Conservation of Eubacterial Replicases
Gene Wijffels1, Brian Dalrymple1, Kritaya Kongsuwan1and Nicholas E. Dixon2
1CSIRO Livestock Industries, Queensland Bioscience Precinct, St. Lucia, Queensland, and
2Research School of Chemistry, Australian National University, Canberra, ACT, Australia
The last 15 years of effort in understanding bacterial DNA
replication and repair has identified that the donut shaped b2sliding
clamp is harnessed by very functionally different DNA polymerases
throughout the lifecycle of the bacterial cell. Remarkably, the sites
of binding of these polymerases, in most cases, appear to be the
same shallow pocket on the b dimer. In every case, binding of b2by
the polymerase enhances their processivity of DNA synthesis. This
binding site is also the same point of interaction between b2and the
clamp loader complex, which binds b2, opens and places it onto the
DNA strand and then vacates the site. b2may also be involved in the
initiation of DNA replication again via contact through this same
site. While much of the research effort has focused on Escherichia
coli and Bacillus subtilis, conservation of this complex system is
becoming apparent in diverse bacteria.
IUBMB Life, 57: 413–419, 2005
Bacterial DNA replication; sliding clamp; clamp loader;
DNA replication, repair and recombination take place
within large, transient and interactive protein complexes in all
organisms. The assembly, disassembly and function of the
protein and DNA components of these complexes are being
elucidated. Many of the mechanisms and structures involved
in these processes are common to prokaryotes, archea,
eukaryotes and even phages and viruses. Much of the ‘action’
occurs around a DNA sliding clamp which moves freely on
intact duplex DNA. The sliding clamp of the T4 phage (gp45),
prokaryotes (b2) and eukaryotes (PCNA) all form donut
shaped rings. It is homodimeric in prokaryotes and homo-
trimeric in phages and eukaryotes. Structurally, the clamps are
virtually identical, but they share little sequence similarity. In
all cases, a clamp loader complex of several proteins ‘clips’ the
stable dimer or trimer onto DNA by opening of one the clamp
subunit interfaces in close proximity to the DNA strand, and
on releasing the loaded clamp, the interface is closed again.
The clamp loader complex vacates its site on the sliding clamp
making the site available to other proteins or protein
complexes involved in various DNA processing activities.
The sliding clamp in all strata of life appears to be a key node
in the protein interaction network associated with these
functions. Recent reviews of DNA replication are available
The eubacterial DNA replicases have a highly conserved
organization with three main components, the replicative
DNA polymerase, the sliding clamp (b2) and the clamp loader
(g complex) which together constitute the DNA polymerase III
(pol III) holoenzyme (see fig. 2 in Schaeffer et al. (1)). The
sliding clamp’s interactions with a large number of other
enzymes involved in DNA replication and repair is summar-
ized in Fig. 1. Despite the lack of sequence similarity among
sliding clamps in prokaryotic and eukaryotic organisms, there
is good primary sequence conservation of the b2 subunit
within eubacterial species, and consistent with this, all b2
subunits isolated or expressed thus far have been shown to be
stable homodimers (4–6; Kongsuwan and Wijffels, unpub-
CLAMP LOADER COMPLEXES
The sliding clamp, b2, is loaded by the clamp loader, which
in E. coli consists of a minimal assembly of five subunits:
(g/g)173dd’(see Fig. 2, Schaeffer et al. (1)). Orthologues of the
d’ and t/g subunits have been easily identified in all eubacteria,
but it is only very recently that orthologues of the d subunit
have been recognized in eubacteria outside of the gamma and
beta subdivisions of the proteobacteria (4, 7, 8). Indeed,
although the d subunit is required for loading of b2 onto
DNA, it is the least conserved of the essential components of
the Pol III holoenzyme.
Received 21 December 2004; accepted 23 March 2005
Address correspondence to: Gene Wijffels, CSIRO Livestock
Industries, Queensland Bioscience Precinct, 306 Carmody Road., St.
