FEMS Microbiology Reviews 26 (2003) 533^554
www.fems-microbiology.org
Common domains in the initiators of DNA replication in Bacteria,
Archaea and Eukarya: combined structural, functional and
phylogenetic perspectives
Rafael Giraldo
Department of Molecular Microbiology, Centro de Investigaciones Biolo¤gicas (CSIC), C/Vela¤zquez 144, 28006 Madrid, Spain
Received 11 October 2002; received in revised form 12 November 2002; accepted 13 November 2002
First published online 7 December 2002
Abstract
Although DNA replication is the universal process for the transmission of genetic information in all living organisms, until very
recently evidence was lacking for a related structure and function in the proteins (initiators) that trigger replication in the three ‘Life
Domains’ (Bacteria, Archaea and Eukarya). In this article new data concerning the presence of common features in the initiators of
chromosomal replication in bacteria, archaea and eukaryotes are reviewed. Initiators are discussed in the light of: (i) The structure and
function of their conserved ATPases Associated with various cellular Activities (AAA+) and winged^helix domains. (ii) The nature of the
macromolecular assemblies that they constitute at the replication origins. (iii) Their possible phylogenetic relationship, attempting to
sketch the essentials of a hypothetical DNA replication initiator in the micro-organism proposed to be the ancestor of all living cells.
4 2002 Federation of European Microbiological Societies. Published by Elsevier Science B.V. All rights reserved.
Keywords : DNA replication initiators; Origin binding proteins ; AAA+ domain ; Winged^helix domain ; Phylogeny
Contents
1.
2.
3.
4.
5.
Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
The structure and function of OBPs in Gram-negative bacteria . . . . . . . . . . . . . . . . . . . .
2.1. DnaA, the universal initiator of chromosomal replication in Bacteria, is an origin-speci¢c
unwinding AAA+ . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
2.2. Plasmid Rep OBPs as minimal initiators consisting in origin binding WH modules . . .
The structure and function of OBPs in Archaea and Eukarya . . . . . . . . . . . . . . . . . . . . . .
3.1. Eukaryal ORC and Cdc6 as combined AAA+/WH machines in origin binding and
remodelling . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
3.2. Archaeal Orc1/Cdc6 OBPs are simpli¢ed versions of eukaryal AAA+/WH . . . . . . . . .
Phylogeny of OBPs domains: Outlining the ancestral initiator . . . . . . . . . . . . . . . . . . . . .
4.1. The three-dimensional structures of a few replication proteins unveil missed sequence
similarities . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
4.2. Possible alternative scenarios for the phylogenetic origin of OBPs . . . . . . . . . . . . . . .
Concluding remarks . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
533
534
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540
540
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544
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547
547
Acknowledgements . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
549
References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
549
1. Introduction
* Tel. : +34 (91) 5611800 (ext. 4269) ; Fax : +34 (91) 5627518.
E-mail address : [email protected] (R. Giraldo).
Since the early times of Molecular Biology, the universality of the processes involved in the transmission of ge-
0168-6445 / 02 / $22.00 4 2002 Federation of European Microbiological Societies. Published by Elsevier Science B.V. All rights reserved.
PII : S 0 1 6 8 - 6 4 4 5 ( 0 2 ) 0 0 1 4 5 - 6
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netic information has been interpreted to re£ect the common evolutionary origin of all known organisms. Thus, it
is currently believed that life emerged from the con£uence
between self-replicating RNA molecules (the prebiotic
‘RNA world’) [1,2] and self-assembling lipids [3,4], with
the energy provided by organometallic chemistry [5,6].
RNA became then able to direct protein synthesis (a primitive translation system) [7] and was later replaced by
DNA (reverse transcription) as the kind of genetic material ubiquitous in all extant living cells [8]. Therefore DNA
replication, compared with transcription and translation,
would be a more recently developed process [8^10] providing us with one of the latest landmarks to de¢ne, in molecular terms, the Last Universal Common Ancestor
(LUCA) of cellular life on Earth [11^15].
In the classic replicon model [16], regulated DNA replication requires a trans-acting factor (the initiator) able to
speci¢cally bind to a cis-acting DNA sequence (the replicator, or replication origin), thus resembling the proposal
made shortly before for the regulatory circuits in gene
expression. For the last 40 years, the replicon has consistently received multiple experimental support (reviewed in
[17]). In evolutionary terms, for the primordial forms of
cellular life the appearance of both initiator proteins and
origins of replication might be the answer to the requirement for a controlled copy of the genetic information.
This is thus integrated with the rest of cellular functions,
being genomes replicated once, and only once, each cell
cycle. This need, risen by the appearance of the cellular
level of organization, did not previously occur in the prebiotic world of self-replicating molecules.
DNA replication initiators are either single proteins, or
multisubunit complexes, that bind sequence-speci¢cally to
the origins of replication (thus they are also known as
Origin Binding Proteins, OBPs), where they usually assemble into oligomers [18^20]. Initiators play two roles: (i)
they melt the two strands of DNA and (ii) they bring to
the resulting replication bubble other protein factors essential for replication. These are responsible for the extension of the replication fork (helicases), synthesis of an
RNA primer (primases) and copying the template with
high ¢delity and processivity (DNA polymerases) [17,18].
It is a common trait that all OBPs, in spite of their di¡erent nature and precise mechanisms of action (Table 1) [17^
29], require to be activated in order to exert their triggering role in DNA replication [30,31]. Thus ATP binding
and hydrolysis can de¢ne active or silent conformational
states [19]. In addition, posttranslational modi¢cation
(e.g., by phosphorylation) [31^33] and/or down-regulation
by proteolysis [34^36] often are key events in controlling
initiator function. Apart from the speci¢c sequences where
OBPs bind, replication origins include conserved AT-rich
repeats with enhanced tendency to be unwound under
superhelical stress, or by the action of non-sequence speci¢c DNA binding proteins [17].
The mechanisms to achieve DNA replication initiation,
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astonishingly diverse, are summarized in Table 1. The
structure and function of the OBPs in a number of viruses
with miscellaneous types of initiation [23,25^29] and in
plasmids replicating through a rolling circle-type mechanism (that implies origin nicking and extension of the resulting 3P-end, displacing the non-template strand) [20^22]
have been recently reviewed elsewhere. The same occurs
with details concerning the macromolecular machines directly involved in DNA synthesis at the replication fork
[18,37]. Therefore, this review is focussed in those OBPs
that initiate DNA replication by a functionally analogous
mechanism, implying origin binding and DNA melting,
namely OBPs in Gram-negative bacteria plasmids and in
all bacterial, archaeal and eukaryal chromosomes. In particular, the presence of common structural domains in this
kind of OBPs, beyond their marginal sequence similarities,
leaves the way open to discuss their possible phylogenetic
relationships [8,9]. With the knowledge already available
on present OBPs, maybe it is time to start outlining the
structure and properties expected for the primordial initiator of DNA replication in the hypothetical ancestral
micro-organism known as LUCA.
2. The structure and function of OBPs in
Gram-negative bacteria
All known bacteria, either Gram-negatives or -positives,
replicate the DNA of their single chromosome by means
of an essentially identical mechanism that, in terms of
OBPs, is triggered by binding of the DnaA initiator protein to a conserved, unique origin of replication (oriC) [38^
41]. However, initiation of plasmid extrachromosomal
DNA elements follows a variety of mechanisms, classi¢ed
according to the shape of the resulting replication intermediates: rolling circle (c), strand displacement (D-loop)
and ‘theta’ (a) [20,21]. The last type of mechanism is the
most common in Gram-negative bacteria. A number of
such plasmid replicons have been intensively used over
the last 25 years, apart from their intrinsic interest as
vectors of threatening antibiotic resistances and in biotechnological and environmental applications, as valid model
systems to get hints on chromosomal replication. This is
due to the fact that, albeit many plasmids encode for their
own OBP (termed Rep), they still depend on the cellular
factors required for DnaA/oriC replication [20,21].
