REVIEW ARTICLE
DNA replication fidelity in Escherichia coli: a multi-DNA
polymerase affair
Iwona J. Fijalkowska1, Roel M. Schaaper2 & Piotr Jonczyk1
1
Institute of Biochemistry and Biophysics, Polish Academy of Sciences, Warsaw, Poland; and 2Laboratory of Molecular Genetics, National Institute
of Environmental Health Sciences, Research Triangle Park, Durham, NC, USA
Correspondence: Piotr Jonczyk, Institute of
Biochemistry and Biophysics, Polish Academy
of Sciences, Pawinskiego 5A, 02-106
Warsaw, Poland. Tel.: +48 22 592 1113;
fax: +48 22 592 2190;
e-mail: piotrekj@ibb.waw.pl
Received 11 November 2011; revised 29
February 2012; accepted 1 March 2012. Final
version published online 5 April 2012.
DOI: 10.1111/j.1574-6976.2012.00338.x
MICROBIOLOGY REVIEWS
Editor: Grzegorz Wegrzyn
Keywords
DNA replication fidelity; accessory DNA
polymerases; polymerase switching;
untargeted mutagenesis; leading and lagging
DNA replication; exonucleolytic proofreading.
Abstract
High accuracy (fidelity) of DNA replication is important for cells to preserve
the genetic identity and to prevent the accumulation of deleterious mutations.
The error rate during DNA replication is as low as 109 to 1011 errors per
base pair. How this low level is achieved is an issue of major interest. This
review is concerned with the mechanisms underlying the fidelity of the chromosomal replication in the model system Escherichia coli by DNA polymerase
III holoenzyme, with further emphasis on participation of the other, accessory
DNA polymerases, of which E. coli contains four (Pols I, II, IV, and V).
Detailed genetic analysis of mutation rates revealed that (1) Pol II has an
important role as a back-up proofreader for Pol III, (2) Pols IV and V do not
normally contribute significantly to replication fidelity, but can readily do so
under conditions of elevated expression, (3) participation of Pols IV and V, in
contrast to that of Pol II, is specific to the lagging strand, and (4) Pol I also
makes a lagging-strand-specific fidelity contribution, limited, however, to the
faithful filling of the Okazaki fragment gaps. The fidelity role of the Pol III τ
subunit is also reviewed.
Introduction
The mechanisms by which organisms are able to efficiently and faithfully replicate their DNA are of critical
importance in genetics, biology, and medical science, and
research into these mechanisms has received significant
attention. Overall, DNA replication is a high-accuracy
process that enables cells and organisms to maintain their
genetic identity. On the other hand, the replication process is not without error and mutations will occur with
defined frequencies. While mutations are essential factors
in mediating long-term processes of adaptation and evolution or in generating antibody diversity, in the short
run mutations are generally deleterious and may be a
cause of human disease, like birth defects or cancer
(Loeb, 1991; Jackson & Loeb, 1998; Preston et al., 2010;
Tuppen et al., 2010). Studies of spontaneous mutation
rates in various systems have provided a baseline value
for the in vivo replication error rate. When expressed per
base pair (per round of replication), the rates can be
5 9 1010 for bacterial systems (Escherichia coli) or
FEMS Microbiol Rev 36 (2012) 1105–1121
5 9 1011 for mammalian systems (Drake, 1991; Drake
et al., 1998). It is clear that such low error rates must be
the outcome of multiple fidelity pathways acting in parallel and/or consecutively.
In broad outline, high fidelity must be based on two
major components. First, the DNA must be kept intact
and, to the extent possible, free of any damage before
DNA replication is to proceed. Obviously, damaged DNA
is unlikely to be copied accurately. The multitude of
DNA surveying and repair systems used by cells to monitor their DNA attest to the critical importance of this
issue (Friedberg et al., 2006). Second, the intrinsic accuracy of DNA replication, when performed on essentially
damage-free DNA, has its limits. Therefore, unavoidably,
DNA polymerases will commit errors, leading to mutations. The present review focuses on this particular aspect
of DNA replication fidelity and will cover insights
obtained regarding this process from the model system
E. coli. The data used are derived from mutation frequencies in actively growing E. coli cultures under aerobic conditions in the absence of external stresses.
ª 2012 Federation of European Microbiological Societies
Published by Blackwell Publishing Ltd. All rights reserved
1106
Nevertheless, spontaneous DNA damage (e.g. hydrolytic
DNA damage leading to deaminated bases or abasic sites)
does occur under these conditions, which, if unrepaired,
may contribute to observed mutations. Thus, an absolute
distinction between the two types of mutation production
may not be possible. In our studies, we have minimized
the possible contribution of spontaneous DNA damage
by performing most experiments in DNA mismatchrepair-defective strains, in which uncorrected replication
errors are assumed to predominate (see below).
As a general outline, we present in Fig. 1 a cartoon
showing how in most organisms the overall accuracy of
DNA replication can be considered as the outcome of
three (highly conserved) components acting sequentially:
(1) selection of the correct DNA nucleotide by the replicative DNA polymerase, (2) removal of any misinserted
nucleotides by the 3′?5′ exonuclease proofreading activity associated with the polymerase, and (3) postreplicative
correction of polymerase errors that have escaped proofreading by the DNA mismatch repair system (MMR)
(Schaaper, 1993a; Kunkel & Bebenek, 2000; Kunkel, 2004;
Kunkel & Bebenek, 2004). In E. coli, the mismatch repair
step is performed by the mutHLS system, which is capable of distinguishing the (incorrect) newly synthesized
strand from the (correct) parental strand based on the
transient undermethylation (at 5′-GATC-3′ sequences)
of the newly made strand (reviewed in Modrich, 1987;
I.J. Fijalkowska et al.
Kunkel & Erie, 2005). Roughly, the contribution of each
of these components to the error rate can be estimated
at 105 (insertion), 102 (proofreading), and 103
(mismatch repair), accounting for the 1010 overall rate,
although each contribution is highly dependent on the
precise type of error under question (Schaaper, 1993a;
Kunkel & Bebenek, 2000; Kunkel, 2004).
Chromosomes are replicated generally by large, multisubunit, dimeric complexes – termed replicases, which
are capable of copying the two strands, leading and lagging, at a replication fork in a coordinated fashion. In
E. coli, the replicase is DNA polymerase III holoenzyme
(Pol III HE), a dimeric enzyme that contains two copies
of DNA polymerase III, one for each strand. A schematic
depiction of HE containing its various associated subunits
at a replication fork (replisome) is shown in Fig. 2. Replicases and their functioning at the replication fork are
subjects of intense current interest (e.g. McHenry, 2011).
Specific questions regarding the functioning and fidelity
of chromosomal replisomes center around: (1) what is
the precise composition of the complex, (2) which DNA
polymerase copies which strand, (3) is there differential
fidelity between leading and lagging strands, (4) what is
the contribution to fidelity of the noncatalytic subunits
within the replicase, and (5) what is the fidelity contribution, if any, of the additional DNA polymerases that cells
possess? The present review will focus, in part, on the latter aspect, namely the possible role of the additional
polymerases, often referred to as ‘polymerase switching’.
Multiple DNA polymerases in the cell
Fig. 1. General scheme indicating how three serial fidelity steps
during chromosomal replication can produce the low error rate of
~ 1010 errors per base per round of replication. The steps are (a)
discrimination by the polymerase against inserting an incorrect base
(error rate ~ 105); (b) proofreading (editing) of misinserted bases
(e.g. T·T) by the 3′?5′ exonuclease associated with the polymerase
(escape rate ~ 102); and (c) removal of remaining mismatches (e.g.
T·C,) by postreplicative DNA mismatch repair (MMR) (escape rate
~ 103).
ª 2012 Federation of European Microbiological Societies
Published by Blackwell Publishing Ltd. All rights reserved
Escherichia coli encodes five distinct DNA polymerases:
Pol I, Pol II, Pol III, Pol IV, and Pol V. These polymerases, along with some of their relevant properties, are listed
in Fig. 3. The presence of multiple DNA polymerases in
the cell is a general phenomenon: for example, the yeast
Saccharomyces cerevisiae has eight, while human cells possess at least 16 DNA-template-dependent DNA polymerases (Friedberg et al., 2006; Shcherbakova & Fijalkowska,
2006; Kunkel, 2009). Some of these, functioning usually
as replicative DNA polymerases, are high-fidelity enzymes
possessing a 3′?5′ exonuclease proofreading activity. But
other polymerases are error-prone enzymes, lacking a
proofreading activity and having an adjusted (or
enlarged) active site permitting higher frequencies of base
misinsertions (reviewed in Friedberg et al., 2002; Goodman, 2002; Nohmi, 2006; Shcherbakova & Fijalkowska,
2006; Jarosz et al., 2007; Lehmann et al., 2007; McCulloch
& Kunkel, 2008). Most of the error-prone polymerases,
referred to as translesion synthesis polymerases (TLS
polymerases), may have evolved to bypass replicationblocking template lesions. TLS allows strand extension
FEMS Microbiol Rev 36 (2012) 1105–1121
1107
DNA replication fidelity in Escherichia coli
Fig. 2. A model of the Escherichia coli DNA Pol III HE at the
chromosomal replication fork, synthesizing, simultaneously, leading
and lagging strands in, respectively, continuous (leading-strand) and
discontinuous (lagging-strand) fashion. The aeh complex represents
the Pol III core, in which a is the polymerase, e is the exonuclease
(proofreading) subunit, and h is a stabilizing subunit. See text for
details. Not shown is the DnaG primase, which, in association with
the DnaB helicase, produces lagging-strand RNA primers supporting
the discontinuous synthesis in this strand. The c complex (cdd′vw)
conducts the cycling (loading and unloading) of the b2 processivity
clamps, which is particularly important for the cycling of the
polymerase in the lagging strand. Recent studies have suggested that
the relevant form of the DnaX assembly (τ2cdd′vw as shown here)
may be τ3dd′vw (noting that c and τ are both products of the dnaX
gene and differ only in their C-termini). As τ contains the extra
C-terminal extension that mediates the τ–a interaction, the τ3dd′vwcontaining HE is capable of binding a third Pol III core (McInerney
et al., 2007; Reyes-Lamothe et al., 2010; Georgescu et al., 2012).
This third Pol III core (not shown) may participate in polymerase
switching and hence contribute to the chromosomal replication
process (see text).
when the replicative DNA polymerase is stalled at a DNA
lesion, a subject that has been extensively covered (reviewed
in Friedberg et al., 2002; Goodman, 2002; Nohmi, 2006;
Shcherbakova & Fijalkowska, 2006; Jarosz et al., 2007;
Fuchs & Fujii, 2007; Lehmann et al., 2007; Guo et al., 2009;
Takata & Wood, 2009; Sutton, 2010; Andersson et al.,
2010; Livneh et al., 2010; Ho & Scharer, 2010).