Lucia, Queensland 4068, Australia. E-mail: Gene.Wijffels@csiro.au
IUBMBLife, 57(6): 413–419, June 2005
ISSN 1521-6543 print/ISSN 1521-6551 online ª 2005 IUBMB
The assembly of the d, d’ and g/t subunits into clamp
loaders has been demonstrated for an expanding number of
organisms – Streptococcus pyogenes and Staphylococcus
aureus, both Gram positive organisms (4, 9), the two Gram
negative organisms, Helicobacter pylori (Kongsuwan, unpub-
thermophiles, Thermus thermophilus (6) and Aquifex aeolicus
(5). The distribution of the g subunit (a truncated version of t
which is also encoded by the dnaX gene) is variable. While E.
coli expresses g and t subunits, S. pyogenes, S. aureus, P.
aeruginosa, A. aeolicus and B. subtilis express only the
complete DnaX product, the t subunit (4, 5, 8–10). On the
other hand, the g subunit is seen in T. thermophilus and a
number of Enterobacteriaceae, while Aeromonas and Vibrio
may express an intermediate form (6, 11). The use of
combinations of t and g subunits in the clamp loaders may
impact on the clamp loaders’ interactions with the replicative
polymerases within the replisome complex.
Orthologues of the two smaller subunits of the E. coli
clamp loader,wandc, or proteins taking a similar role, have
only been observed in eubacterial species very closely related
to E. coli. Recent work from Jarvis and colleagues (8) has
demonstrated increased proficiency of the P. aeruginosa
holoenzyme and clamp loader functions in the presence of
E. coliwandcsubunits, and suggest that this indicates that
similar subunits may exist in Pseudomonas.
THE DNA POLYMERASE SUBUNIT
The a subunit (expressed from the dnaE gene) is the main
replicative DNA polymerase in E. coli. It forms a core enzyme
with the y and e (proofreading exonuclease) subunits (see Fig.
2, Schaeffer et al. (1)). A. aeolicus and P. aeruginosa a subunits
complex with an e subunit (5, 8), whereas in T. thermophilus,
the a subunit is devoid of both e and y subunits (6). The y
subunit is not widespread in bacteria, but the e subunit (or
similar proteins) is common.
A SECOND MAJOR REPLICATIVE DNA POLYMERASE
A related DNA polymerase, PolC, appears to be the major
replicative polymerase in many Gram-positive species. This
enzyme possesses its own proofreading activity, is highly
processive, and is essential in organisms containing the gene.
The interaction of PolC with t would appear to be weaker
than the binding between the a and t subunits (4, 8, 9),
suggesting that t does not secure this polymerase as tightly to
the replisome. In E. coli, it appears that the t subunit might
Figure 1. Protein inter-relationships in the Pol III complex. Functions, families and b2binding motifs of b2binding proteins are
shown schematically. Numbers in arrows indicate the number of identical subunits in multi-subunit complexes. Component
proteins are named according to families and subfamilies as defined in Dalrymple et al., 2003 (22).
414 WIJFFELS ET AL.
sequester the a subunit from b2until it ‘senses’ the correct
DNA context for its binding to b2(12).
In low GC-content Gram-positive bacterial species, both
polymerases are essential. PolC is required for leading strand
synthesis, while the DnaE polymerase, which is closely related
to the E. coli a subunit, is proposed to be responsible for
synthesis of the lagging strand (13). Furthermore, the DnaE
enzyme has been observed to be far less processive and more
error prone in these species (a very different profile to the a
subunit of non-PolC containing bacteria), suggesting it has an
additional role in DNA repair (14, 15).
THE b2SLIDING CLAMP IS USED BY MANY DNA
POLYMERASES AND DNA REPAIR PROTEINS
It is now recognized that throughout the life of a cell, the b2
sliding clamp is harnessed by many DNA polymerases and
other proteins involved in the synthesis and integrity of
double-stranded DNA. In fact, b2, with its ability to slide
along duplex DNA and to stall at sites of DNA damage, may
act as a physical screening device that stops to recruit the
appropriate repair complex wherever DNA integrity is
compromised (16). The b dimer interacts with several enzymes
active in excision repair, mismatch repair and translesion
DNA synthesis. Binding of b2increases the processivity of
these polymerases (11, 14, 16) and, in the case of Pol IV, an
increase in the affinity for dNTPs (17). These protein families
(Fig. 1 and Table 1) include Pol I, Pol II (or PolB), Pol IV (or
DinB) and Pol V (UmuD and UmuC subunits), as well as
MutS, a mismatch recognition protein (reviewed in refs. 2, 3).