2.1. DnaA, the universal initiator of chromosomal
replication in Bacteria, is an origin-speci¢c
unwinding AAA+
DnaA is the OBP functional in all bacteria characterized
so far. DnaA has been extensively studied in Escherichia
coli, both from the biochemical and genetic points of view,
illustrating that it is not only central to initiation, but a
key regulator of bacterial cell cycle [41,42]. Since there are
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535
Table 1
A summary of the distinct macromolecular assemblies, also termed SNUPS (Specialized NUcleoProtein complexeS, displayed as cartoons), and initiation
mechanisms for the diverse OBPs (shadowed ovals) found in viruses, plasmids and chromosomes across the three ‘Life Domains’
Dashed horizontal lines mark the boundaries between Bacteria, Archaea and Eukarya, being the former thicker to indicate that the OBPs in Archaea/
Eukarya are thought to be more similar each other than to those in Bacteria [8]. The initiator proteins that are the subject of this review are typed outlined. Data to elaborate this table were taken from references [17^29].
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Fig. 1. The structures of the OBPs of: (A) a bacterial chromosome, AeDnaA from A. aeolicus (Protein Data Bank, PDB, entry 1L8Q) [56], (B) a
Gram-negative bacteria plasmid, RepE54 from E. coli mini-F plasmid (PDB entry 1REP) [94], and (C) an archaeal chromosome, PaCdc6 from P. aerophilum (PDB entry 1FNN) [60]. In the AeDnaA model (A), the AAA+ domain III (in purple) has been displayed, as a ribbon model of the peptide
backbone, with the same orientation shown for the equivalent domain in PaCdc6 (C). The Mg2þ -ADP cofactor (in red) is sandwiched between the two
characteristic K/L plus all K-helical subdomains [58]. The DBD IV (HTH), structurally similar to Trp repressor [62] is in grey. In RepE54 model (B),
corresponding to the monomeric species active in origin binding and initiation [84], DNA (present in the original crystal structure) has been removed
for clarity. Two di¡erent orientations of RepE54 have been displayed to highlight : Left hand, the pseudo-two-fold axis (dashed line) relating the two
WHs (three K-helices bundle, plus a three-stranded antiparallel L-sheet), in orange and pink, respectively, for the N-terminal and C-terminal domains
(additional secondary structure elements are in grey). Right hand, the same WH1 orientation shown below for the WH in PaCdc6. In the PaCdc6 model (C), the N-terminal AAA+ domain has been colored in green, with its Mg2þ -ADP cofactor in red. The C-terminal WH is shown in blue. Models
were rendered with Swiss-Pdb Viewer (http://us.expasy.org/spdbv).
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a number of excellent recent reviews on its function [38^
41], this article deals with the structure of DnaA domains.
Based on sequence similarities across di¡erent bacterial
species [38], four domains have been characterized in
DnaA, involved in a number of speci¢c functions. In
E. coli (Ec), the protein (52.5 kDa) consists in (from its
N-terminus) : (I) A conserved domain (residues 1^57), including a Leucine Zipper (LZ)-like oligomerization motif
(1^23) [43], required to support both chromosomal and
plasmid replication [44], and a tight binding site for the
DnaB helicase (24^86) [45,46]. This overlaps with (II), the
less conserved region (residues 57^129), carrying various
insertions in di¡erent bacterial species [38,39]. (III) An
ATP binding domain (residues 130^350), essential for
chromosomal initiation, that includes canonical WalkerA/B motifs [47] (see below), the residues (135^148) initially
contacted by DnaB [45] and two potential K-helices (327^
344 and 357^374) that bind acidic phospholipids, altering
537
the conformation of the domain, to release ADP [48^51].
(IV) A DNA binding region (374^467) [52^54] that, based
on secondary structure predictions, was proposed to consist in an K-helical bundle [55].
The crystal structure of a fragment, including domains
III and IV, from Aquifex aeolicus (Ae) DnaA has been
recently solved [56] (Fig. 1A). As it had been previously
modelled [55], the fold of the ATP binding domain III
belongs to the AAA+ superfamily [57^59]. The AAA+
group includes chaperones (often coupled to proteolytic
subunits) that unfold protein targets, inducers of membrane fusion, motor proteins and remodellers of DNA.
The structure of AAA+ consists in two subdomains: an
N-terminal K/L RecA-like fold (a ¢ve-stranded parallel
L-sheet £anked, respectively, by two and at least three
K-helices) plus a C-terminal K-helical bundle [56,60]. The
N-terminal subdomain includes two Walker motifs, for
nucleotide binding and hydrolysis, respectively: A (or
Fig. 2. Functional similarities between the OBPs in bacterial chromosomes (A, DnaA initiator in E. coli oriC) [38^40], Gram-negative bacteria plasmids
(B, RepA initiator in Pseudomonas pPS10 oriV) [86], and eukaryotic chromosomes (C, ORC initiator in S. cerevisiae ARS) [130]. For the three OBPs,
initiator function requires two steps: (I) dissociation and conformational activation; (II) speci¢c binding to origin DNA repeats (blue boxes on the double helix). For DnaA-dependent initiation at oriC, the subsequent unwinding step (III) of the A+T-rich origin repeats (red boxes) has been also depicted, but analogous steps have been shown to occur in plasmid and eukaryotic initiation. In the absence of origin sequences, OBPs tend to form inactive aggregates (colored in salmon). These are either dimers, as described for RepA repressor species (established through its N-terminal WH) [90] or
for the isolated Orc4 subunit (through its C-terminal WH) [187], or oligomers as characterized for ADP-bound (magenta) DnaA [75,76] and the N-terminal AAA+ domain of Orc4 [187]. Protein chaperones of the Hsp70/DnaK family (yellow) [110,111,113^116,77^78,187], or the allosteric e¡ect of speci¢c origin DNA sequences (oriV iterons, for RepA) [99], untangle those aggregates (grey arrows) to yield the OBPs species active in initiation. This implies a conformational change (RepA N-terminal WH) and/or the assembly of other OBP subunits (Orc1-3,5-6, in green). Speci¢c origin recognition
requires ATP (blue) in the nucleotide binding sites (white pockets) of the AAA+ domains in DnaA (purple) [56,69^70] and Orc1/5 [134]. For DnaA
[48^51] and ORC [148], acidic phospholipids (PLs) contribute to ADPCATP interchange. The modules responsible for origin DNA binding are the
C-terminal K-helical bundle in DnaA (grey) and the activated WHs in RepA and Orc4 (orange), together with the C-terminal WH in RepA (pink) or
the DBDs in other ORC subunits.
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P-loop), GX4 GKT (residues 172^179 in E. coli DnaA) and
B, LLIDD (in DnaA, 232^236). In Walker-A the conserved Lys contacts the L- and Q-phosphates of ATP,
whereas the Thr contributes to coordinate the essential
Mg2þ [56,60]. In Walker-B the two Asp carboxylates act
(i) as partners for the metal and (ii) as a base activating a
water molecule for nucleophilic attack on the Q-phosphate
[56,60]. Residues responsible for ATP binding are not only
located in the Walker-A motif at the N-terminal subdomain, but also in two additional motifs, termed sensor-1
and 2. More speci¢cally, a conserved Arg residue in sensor-2, found at the N-terminus of the third K-helix in the
C-terminal subdomain, senses the presence or absence of
the Q-phosphate [56,60]. In addition, the purine and ribose
rings of ATP stay in a pocket constituted by residues from
both subdomains. Therefore, nucleotide binding and hydrolysis result in a change in the position of each subdomain respect to the other [56,61]. Furthermore, in AeDnaA, a long K-helix (K12) connects domain III with the
C-terminal DNA binding domain IV [56]. It probably
would act as an e¡ector, transmitting to the later the conformational changes occurring in the former upon ATP
hydrolysis [61]. These movements, coordinated across the
distinct subunits of the assemblies that they usually establish [58], appear to be crucial for the function of AAA+
proteins. Thus, among others, in pulling apart the two
strands of DNA (unwinding, in replication or recombination), unravelling the secondary structure elements in a
protein (unfolding, often for proteolytic degradation) or
prying subunits apart (in disassembling oligomers) [59].
In AeDnaA crystal structure [56], the C-terminal domain IV consists in a classic, all K-helical, Helix^Turn^
Helix (HTH) motif related to that found, among many
other DNA Binding Domains (DBDs), in the Trp repressor [62] (Fig. 1A). In addition to the HTH motif, an extra
basic loop (including a conserved Arg residue) could contact DNA minor groove or the phosphate backbone [56].