In addition to lesion bypass performed by specialized
TLS polymerases, evidence has accumulated, from both
in vitro and in vivo experiments, that the replicase is a
dynamic entity, in which DNA polymerase exchanges can
occur, thus providing access of accessory polymerases to
the replication fork also during the synthesis of undamaged DNA (Banach-Orlowska et al., 2005; Furukohri
et al., 2008; Gawel et al., 2008; Uchida et al., 2008; Curti
et al., 2009; Heltzel et al., 2009a; Makiela-Dzbenska et al.,
FEMS Microbiol Rev 36 (2012) 1105–1121
2009). These observations raise important fidelity questions. In some cases, this participation of accessory
polymerases may lead to improved fidelity, when the
accessory DNA polymerase is accurate (e.g. a proofreading-proficient polymerase), while in other cases this may
lead to lower fidelity when the polymerase is error-prone.
In this review, we will summarize the results from
work conducted in the bacterium E. coli, showing that
during normal DNA replication, both replicative and
accessory DNA polymerases contribute to the overall
fidelity of DNA replication. Furthermore, we will describe
studies suggesting that certain noncatalytic subunits of
HE, like the τ subunit, have a role in controlling the
access of these polymerases to the fork, affecting, in this
manner, the fidelity of replication. We will first discuss
the major replicase, DNA Pol III HE, then accessory
polymerases II, IV, and V, which may have occasional
access to the replication fork, and finally Pol I, whose
fidelity role may be limited to the processing of Okazaki
fragments.
DNA Pol III HE
The major replicative DNA polymerase in E. coli cells is
DNA Pol III HE that can synthesize DNA with high processivity (over 50 kb per binding event) (Yao et al., 2009)
and with high speed (up to 1000 nucleotides per second).
As shown in Fig. 2, Pol III HE in E. coli is an asymmetric, dimeric enzyme and can perform coupled, high-speed
replication of the two, leading and lagging, DNA strands
at the replication fork (for reviews, see: McHenry, 2003;
O’Donnell, 2006; Pomerantz & O’Donnell, 2007; Yao &
O’Donnell, 2008; Langston et al., 2009; McHenry, 2011).
Pol III HE is a complex of 17 subunits (10 distinct proteins) with an overall composition (aeh)2(b2)2τ2cdd′vw.
It contains two Pol III core subassemblies, one for each
strand, each consisting of three tightly bound subunits: a
(dnaE gene product, the DNA polymerase), e (dnaQ gene
product, the 3′?5′ proofreading exonuclease), and h
(holE gene product, stabilizer for e subunit) (Gefter et al.,
1971; Takano et al., 1986; Taft-Benz & Schaaper, 2004).
Each core is tethered to the DNA by a ring-shaped sliding-clamp processivity factor (b2 homodimer) (Kong
et al., 1992). Additionally, Pol III HE contains the sevensubunit (τ2cdd′vw) DnaX complex (Pritchard et al.,
2000). This complex contains the τ2 dimer, which
connects the two Pol III a subunits, creating the dimeric
polymerase unit capable of coupled synthesis of leading
and lagging strands. The DnaX complex is also responsible, because of the activity of the d and d′ subunits, for
the loading and unloading of the b2 sliding-clamp (Jeruzalmi et al., 2001). This important activity is especially
critical for the lagging-strand polymerase, which needs to
ª 2012 Federation of European Microbiological Societies
Published by Blackwell Publishing Ltd. All rights reserved
1108
I.J. Fijalkowska et al.
Fig. 3. The five DNA polymerases of Escherichia coli and some of their relevant properties.
cycle on and off during Okazaki fragment synthesis
(McHenry, 2011).
The form of Pol III HE depicted here, with the DnaX
complex containing two τ subunits and one c subunit, is
the form of HE historically found when isolated from
cells (McHenry, 2011). More recently, a form of Pol III
has been described, upon reconstitution of HE in vitro, in
which the DnaX complex contains three τ subunits and
no c subunit (τ3dd′vw) leading to incorporation of three
pol III core units (McInerney et al., 2007; Reyes-Lamothe
et al., 2010; Georgescu et al., 2012). As τ and c are products of the same (dnaX) gene and are identical for the
first 430 amino acids, they share several functions and
can partially substitute for each other (Flower &
McHenry, 1986; Blinkowa & Walker, 1990; McHenry,
2011). However, τ as the longer, full-length dnaX gene
product (643 residues) has additional capabilities not
present in c, including its interaction with the a subunit,
which is responsible for the dimerization of the polymerase. The alternative τ3dd′vw DnaX complex is, however,
capable of binding three pol III cores, and this trimeric
form of HE has been demonstrated to operate efficiently
in vitro (McInerney et al., 2007; Reyes-Lamothe et al.,
2010; Georgescu et al., 2012). While a trimeric form of
HE may have certain advantages over the dimeric form
(Georgescu et al., 2012), including possibly its fidelity, the
ª 2012 Federation of European Microbiological Societies
Published by Blackwell Publishing Ltd. All rights reserved
significance of the trimeric form for HE operating in vivo
remains to be established (McHenry, 2011).
The fidelity properties of Pol III have been investigated
in vivo and in vitro. The in vivo experiments have suggested that indeed HE is highly accurate, as expected for
the major replicating enzyme (Schaaper, 1993a). The
importance of Pol III for controlling the mutation rate is
also evidenced by the properties of several E. coli mutator
mutants (which have enhanced mutation rates) carrying a
defect in the dnaE gene encoding the Pol III a subunit
(Sevastopoulos & Glaser, 1977; Fijalkowska et al., 1993;
Oller et al., 1993; Vandewiele et al., 2002). Strong mutators have also been found residing in the dnaQ gene,
encoding the e proofreading subunit (Fijalkowska & Schaaper, 1996; Taft-Benz & Schaaper, 1998), establishing the
importance of the proofreading process for the chromosomal replication process. A modest mutator effect was
also detected for strain lacking the holE gene, encoding
the h subunit (Taft-Benz & Schaaper, 2004). This mutator
effect likely reflects the stabilizing role that h subunit
plays for e subunit (the three subunits are arranged in
the linear order a-e-h) (Taft-Benz & Schaaper, 2004;
Chikova & Schaaper, 2005).
While the studies on E. coli mutator mutants described
above bear evidence to the important role that (wildtype) Pol III HE plays in preventing replication errors,
FEMS Microbiol Rev 36 (2012) 1105–1121
1109
DNA replication fidelity in Escherichia coli
they do not necessarily prove that errors made by the
wild-type enzyme contribute to the observed replication
error rate or bacterial mutation rate. That this is indeed
the case was evidenced by the isolation of a series of dnaE
antimutator mutants (Fijalkowska & Schaaper, 1993; Fijalkowska et al., 1993; Oller & Schaaper, 1994; Schaaper,
1993, 1996, 1998). In these strains, replication has
become more accurate because of the altered a subunit of
HE, resulting in a detectably lower mutation rate. The
mechanism underlying the antimutator effects could be
enhanced fidelity at the insertion step or, more likely, an
effect on the interplay between polymerase and associated
proofreading exonuclease. Following a nucleotide misinsertion, competition arises between (1) the forward polymerization rate, extending the mismatch and hence fixing
the error as a potential mutation, and (2) the reverse
reaction by the movement of the incorrect base from the
polymerase active site to the exonuclease site leading to
the removal of the base (proofreading). Obviously,
impairment in the polymerase forward rate will lead to a
different partitioning between the two reactions, leading
to enhanced proofreading and a reduction in the error
rate. This enhanced-proofreading mechanism for antimutators was first demonstrated in studies with the bacteriophage T4 DNA polymerase (Drake & Allen, 1968; Gillin
& Nossal, 1976; Drake, 1993; Reha-Krantz & Nonay,
1993; Reha-Krantz, 1995).
Makiela-Dzbenska et al., 2009; Gawel et al., 2011). The
conclusion reached from these experiments is that indeed
there is a significant difference in the error rate depending
on the gene orientation. This difference is generally 2- to
6-fold, with the lagging-strand replication being more
accurate. Therefore, the majority of replication errors result
from leading-strand replication (Fijalkowska et al., 1998).
It should be noted that the critical experiments were all
performed in a mismatch-repair-defective (mutL) background (see Fig. 1), so that the outcomes reflect the direct
production of the errors without interference of the subsequent mismatch repair step, which may have its own specificity.
Differential leading- and lagging-strand replications
have also been described in eukaryotic systems, like S. cerevisiae (Pavlov et al., 2002; Pursell et al., 2007; Nick
McElhinny et al., 2008). In this case, the fidelity differences are ascribed to replication being performed by different enzymes in the two strands (e.g. Pol e and Pol d in
leading and lagging strands, respectively). However, in the
E. coli case, both strands are copied by the same polymerase (Pol III) and the differential outcome must reflect a
different behavior of the same enzyme in its two configurations. The reason why in E. coli leading and lagging
strands are copied with differential fidelity is still open at
this time, but some clues may be found in the experiments focusing on the role of the accessory DNA polymerases, as described in the following sections.
Differential fidelity of Pol III HE in
leading and lagging strands
DNA polymerase switching
Based on the differential enzymology of leading- and lagging-strand polymerization (continuous synthesis in the
leading strand and discontinuous in the lagging strand),
it is a relevant question to ask whether replication fidelity
is identical in the two strands or whether they might differ. In the latter case, most of the replication errors may
result predominantly from one of the two strands. This
question has been addressed in E. coli by investigating the
reversion of certain lacZ alleles when placed in the two
possible orientations on the E. coli chromosome. The
logic for this type of experiment is that when a given
base substitution is mediated by a known mispair (e.g.