Synthesis of many of these proteins is induced during the SOS
(DNA damage) response.
b2IN SWITCHING FROM INITIATION TO
ELONGATION IN DNA REPLICATION
In E. coli DNA replication starts at a specific initiation site
(oriC) where the initiator protein, the active form of DnaA
(ATP-DnaA) binds to oriC, and this allows a local opening of
the duplex DNA. DnaB helicase moves in to generate a more
extensive unwinding of the DNA and expand the single-
stranded region. b2is then loaded onto primed single stranded
DNA by the clamp loader allowing the Pol III holoenzyme to
assemble around it. This complex rapidly replicates large
stretches of DNA along the single DNA strands. On
termination of replication, the Pol III holoenzyme falls away
from b2. The free b2sitting on this duplex DNA at the end of
the newly synthesised fragment interacts indirectly with ATP-
DnaA through a bridge protein called Hda (18, 19). Hda
forms homodimers and two Hda homodimers can bind a
single b2 (20). The interactions to form DnaA-Hda2-b2
promotes the conversion of ATP-DnaA to an inactive form
(ADP-DnaA) which in turn inhibits inappropriate re-initiation
at oriC during replication.
By combining the experimental identification of b2-binding
proteins with the extensive protein sequence information
provided by the eubacterial emerging genome sequences, and
by analogy with known clamp-binding motifs in the eukar-
yotes and Archea, we predicted that many b2-binding proteins
attach to b2via a short pentameric peptide segment with the
consensus sequence, QL[S/D]LF (21, 22). Small regions of the
a subunit and several other proteins containing putative b2
binding sequences were shown to bind b2using a yeast two-
hybrid system, and synthetic peptides containing the b2
binding sequences inhibited a–b2and d–b2interactions in
vitro. Based on the identification of this sequence, a conserved
SLF motif in the d subunit was proposed to be involved in
binding of d to b2during clamp loading (21). The subsequent
crystallographic structure of the b–d complex confirmed that
residues L73and F74of the sLF motif in the E. coli d subunit
penetrate a hydrophobic pocket on the b2 surface (23).
Experimental evidence suggests that the a subunit binds at
the same site in a similar manner (24). Binding of the two
proteins to overlapping sites underpins the switch in replica-
tion whereby b2is passed in the clamp loader to a.
Having identified and experimentally validated the penta-
b2-binding proteins for conserved related conserved motifs and
for new families with such motifs located at the amino- and
carboxyl-termini. Using this search strategy we identified
several new families of putative b2-binding proteins, including
new families of probable DNA repair polymerases DnaE2 and
been experimentally demonstrated. Many species, such as
Streptomyces species, P. aeruginosa and Agrobacterium tume-
faciens C58, contain one or more copies of members of DnaE2.
Mycobacterium tuberculosis its expression is linked with the
development of drug resistance (25). Mycobacterium species
lack many of the inducible DNA repair enzymes.
During the course of the bioinformatics analysis a second
motif emerged as a possible b2-binding motif, a hexapeptide
QLPLPL which is related to the pentapeptide motif and found
in the Hda family. With the exception of a few instances, the
hexapeptide motif is close to the amino-terminus. The role of
the hexapeptide motifs from Hda (18, 19) and DinB (26) has
been confirmed experimentally. Mutagenesis of the proposed
b2-binding sites of the repair polymerases Pol II, Pol IV and
Pol V ablated translesion synthesis in vivo (27). Recent
structures of fragments of Pol IV, containing a hexameric
b2-binding motif revealed that two leucines are involved in
similar interactions with the b-binding surface as the L73F74of
the d subunit (28, 29). Thus, all three types of b-binding motif
(from d, pentapeptide and hexapeptide) have a conserved core
of interaction at the same site on b2. These small loops of
sequence may act as ‘guiding pins’ in the recognition,
CONSERVATION OF BACTERIAL REPLICASES415
Table 1 Families of b2-binding proteins, and families of proteins apparently containing pentapeptide b2-binding motifs
All species Clamp loading Single copy
All species not containing PolCMajor replicative DNA polymer-
Lagging strand replicative DNA
polymerase and repair?