ATP is not strictly required for the sequence-speci¢c binding of DnaA monomers to ¢ve, double-stranded, 9-mer
repeats (DnaA boxes R1-R4 and M: 5P-TTA/TTNCACA)
found at oriC [63^65], where up to 20^30 DnaA molecules
¢nally form an oligomeric nucleoprotein complex [66^68]
(Fig. 2A). However, it is essential for the speci¢c binding
of DnaA to six secondary sites (ATP-DnaA boxes: 5PAGatct) found in the AT-rich repeats adjacent to the
high-a⁄nity R1 box [69,70]. Cooperative binding of
DnaA to the R1 and ATP-DnaA boxes, involving its oligomerization domain I [43], results in DNA unwinding,
stabilization of the single strands by ATP-DnaA [70] and
then in loading the DnaB helicase by displacing its loader
DnaC (both are hexameric ATPases) [71]. Subsequent
ATP hydrolysis, stimulated by the processivity factor of
DNApol III (L-clamp) and Hda [72,73], exerts a conformational change in DnaA [74] that becomes unable to
initiate further replication rounds, a process termed
RIDA, standing for ‘Regulatory Inhibition of DnaA’
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[41,75,76]. DnaA can then be re-activated either by acidic
phospholipids [48^51] or DnaK chaperone [77,78] (Fig.
2A), that exchange ADP by ATP. It is noteworthy that
DnaB, although it stabilizes its oligomeric state in a similar ATP-Mg2þ dependent way than the hexameric/heptameric assemblies characteristic of some AAA+ proteins
[57^59], is not a member of this family, on the contrary
to its adaptor DnaC [19].
2.2. Plasmid Rep OBPs as minimal initiators consisting in
origin binding WH modules
A number of plasmid replicons have been studied in
their functional details by means of genetic and biochemical approaches [20,21]. Thus P1 [79], R6K [80], RK2 [81]
and pSC101 [82] replicons, among others, contribute in
great manner to our current view of plasmid replication
in Gram-negative bacteria. This section is focussed in
RepA (26.6 kDa), the initiator protein of pPS10, a plasmid isolated from the phytopathogen Pseudomonas savastanoi [83], and RepE, the initiator of E. coli mini-F plasmid [84], since both proteins are the best characterized in
structural terms. There are clear sequence similarities between pPS10 RepA, mini-F RepE and other plasmid Rep
proteins [20,21]. Thus both OBPs are valid general model
systems to study the structure and function of plasmid
initiators and their relation with chromosomal DNA replication.
Rep proteins usually bind to directly repeated sequences
(iterons) found at their respective replication origins (oriV)
to establish the initiation complex [84^86] (Fig. 2B). In
addition, protein^protein interactions between Rep molecules bound to two iteron tracks, located distant in either
the same or in di¡erent DNA molecules, have been proposed to be a means of negative control of initiation
(termed ‘handcu⁄ng’) by pairing origins together [87^
89]. Some Rep proteins also bind to an inversely repeated
sequence (operator) that overlaps with the promoter of the
rep genes, thus acting as self-repressors [84,90]. An HTH
motif at the protein C-terminus is the main determinant of
Rep binding to both operator and iteron DNA sequences
[91,92]. The most abundant Rep protein species in solution
are dimers, that bind to the operator, whereas Rep monomers bind to the iterons [84^86,90]. In pPS10 RepA, mutations in a LZ sequence motif found at its N-terminus
(residues 12^33) [93] enhance dimer dissociation [85]. It
was predicted [86] that RepA consists of two WH domains
(residues 1^132 and 133^230, respectively), a proposal
then con¢rmed by the crystal structure of RepE54 (Fig.
1B), a mutant monomer of the related mini-F OBP, bound
to iteron DNA [94].
WH are a large family of DBDs, found in proteins of
both prokaryotic and eukaryotic sources, which ¢rst members to be identi¢ed, on the basis of their crystal structure,
were the ‘Fork-Head’ transcription factor HNF-3Q, the
globular core of the linker histone H5, CAP and LexA
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(reviewed in [95]). WH domains are composed of a bundle
of three K-helices, plus an extra three-stranded antiparallel
L-sheet [95]. In a quite relaxed way, members of the family
present diverse topologies to link their secondary structure
elements into a WH fold, but the canonical order is: K1,
L1, K2, K3, L2, L3, plus two loops (‘the wings’) interconnecting the last three bits [95]. K-Helices 2 and 3 constitute
a HTH DNA recognition module (with the interhelical
angle ranging 100^150‡), with K3 binding to the major
groove in DNA whereas the loop linking L2 and L3 binds
to the minor groove [95]. However, in a recently described
WH member, RFX1, it is the loop between K3 and L2, the
element that contacts DNA major groove, instead of K3,
which follows the minor groove [96]. In addition, charged
and hydrophobic residues exposed in both the helical bundle and the L-sheet have been implied in dimerization [97]
and in interactions with other proteins [98].
It has been shown that dissociation of pPS10 RepA
dimers into monomers results in a structural change from
a compact arrangement of the two WH domains into a
more elongated form [86]. In the dimers, the C-terminal
domain (WH2) in each protomer binds to an 8-bp arm of
the operator inverted repeat (5P-GGACAGGG) through
the major groove, whereas the N-terminal domains
(WH1) form the dimerization interface [86]. In the monomers, WH2 binds to the 3P-half of each 22-bp iteron DNA
repeat (5P-GGGTTTAAAGGGGACAGATTCA), recognizing the same sequence found in the operator (both
underlined), while WH1 changes its structure and becomes
able to contact the 5P-iteron end, through both the phosphodiester backbone and the minor groove [86]. The idea
that Rep proteins should have two domains was independently inferred by means of sequence logo analysis of the
informational content of the iteron repeats in a number of
plasmids, concluding that iterons are composed of two
conserved halves [79]. In the RepE54 monomer structure
(Fig. 1B), the conserved N-terminal leucines are clustered
along two K-helices, buried in the hydrophobic core of
WH1, that resemble a folded jack-knife. The shorter helix
(K1) includes the ¢rst Leu residue (Leu12 in RepA) and
the larger (K2) the third and fourth (Leu26 and Leu33),
whereas the second (Leu19) is found in the intervening
turn [94]. Both WH domains in RepE54 are related by a
pseudo-two-fold symmetry (Fig. 1B) [94]. Apart from
these facts, the predictions made for pPS10 RepA [86]
proved to be correct : thus the polarity of both WH domains when bound to iteron DNA, the recognition by
WH1 of the phosphodiester backbone (rather than contacting bases in the major groove) and the requirement of
a conformational change in WH1 upon dimerization [86].
The later was con¢rmed after docking studies attempting
to model RepE54 dimers based on the crystal structure of
the monomers, concluding that severe steric clashes would
occur if WH1 does not change its structure in the dimers
[94].
Recently the detailed biophysical characterization of
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539
RepA-2L2A, a mostly monomeric species of RepA in
which the two ¢rst conserved Leu residues were changed
to Ala [99], suggests that this mutant resembles a transient
folding intermediate in the way from dimers to active
monomers. The mutated K1 is disabled to fold-back into
the core of the WH1, where the key hydrophobic interactions between the original leucines and Trp94 were disrupted, thus allowing the K-helix to move freely, resulting
in an extended conformation of the proposed jack-knife
[99]. If the N-terminal leucine residues have a direct contribution to the Rep dimerization interface or if they favour protein association indirectly (e.g., through the stabilization of the dimeric conformation) remains to be
determined. In vitro, micromolar amounts of a single iteron DNA sequence actively induce in RepA both the dissociation of dimers into monomers and the predicted (see
above) conformational change in the WH1 domain, consisting in a signi¢cant increase of the overall L-sheet component at the expense of the K-helical one [99]. On the
contrary, binding of RepA dimers to the operator sequence neither dissociates them, nor changes their conformation [99]. The ligand-induced monomerization of RepA
dimers, with a coupled conformational change, would thus
be a case for the allosteric e¡ect of a DNA substrate in the
structure of its protein DBD [100^102].