Ttemplate·G errors causing A·T?G·C base substitutions),
inversion of the gene orientation will place the causative
error from, for example, the leading strand to lagging
strand or vice versa. Thus, any difference observed in the
mutation rate between the two orientations may be indicative of strand-dependent replication fidelity. These
experiments have been performed with several lacZ alleles
under a large series of circumstances (Fijalkowska et al.,
1998; Maliszewska-Tkaczyk et al., 2000; Gawel et al.,
2002a, b; Banach-Orlowska et al., 2005; Kuban et al., 2005;
The question whether the E. coli accessory DNA polymerases have access to the DNA replication process and
hence may contribute to the observed error rate may be
raised for several reasons, but most directly based on the
relatively high intracellular concentrations of Pol I (400
molecules per cell), Pol II (50 molecules per cell), or Pol
IV (200–250 molecules per cell), compared to those estimated for Pol III HE (10–20 molecules per cell) (Kornberg & Baker, 1992; Qui & Goodman, 1997; Kim et al.,
2001; McHenry, 2003). The term ‘DNA polymerase
switching’ is used to describe a process by which one
DNA polymerase (e.g. the major replicative DNA polymerase) is replaced, temporarily, by another DNA polymerase at the 3′-OH end of growing chain, possibly in a
coordinated manner. This type of DNA polymerase
exchange occurs normally during DNA replication of lagging-strand DNA synthesis. Lagging-strand replication
starts with the synthesis of a short RNA primer by DNA
primase (DnaG in E. coli) followed by a switch replacing
the primase by the main replicating polymerase for the
elongation of the strand. When the polymerase reaches
the end of the Okazaki fragment, a second switch occurs,
FEMS Microbiol Rev 36 (2012) 1105–1121
ª 2012 Federation of European Microbiological Societies
Published by Blackwell Publishing Ltd. All rights reserved
1110
replacing the polymerase by an enzyme (Pol I in E. coli)
capable of removing the RNA primer and filling the
remaining gap.
Do other, similar types of DNA polymerase exchanges
occur at the replication fork during ongoing DNA replication, allowing alternative polymerases to synthesize
parts of the chromosome and contribute to the overall
replication fidelity? The possible mechanisms that promote or enable polymerase switches have been the subject
of several investigations. In vivo data clearly indicate that
in prokaryotic cells the b sliding-clamp, like PCNA
(Proliferating Cell Nuclear Antigen) in eukaryotes, is the
important platform that coordinates the participation of
different polymerases (Becherel et al., 2002; Lenne-Samuel
et al., 2002; Heltzel et al., 2009a; reviewed in Sutton,
2010). In vitro data also demonstrate the possibility of
polymerase exchanges during ongoing DNA synthesis,
showing that DNA polymerases compete depending on
polymerase concentrations and their affinity to the bclamp (Indiani et al., 2005, 2009; Furukohri et al., 2008;
Heltzel et al., 2009b).
Role of DNA polymerase II (Pol II)
DNA polymerase II, encoded by the polB (dinA) gene,
was discovered in 1970 (Knippers, 1970). Some of its
properties are presented in Fig. 3. Pol II is a prototype
for the B-family polymerases, which mostly contain replicative DNA polymerases, including the major eukaryotic
DNA polymerases a, d, e, as well as DNA polymerase ζ
(Bonner et al., 1990). Purified Pol II protein is a single
90-kDa polypeptide possessing both a DNA polymerase
and a 3′?5′ exonuclease (Cai et al., 1995). There are 30–
50 molecules of Pol II per cell, which is several times
more than the number of Pol III HE molecules (Qui &
Goodman, 1997). The level of Pol II increases about sevenfold following SOS induction (global response to DNA
damage) (Bonner et al., 1990; Iwasaki et al., 1990).
Genetic studies indicate that Pol II may be involved in a
variety of cellular activities including repair of intrastrand
cross-links (Berardini et al., 1999), repair of DNA damaged by UV irradiation and replication restart after UV
irradiation (Masker et al., 1973; Rangarajan et al., 1999),
repair of DNA damaged by oxidation (Escarceller et al.,
1994), stress-induced mutagenesis (Foster et al., 1995;
Hastings et al., 2010), and long-term survival (Yeiser
et al., 2002). It has been also shown that although Pol II
is a proofreading-proficient enzyme, it can carry out
error-prone TLS at certain lesions (Becherel & Fuchs,
2001; Wang & Yang, 2009). In vitro studies have shown
that Pol II interacts with the b-clamp (b2) and the
clamp-loading complex (cdd′vw), both major Pol III
accessory proteins, to become competent for synthesizing
ª 2012 Federation of European Microbiological Societies
Published by Blackwell Publishing Ltd. All rights reserved
I.J. Fijalkowska et al.
DNA with high fidelity and processivity (Wickner & Hurwitz, 1974; Hughes et al., 1991; Bonner et al., 1992; Heltzel et al., 2009a). Thus, E. coli, besides Pol III, possesses
another DNA polymerase capable of participating, at least
in principle, in chromosomal DNA synthesis.
The above properties of Pol II have given rise to the
concept that this enzyme may function as a back-up polymerase for Pol III HE (Banach-Orlowska et al., 2005). In
this model, Pol II may occasionally take the place of Pol
III through polymerase switching. Initially, this concept
was rather difficult to accept, as experiments with E. coli
strains deleted for Pol II did not show significant effects
on the chromosomal error rate (Rangarajan et al., 1997;
Banach-Orlowska et al., 2005). However, a role of Pol II
in chromosomal DNA synthesis was revealed through
experiments using mutants carrying a proofreading-deficient form of Pol II (PolBex1). The underlying rationale
for the experiments replacing the accurate, proofreadingproficient form of Pol II by its proofreading-deficient and
error-prone form (PolBex1) is that in a background of
high-fidelity DNA synthesis (by Pol III HE), even a small
amount of Pol II-mediated synthesis may be detectable
through an increase in the mutation frequency (mutator
phenotype). Initially, this strategy was used to demonstrate a polBex1-mediated increase in chromosomal rpoB
mutations in the dnaE915 antimutator background
(Rangarajan et al., 1997). The dnaE915 allele encodes a
mutant Pol III a-subunit that promotes higher accuracy
of chromosomal replication (Fijalkowska & Schaaper,
1993; Fijalkowska et al., 1993), and it has been assumed
that the more prominent apparent role of the polBex
allele in the dnaE915 background is a consequence of
either the reduced background of HE-mediated mutations, making it easier to see the Pol II effect, or a possibly enhanced tendency of the DnaE915-encoded
polymerase to dissociate from the template and hence to
exchange places with Pol II (Fijalkowska et al., 1993; Fijalkowska & Schaaper, 1995; Rangarajan et al., 1997).
Regardless of the precise mechanisms, these experiments
highlight the potential of Pol II to participate in chromosomal DNA synthesis.
Using the lacZ leading/lagging system described earlier,
Banach-Orlowska et al. (2005) also demonstrated that the
presence of the proofreading-deficient Pol II (PolBex1) in
the cell significantly increased the mutant frequencies,
however, not only in cells carrying dnaE915 but also in
dnaE+ cells. Even more interestingly, the polBex1 mutator
effect was more pronounced for the lagging strand. These
results suggested that Pol II has access to the chromosomal replication fork and participates in the replication
process, possibly in a strand-preferential way. Interestingly, no significant changes in mutability were observed
in cells lacking Pol II (DpolB strains), suggesting that any
FEMS Microbiol Rev 36 (2012) 1105–1121
DNA replication fidelity in Escherichia coli
DNA synthesis performed by wild-type (proofreadingproficient) Pol II is generally error-free.
We have proposed that the role of Pol II in DNA replication is that of a back-up polymerase to Pol III HE.
There are likely to be situations where HE may temporarily dissociate from the primer terminus, possibly randomly, but more plausibly only in special situations
where HE could temporarily stall, such as at DNA lesions
or secondary structures. We have proposed that the fidelity role of Pol II is most easily explained by a switch-over
from HE to Pol II at terminal mismatches created by a
HE misinsertion error. Extension of terminal mismatches
is generally difficult for DNA polymerases (Perrino &
Loeb, 1989; Mendelman et al., 1990; Joyce et al., 1992),
and in vitro data on a subunit and HE have confirmed
this to be the case also for Pol III (Kim & McHenry,
1996; Mo & Schaaper, 1996; Pham et al., 1998, 1999).
While a majority of terminal mismatches are expected to
be removed by the proofreading activity of Pol III (e subunit), a certain fraction of errors will escape the exonuclease, presenting a critical decision point for the
polymerase. Extension of the mismatch would fix the error
into a potential mutation, while Pol III dissociation from
the mismatch would allow for additional scenarios. Takeover of the terminal mismatch by Pol II is expected to lead
to the removal of the mismatch by the Pol II 3′-exonuclease, a clear fidelity function. On the other hand, a take-over
by the exonuclease-deficient Pol II would likely lead to the
extension of the mismatch – fixing the error – consistent
with the observed polBex mutator effect. This model has
the added advantage that extensive chromosomal synthesis
by Pol II need not be invoked to explain the polBex mutator
effect (Banach-Orlowska et al., 2005). The possibility that
the proofreading activity of one polymerase can correct
errors made by another polymerase has been shown in
S. cerevisiae cells (Pavlov et al., 2004).
Further support for the involvement of Pol II in DNA
replication and the proposed sequential action of Pol III
and Pol II came from experiments showing that the polBex
allele synergistically enhanced a Pol III proofreading defect
(dnaQ mutant) or Pol III mutator effect (dnaE486 or
dnaE511 mutants) (Banach-Orlowska et al., 2005). Again,
the simplest interpretation of these results is that when
Pol III HE has problems (e.g. stalling owing to increased
mismatch production and/or defective polymerase) and
the frequency of its dissociation increases, the access of Pol
II to the terminal mismatch also increases. This model for
the participation of Pol II is presented in Fig. 4.
Role of Pol IV at the replication fork
DNA Polymerase IV (Pol IV) is one of the two main TLS
polymerases of E. coli, the other being Pol V (see section
FEMS Microbiol Rev 36 (2012) 1105–1121
1111
below), both members of Y family of polymerases (Reuven et al., 1999; Tang et al., 1999; Wagner et al., 1999).
Pol IV, encoded by the damage-inducible dinB gene, is a
single-polypeptide enzyme lacking intrinsic proofreading
activity, and its fidelity is considered to be relatively low
(Tang et al., 2000; Goodman, 2002). The constitutive
intracellular concentration of Pol IV is fairly high (250
molecules per cell) and increases by about 10-fold upon
SOS induction (Kim et al., 2001). The high constitutive
presence of this polymerase in the cell raises the question
what its role might be and whether it might have occasional access to the replication fork and affect the overall
chromosomal fidelity. Owing to the lack of exonucleolytic
proofreading activity, its involvement in replication, if
any, is expected to be error-prone.