Repair DNA Polymerase
DnaE1 All PolC containing speciesSingle copyxxSLFyes4
DnaE2 Sporadic, primarily in Proteobac-
teria linked to DinB3
May be multiple
QLPLF nolow Internal
PolC Limited, primarily low GC-gram
Sporadic, primarily Proteobacteria
Major replicative DNA polymer-
ase – leading strand?
Repair DNA polymerase
Sporadic, primarily Proteobacteria
Repair DNA polymerase
Repair DNA polymerase
DinB2Limited, Primarily low-GC gram
Sporadic, primarily in Proetobac-
teria, linked to DnaE2
Repair DNA polymerase Some episomalQLSLF nohigh
DinB3Repair DNA polymerase? May be multiple
QLGLD nolow Internal
Initiation of replication
Duf72 Broad distribution, motif only
species closely related to E. coli
and B. subtilis
Sporadic distribution, primarily
Initiation of replication
Repair DNA polymerase subunit
Initiation of replication
Repair DNA polymerase
The proteins are listed by family, function, gene location and copy number, the consensus b2binding motif in their family, the ranking of motifs found in members of the family versus the whole
set of motifs (see ref. 22), and the location of the predicted b2-binding motif in the protein. n.a.: not applicable.
WIJFFELS ET AL.
orientation and interaction of large proteins with each other.
Once the interaction is initiated, other weakly attractive
surfaces of the protein partners may interact to strengthen
the complex. Furthermore, the confirmation of the proteins in
context of DNA may also affect their affinity for b2 as
exemplified by the a subunit (30).
However, three proteins, DNA ligase, Pol I and UmuD
(and its proteolysed form, UmuD’), have been shown to bind
b2but do not contain the b2binding motif(s) (31, 32). Pol I can
compete for binding to b2 with the a subunit and with a
peptide bearing a b2binding motif (33). UmuD and UmuD’
also bind in the same region of b2as most of the other b2
binding proteins (34).
ARE ALL b2-BINDING PEPTIDES EQUAL?
The conservation of the b2-binding peptides and the
binding site on b2allows the proteins from different species
to interact. S. aureus, S. pyogenes and B. subtilis PolC
subunits can use E. coli b2as their sliding clamp (4, 35, 36).
However, the interspecies exchange does not always apply.
The E. coli polymerase (a) cannot use b2 from the other
species (36), and the H. pylori d subunit does not interact
with E. coli b2. The E. coli clamp loader cannot load
S. aureus b2(35) and the S. pyogenes clamp loader cannot
load E. coli b2 (4). These results indicate that underlying
mechanisms associated with some structural features of the
E. coli b2 clamp may not apply to bacteria outside the
Experimental data with peptides with native sequences
derived from a number of b2-binding proteins and modified
peptides have shown that the peptides with the sequences
closest to the consensus sequence have the strongest b2binding
We have attempted to correlate the ranking of the b2-
binding sequences in the different families of b2 binding
proteins with their potential function and competition for
the b2-binding site. The distribution of conservation to the
b2 binding sequences compared to the consensus sequence
was analysed on a protein family by family basis. Some
protein families, PolC, PolB and DinB2, for example, had
very high ranking conserved motifs, whilst other families,
such as MutS and DnaE2 had generally poorly conserved
motifs (Table 1).