By speci¢c binding to the iteron sequence repeats at
oriV, Rep OBPs establish homo-oligomeric nucleoprotein
complexes (Fig. 2B) similar to those formed by DnaA at
oriC [63^68] (Fig. 2A). However, in most plasmid replicons, these Rep^oriV complexes are insu⁄cient to trigger
DNA replication by themselves. Thus DNA supercoiling
and other protein factors borrowed from the host (DnaA
and the pseudo-histones HU/IHF) are required to melt the
AT-rich repeats adjacent to iterons [103^106]. A feature of
Rep initiators is that, opposite to DnaA (see above), they
do not bind ATP. Although Rep proteins can promote
some structural transitions in DNA [107], most plasmids
still require DnaA to aid in origin unwinding [103^106]
and DnaB helicase loading [44,108]. This would explain
the ubiquitous presence of one or more 9-bp DnaA boxes
in most plasmid replicons [20,21,81]. The precise role of
DnaA in plasmid origin unwinding seems not to be the
same discussed for oriC melting (see above) since the
ADP-bound DnaA species [109], and even a deletion mutant lacking its AAA+ domain [44], are functional in plasmid replication. As noted before, Rep OBPs experience
conformational activation in a di¡erent way than DnaA.
Besides the allosteric e¡ect of iterons on RepA (see above)
[99], molecular chaperones, either the triad DnaK^DnaJ^
GrpE or ClpA, have been implicated in dimer dissociation, and coupled conformational activation, of the Rep
OBPs (Fig. 2B) in plasmids P1 [110^115] and F [116].
ClpX [117] and DnaK plus ClpB [118] chaperones have
a role in the activation of the initiator of RK2 plasmid. In
addition, the ClpA hexamer unfolds P1 Rep OBP, threading it into the proteolytic chamber formed by ClpP hep-
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tamers [119^121]. Interestingly, ClpA, ClpX and ClpP belong to the AAA+ superfamily [58^59].
In summary, plasmid Rep OBPs are examples of the
viability of a DNA binding module (a duplicated WH
domain) as a minimalist DNA replication initiator, provided it is able to experiment origin-speci¢c conformational activation and then to recruit other proteins (HU,
DnaA) that help with DNA unwinding and helicase loading.
3. The structure and function of OBPs in Archaea and
Eukarya
The study of DNA replication initiation in eukaryotic
replicons initially followed a path slower than in bacteria
[24]. Viral replicons, such as SV40 (reviewed in [122]), were
among the very ¢rst to be studied, before Saccharomyces
cerevisiae replication became a star due to the isolation of
yeast replicators, the Autonomous Replication Sequences
(ARS) (see [123] for a recent review). Eukaryal linear chromosomes, on the contrary to the circular bacterial one,
present multiple origins of replication (in S. cerevisiae haploids, 300^400 ARS spread across 16 chromosomes [124,
125]) to allow copying the complete genome timely along
the S-phase of the cell cycle. This compensates for the fact
of having eukaryotic replicative DNA polymerases lower
processivity than bacterial DNApol III [37]. The existence
of many replication origins introduces further complexity
in the mechanisms that control eukaryotic replication
[31,126,127]. During the past 10 years the ¢eld of eukaryotic DNA replication has expanded to a maturity similar
to that achieved in bacteria, founded on the discovery
of the initiator, the Origin Recognition Complex (ORC)
[128,129]. ORC was ¢rst characterized in S. cerevisiae and
then in any other eukaryote checked for (reviewed in
[130]), soon followed by other components of the machinery responsible for DNA replication initiation. More recently, studies on DNA replication in Archaea, ‘the third
Domain of Life’ [131], are growing at an impressive path,
to show that these prokaryotic organisms provide simpli¢ed, yet more robust, model systems to get insights into
the structure and function of the eukaryotic replication
machinery. Several excellent reviews can be found in the
recent literature, discussing the mechanisms and regulatory circuits of DNA replication, both in Eukarya
[31,126,127] and Archaea [132,133]. Therefore this section
focuses in the structure and function of their OBPs.
3.1. Eukaryal ORC and Cdc6 as combined AAA+/WH
machines in origin binding and remodelling
ORC initiator is composed of six protein subunits, labelled Orc1-6 (in S. cerevisiae: 120, 72, 62, 56, 53, 50 kDa,
according to SDS^PAGE), that speci¢cally bind to replicators in an ATP-dependent way [128,134] (Fig. 2C). In
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S. cerevisiae (Sc) all six ORC subunits are essential for
viability and remain associated with ARS through all the
cell cycle [135,136,125]. Yeast ORC seems to include a
single copy of each subunit [130] but subcomplexes defective in Orc1 [137] and Orc6 [138] have been isolated in
higher eukaryotes, together with others including extra
copies of some subunits [139], being proposed to control
DNA replication and cell cycle progression. Thus those
two subunits would be associated in a looser way with
an Orc2-5 core [140,141]. ARS are composed of two kinds
of conserved elements: A (also termed ACS, ARS Consensus Sequence, 5P-A/TTTTAT/CA/GTTTA/T) and B1^
B3 [123,126]. ORC binds to the A and B1 elements
[142,143] where, according to crosslinking experiments,
all its subunits except Orc3 and Orc6 contact the DNA
double helix in two patches separated by about 50 bp
[144]. The role of B2 sequence is under discussion, being
either the place where DNA is unwound or the binding
site for other components of the pre-replicative complex,
such as Cdc6 and Mcm2-7 [145]. B3 is the binding site for
Abf1, a transcription factor enhancing DNA replication
[146]. ORC interacts preferentially with one of the strands
in double-stranded ARS [144]. ATP binds to Orc1 and
Orc5 subunits (in higher eukaryotes, also to Orc4), but
only the site in Orc1 presents some hydrolytic activity
on the nucleotide and it is essential for ORC function
[134]. Single-stranded DNA, generated after ARS melting,
stimulates ATP hydrolysis and exerts in ORC a change
from an elongated to a curved shape [147], somehow resembling the conformational changes described in the bacterial OBPs DnaA [70] and Rep [99] (see above). Moreover, it has been recently found that, as in bacterial DnaA
[49^51], acidic phospholipids also bind to ORC interfering
with ARS binding, most probably through the release of
ATP [148]. ORC positions nucleosomes around ARS in a
way suitable for initiation [149] and contributes to repress
the yeast mating type loci HML/HMR-E [150]. In the
replication of the Epstein-Barr virus, ORC cooperates
with the viral initiator (EBNA1) in assembling the components of the replication fork at the viral origin (oriP)
[151,152].
The regulation of the assembly and activation of replicative complexes across S. cerevisiae cell cycle is extremely
elaborated, but leans on the initial ORC^ARS complex, a
landing pad for a number of essential replication factors
[130]. In G1, under low Cdc28-Clb5/6 Cyclin-Dependent
Kinases (CDKs) activity [153,154], due to the AnaphasePromoting Complex/Cyclosome (APC/C) [155] and to the
presence of the Cdc28 inhibitor Sic1 [156], pre-replicative
complexes assemble at ARS, containing ORC and the
newly synthesized co-initiator Cdc6 [157]. The ATP-bound
form of Cdc6 is required for its association with ORC
[158], enhancing the speci¢city and a⁄nity of the later
for ARS [159]. The complex formed by Cdt1 [160] and
Mcm2-7 [161] then enters into the nucleus (‘licensing’)
and binds to ARS [162], interacting with ORC and Cdc6
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[163,164]. During late G1, Cdc28-Cln1/2 CDKs phosphorylate Sic1, that is then poly-ubiquitinated by SCFCdc4 , to
promote its degradation in the 26S proteosome, and the
APC/C becomes inhibited [165^167]. Thus, at the G1-S
transition, origins are ¢red when Cdc28-Clb5/6 phosphorylate Cdc6 at its N-terminus, now becoming tagged for
quick proteolysis [168], and promote Cdct1 exportation
out of the nucleus [162]. These two processes, together
with phosphorylation by CDKs of Orc2 and Orc6 subunits and MCMs [154], co-operate to prevent re-initiation
until the next cell cycle. The crucial step for the transition
towards DNA synthesis is phosphorylation of Mcm2 by
the Cdc7-Dbf4 kinase (DDK), resulting in a conformational change in Mcm2-7 [169]. Cdc45 becomes then associated with Mcm10 [170] and Mcm2-7 [171]. The later,
detached from the pre-replicative complex by Cdc45
[136], becomes an active hetero-hexameric helicase ring,
functionally similar to bacterial DnaB (see above).