Goodman (2002) proposed that in certain situations,
for example at blocked replication forks, Pol IV might
Fig. 4. Proposed scheme for the involvement of Escherichia coli DNA
polymerases in error production and prevention at the replication
fork, as based on the work described in this review. Events start
when Pol III commits a misinsertion error (indicated as T:T or A:A) in
either DNA strand. While Pol III may handle the mismatch by itself by
either extending the mismatch, yielding a potential mutation, or
removing it using its proofreading activity (not shown), in a fraction
of cases Pol III may dissociate from the mismatch, leading to a
possible polymerase exchange (arrows). The data suggest that, in
both strands, Pol II is the primary enzyme for such a polymerase
exchange, leading to the removal of the terminal mismatch by the
3′ exonuclease of Pol II (back-up proofreading function). When
dissociation occurs in the lagging strand, Pol IV and Pol V may also
compete for the primer terminus, leading to the extension of the
mismatch and fixing the error as a potential mutation. In contrast, Pol
I does not compete for the primer terminus in either strand. Instead,
its fidelity function is limited to the error-free filling of the Okazaki
fragment gaps. We have also indicated in the scheme the third Pol III
core (shaded green) that may be present in HE if the indicated DnaX3
assembly were composed of three τ subunits (McInerney et al., 2007;
Reyes-Lamothe et al., 2010; Georgescu et al., 2012). If present, this
third core might be expected to behave similarly to Pol II, leading,
upon binding to the terminus, to mismatch removal (preferential
binding with exonuclease site). See text for further details and
justification.
ª 2012 Federation of European Microbiological Societies
Published by Blackwell Publishing Ltd. All rights reserved
1112
gain access to the nascent 3′-end and perform remedial
DNA synthesis. This possibility again raised the question
of any possible Pol IV contribution to spontaneous mutagenesis. However, three independent studies demonstrated
that in growing cells the lack of Pol IV did not significantly reduce the level or types of spontaneous chromosomal mutations (McKenzie et al., 2003; Kuban et al.,
2004; Wolff et al., 2004). This indicated that access of Pol
IV to the replication fork must be limited under normal
growth conditions, at least as far as any error-prone role
is involved (an error-free role would not be readily
detectable in these assays). On the other hand, overproduction of Pol IV, from low- or medium-copy number
plasmids, clearly resulted in a mutator phenotype (Kim
et al., 1997; Wagner & Nohmi, 2000; Kuban et al., 2005).
Interestingly, our group also showed that this Pol IV
mutator effect occurs preferentially during lagging-strand
synthesis, similar to the mutator effect of polBex1 strains
(Banach-Orlowska et al., 2005; Kuban et al., 2005). Furthermore, the expression of dinB from a medium-copy
plasmid led to a threefold decrease in viability, indicative
of some deleterious effects of Pol IV overproduction (Kuban et al., 2005), consistent with experiments in which
Pol IV was overexpressed from an arabinose-inducible
promoter (Uchida et al., 2008), which showed the inhibition of DNA synthesis and a dramatic loss of viability
when Pol IV levels were increased up to 15-fold higher
than observed in SOS-induced cells (Uchida et al., 2008).
Clearly, Pol IV can compete with HE when its concentration is sufficiently elevated.
To account for the mutator effect resulting from Pol
IV overproduction, two simple models can be proposed.
In the first model, the increased number of mutations
results from errors made by Pol IV during ongoing synthesis. However, this would require extensive synthesis by
Pol IV, especially in view of the low processivity of Pol
IV (even when supported by the b-clamp) (Wagner et al.,
2000; Kobayashi et al., 2002). For example, assuming that
Pol IV is 100-fold less accurate than Pol III, the 10-fold
mutator effect would require 10% of the chromosome to
be replicated by Pol IV. In the second, probably more
plausible model, the Pol IV mutator activity results from
the error-prone extension of mismatches made by Pol III
HE, similar to the proposed model for the PolBex mutator effect (see above). Again, terminal mismatches present
a logical step-up point for accessory polymerases, and
their effect could be either mutagenic or antimutagenic
depending on the nature of the accessory polymerase.
Support for this view of Pol IV-mediated mismatch
extension is found in experiments where greater than
additive effects were observed when Pol IV overproduction is performed in dnaQ928, dnaE486, or dnaE511
mutator strains, which contain functionally impaired e
ª 2012 Federation of European Microbiological Societies
Published by Blackwell Publishing Ltd. All rights reserved
I.J. Fijalkowska et al.
(dnaQ) or a (dnaE) subunits (Kuban et al., 2005). As the
dnaQ or dnaE mutator alleles are expected to increase the
number of replication errors (i.e. terminal mismatches), a
multiplicative or at least a greater than additive effect is
expected.
The experiments with the dnaQ or dnaE mutator
alleles, where replication produces increased number of
terminal mismatches, provide a useful testing ground for
probing the competition between various polymerases, in
particular competition between accessory polymerases Pol
II and Pol IV. These experiments were performed in dnaE
or dnaQ mutator strains lacking Pol II, Pol IV, or both
(Banach-Orlowska et al., 2005; Makiela-Dzbenska et al.,
2009). In contrast to what is observed in dnaE+ strains,
the lack of Pol II (DpolB) in dnaE486 or dnaE511 strains
led to a 10- or 7-fold increase in the mutator effect,
respectively. The lack of Pol IV (DdinB) reduced the
mutator activity of dnaE486 or dnaE511 by twofold. Both
results are consistent with an important role of the two
accessory polymerases in establishing the replication error
rate: Pol II in a mutation-preventive mode and Pol IV in
a mutation-enhancing mode. When both accessory
polymerases were deleted, the mutability of dnaE strains
was similar to that observed when only Pol IV was lacking. The results are fully consistent with a model in which
access of Pol IV leads to mutagenesis, and this is a major
component of the dnaE or dnaQ mutator effects, whereas
Pol II is strongly antimutagenic, most simply envisaged
by competing out Pol IV for access to the mismatch.
Interestingly, the (dinB-dependent) 10- or 7-fold
increase in mutagenesis of dnaE486 DpolB or dnaE511
DpolB strains is much lower than the increase caused by
the presence of polBex1 allele (55- to 280-fold) (polBex
dnaE486 or polBex dnaE511). This large 55- to 280-fold
effect indicates that Pol II’s role in editing mismatches in
dnaE486 or dnaE511 cells is much larger than anticipated
from the DpolB or DpolB DdinB experiments. Presumably,
in the absence of Pol II, Pol IV still has limited access
and will need to compete with other factors, most likely
Pol III HE. Taken together, these in vivo experiments
indicate that Pol II along with its proofreading activity
serves as an important back-up system for error removal
and at the same time protects primer termini against the
activity of error-prone Pol IV. These functions become
particularly important under conditions of a malfunctioning of Pol III HE. The role of Pol IV in lagging-strand
replication and its competition with Pol II is schematically presented in Fig. 4.
Role of DNA polymerase V (Pol V)
Pol V, encoded by the umuDC operon, is, like Pol IV, a
member of the Y family of polymerases (Reuven et al.,
FEMS Microbiol Rev 36 (2012) 1105–1121
1113
DNA replication fidelity in Escherichia coli
1999; Tang et al., 1999; Wagner et al., 1999). See Fig. 2
for some of its properties. Pol V is a heterotrimeric complex (UmuD02 C), containing the umuC gene product representing the catalytic subunit, and two copies of UmuD′,
a product of RecA-facilitated autodigestion of UmuD
protein (Nohmi et al., 1988; Shinagawa et al., 1988;
McDonald et al., 1998). Pol V is clearly an error-prone
enzyme, as it lacks proofreading activity and has a promiscuous base insertion fidelity that allows it to copy
across a variety of DNA lesions (TLS polymerase) at the
expense of high levels of mutagenesis (Reuven et al.,
1999; Tang et al., 1999, 2000). The level of Pol V in
normal (noninduced) cells is low and largely undetectable (Woodgate & Ennis, 1991), although certain genetic
experiments have suggested that it is not entirely absent
(e.g. Bhamre et al., 2001). However, importantly, upon
SOS induction, the level of Pol V increases dramatically, producing 2400 molecules of UmuD and 200
molecules of UmuC (Woodgate & Ennis, 1991; Kim
et al., 2001).
As normally Pol V is rarely expressed in the cell, it
does not play a significant role in determining the chromosomal replication fidelity under normal (non-SOSinduced) growth conditions (Nowosielska et al., 2004).
On the other hand, its potential of interfering with ongoing chromosomal replication can be seen in strains in
which the SOS system (and hence Pol V) is induced constitutively in the absence of exogenous DNA damage.
This condition of ‘spontaneous SOS induction’ is
achieved in certain recA strains (recA441, recA730) in
which the RecA regulatory protein is constitutively active,
leading to constitutive expression of the SOS system,
including the overproduction of Pol V as well as Pol IV
(Witkin, 1974; Witkin et al., 1982; Tessman & Peterson,
1985). In these strains, increased mutagenesis is observed
in the absence of any DNA-damaging treatment (Castellazzi et al., 1972; Ciesla, 1982; Witkin & Kogoma, 1984;
Sweasy et al., 1990). This phenomenon of increased spontaneous mutagenesis is named the ‘SOS mutator phenotype’ or ‘untargeted SOS mutagenesis’ (Caillet-Fauquet &
Meanhaut-Michel, 1988). Genetic studies on the recA730induced SOS mutator phenotype led to a postulated
model in which the Pol V-mediated increase in mutagenesis results from Pol V displacing HE at HE-generated
terminal mismatches (insertion errors) leading to errorprone extension of these mispairs (Fijalkowska et al.,
1997). This type of mispair extension is likely further
aided by the observed increases in cellular dNTP levels in
SOS-induced cells (Gon et al., 2011). As noted above for
Pol II and Pol IV, error-prone extension of pre-existing
mispairs is an economical way of accounting for the
increased mutation rates, as opposed to postulating
extensive synthesis by Pol V and accounting for the high
FEMS Microbiol Rev 36 (2012) 1105–1121
error rate by Pol V-mediated synthesis errors (Maliszewska-Tkaczyk et al., 2000).
Among the base substitution mutations that are
enhanced in recA730 strains, transversions predominate
(Fijalkowska et al., 1997; Maliszewska-Tkaczyk et al., 2000;
Kuban et al., 2006). This finding is consistent with in vitro
data showing that transversion mismatches are most difficult to extend by HE (and more difficult to proofread) and
result in DNA polymerase stalling (Kim & McHenry,
1996). It was suggested that the transient stalling of Pol III
HE at terminal mismatches may stimulate the dissociation
of HE, allowing extension of mismatches by Pol V (Fijalkowska et al., 1997). Overall, the data strongly support
the idea that in recA730 strains, and in SOS-induced cells
in general, there is a competition between the major replicase (HE) and error-prone Pol V (Fijalkowska et al., 1997;
Timms & Bridges, 2002; Vandewiele et al., 2002).
