdc6 homologues. The
precise number of putative Cdc6s differs between species;
for example M. thermautotrophicus contains two copies,
Pyrococcus species contain one, the Sulpholobales contain
three and Halobacterium sp. has up to 13 genes. This may
have evolutionary implications, with the Cdc6 protein being
responsible for multiple roles within the archaeal pre-RC
formation. Cdc6 and some ORC subunits do share a certain
degree of homology, including some structural homology
with a conserved winged helix domain in both Cdc6 and
Orc1 [22]. Thus Cdc6 may play a dual role in both origin
recognition and subsequent loading of the MCM proteins.
It should be noted that the archaeal Cdc6 has structural
similarity to the bacterial origin-recognizing protein, DnaA
[23].
The second fundamental difference between eukaryotes
and Archaea is the lack of any Cdt1 homologue, responsible
for loading eukaryotic MCMs at the pre-RC. However, the
sequence of Cdt1 in eukaryotes appears highly divergent,
hence the Saccharomyces cerevisiae homologue has only
Molecular Mechanisms and Manipulation in Archaea
Figure 2 Schematic diagram of motif arrangements in MCM proteins
The core catalytic complex is made up of MCMs 4/6/7. This sub-complex possesses helicase activity in vitro. The regulatory
subunits (Reg s.u and Reg Dimer) MCM2 and MCM3/5 abolish this activity in vitro. MCMs 2, 4, 6 and 7 possess zinc-finger
domains which can also be identified in some archaeal MCMs. NLS, nuclear localization sequence.
recently been identified [11,24,25]. Before Cdt1 was identified
in this simple eukaryote, it was speculated that the absence
of a Cdt1 implied that its function was only required
in those eukaryotes with more complex origins, such as
Schizosaccharomyces pombe and metazoans [11,24]. Since the
identification of Cdt1 in the simplest of eukaryotes, it may
only be a matter of time before an archaeal homologue of
Cdt1 is discovered.
The conservation of the MCM helicase proteins, which
are probably required for DNA unwinding and replicationfork progression is at present of most interest. Much of
the biochemical information we have relating to archaeal
replication concerns this protein; these data demonstrate
activities consistent with those found in eukaryotic MCM
complexes. This substantiates the use of the Archaea as a
model system for understanding the molecular mechanisms
of DNA unwinding in eukaryotes.
The MCM helicase
Helicases are an integral part of many cellular processes
and defects in helicase activity have been linked to various
cancer-prone conditions and aging disorders [26]. The precise
function of a helicase is the coupling of NTP hydrolysis to
nucleic-acid unwinding by using the chemical energy derived
from the hydrolysis activity to catalyse the separation of
the DNA and/or RNA duplex [26]. All helicase enzymes
contain a series of highly conserved protein sequences, the
‘helicase motifs’, which have been shown to be essential in
the functioning of this class of enzymes [27]. Helicases can
be divided into four distinct superfamilies. The only motifs
common to all superfamilies are motifs I and II, the Walker A
and B motifs that are involved in NTP binding and hydrolysis
respectively. Helicases have been described that differ in
both the numbers of subunits that make up an active protein
and the mechanism of action by which they work. Monomeric, dimeric and hexameric helicase complexes have all
been described [27]. Models described for mechanisms based
on varying subunit compositions include the ‘inchworm’
model for monomeric helicases and a ‘rolling’ method for
dimers [27]. Various models have been proposed in the case of
hexameric helicases due to the apparent topological problems
in loading a ring-shaped complex on to nucleic acid. These
include disassembly and reassembly of the hexamer around
the substrate, and a threading of the free substrate ends
into the central cavity of the helicase [26]. Experimental
evidence would suggest that the most likely model is of a
facilitated ‘ring opening’ step prior to loading. This is seen
for both the DnaB helicase and the T7gp4A helicase-primase
hexamer [26,28,29].
The MCMs were first described in yeast by a genetic
screen that identified genes containing mutations that resulted
in strains that were unable to maintain mini-chromosomes,
indicating a role in DNA replication [30]. There are six
MCMs in all eukaryotes, MCMs 2–7 [31,32] (see Figure 2).
It is believed that a heterohexameric MCM complex is
responsible for initiating unwinding at the origin and also for
elongation of the replication fork by processive unwinding
[33]. In addition, at least one MCM homologue has been
identified in all sequenced archaeal genomes. A single copy
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is found in most Archaea, with notable exceptions being
M. jannaschii, which has four, and Methanosarcina acetivorans and M. kandleri, both of which have two annotated
genome copies, although only one gene in each case contains
functional Walker motifs (J.P.J. Chong, unpublished work).
All MCMs share a great deal of homology, particularly
in the central domain of the protein, which spans some
200 amino acids and includes the NTP-binding and hydrolysis domains [27].
MCM complex formation
In addition to the pre-dominant heterohexamer containing
MCMs 2–7, the eukaryal MCMs can form a series of smaller
sub-complexes [34,35]. The heterohexamer is probably the
species recruited to the origin. However, in vitro the only
helicase activity associated with eukaryotic MCMs has been
found in a double trimer of MCMs 4/6/7; the heterohexamer
itself exhibits no detectable activity [36–38]. The MCM4/6/7
double trimer has a 3 →5 helicase activity in the presence
of ATP and dATP, but in vitro processivity is limited to
unwinding some 30 bp of duplex [37]. Processivity is greatly
enhanced by the addition of single-stranded DNA-binding
protein (SSB) and the use of forked substrates where up
to 600 bp of duplex may be unwound [39]. Other subcomplexes that have been identified include a dimer of
MCMs 3 and 5, which appears to function as a regulatory
subunit, inhibiting the helicase activity of the MCM4/6/7
double trimer [36,40]. Similarly, formation of a MCM2/4/6/7
tetramer abolishes helicase activity, indicating a regulatory
role for the MCM2 monomer [37]. One model that would
explain this data is that an MCM4/6/7 catalytic core is
regulated by both the MCM2 monomer and the peripheral
MCM3/5 dimer, with helicase activity requiring the removal
of both MCM2 and MCM3/5 once the hexamer has loaded
on to DNA. On the other hand, data regarding degron
mutants of each of the MCMs in S. cerevisiae demonstrate
a requirement for all six MCMs before, during and after
initiation steps [41]. Whether the in vitro helicase activity
is representative of the in vivo MCM activity and the exact
in vivo composition of the functional complex is a subject for
further investigation.