Mcm4, 6 and 7 would work as catalytic subunits, whereas
Mcm2, 3 and 5 would have a regulatory function [172^
174], resembling the cycle of ATP synthesis in F1-ATPase
[175]. Cdc45 also contributes to load the single-stranded
DNA binding protein RPA, the PCNA clamp and the
DNApol-K that, with the help of its primase activity,
starts DNA synthesis (S-phase) [176]. It has been recently
found that DNA replication and cell proliferation (ribosome assembly and synthesis of proteins) may be linked
through the interaction between ORC and a nucleolar
protein, Yph1 [177].
541
Regarding to the structure of the ORC subunits, it has
been found that Orc1 and Orc5 from di¡erent eukaryal
organisms share sequence similarity with Orc4 and altogether with eukaryotic and archaeal Cdc6 [178] (see below). Although the three-dimensional structure of any entire ORC subunit is still lacking, two independent studies
have recently addressed this issue. On one hand, the crystal structure of Cdc6, an Orc1,4,5 orthologue, from the
Crenarchaea Pyrobaculum aerophilum (PaCdc6, 45 kDa)
[60] shows that these OBPs are composed of an N-terminal AAA+ domain, as previously proposed [158], linked to
an unexpected C-terminal WH domain (Fig. 1C). This
study also included the mutational analysis of both domains in the homologous Cdc6 from Schizosaccharomyces
pombe (SpCdc18), followed soon by a similar report in
ScCdc6 [179]. However, if the WH domain has a role in
DNA binding and/or in protein^protein interactions
[95,97,98] remains to be determined, although it has
been shown that it modulates autophosphorylation in
archaeal and eukaryal Cdc6 [180]. The AAA+ domains
in bacterial DnaA and eukaryotic/archaeal Cdc6 are
nearly identical (Fig. 3A). Some ORC subunits include
characteristic extra domains. Thus, SpOrc4 bears at its
N-terminus nine repeats of an AT-hook motif for DNA
binding through the DNA minor groove [181,182], shown
to be su⁄cient for speci¢c origin recognition by ORC
[183,184]. A portion of the N-terminus of Orc1 shares
similarities with Sir3 and its three-dimensional structure
has been recently shown that it is composed of Bromo-
Fig. 3. Stereo views of the peptide backbones of similar OBP domains superposed by least squares. A: The AAA+ domains in AeDnaA [56] (Fig. 1A)
V . B: The WHs in RepE54 [94] (Fig. 1B) and PaCdc6 [60] (Fig. 1C). Rmsd (31 CK
and PaCdc6 [60] (Fig. 1C). Rmsd among 108 CK atoms: 1.58 A
V . The ¢gure stresses the nearly identical three-dimensional folds and topologies of the compared OBP domains, in spite of the extreme
atoms): 1.66 A
phylogenetic distances separating Bacteria and Archaea/Eukarya. Models were built using Swiss-Pdb Viewer.
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Adjacent Homology plus K-helical subdomains [185]. The
later is su⁄cient for binding to Sir1, being thus responsible
for the role of ORC in chromatin silencing [185,150]. It is
noteworthy that the Mcm proteins, as well as the subunits
of the DNA polymerase clamp loading NP:Q3:N complex
[186], are also members of the AAA+ superfamily
[58,59], that thus becomes the most versatile domain
among those involved in remodelling nucleoprotein complexes for DNA replication.
An independent biophysical and biochemical study on
the ScOrc4 subunit arrived to the same proposal made for
the structure of its orthologue PaCdc6 [60] (see above),
correctly assigning a WH fold to its C-terminal domain
[187]. In addition, the same study also managed to underline functional similarities between the WHs of ScOrc4
and the Rep OBPs in Gram-negative bacteria plasmids
(see above), such a role in protein^protein interactions
[187]. More in detail, ScOrc4 was found to consist in
two domains (residues 1^365, including the AAA+, and
366^529). The C-terminal one, although sharing low sequence similarity (19% identity) with the N-terminal domain of pPS10 RepA OBP, was proposed to fold into a
similar WH, based on the conservation in ScOrc4 of key
residues in the hydrophobic core of RepA/RepE54 WH1
[94] (Fig. 3B). These structural similarities between eukaryotic and plasmid initiators were not pointed out in
the crystallographic study on PaCdc6 [60].
543
The AAA+ N-terminal domain of ScOrc4 shows a functional feature of the RepA-type prokaryotic initiators
[110,111,113,114,116,118] (Fig. 2B,C): binding to chaperones of the Hsp70 family [187]. This kind of interaction
has been also reported for the initiators E1 (human papilloma virus) [188^189] and UL9 (herpes simplex virus)
[190] (Table 1). Yeast Hsp70 chaperone modulates the
association state of the ScOrc4 subunit by dissociating
oligomers into dimers [187]. Thus it could be a step towards the assembly of the complete multisubunit ORC
(Fig. 2C). The conformation of some ORC subunits has
been proposed to be di¡erent when the complex is assembled free or associated with ARS DNA, a process in
which ATP could be the allosteric e¡ector [147]. By analogy with its prokaryotic DnaK homologue on Rep proteins [115,86], Hsp70 could also control the compactness
between the N- and C-terminal domains of ScOrc4, thus
switching between di¡erent functional states of this subunit in the replication complex. It is noteworthy that in
AeDnaA, RepE54 and PaCdc6 OBPs the ¢rst long K-helix
in their DBDs (K14, K2 and K16, respectively) is preceded
by a shorter one (namely, K13, K1 and K15) (Fig. 1A) [56].
In pPS10 RepA (see above, on the jack-knife mechanism)
[99], as well as in the loosely related Ets-1 WH [191], it has
been proposed that changing the relative orientation of
those K-helices is a key step in the conformational activation of their DBDs.
6
Fig. 4. Phylogenetic relationships between OBPs, based on their conserved AAA+ domains. A: 82 sequences of OBPs from Bacteria (DnaA), Eukarya
(Orc1,4,5 and Cdc6) and Archaea (Orc1/Cdc6) were retrieved from the NCBI web site (http://www.ncbi.nlm.nih.gov :80/entrez/query.fcgi?db = Protein ;
updated in May 2002). Multiple alignments were performed separately on the DnaA, ORCs, Cdc6 and archaeal sequence datasets with CLUSTAL-X
(BLOSUM series protein weight matrix; gap penalties: 10.0 for opening and 1.0 for extension) [202]. Then pro¢le alignments were performed between
those pre-aligned clusters (penalties: 20.0 opening, 1.0 extension) prior to a ¢nal round of multiple alignment (penalties : 10.0 opening, 1.0 extension)
with all sequences, followed by minor manual adjustments. Outlined sequence names correspond to those OBPs extensively discussed in this review.