As SOS induction leads to enhanced levels of both Pol
V and Pol IV, the participation of the latter in the SOS
mutator activity also requires consideration. To address
this question, our group showed that deletion of Pol IV
from recA730 cells (recA730 DdinB::kan) reduced mutagenesis relative to the dinB+ recA730 strain by at least
twofold (Kuban et al., 2006). As loss of Pol V (DumuDC
recA730 strain) completely abolishes the recA730 mutator
effect (Witkin & Kogoma, 1984; Sweasy et al., 1990), it
was concluded that both Pol V and Pol IV are required
for full expression of the mutator effect, with Pol V playing the critical role, presumably in extending the primary
mismatch. Pol IV may participate in further extension of
the Pol V-generated product, thereby improving the likelihood that the mismatch-containing DNA is ultimately
converted into a mutation (Kuban et al., 2006).
Studies of the strandedness of the Pol V-mediated SOS
mutator effect clearly indicated that the effect results primarily, if not exclusively, from events during lagging-strand
replication (Maliszewska-Tkaczyk et al., 2000; Kuban et al.,
2005). This preference mimics that of the Pol IV mutator
effect and, to some extent, the PolBex mutator effect (see
above). Clearly, the lagging strand is more accessible for
accessory polymerases. This is consistent with the greater
dissociativity of HE in the lagging strand. See also Fig. 4.
Role of DNA polymerase I (Pol I)
DNA polymerase I, encoded by the polA gene, was the
first polymerase ever identified (Bessman et al., 1956; De
Lucia & Cairns, 1969). Pol I is the most abundant DNA
polymerase in E. coli (~ 400 molecules per cell) (Kornberg & Baker, 1992). Pol I, a 103-kDa protein, possesses
three enzymatic activities: the 5′?3′ polymerase, a 5′?3′
exonuclease, and a 3′?5′ proofreading exonuclease (Joyce
& Grindley, 1984). The polymerase and 5′?3′ exonuclease
ª 2012 Federation of European Microbiological Societies
Published by Blackwell Publishing Ltd. All rights reserved
1114
activities enable Pol I to fulfill its primary role in DNA
replication: to remove RNA primers and fill the resulting
gap during the maturation of Okazaki fragments in the
lagging strand (Okazaki et al., 1971). Pol I also fills gaps
generated during DNA repair and recombination (Cooper
& Hanawalt, 1972; Kornberg & Baker, 1992; Friedberg
et al., 2006). Pol I, which is presumably capable of interacting with the b-processivity clamp, is a competent
high-fidelity enzyme and should be capable, in principle,
of competing with HE, similar to Pol II and Pol IV
(López de Saro & O’Donnell, 2001).
The role of Pol I in chromosomal replication fidelity
has been investigated (Curti et al., 2009; MakielaDzbenska et al., 2009, 2011), most productively using a
proofreading-deficient variant of Pol I (polAexo mutant),
analogously to the polBex mutant described above. The
logic behind these experiments is again that small, normally error-free, contributions of Pol I would be observable as an increase in the mutation frequency when
substituted by its error-prone proofreading-deficient
counterpart. A 1.5- to 4-fold increase in mutant frequencies was observed in polAexo cells, indicating that Pol I
indeed plays a role in fidelity. Three separate observations
in these studies led us to further define this role: (1) the
polAexo mutator effect has a lagging-strand preference,
(2) there is no synergism between the polAexo mutator
effect and the mutator effects exerted by other DNA
polymerases or replication defects, and (3) no competition could be demonstrated between Pol I, on the one
hand, and Pol II and Pol IV on the other. Based on these
results, we concluded that Pol I does not compete directly
at the growing point in the replication fork. Instead, the
fidelity role of Pol I is concerned primarily with the
error-free processing of the Okazaki fragment gaps,
consistent with the established role of Pol I in the maturation of Okazaki fragments. In E. coli, Okazaki fragments average between 1000–2000 nucleotides, while
the length of RNA primers is 10–20 nucleotides (Zechner et al., 1992a, b). Thus, Pol I is likely responsible
for 1–2% of DNA synthesis in the lagging strand.
However, if even such a relatively small fraction of
chromosomal DNA is synthesized by an enzyme that is
50- to 100-fold less accurate (polAexo) than Pol III,
then a 2% contribution in DNA replication may produce a twofold increase in the chromosomal error rate.
See Fig. 4 for inclusion of Pol I in the overall chromosomal fidelity scheme.
Role of the Pol III HE τ subunit in
fidelity and polymerase switching
The dnaX gene encodes both the τ and c subunits of HE
(Fig. 2). τ is the gene’s full-length (643 amino acid)
ª 2012 Federation of European Microbiological Societies
Published by Blackwell Publishing Ltd. All rights reserved
I.J. Fijalkowska et al.
product, while c is a truncated product lacking the C-terminal residues because of a translational frameshifting
event at residues 428–430 (Flower & McHenry, 1986;
Blinkowa & Walker, 1990). While c, as part of the cdd′
complex, is responsible for loading and unloading the
b-clamps from DNA (reviewed in Bowman et al., 2005),
τ fulfills several additional critical functions, both structurally and mechanistically, owing to its unique C-terminus (for a review, see McHenry, 2011). Foremost, τ
dimerizes the two pol III cores within HE, creating the
dimeric polymerase that is capable of copying the two
DNA strands simultaneously. τ interacts with the DnaB
helicase (Gao & McHenry 2001) and regulates in this
manner the speed of the replication fork (Dallmann et al.,
2000). τ also controls the cycling of the lagging-strand
polymerase, as it mediates the release of the Pol III core
from its b-processivity clamp upon the completion of
Okazaki fragments (Leu et al., 2003; López de Saro et al.,
2003). Based on the central role of τ within HE, including its direct interaction with both the leading- and lagging-strand polymerase, a role of τ in determining the
fidelity of HE, either directly or via an effect on polymerase switching, might be expected. Indeed, a survey of several dnaX(Ts) mutants revealed a mutator phenotype, at
permissive temperatures, for the dnaX36 and dnaX2016
alleles (Pham et al., 2006). The dnaX36 mutator was considered particularly interesting because its underlying
mutation (E601K) is located in the C-terminus of DnaX
and is hence unique to τ (Pham et al., 2006). Analysis of
the specificity of mutagenesis of the dnaX36 mutator
revealed a unique pattern: transversion base substitutions
as well as (1) frameshifts in nonrun sequences are specifically enhanced (Pham et al., 2006). Based on these
and other data, it was concluded that τ subunit is an
important factor in controlling the accuracy of chromosomal replication. Further, its effect is likely mediated at
the processing stage of HE-made replication errors –
rather than the initial error production by HE.
Studies on the strandedness of the dnaX36 mutator
effect revealed that the effect applied to both strands,
although with a slight preference for the lagging strand
(Gawel et al., 2011). It was also found that the dnaX36
mutator effect was largely independent of dinB and that
hence it is not mediated by an increased switch-over of
HE to error-prone Pol IV. On the other hand, an
enhancement of the dnaX36 mutator effect was observed
in the DpolB background, most pronounced for the lagging strand (Gawel et al., 2011). These results indicate
that Pol II plays a role in preventing mutations in dnaX36
strains and that this role may be more important in lagging-strand replication. This is in contrast to the situation
in dnaX+ strains, where the DpolB has no effect (Rangarajan et al., 1997; Banach-Orlowska et al., 2005). This
FEMS Microbiol Rev 36 (2012) 1105–1121
DNA replication fidelity in Escherichia coli
suggests that in the dnaX36 mutant Pol II has increased
access to the replication fork and that in this case Pol II
acts to prevent mutations (Gawel et al., 2008; 2011). Part
of this role of Pol II is to outcompete access by Pol IV,
because the dnaX36 DpolB DdinB triple mutant has
reduced mutability relative to the dnaX36 DpolB strain
(Gawel et al., 2008, 2011).
The important role of Pol II in preventing replication
errors was demonstrated most dramatically when dnaX36
was combined with the polBex1 proofreading deficiency.
In this case, the polBex1 deficiency increased the mutant
frequencies dramatically (20- to 50-fold) above the
dnaX36 level, and this was true for both leading and lagging strands. The strong polBex1 mutator effect resulting
from the Pol II proofreading deficiency is not dependent
on the action of Pol IV, because the dnaX36 polBex1
DdinB triple mutant behaved essentially as the dnaX36
polBex1 strain (Gawel et al., 2008; 2011). This again indicates that Pol II is very effective in preventing access by
Pol IV. Overall, these results highlight the important role
that Pol II can play in controlling the fidelity. It should
be noted that within the favored model of Pol II as a
back-up proofreader for HE-made insertion errors, a
50-fold mutant frequency increase by the polBex1 allele
could indicate that some 98% of Pol III errors become a
substrate for Pol II. When the normal, wild-type Pol II is
present, these errors are efficiently removed by Pol II’s
proofreading activity, while in case of the polBex1 allele,
they have a high chance of being extended by Pol II to
fix the error into a potential mutation (Gawel et al.,
2008, 2011).
What do these results tell us about the fidelity function
of τ subunit? The defect in dnaX36 is in Domain V of
DnaX, which is responsible for the a–τ interaction (Gao
& McHenry 2001; Pham et al., 2006; Su et al., 2007). The
relevance of this location to the mutator effect was further evidenced by the isolation of an additional set of
dnaX mutators with properties similar to dnaX36 and all
located in Domain V (Pham et al., 2006). Thus, an
altered or impaired a–τ interaction underlies the
observed mutator effect. It may be that the impaired a–τ
interaction leads to more frequent Pol III dissociations. If
so, the sole fidelity function of τ would be to keep HE
together in its dimeric state and on the primer templates;
upon dissociation, less accurate polymerases may take a
turn, accounting for the mutator phenotype. While this is
a possibility, the model does not readily account for (1)
the strength of the dnaX36 mutator effect (too much synthesis by error-prone enzymes required), (2) the independence of the mutator effect of Pol IV, the major
candidate for error-prone interference, (3) the substantial
mutator effect of dnaX36 DpolB DdinB strains (i.e. even
in the absence of Pol II and Pol IV), as well as (4) the
FEMS Microbiol Rev 36 (2012) 1105–1121
1115
unusual specificity of the mutator effect: preferential
enhancement of transversion base substitutions as well as
1 frameshifts in nonrun sequences (Pham et al., 2006;
Gawel et al., 2008, 2011).