All the archaeal MCM homologues are more closely related
to eukaryotic MCM4 than any of the other MCMs. Archaeal
MCMs form complexes consistent with homohexamers.
In the case of S. solfataricus, the active species has been
shown to consist of a single homohexamer [42]. The
M. thermautotrophicus MCM protein has been more widely
studied and apparently forms a homododecameric structure
[43–45], although recent electron microscopy evidence points
to the possible formation of a seven-membered ring [46].
Studies of this complex have led to the widely held belief
that despite the weak eukaryal helicase activity, the MCM
complex is in fact responsible for DNA unwinding during
replication. The M. thermautotrophicus MCM complex has
been shown to possess ATP hydrolysis activity stimulated
by the presence of DNA. Mutations of the conserved lysine
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residue in the Walker A motif abolish this activity [43,45]. The
M. thermautotrophicus MCM complex has also been shown
to display processive 3 → 5 helicase activity, displacing
up to 500 bp of DNA in vitro. This activity requires the
presence of ATP or dATP but no other additional co-factors
[43–45]. In contrast, studies of the S. solfataricus MCM resulted in an ATP-dependent 3 → 5 helicase activity that was
stimulated greatly by the addition of an archaeal SSB. Further
characterizations revealed a physical association between SSB
and the S. solfataricus MCM hexamer [42]. Furthermore,
ATPase activity of the S. solfataricus MCM complex was not
stimulated by the presence of DNA [42], an observation in
contrast with that seen for other helicase and MCM enzymes.
These differences may reflect a modulation of activity in
the crenarchaeota as opposed to eukaryotes and the euryarchaeotes.
A role for the zinc-finger motif in
MCM function
Zinc-finger motifs have been identified in many proteins
requiring DNA or protein contacts. They have been found
in both eukaryotic and archaeal MCM proteins. In particular,
the zinc-finger motif is highly conserved between yeast and
higher-eukaryotic MCMs 2, 4, 6 and 7, suggesting that
this motif may play an important role in the functioning
of these proteins. Mutation of the zinc-finger motif of
MCM4 resulted in disruption of the MCM4/6/7 trimer–
trimer interactions in the active MCM4/6/7 hexamer and
resulted in single trimeric complexes [47]. This suggests
that the zinc-finger motif in MCM4 is required for stable
hexameric subunit interactions. The MCM2 zinc finger has
been implicated in protein–DNA contacts. Phosphorylation
of this subunit in the pre-RC may cause conformational
changes and could be responsible for releasing the MCM
complex from the origin and allowing replication to proceed
[48]. The M. thermautotrophicus MCM protein also contains
the zinc-finger motif that is found within a non-conserved
region of the eukaryotic MCM sequence. This motif is also
seen in the Archaeoglobus fulgidus and the S. solfataricus
MCMs but in only one of the four M. jannaschii MCM
sequences [49]. The archaeal zinc finger is comparable with
that of eukaryotic MCM2 (Cys-Xaa2 -Cys-Xaan -Cys-Xaa2 Cys). This zinc finger is apparently dispensable for the
M. thermautotrophicus MCM protein–protein interactions
and complex formation, as mutations in this motif in
M. thermautotrophicus MCM do not affect multimerization
[50]. However, a defect in Zn2+ -binding ability affects DNAdependent ATPase activity, single-stranded-DNA-binding
ability and helicase activity, indicating a role for this motif
in MCM function [50].
Are the Archaea a good model system for
understanding replication in eukaryotes?
The molecular mechanisms of the proteins required for
initiation of DNA replication, in particular the biochemistry
of the MCM complex, appear to be conserved between
Molecular Mechanisms and Manipulation in Archaea
Archaea and eukaryotes. Both domains of life contain MCM
and Cdc6 homologues and strong similarities exist between
eukaryotic and archaeal MCM activities in vitro. In addition,
although not discussed here, many of the archaeal proteins
likely to be involved in replication-fork progression also share
homology with their eukaryotic equivalents. Regulation of
the timing and activation of DNA replication is likely
to be under an entirely different form of control in the
Archaea. In eukaryotes, an additional ‘layer’ of regulation
controls the firing of origins possessing assembled pre-RCs.
This regulation involves Dbf-dependent kinase and cyclindependent kinase activity, two classes of enzyme for which no
archaeal homologues have yet been identified. An interesting
question therefore is how the replication competency of
an archaeal cell is controlled. Modulation of proteins by
post-translational modifications such as phosphorylation
or adenylation may play a role. Many of these control
mechanisms are yet to be understood and the identification
of more proteins specific to archaeal DNA replication will no
doubt follow.
We thank Dr Alan Majernı́k and Dr Setareh Sepehri Chong for critical
reading of this review. J.P.J.C. is a Biotechnology and Biological
Sciences Research Council David Phillips Research Fellow.
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