Boxes are color-coded according to the chemical nature of conserved (in at least 20% of sequences) amino acid residues: yellow (hydrophobic and aromatic: A, V, L, I, M, C, F, Y, W); green (polar: S, T, N, Q, H, A, C); red (acidic: D, E, plus similarly shaped N, Q); blue (basic : K, R, plus N, Q,
H); pink (P); orange (G). For simpli¢ed display, three regions showing large insertions in a small subset of sequences have been removed (their position
and extension are indicated between brackets on the EcDnaA sequence). The secondary structure elements (K-helices and L-strands) found in PaCdc6
crystal structure [60] are shown over the sequence alignment. The two subdomains in the AAA+ fold (Fig. 1) are contoured with a dashed black line,
whereas relevant sequence motifs (Walker-A/B and sensor-1/2) are in grey boxes. Histogram below the alignment re£ects the degree of residue conservation for each position. OBPs sequences and accession numbers: DnaA (Ec, Escherichia coli P03004; Tht, Thermus thermophilus Q9X9D5; Cht, Chlamydia trachomatis O84252; Al, Acholeplasma laidlawii Q9KHU8 ; Ae, A. aeolicus O66659; Bs, Bacillus subtilis P05648; Bob, Borrelia burgdorferi P33768;
Bua, Buchnera aphidicola P29434; Cj, Campylobacter jejuni Q9PJB0; Cac, Caulobacter crescentus P35887 ; Der, Deinococcus radiodurans Q9RYE7 ; Hi,
Haemophilus in£uenzae P43742; Hp, Helicobacter pylori Q9ZJ96; Ll, Lactococcus lactis Q9CJJ2; Ml, Micrococcus luteus P21173; Myt, Mycobacterium
tuberculosis P49993; Myg, Mycoplasma genitalium P35888 ; Nm, Neisseria meningitidis Q9JW45; Pm, Pasteurella multocida Q9CLQ4; Prm, Prochlorococcus marinus Q51896 ; Pmi, Proteus mirabilis P22837 ; Psa, Pseudomonas aeruginosa Q9I7C5; Rm, Rhizobium meliloti P35890 ; Rp, Rickettsia prowazekii
Q59758 ; Spc, Spiroplasma citri P34028 ; Sta, Staphylococcus aureus P49994 ; Spn, Streptococcus pneumoniae O08397 ; Sco, Streptomyces coelicolor
P27902 ; Sy, Synechocystis sp. P49995 ; Tm, Thermotoga maritima P46798; Trp, Treponema pallidum O83047 ; Up, Ureaplasma parvum Q9PRE2; Vc, Vibrio cholerae Q9KVX6; Xf, Xylella fastidiosa Q9PHE3; Zym, Zymomonas mobilis Q9S493. Orc1 (Sc, Saccharomyces cerevisiae P54784; Ca, Candida albicans O74270 ; Sp, Schizosaccharomyces pombe P54789 ; Ce, Caenorhabditis elegans Y39A1A.12 ; Dm, Drosophila melanogaster O16810 ; Mm, Mus musculus Q9Z1N2; Hs, Homo sapiens Q13415). Orc4 (Sc, P54791 ; Sp, Q9Y794; Dm, NP_477320; Xl, Xenopus laevis O93479; Mm, O88708; Hs, O43929).
Orc5 (Sc, P50874; Sp, O43114 ; Dm, Q24169 ; Mm, Q9WUV0 ; Hs, O43913 ; Zm, Zea mays AAL91670). Cdc6 (Sc, P09119; Ca, T46606; Sp, P41411;
Stp, Strongylocentrotus purpuratus AAL37208 ; Xl, T46977; Mm, XP_122302; Hs, AAH25232 ; At, NP_565686 ; Os, Oryza sativa BAC03316). Archaeal
Orc1/Cdc6 (Pa, Pyrobaculum aerophilum AAL62992; Ap-1, Aeropyrum pernix APE0152 ; Ap-2, APE0475; Ss-1, Sulfolobus solfataricus NP_341806; Ss-2,
NP_342278; Ss-3, NP_343568 ; Af-1, Archaeoglobus fulgidus AF0244; Af-2, AF0695 ; Mt-1, Methanobacterium thermoautotrophicum AAB85889 ; Mt-2,
AAB86072; pFZ1, Mt plasmid X68367; Mta-1, Methanosarcina acetivorans AAM03539 ; Mta-2, AAM03455; Ta, Thermoplasma acidophilum
CAC11593; Tv, Thermoplasma volcanium BAB60645; Pyh, Pyrococcus horikoshii PH0124; Pya, Pyrococcus abysii PAB2265; Hh, Halobacterium sp. plasmid NRC-1 NC002607 ; Fea, Ferroplasma acidarmanus NC_002709). B: An unrooted neighbor-joining phylogenetic tree calculated by means of CLUSTAL-X [202] on the sequence alignment shown in (A). Numbers in nodes are the reliability values obtained after 100 bootstrapping trials. Tree was
plotted with TreeView (http://taxonomy.zoology.gla.ac.uk/rod/treeview.html). Bacterial sequences are displayed in purple, whereas archaeal ones are in
green and eukaryal in red.
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3.2. Archaeal Orc1/Cdc6 OBPs are simpli¢ed versions of
eukaryal AAA+/WH
Well before the crystal structure of PaCdc6 (see above)
made explicit the similarities between archaeal and eukaryal OBPs [60], they had been proposed based on sequence comparison analyses [158]. A number of searches
for ORC/Cdc6 homologues in the genomes annotated so
far have resulted in that most Archaea have a single
ORC1/CDC6 gene albeit some, as Sulfolobus solfataricus,
have up to three and Methanococcus jannaschii seems to
have none [132,133] (Fig. 4). Thus their encoded proteins
might exert all the functions described for eukaryal ORC
and Cdc6 OBPs (see above). A unique replication origin,
oriC-like, was identi¢ed in the single circular chromosome
of the Euryarchaea Pyrococcus abysii (Pya) after cumulative analysis of the G+C skew in each strand, a signature
for prokaryotic replication origins, and veri¢ed to be the
earliest replicated region [192]. oriC is bound by the Orc1/
Cdc6 initiator in vivo [193]. Nevertheless, the possibility
that some other Archaea (M. jannaschii among them) also
could have more than one replication origin can not be
excluded yet [194]. As for their bacterial chromosomal and
plasmid counterparts, ORC1/CDC6 OBPs genes are usually clustered around the putative replication origin, together with those for the two subunits of the euryarchaeal
DNA polymerase D [132,133,192]. However, this is not
the case in Archaeoglobus fulgidus [194]. It is noteworthy
that all the archaeal genomes analyzed so far, again with
the exception of M. jannaschii, also contain a single MCM
gene [10]. It has been shown that the Mcm protein of
Methanobacterium thermoautotrophicum (Mt) assembles
into a double hexameric ring with helicase activity
[195,196], each one resembling the functional form of the
hetero-hexameric eukaryal Mcm2-7 complex [174] (see
above).
Overall, it seems that the archaeal replication machinery
is a simpli¢ed version of that functional in eukaryotes,
since homo-oligomers of a single initiator protein in Archaea manage to perform the job of the more complex
hetero-oligomeric assemblies found in Eukarya, but probably working on a prokaryotic type replicator. The advantage of having multiple di¡erent subunits in eukaryotic
OBPs might be that these would thus be more suitable
for ¢ne, cell cycle-coupled, post-translational regulation.
As pioneered by the studies on PaCdc6 [60], PyaoriC
[192,193] and MtMcm [195,196], archaeal replicons are
amenable model systems for biochemical and structural
analyses, leaving aside the standing di⁄culties for genetic
manipulation of Archaea.
4. Phylogeny of OBPs domains: Outlining the ancestral
initiator
Sequence comparisons of the proteins involved in the
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processes relevant for the transmission of the genetic information in the three ‘Life Domains’ [131] have arrived to
a paradox. Namely, similarities can be universally recognized between the components of the transcriptional and
translational machines, but the proteins involved in DNA
replication cluster in two clearly di¡erent groups, sharing
those from Archaea and Eukarya clear similarities, whereas their functional counterparts in Bacteria seem unrelated
[8^10]. The former specially applies to OBPs, but also to
helicases, single-stranded DNA binding proteins, primases,
DNA polymerases and their accessory factors [132,133].
This observation has settled down the basis for the recent
proposal that DNA replication was ‘invented’ twice independently, a scenario compatible with having LUCA a
mixed RNA^DNA genome whose replication would be
worked out by an RNA polymerase and a reverse transcriptase [9]. However, this view runs against the common
wisdom derived from decades of studies on Molecular Biology, concluding that the essential processes share common molecular bases in all organisms (as re£ected in an
old biochemical adagio : ‘What is true for E. coli is true for
elephants’) [197]. Among all those replication proteins just
mentioned above, OBPs phylogenies are particularly signi¢cant since initiators are likely the answer to the early
need, inherent to the development of a cellular organization, for a controlled replication of DNA. This is required
to synchronize duplication of the genetic material with the
rest of cellular processes, by means of regulating initiator
expression and its biochemical activities. Control was
likely not a problem for self-replicative nucleic acids in
the pre-existent ‘RNA world’ [1].
With the advent of ‘the Genomic Era’, having already
hundreds of genomes completely sequenced and millions
of new entries in protein databases (just check it ‘on line’
at http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db = Genome), we are beginning to exceed our ability for naming
most of them with functionally signi¢cant names [198]. It
is time to wonder if our current tools to annotate genomes
and to identify functions are su⁄cient by themselves to
handle such a vast amount of data in a meaningful way
and to drive Biology successfully through the XXIst century. Fortunately, besides spectacular recent improvements in bio-informatic tools, experimental Science is, as
always, to the rescue by means of three main approaches.