As a preferred model, we have suggested that the fidelity function of τ results from its role in assisting HE
when it is faced with terminal mismatches created by the
a subunit (Maliszewska-Tkaczyk et al., 2000; BanachOrlowska et al., 2005; Kuban et al., 2005; Gawel et al.,
2008; Makiela-Dzbenska et al., 2009). Terminal mismatches are more difficult to extend (Perrino & Loeb,
1989; Mendelman et al., 1990; Joyce et al., 1992; Kim &
McHenry, 1996) and temporary stalling will ensue. In
most cases, this stalling will lead to a conformational
switch moving the primer terminus to the exonuclease
(proofreading) subunit leading to the removal of the
incorrect nucleotide. However, a subset of errors may be
refractory to this mechanism (Kim & McHenry, 1996;
Pham et al., 2006), leading to a longer-lived type of stalling, which may impede the progress of the replication
fork. Evidence for this type of HE stalling has been
obtained in in vitro fidelity experiments (Pham et al., 2006;
R. M. Schaaper, unpublished data). We propose that,
in vivo, this type of stalling is sensed and remedied by τ
subunit, leading to the removal of the error and continuation of DNA synthesis. This role of τ as sensor for a stalled
polymerase is similar to, and may share features with the
proposed sensing role of τ in the lagging strand that leads
to the release of the polymerase in this strand when it
reaches the end of the Okazaki fragment (Leu et al., 2003;
López de Saro et al., 2003; McInerney et al., 2007).
This τ-mediated fidelity mechanism operates on both
leading and lagging strands (Gawel et al., 2011). How τ
promotes error removal is not yet known, but in the simplest case it may involve enforcing the required conformational switch in a that places the erroneous terminal
nucleotide in the e exonuclease site. Alternatively, τ may
dislodge Pol III and channel the mismatch toward the
exonuclease of Pol II or, possibly, to the third Pol III core
if present at the replication fork (Georgescu et al., 2012).
Obviously, in the absence of this τ function (as in the
dnaX36 mutant), this mechanism is inoperative, and Pol
III may eventually extend the mismatch, accounting for
the dnaX mutator effect. This model is supported by the
specificity of the dnaX mutator effect: transversions and
nonrun (1) frameshifts are specifically enhanced. Polymerase stalling is most pronounced at transversion
mismatches (purine–purine or pyrimidine–pyrimidine
mispairs) (Perrino & Loeb, 1989; Mendelman et al., 1990;
Joyce et al., 1992; Kim & McHenry, 1996), and their
ability to be proofread may also be limited (Kim &
McHenry, 1996; Pham et al., 1998, 1999). Forced extension
of such mismatches will naturally lead to transversion
ª 2012 Federation of European Microbiological Societies
Published by Blackwell Publishing Ltd. All rights reserved
1116
base-pair substitutions or, a preferred option in permissible sequence contexts, (1) frameshifts by misalignment of the incorrect base on the next complementary
template base (Mo & Schaaper, 1996; Pham et al., 1998,
1999).
Summary and concluding remarks
The results of the studies clearly indicate that the accessory DNA polymerases of E. coli can, at least occasionally,
obtain access to the replication fork and participate in
chromosomal replication, affecting in this manner the
overall replication fidelity and the resulting bacterial
mutation rate. Depending on the polymerase, the effect
may be to improve the fidelity (for the proofreading-proficient enzymes, such as Pol I or Pol II) or to produce an
increased number of mutations (for the error-prone
polymerases, Pol IV or V). Our data and interpretations
are presented most straightforwardly using the cartoon of
Fig. 4.
In a most parsimonious model, DNA Pol III HE, as an
efficient and high-speed polymerase machine, is not
expected to give way to accessory polymerases unless provoked by conditions impeding its progress. One such
case, most relevant to the fidelity issue, is the expected
stalling on terminal mismatches, from which it is difficult
to continue synthesis especially for high-efficiency
enzymes with ‘tight’ catalytic sites (Beard & Wilson
2003). We have indicated this in Fig. 4, where HE is temporarily stalled on a (e.g. T:T or A:A) mismatch. If the
mismatch cannot be rapidly proofread by HE, the enzyme
may temporarily release the 3′ terminus, providing access
opportunities for accessory polymerases. Entry by an exonuclease-proficient enzyme like Pol II would lead to the
removal of the terminal mismatch, especially because
DNA polymerases – when first binding to a primer terminus – do so with their exonuclease site rather than the
polymerase site (Johnson, 1993). This represents the basis
of the postulated role of Pol II as a back-up proofreader
for Pol III-mediated replication errors. Importantly, our
data indicate that Pol II performs this role in both the
leading and lagging strands.
Under conditions where Pol IV or Pol V is present at
elevated levels, they too can gain access to the abandoned
primer termini. However, they have to compete with Pol
II. Our results clearly indicate that, for example, the
mutagenic potential of Pol IV is strongly limited by the
presence of Pol II (Banach-Orlowska et al., 2005; Gawel
et al., 2008, 2011). However, the issues are clearly more
complicated than the simple model might suggest, as Pol
IV and Pol V, in contrast to Pol II, appear restricted in
their actions primarily to the lagging strand. Thus, the
ª 2012 Federation of European Microbiological Societies
Published by Blackwell Publishing Ltd. All rights reserved
I.J. Fijalkowska et al.
dissociative events at terminal mismatches in the two
strands are likely to be different mechanistically, perhaps
reflective of the normally different behavior of the Pol III
core in the two strands (highly processive vs. discontinuous in leading vs. lagging strands, respectively). It may be
that the polymerase switch in the leading strand follows a
prescribed and controlled pattern that favors the recruitment of Pol II or a return of Pol III itself, while the
switch in the lagging strand may resemble more a ‘freefor-all’ type of switch.
The precise nature of these events remains to be determined, although, as discussed above, the action of τ subunit is likely to be a critical factor, acting as a sensor for
stalled polymerases and/or as a switch that remedies the
stalled situation. One particular class of dnaX mutants,
exemplified by dnaX36, displays a unique mutator effect
that appears fully consistent with a malfunctioning of this
τ-mediated sensing and/or switching mechanism, and in
these strains, Pol III might be forced to extend errors that
would normally be resolved. Additional genetic experiments as well as biochemical approaches will be needed to
provide further insight into these intriguing mechanisms.
The other important issue that needs to be further
addressed is why in normal, wild-type cells the fidelity of
lagging-strand replication is several-fold higher than that
of the leading strand. It was suggested that the laggingstrand replication has at least one additional mechanism
available for error correction, possibly related to more
facile polymerase dissociation in this strand (Fijalkowska
et al., 1998). If enhanced dissociation leads to more frequent engagement of Pol II, the greater fidelity of lagging-strand replication could be readily explained.
Consistent with this idea is the stronger lagging-strand
mutator effect associated with the exonuclease-deficient
polBex1 allele (Banach-Orlowska et al., 2005). However,
the situation is not so simple, as in the polBex1 strain an
inversion of strand bias is actually observed for some of
the tested lacZ markers (i.e. leading-strand replication is
now more accurate). Thus, this question will also require
further investigation.
Acknowledgements
The authors thank Drs K. Bebenek and A. Clausen for
their careful reading of the manuscript for this paper.
The work described in this review was supported by grant
2 PO4A 061 30 (to I.J.F. and P.J.) from the Polish Ministry of Science and Higher Education, and project number
Z01 ES065086 of the Intramural Research Program of the
NIH, National Institute of Environmental Health Sciences
(to RMS), and by the Foundation for Polish Science
TEAM Program (to I.J.F.).
FEMS Microbiol Rev 36 (2012) 1105–1121
DNA replication fidelity in Escherichia coli
Authors’ contribution
I.J.F., R.M.S. and P.J. contributed equally to this paper.
References
Andersson DI, Koskiniemi S & Hughes D (2010) Biological
roles of translesion synthesis DNA polymerases in
eubacteria. Mol Microbiol 77: 540–548.
Banach-Orlowska M, Fijalkowska IJ, Schaaper RM & Jonczyk
P (2005) DNA polymerase II as a fidelity factor in
chromosomal DNA synthesis in Escherichia coli. Mol
Microbiol 58: 61–70.
Beard WA & Wilson SH (2003) Structural insights into the
origins of DNA polymerase fidelity. Structure 11: 489–496.
Becherel OJ & Fuchs RP (2001) Mechanism of DNA
polymerase II-mediated frameshift mutagenesis. P Natl Acad
Sci USA 98: 8566–8571.
Becherel OJ, Fuchs RP & Wagner J (2002) Pivotal role of the
beta-clamp in translesion DNA synthesis and mutagenesis in
E. coli cells. DNA Repair (Amst) 1: 703–708.
Berardini M, Foster PL & Loechler EL (1999) DNA polymerase
II (polB) is involved in a new DNA repair pathway for DNA
interstrand cross-links in Escherichia coli. J Bacteriol 181:
2878–2882.
Bessman MJ, Kornberg A, Lehman IR & Simms ES (1956)
Enzymatic synthesis of deoxyribonucleic acid. Biochim
Biophys Acta 21: 197–198.
Bhamre S, Gadea BB, Koyama CA, White SJ & Fowler RG
(2001) An aerobic recA-, umuC-dependent pathway of
spontaneous base-pair substitution mutagenesis in
Escherichia coli. Mutat Res 473: 229–247.
Blinkowa AL & Walker JR (1990) Programmed ribosomal
frameshifting generates the Escherichia coli DNA polymerase
III c subunit from within the τ subunit reading frame.
Nucleic Acids Res 18: 1725–1729.
Bonner CA, Hays S, McEntee K & Goodman MF (1990) DNA
polymerase II is encoded by the DNA damage inducible
dinA gene of Escherichia coli. P Natl Acad Sci USA 87:
7663–7667.
Bonner CA, Stukenberg PT, Rajagopalan M, Eritja R,
O’Donnell M, McEntee K, Echols H & Goodman MF
(1992) Processive DNA synthesis by DNA polymerase II
mediated by DNA polymerase III accessory proteins. J Biol
Chem 267: 11431–11438.
Bowman GD, Goedken ER, Kazmirski SL, O’Donnell M &
Kuryan J (2005) Clamp loaders and replication initiations.
FEBS Lett 579: 863–867.
Cai H, Yu H, McEntee K, Kunkel TA & Goodman MF (1995)
Purification and properties of wild-type and exonucleasedeficient DNA polymerase II from Escherichia coli. J Biol
Chem 270: 15327–15335.
Caillet-Fauquet P & Maenhaut-Michel G (1988) Nature of the
SOS mutator activity: genetic characterization of untargeted
mutagenesis in Escherichia coli. Mol Gen Genet 213: 491–
498.