Although the a subunit (DnaE) is the major replicative
DNA polymerase in most species, its b2-binding sequence has
an intermediate ranking or conservation of the consensus b2-
binding sequences relative to PolC proteins (Table 1). This was
rather surprising as the DnaE enzyme probably occupies b2
for more time than any other enzyme in the cell. In contrast to
all of the other enzymes the b2-binding peptide motif in the
DnaE proteins is not located close to, or at, the amino- or
carboxyl-terminus. In fact, the b2-binding motif is flanked by
two DNA-binding domains, suggesting that the polymerase
subunit could contact DNA on both sides of b2to provide
additional stability to the complex. Binding at this site may
also be supported by a second b2-binding site located at the
carboxyl-terminus of the a subunit (12). However, this site is
not conserved across a wide range of species. In contrast, the
high ranking b2-binding peptide in the PolC enzyme is located
right at the carboxyl-terminus of the protein. The DnaE
enzymes in PolC-containing species have an even weaker
(predicted) b2-binding motif than the other DnaE enzymes. It
is interesting to speculate that the distinct motif in most DnaE
proteins represents a compromise between the b2-binding
requirements for leading and lagging strand synthesis. In
PolC-containing species, the weaker interaction of DnaE with
b2is allowed because of its lagging-strand-specific role.
PLASMID-ENCODED b2-BINDING PROTEINS
A number of families of genes that encode replication
initiator (Rep) proteins are carried on mobile genetic elements
such as plasmids, phages and transposons (38, 39). Several of
these proteins contain high ranking potential b2-binding
motifs (21). The distribution of families of Rep proteins
containing potential b2-binding sites is sporadic, but the
sequences are conserved across members of a family or
subfamily. To the best of our knowledge, the binding of
plasmid or phage Rep proteins to b2has not been investigated,
although many Rep proteins have been shown to bind to other
components of the DNA replication apparatus. No episome
encoding both a Rep protein with a putative b2-binding site
and a repair DNA polymerase (of either the DinB/UmuC/
Rad30/Rev1 or the DnaE family) has been described. It is not
clear what benefit accrues to plasmids encoding repair DNA
There is clear conservation of the general architecture of the
clamp loader and the sliding clamp across all eubacterial
species. The two, apparently interchangeable, peptide b2-
binding motifs, and their common binding site on b2appear to
represent a universal system across all eubacteria, the many
interacting partners providing a strong selective pressure for
conservation. The multiplicity of b2-binding proteins in one
cell, all predicted to bind at the same site on b2, raise questions
concerning the nature of the regulation of these interactions
(2, 33). The biological significance of some the interactions, for
example with MutS and DNA ligase, have yet to be
demonstrated, and may vary in utility between species and
environmental or ecological contexts. The extent of use of the
b2sliding clamp by polymerases involved in replication, repair
or recombination of duplex DNA is quite different among
Factors influencing competition for the site on b2may be:
(a) the context of the site on DNA at which b2is located/
CONSERVATION OF BACTERIAL REPLICASES417
recruited – a lesion, the site of a mismatch, and a replicative
primer are examples; (b) the dynamics of other proteins in
proximity to the site – as part of a transient active complex or
a stalled, dissembling complex; and (c) the intrinsic ‘competi-
tiveness’ of the b2-binding motif of the protein. Peptides with
sequences closest to the pentameric consensus motif QL[D/
S]LF or the hexameric consensus motif QLXLXL appear to be
bind b2most effectively (37).
These and other factors affecting the role of b2in DNA
metabolism may be studied in the various repertoires of b2-
binding proteins in different bacterial species. For example, H.
pylori has only two identifiable b2-binding proteins: the a and
d subunits of the replicase, the bare minimum required for
DNA replication. M. tuberculosis lacks inducible DNA repair
enzymes. Its DnaE2 protein may have evolved to assume part
of this role. Both these bacteria are fastidious, slow growing
In contrast, E. coli, A. tumefaciens and B. subtilis have large
cohorts of repair enzymes, some encoded by plasmids. All
these species are well adapted to diverse environments. For
DNA replication in the Gram-positive organisms, PolC has
acquired the role of a highly processive, accurate, replicative
polymerase and has generally highly-ranked b2-binding
motifs. The DnaE enzyme in these organisms appears to have
assumed the role of an error prone, less processive polymerase
dedicated to the lagging strand.
Thus far, DNA replication, repair and recombination of
few organisms have been studied in any depth. The
environmental pressures on the evolution, selection and
usage of the b2-binding proteins are not understood.
Uncovering the roles of these factors by interrogating the
DNA replication and repair systems of diverse bacterial
species will provide models of other ‘solutions’ and will
become essential in understanding
evolution of new phenotypes in bacteria and their plasmids
in medical and agricultural environments.
and predicting the
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