(i) Proteomics, aiming to the complete description of the
patterns of expression and interaction for all proteins in
any cell type of an organism [199]. (ii) Structural Genomics provide the hope that solving the three-dimensional structures of ‘unknown protein entries’ in a few model
genomes will provide a hint on their function, due to the
existing intimate correlation between the structure and
function of biological macromolecules [200]. (iii) The classic fashioned ‘curiosity driven’ research in academic
groups that will continue supplying precious knowledge
and human expertise on concrete biological problems,
now in danger to be left aside by the other two, more
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fashionable, ‘discovery driven’ enterprises. In this sense
experimental research on micro-organisms, in addition to
allowing the possibility of sampling biodiversity through
their genomes [201], has recently provided clues towards
unravelling the phylogenetic origin of DNA replication [8^
10], and thus about one of the essentials for de¢ning a
living cell.
4.1. The three-dimensional structures of a few replication
proteins unveil missed sequence similarities
The most common way to establish phylogenetic relationships between proteins is to compare their sequences
by means of multiple alignments, in which identical residues and conservative substitutions, as well as insertions
and gaps, are taken into account. The scores thus obtained
are transformed into pairwise distances among the compared sequences, that rank them in a way feasible to be
represented as an schematic tree [202]. Although such approach is invaluable in most of the cases, it has a limited
value if the sequences of two truly homologous proteins
show a low degree of identity (say 6 20%), sometimes
even beyond recognition ( 9 9%). This can be the consequence of having evolved (at least) one of the proteins at a
fast rate after their divergence [203]. In these cases, the
exclusive use of sequence alignments as phylogenetic criteria has the potential pitfall of concluding that two given
proteins are unrelated, in spite of the existence of experimental evidence on having identical function, and even
the same three-dimensional structure. Structures have the
advantage of being more conserved through evolution
than sequences. With the current exponential growth in
the number of entries in the database of protein atomic
coordinates (PDB, http://www.rcsb.org/pdb) [200], the later possibility is not exceptional anymore. It is still open to
debate if we are approaching the complete coverage of
protein architectures or if such a catalogue will expand
further [204]. Therefore, any attempt to describe the phylogeny of a protein family should take into account the
(common) three-dimensional structure of at least one of its
members (ideally also those for the most divergent ones).
This puts sequence features in a structural framework,
bringing the ‘homology’ concept to its physico-chemical
ground.
To outline just a few examples among many recently
reported, the processivity subunits of the replicative DNA
polymerases (L-clamp in Bacteria and PCNA in Eukarya/
Archaea), as well as their loading factors (NP:Q3:N complex
and Replication Factor C (RFC), respectively), have
nearly identical three-dimensional structures, in spite of
showing only marginal sequence similarities [186]. Structural similarity is usually expressed as the root mean
V ) between a discrete number
square deviation (rmsd, in A
V for
of overlapped CK atoms (in the example above, 0.9 A
55 residues in both clamps) [186]. Out of the group of
proteins involved in DNA replication, the single-stranded
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545
DNA binding proteins that pack telomere ends in yeast
(Cdc13) and ciliated protozoa (K-subunit) share the same
V for 113 CK atoms), although seOB-fold (rmsd = 2.2 A
quence similarities were beyond recognition [205]. Also
based on structural similarity, it has been found that prokaryotes have two proteins (FtsZ, MreB) related to euV
karyotic cytoskeletal components: tubulin (rmsd = 2.4 A
V
for 178 CK) [206] and actin (rmsd = 3.7 A for 310 CK)
[207], respectively. The same kind of structural homology
criterion, applied in this review to the AAA+ domains of
DnaA and Orc1,4,5/Cdc6 on one hand and to the WHs in
Rep and Orc1,4,5/Cdc6 on the other (see above, Fig. 3),
can be used to discriminate cases of functional analogy in
non-phylogenetically related proteins. Thus, DNA primases in Bacteria and Archaea/Eukarya (DnaG and Pri,
respectively) are unrelated (that is, are a case for convergent evolution) since, leaving aside a cluster of acidic residues similarly arranged to position two catalytic Zn2þ /
Mg2þ ions, they have completely di¡erent three-dimensional folds [208]. The power of comparing protein architectures is evident in more subtle examples, such as the
WHs domains of plasmid Rep [94] and P4 phage gpK
[98] OBPs. In this case the same overall fold is found,
albeit with two distinct topologies (connectivities) of their
K-helical and L-strand elements, pointing to another case
of convergent evolution.
With the concerns just outlined in mind, the analyses of
phylogenetic trees for bacterial, archaeal and eukaryal
OBPs, based either on sequence alignments of their
AAA+ (Fig. 4) or WH (Fig. 5) domains, must be approached with caution. Except for closely related species,
long length branching is a common feature in both cases
(specially for WHs), possibly pointing to quick rates of
evolution after divergence from the ancestor of each domain [203]. However, OBPs from the same ‘Life Domain’
cluster together, indicating that divergence has not proceeded too far for allowing the recognition of a few conserved characteristic residues. These are particularly evident in AAA+ domains, where more key functional
residues have been identi¢ed (see above) and appear consistently conserved. It is noteworthy that the topology
(branching order) of the Orc1 and Cdc6 groups is inverted
in both trees, being contiguous, besides to the prokaryotic
sequences (DnaA’s AAA+ or Rep’s WH), either to the
archaeal group (AAA+) or to Orc4 (WHs). As further
discussed below (Fig. 6), this could re£ect Horizontal
Gene Transfer (HGT) [209] events a¡ecting both OBP
domains throughout the divergent evolution of initiators
from their common ancestor, but this hypothesis requires
further rigorous testing since deep-rooting branches are not
supported by high bootstrap values (Figs. 4B, 5B) [210].
WH and AAA+ domains are ubiquitous as DNA binding and remodelling protein modules, raising the question
on their appearance in evolution. In each of the annotated
whole genomes available is common to ¢nd tens of proteins having a putative domain with the AAA+ signature.
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A recent study on the occurrence of individual domains in
bacterial, archaeal and eukaryal genomes showed that
WHs are the third most common protein modules (only
after P-loop NTP-hydrolases and Rossmann folds) [211].
Moreover, WH is used as a basic module, besides DNA
replication, in proteins related with the other processes
universally involved in the transmission of genetic information. Thus, in translation the S4 protein, including a
WH of the ETS type, holds together ¢ve RNA helices in
the small ribosomal subunit (30S) [212]. S4 is a key protein
in the early steps of the assembly of the 30S particle,
strongly interacting with the DnaK chaperone [213], as
described for the WH domains in plasmid OBPs. In transcription WHs are present in a number of general factors
of RNA polymerase II, such as the C-terminal domains of
Rap54 and Rap30 (TFIIF) and the central domain of
TFIIE (reviewed in [214]). The RuvB motor protein, involved in prokaryotic homologous recombination, has the
same combination of domains (AAA+ followed by WH)
discussed above for Orc1,4,5/Cdc6 [215]. These facts point
out to the ancestral origin of both domains, most likely
traceable back to LUCA [216].
4.2. Possible alternative scenarios for the phylogenetic
origin of OBPs
The possibility that the AAA+ domains in DnaA/
Orc1,4,5/Cdc6, and the WHs in Rep and Orc1,4,5/Cdc6,
OBPs emerged by convergent evolution (Fig. 6A) is very
unlikely, given their astonishingly high degree of structural
similarity (Fig. 3). Another possible scenario (Fig. 6B)
takes advantage of the predominant role of plasmids in
HGT among micro-organisms [209]. The gene coding for
an ancestral AAA+ initiator in Archaea/Eukarya could
have been fused with a WH gene of plasmid origin, transferred from a primordial member of Bacteria, to constitute
distinct domains in a chimeric OBP that would then duplicate and diverge into the multisubunit ORC and Cdc6.
This hypothesis provides an example on how bacteria
could have contributed to build the eukaryal nuclear genome [217] an also implies that DnaA would be closer to
the ancestral initiator, since it would conserve the original
(non-WH) DBD. In addition, it would constitute a case
for HGT a¡ecting ‘informational’ genes, rather than the
547
most common examples involving ‘operational’ (metabolic) ones [218]. Alternatively (Fig. 6C), as Woese proposes,
LUCA might have been a community of proto-cells (‘progenotes’), rather than a proper cellular entity with de¢ned
components and lineage [11,15]. Progenotes would interchange their genomes that, on their side, ‘more resembled
mobile genetic elements than typical modern chromosomes’ [11]. Ancestral genomes could be thus conceived
as consisting in a number of plasmid-like replicons, some
of which would contribute with a gene coding for a distinct domain (AAA+ and WH) of the ancestral initiator.