FEMS Microbiol Rev 36 (2012) 1105–1121
1117
Castellazzi M, George J & Buttin G (1972) Prophage induction
and cell division in E. coli. I. Further characterization of the
thermosensitive mutation tif-1 whose expression mimics the
effect of UV irradiation. Mol Gen Genet 119: 139–152.
Chikova AK & Schaaper RM (2005) The bacteriophage P1 hot
gene can substitute for the Escherichia coli DNA polymerase
III h subunit. J Bacteriol 187: 5528–5536.
Ciesla Z (1982) Plasmid pKM101-mediated mutagenesis in
Escherichia coli is inducible. Mol Gen Genet 186: 298–300.
Cooper PK & Hanawalt PC (1972) Role of DNA polymerase I
and the rec system in excision-repair in Escherichia coli.
P Natl Acad Sci USA 69: 1156–1160.
Curti E, McDonald JP, Mead S & Woodgate R (2009) DNA
polymerase switching: effects on spontaneous mutagenesis in
Escherichia coli. Mol Microbiol 71: 315–331.
Dallmann HG, Kim S, Pritchard AE, Marians KJ & McHenry
CS (2000) Characterization of the unique C terminus of the
Escherichia coli tau DnaX protein: monomeric C-tau binds
alpha and DnaB and can partially replace tau in the
reconstituted replication fork. J Biol Chem 275: 15512–
15519.
De Lucia P & Cairns J (1969) Isolation of an E. coli strain with
a mutation affecting DNA polymerase. Nature 224: 1164–
1166.
Drake JW (1991) Spontaneous mutation. Annu Rev Genet 25:
125–146.
Drake JW (1993) General antimutators are improbable. J Mol
Biol 229: 8–13.
Drake JW & Allen EF (1968) Antimutagenic DNA polymerase
of bacteriophage T4. Cold Spring Harb Symp Quant Biol 35:
339–344.
Drake JW, Charlesworth B, Charlesworth D & Crow JF
(1998) Rates of spontaneous mutation. Genetics 148: 1667–
1686.
Escarceller M, Hicks J, Gudmundsson G, Trump G, Touati D,
Lovett S, Foster PL, McEntee K & Goodman MF (1994)
Involvement of Escherichia coli DNA polymerase II in
response to oxidative damage and adaptive mutation.
J Bacteriol 176: 6221–6228.
Fijalkowska IJ & Schaaper RM (1993) Antimutator mutations
in the a subunit of Escherichia coli DNA polymerase III:
identification of the responsible mutations and alignment
with other DNA polymerases. Genetics 135: 1039–1044.
Fijalkowska IJ & Schaaper RM (1995) Effects of Escherichia coli
DNA antimutator alleles in proofreading deficient mutD5
strain. J Bacteriol 177: 5979–5986.
Fijalkowska IJ & Schaaper RM (1996) Mutants in the Exo I
motif of E. coli dnaQ: defective proofreading and inviability
due to error catastrophe. P Natl Acad Sci USA 93: 2856–
2861.
Fijalkowska IJ, Dunn RL & Schaaper RM (1993) Mutants of
Escherichia coli with increased fidelity of DNA replication.
Genetics 134: 1023–1030.
Fijalkowska IJ, Dunn RL & Schaaper RM (1997) Genetic
requirements and mutational specificity of the Escherichia
coli SOS mutator activity. J Bacteriol 179: 7435–7445.
ª 2012 Federation of European Microbiological Societies
Published by Blackwell Publishing Ltd. All rights reserved
1118
Fijalkowska IJ, Jonczyk P, Tkaczyk MM, Bialoskorska M &
Schaaper RM (1998) Unequal fidelity of leading and lagging
strand DNA replication on the Escherichia coli chromosome.
P Natl Acad Sci USA 95: 10020–10025.
Flower AM & McHenry CS (1986) The c subunit of DNA
polymerase III of Escherichia coli is produced by ribosomal
frameshifting. P Natl Acad Sci USA 87: 3713–3717.
Foster PL, Gudmundsson G, Trimarchi JM, Cai H &
Goodman MF (1995) Proofreading-defective DNA
polymerase II increases adaptive mutation in Escherichia
coli. P Natl Acad Sci USA 92: 7951–7955.
Friedberg EC, Wagner R & Radman M (2002) Specialized
DNA polymerases, cellular survival, and the genesis of
mutations. Science 296: 1627–1630.
Friedberg EC, Walker GC, Siede W, Wood RD, Schult RA &
Ellenberger T (2006) DNA Repair and Mutagenesis, 2nd edn.
ASM Press, Washington, DC.
Fuchs RP & Fujii S (2007) Translesion synthesis in Escherichia
coli; lessons from the NarI mutation hot spot. DNA Repair
(Amst) 6: 1032–1041.
Furukohri A, Goodman MF & Maki H (2008) A dynamic
polymerase exchange with Escherichia coli DNA polymerase
IV replacing DNA polymerase III on the sliding clamp.
J Biol Chem 283: 11260–11269.
Gao D & McHenry (2001) tau binds and organizes Escherichia
coli replication proteins trough distinct domains. Domain
IV, located within the unique C terminus of tau, binds the
replication fork helicase, DnaB. J Biol Chem 276: 4441–4446.
Gawel D, Bialoskorska M, Jonczyk P, Schaaper RM &
Fijalkowska IJ (2002a) Asymmetry of frameshift mutagenesis
during leading and lagging strand replication in Escherichia
coli. Mutat Res 501: 129–136.
Gawel D, Maliszewska-Tkaczyk M, Jonczyk P, Schaaper RM
& Fijalkowska IJ (2002b) Lack of strand bias in UVinduced mutagenesis in Escherichia coli. J Bacteriol 184:
4449–4454.
Gawel D, Pham PT, Fijalkowska IJ, Jonczyk P & Schaaper RM
(2008) Role of accessory DNA polymerases in DNA
replication in Escherichia coli: analysis of the dnaX36
mutator mutant. J Bacteriol 190: 1730–1742.
Gawel D, Jonczyk P, Fijalkowska IJ & Schaaper RM (2011)
dnaX36 mutator of Escherichia coli: effects of the tau
subunit of the DNA polymerase III holoenzyme on
chromosomal DNA fidelity. J Bacteriol 193: 296–300.
Gefter ML, Hirota Y, Kornberg T, Wechsler JA & Barnoux C
(1971) Analysis of DNA polymerases II and III in mutants
of Escherichia coli thermosensitive for DNA synthesis. P Natl
Acad Sci USA 68: 3150–3153.
Georgescu RE, Kurth I & O’Donnell ME (2012) Singlemolecule studies reveal the function of a third polymerase
in the replisome. Nat Struct Mol Biol 19: 113–116.
Gillin FD & Nossal NG (1976) Control of mutation frequency
by bacteriophage T4 DNA polymerase. I. The CB120
antimutator DNA polymerase is defective in strand
displacement. J Biol Chem 251: 5219–5224.
ª 2012 Federation of European Microbiological Societies
Published by Blackwell Publishing Ltd. All rights reserved
I.J. Fijalkowska et al.
Gon S, Napolitano R, Rocha W, Coulon S & Fuchs RP (2011)
Increase in dNTP pool size during the DNA damage
response plays a key role in spontaneous and inducedmutagenesis in Escherichia coli. P Natl Acad Sci USA 108:
19311–19316.
Goodman MF (2002) Error-prone repair DNA polymerases in
prokaryotes and eukaryotes. Annu Rev Biochem 71: 17–50.
Guo C, Kosarek-Stancel JN, Tang TS & Friedberg EC (2009)
Y-family DNA polymerases in mammalian cells. Cell Mol
Life Sci 66: 2363–2381.
Hastings PJ, Hersh MN, Thornton PC, Fonville NC, Slack A,
Frish RL, Ray MP, Harris RS, Leal SM & Rosenberg SM
(2010) Competition of Escherichia coli DNA polymerases I,
II and III with DNA Pol IV in stressed cells. PLoS ONE 5:
e10862.
Heltzel JMH, Scouten-Ponticelli SK, Sanders LH, Duzen JM,
Cody V, Pace J, Snell EH & Sutton MD (2009a) Sliding
clamp-DNA interactions are required for viability and
contribute to DNA polymerase management in Escherichia
coli. J Mol Biol 387: 74–92.
Heltzel JMH, Maul RW, Scouten-Ponticelli SK & Sutton MD
(2009b) A model for DNA polymerase switching involving a
single cleft and the rim of the sliding clamp. P Natl Acad
Sci USA 106: 12664–12669.
Ho TV & Scharer OD (2010) Translesion DNA synthesis
polymerases in DNA interstrand crosslink repair. Environ
Mol Mutagen 51: 552–566.
Hughes AJ, Bryan SK, Chen H, Moses RE & McHenry CS
(1991) Escherichia coli DNA polymerase II is stimulated by
DNA polymerase III holoenzyme auxiliary subunits. J Biol
Chem 266: 4568–4573.
Indiani C, McInerney P, Georgescu R, Goodman MF &
O’Donnell M (2005) A sliding-clamp toolbelt binds highand low-fidelity DNA polymerases simultaneously. Mol Cell
19: 805–815.
Indiani C, Langston LD, Yurieva O, Goodman MF &
O’Donnell M (2009) Translesion DNA polymerases remodel
the replisome and alter the speed of the replicative helicase.
P Natl Acad Sci USA 106: 6031–6038.
Iwasaki H, Nakata A, Walker GC & Shinagawa H (1990) The
Escherichia coli polB gene, which encodes DNA polymerase
II, is regulated by the SOS system. J Bacteriol 172: 6268–
6273.
Jackson AL & Loeb LA (1998) On the origin of multiple
mutations in human cancers. Semin Cancer Biol 8: 421–429.
Jarosz DF, Beuning PJ, Cohen SA & Walker CS (2007)
Y-family DNA polymerases in Escherichia coli. Trends
Microbiol 15: 70–77.
Jeruzalmi D, Yurieva O, Zhao Y, Young M, Stewart J,
Hingorani M, O’Donnell M & Kuriyan J (2001) Mechanism
of processivity clamp opening by the delta subunit wrench
of the clamp loader complex of E. coli DNA polymerase III.
Cell 106: 417–428.
Johnson KA (1993) Conformational coupling in DNA
polymerase fidelity. Annu Rev Biochem 62: 685–713.
FEMS Microbiol Rev 36 (2012) 1105–1121
DNA replication fidelity in Escherichia coli
Joyce CM & Grindley ND (1984) Method for determining
whether the gene of Escherichia coli is essential – application
to the polA gene. J Bacteriol 158: 636–643.