This would be more a macromolecular assembly than a
multidomain OBP. The appearance of the later by gene
fusion would be roughly coincident with the transition of
a particular community of proto-cells to the proper cellular status, giving way to the successive establishment of
each one of the three ‘Life Domains’ [15]. In such a scenario, non-orthologous gene displacement, as proposed by
Forterre [219], might be responsible for the replacement,
just before the emergence of the Bacteria domain, of the
ancestral chromosomal initiator by an analogue gene of
(say) viral origin, including the AAA+ domain and a nonWH DNA binding module. In such a case, archaeal/eukaryal OBPs would resemble the replication initiators
found in LUCA more closely than bacterial DnaA. Modern plasmid Rep OBPs, composed solely of WHs domains,
would be thus a relict from the ancestral modular initiators found in proto-cells.
5. Concluding remarks
The classic integrated view on the processes of transmission of the genetic information has been challenged
by modern genomics, since protein sequence comparisons
conclude that the set of genes for DNA replication in
Bacteria clearly di¡ers from that found in Archaea and
Eukarya [8^10]. This divergence is specially noteworthy
for the proteins that initiate chromosomal replication in
Bacteria (DnaA) [38^40] and Eukarya (the six subunits of
the origin recognition complex, ORC) [130] which, in spite
of their common function in binding to DNA replicators,
lack signi¢cant sequence similarity. Nevertheless, DnaA
[56] and some ORC subunits share an AAA+ module
6
Fig. 5. Phylogenetic relationships between OBPs, based on their conserved WH domains. A: The sequence alignment was generated, as speci¢ed in Fig.
4A, with a number of OBP sequences from Gram-negative bacteria plasmids (those sharing clear similarities with pPS10 RepA [20,21]), archaeas and
eukaryotes, retrieved from the NCBI web site (see Fig. 4 legend, database updated in August 2002). OBPs sequences and accession numbers (not already quoted in Fig. 4 legend): Rep (pPS10, P. savastanoi S20615 ; pECB2, P. alcaligenes Y10829; pRO1614, P. aeruginosa L30112; pCM1, Chromohalobacter marismortui X86092 ; pFA3, Neisseria gonorrhoeae M31727; pSC101, E. coli K00828 ; pminiF, E. coli X00959 ; pR6K, E. coli M65025 ;
pGSH500, Klebsiella pneumoniae Z11775; pCU1, E. coli M18262 ; pXF5823, Xylella fastidiosa AF322908; pL6.5, P. £uorescens AJ250853; pTAV3, Paracoccus versutus AF390867). Orc1 (Kl, Kluyveromyces lactis P54788; Nc, Neurospora crassa T50982; Enc, Encephalitozoon cuniculi CAD26247; Cg, Cricetulus griseus AAF66067). Orc4 (Zm, Zea mays AAL10455). For color and graphic display, refer to Fig. 4. Asterisks mark the positions of the conserved Leu and Trp residues discussed to be key parts of the hydrophobic core of the WHs. B: An unrooted, neighbor-joining phylogenetic tree
calculated by means of CLUSTAL-X [202] on the sequence alignment shown in (A) (see Fig. 4B). Bacterial plasmid Rep sequences are displayed in orange, whereas archaeal Orc1/Cdc6 are in green and eukaryal Orc1,4,5 or Cdc6 are in red.
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[58,59] for ATP binding, which triggers conformational
changes when the initiator complexes are assembled with
origin DNA [70,147]. It has been recently found that the
proteins that initiate DNA replication of plasmids in
Gram-negative bacteria (Rep) and a C-terminal domain
in a subunit of yeast ORC (ScOrc4) are structurally related, in terms of protein sequence motifs, overall secondary structure, three-dimensional fold (a WH domain) and
association state [187]. Furthermore, Rep OBPs also experiment conformational changes upon binding to plasmid
origin sequences [99] but, unlike DnaA and ORC, they
just a¡ect the N-terminal of the two WHs in Rep and
are independent of ATP. Similarities between OBPs, besides other ORC subunits such as Orc1 and Orc5, also
extend to Cdc6, that regulates initiation both in Archaea
and Eukarya [60]. In functional terms, Hsp70 chaperones
might modulate the association state of either Rep or Orc4
in homo- or hetero-oligomeric initiation complexes, respectively [187]. It is noteworthy that interactions with
chaperones are recently becoming relevant for the proper
assembly of a growing number of cell factors into large
functional complexes [210,220]. However such interactions, in spite of being detected with signi¢cant frequency
in recent whole-cell proteomic approaches, are still left
aside dismissed as ‘contaminants’ [221], a statement that
should be revised after the evidence discussed above. Plasmid RepA-type initiators form homo-oligomers when
bound at their replication origins [20]. At the prokaryotic
chromosomal origin oriC, the initiator protein DnaA also
establishes homo-oligomeric assemblies [65^68]. The eukaryotic ORC is an hetero-oligomer of six di¡erent,
although structurally related, subunits [130]. Archaeal ini6
Fig. 6. Some of the di¡erent possible scenarios for the evolution of
OBPs in Bacteria, Archaea and Eukarya. This is a speculative cartoon
that is open to further combinations. According to Woese [11], LUCA
genome is drawn as composed of multiple, single-gene size, DNA molecules. Membranes and cell envelopes a di¡erentially colored to re£ect
their distinct composition [13]. A: Whereas the AAA+ domain in
present day chromosomal initiators (purple in Bacteria and green in Archaea/Eukarya) was already found in the ancestral microorganism
(LUCA, in red), being thus orthologues, the WH domains in archaeal/
eukaryal (blue) and plasmid (orange) branches arose independently by
convergent evolution. B: Ancestral OBP would be as proposed above,
but a precursor of present day Gram-negative bacteria would have
transferred horizontally, through a plasmid, a WH domain (orange) to
an ancestor of the archaeal/eukaryal lineage, replacing its primordial
DNA binding domain to yield a chimeric OBP. Again, the ubiquitous
AAA+ domain would diverge in all branches from its precursor found
in LUCA. C: OBP functions in LUCA might have been performed by
independent polypeptides, including the AAA+ domain and a WH, respectively. They could be then fused into a single OBP shortly before
the divergence of the Archaea/Eukarya domains, remaining the WH isolated in plasmids until present. As proposed by Forterre [219], non-orthologous gene displacement would be responsible for the substitution,
just before divergence of all bacterial lineages, of the ancestral chromosomal initiator by an analogue gene of viral origin, including the required AAA+ and DNA binding modules (in purple). Panels (B) and
(C) are compatible with the phylogenetic trees in Figs. 4B and 5B.
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tiation complex is likely to be a homo-oligomer of a single
Orc1/Cdc6 protein [193]. Thus the prokaryotic and eukaryotic OBPs would result to be variations of a unique
macromolecular assembly, evolved to unwind origin DNA
and then to load the factors that constitute the replication
fork.
As it has been proposed in this review, to ¢nd common
molecular traits among living organisms, with the aim
either of gene annotation (v.g., identifying function from
sequence) [198] or phylogenetic analyses (such as the attempts of de¢ning the nature of LUCA) [11^15], will probably require to combine new approaches. Among others,
improved in silico tools and further expanding the current
set of experimental model systems, specially towards those
micro-organisms deep-branching in the ‘Tree of Life’
[131]. This would result in a broader coverage of the biodiversity, with additional bene¢ts for both basic and applied research. Since the current bioinformatic tools, extensively used in comparative whole genome analyses
[201], often fail to recognize consistent relationships between proteins if they are hidden behind low sequence
similarity scores [203], future phylogenetics should rely
as much on Proteomics (broadly speaking) as nowadays
rest on Genomics.
Acknowledgements
The author is indebted to Dr. Ramo¤n D|¤az-Orejas for
over 15 years of friendship, continuous support and
shared interest in the molecular mechanisms of DNA replication. This work has been ¢nanced by Spanish CICYT
(Grant PM99-0096).
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