Joyce CM, Sun XC & Grindley NDF (1992) Reactions at the
polymerase active site that contribute to the fidelity of
Escherichia coli DNA polymerase I (Klenow fragment). J Biol
Chem 267: 24485–24500.
Kim DR & McHenry CS (1996) In vivo assembly of
overproduced DNA polymerase III. Overproduction,
purification and characterization of the a, a-e and a-e-h
subunits. J Biol Chem 271: 20681–20689.
Kim SR, Maenhaut-Michel G, Yamada M, Yamamoto Y,
Matsui K, Sofuni T, Nohmi T & Ohmori H (1997) Multiple
pathways for SOS-induced mutagenesis in Escherichia coli:
an overexpression of dinB/dinP results in strongly enhancing
mutagenesis in the absence of any exogenous treatment to
damage DNA. P Natl Acad Sci USA 94: 13792–13797.
Kim SR, Matsui K, Yamada M, Gruz P & Nohmi T (2001)
Roles of chromosomal and episomal dinB genes encoding
DNA pol IV in targeted and untargeted mutagenesis in
Escherichia coli. Mol Genet Genomics 266: 207–215.
Knippers R (1970) DNA polymerase II. Nature 228: 1050–1053.
Kobayashi S, Valentine MR, Pham P, O’Donnell M &
Goodman MF (2002) Fidelity of Escherichia coli DNA
polymerase IV. Preferential generation of small deletion
mutations by dNTP-stabilized misalignments. J Biol Chem
277: 34198–34207.
Kong XP, Onrust R, O’Donnell M & Kuriyan J (1992) Threedimensional structure of the beta subunit of E. coli DNA
polymerase III holoenzyme: a sliding DNA clamp. Cell 69:
425–437.
Kornberg A & Baker TA (1992) DNA Replication. W.H.
Freeman & Co, New York, NY.
Kuban W, Jonczyk P, Gawel D, Malanowska K, Schaaper RM
& Fijalkowska IJ (2004) Role of Escherichia coli DNA
polymerase IV in in vivo replication fidelity. J Bacteriol 186:
4802–4807.
Kuban W, Banach-Orlowska M, Bialoskorska M, Lipowska A,
Schaaper RM, Jonczyk P & Fijalkowska IJ (2005) Mutator
phenotype resulting from DNA polymerase IV
overproduction in Escherichia coli: preferential mutagenesis
on the lagging strand. J Bacteriol 187: 6862–6866.
Kuban W, Banach-Orlowska M, Schaaper RM, Jonczyk P &
Fijalkowska IJ (2006) Role of DNA polymerase IV in
Escherichia coli SOS mutator activity. J Bacteriol 188: 7977–
7980.
Kunkel TA (2004) DNA replication fidelity. J Biol Chem 279:
16895–16898.
Kunkel TA (2009) Evolving views of DNA replication (in)
fidelity. Cold Spring Harb Symp Quant Biol 74: 91–101.
Kunkel TA & Bebenek K (2000) DNA replication fidelity.
Annu Rev Biochem 69: 497–529.
Kunkel TA & Erie DA (2005) DNA mismatch repair. Annu
Rev Biochem 74: 681–710.
Langston LD, Indiani C & O’Donnell M (2009) Whither the
replisome: emerging perspectives on the dynamic nature
FEMS Microbiol Rev 36 (2012) 1105–1121
1119
of the DNA replication machinery. Cell Cycle 8: 2686–
2691.
Lehmann AR, Niimi A, Ogi T, Brown S, Sabbioneda S, Wing
JF, Kannouche PL & Green CM (2007) Translesion
synthesis: Y-family polymerase and the polymerase switch.
DNA Repair (Amst) 6: 891–899.
Lenne-Samuel N, Wagner J, Etienne H & Fuchs RP (2002) The
processivity factor beta controls DNA polymerase IV traffic
during spontaneous mutagenesis and translesion synthesis
in vivo. EMBO Rep 3: 45–49.
Leu FP, Georgescu R & O’Donnell M (2003) Mechanism of
the E. coli tau processivity switch during lagging-strand
synthesis. Mol Cell 11: 315–327.
Livneh Z, Ziv O & Shachar S (2010) Multiple two-polymerase
mechanisms in mammalian translesion synthesis. Cell Cycle
9: 729–735.
Loeb LA (1991) Mutator phenotype may be required for
multistage carcinogenesis. Cancer Res 51: 3075–3079.
López de Saro FJ & O’Donnell M (2001) Interaction of b
sliding clamp with MutS, ligase and DNA polymerase I.
P Natl Acad Sci USA 98: 8376–8380.
López de Saro FJ, Georgescu RE & O’Donnell M (2003) A
peptide switch regulates DNA polymerase processivity.
P Natl Acad Sci USA 100: 14689–14694.
Makiela-Dzbenska K, Jaszczur M, Banach-Orlowska M,
Jonczyk P, Schaaper RM & Fijalkowska IJ (2009) Role of
Escherichia coli DNA polymerase I in chromosomal DNA
replication fidelity. Mol Microbiol 74: 1114–1127.
Makiela-Dzbenska K, Jonczyk P, Schaaper RM & Fijalkowska
IJ (2011) Proofreading deficiency of Pol I increases the
levels of spontaneous rpoB mutations in E. coli. Mutat Res
712: 28–32.
Maliszewska-Tkaczyk M, Jonczyk P, Bialoskorska M, Schaaper
RM & Fijalkowska IJ (2000) SOS mutator activity: unequal
mutagenesis on leading and lagging strands. P Natl Acad Sci
USA 97: 12678–12683.
Masker W, Hanawalt P & Shizuya H (1973) Role of DNA
polymerase II in repair replication in Escherichia coli. Nat
New Biol 244: 242–243.
McCulloch SD & Kunkel TA (2008) The fidelity of DNA
synthesis by eukaryotic replicative and translesion synthesis
polymerases. Cell Res 18: 148–161.
McDonald JP, Frank EG, Levine AS & Woodgate R (1998)
Intramolecular cleavage of the UmuD-like mutagenesis
proteins. P Natl Acad Sci USA 95: 1478–1483.
McHenry CS (2003) Chromosomal replicases as asymmetric
dimers: studies of subunit arrangements and functional
consequences. Mol Microbiol 49: 1157–1165.
McHenry CS (2011) DNA replicases from a bacterial
perspective. Annu Rev Biochem 80: 403–436.
McInerney P, Johnson A, Katz F & O’Donnell M (2007)
Characterization of a triple DNA polymerase replisome.
Mol Cell 27: 527–538.
McKenzie GJ, Magner DB, Lee PL & Rosenberg SM (2003)
The dinB operon and spontaneous mutation in Escherichia
coli. J Bacteriol 185: 3972–3977.
ª 2012 Federation of European Microbiological Societies
Published by Blackwell Publishing Ltd. All rights reserved
1120
Mendelman LV, Petrusca J & Goodman MF (1990) Base
mispair extension kinetics. Comparison of DNA
polymerase alpha and reverse transcriptase. J Biol Chem 265:
2338–2346.
Mo JY & Schaaper RM (1996) Fidelity and error specificity of
the alpha catalytic subunit of Escherichia coli DNA
polymerase III. J Biol Chem 271: 18947–18953.
Modrich P (1987) DNA mismatch correction. Annu Rev
Biochem 56: 435–466.
Nick McElhinny SA, Gordenin DA, Stith CM, Burgers PM &
Kunkel TA (2008) Division of labor at the eukaryotic
replication fork. Mol Cell 30: 137–144.
Nohmi T (2006) Environmental stress and lesion-bypass DNA
polymerases. Annu Rev Microbiol 60: 231–253.
Nohmi T, Battista JR, Dodson LA & Walker GC (1988) RecAmediated cleavage activates UmuD for mutagenesis:
mechanistic relationship between transcriptional
derepression and the posttranslational activation. P Natl
Acad Sci USA 85: 4331–4335.
Nowosielska A, Janion C & Grzesiuk E (2004) Effect of
deletion of SOS-induced polymerase II, IV and V, on
spontaneous mutagenesis in Escherichia coli mutD5. Environ
Mol Mutagen 43: 226–234.
O’Donnell M (2006) Replisome architecture and dynamics in
Escherichia coli. J Biol Chem 281: 10653–10656.
Okazaki R, Arisawa M & Sugino A (1971) Slow joining of newly
replicated DNA chains in DNA polymerase I-deficient
Escherichia coli mutants. P Natl Acad Sci USA 68: 2954–2957.
Oller AR & Schaaper RM (1994) Spontaneous mutation in
Escherichia coli containing the dnaE911 DNA polymerase
antimutator allele. Genetics 138: 263–270.
Oller AR, Fijalkowska IJ & Schaaper RM (1993) The
Escherichia coli galK2 papillation assay: its specificity and
application to seven newly isolated mutator strains. Mutat
Res 292: 175–185.
Pavlov YI, Newlon CS & Kunkel CA (2002) Yeast origins
establish a strand bias for replicational mutagenesis. Mol
Cell 10: 207–213.
Pavlov YI, Maki S, Maki H & Kunkel TA (2004) Evidence for
interplay among yeast replicative polymerase alpha, delta
and epsilon from studies of exonuclease and polymerase
active site mutations. BMC Biol 2: 11.
Perrino FW & Loeb LA (1989) Differential extension of 3′
mispairs is a major contribution to the high fidelity of calf
thymus DNA polymerase-alpha. J Biol Chem 264: 2898–2905.
Pham PT, Olson MW, McHenry CS & Schaaper RM (1998)
The base substitution and frameshift fidelity of Escherichia
coli DNA polymerase III holoenzyme in vitro. J Biol Chem
273: 23575–23584.
Pham PT, Olson MW, McHenry CS & Schaaper RM (1999)
Mismatch extension by Escherichia coli DNA polymerase III
holoenzyme. J Biol Chem 274: 3705–3710.
Pham PT, Zhao W & Schaaper RM (2006) Mutator mutants
of Escherichia coli carrying defect in the DNA polymerase III
tau subunit. Mol Microbiol 59: 1149–1161.
ª 2012 Federation of European Microbiological Societies
Published by Blackwell Publishing Ltd. All rights reserved
I.J. Fijalkowska et al.
Pomerantz RT & O’Donnell M (2007) Replisome mechanics:
insights into twin DNA polymerase machine. Trends
Microbiol 15: 156–164.
Preston BD, Albertson TM & Herr AJ (2010) DNA replication
fidelity and cancer. Semin Cancer Biol 20: 281–293.
Pritchard AE, Dallmann HG, Glover BP & McHenry CS
(2000) A novel assembly mechanism for the DNA
polymerase III holoenzyme DnaX complex: association
of dd’ with DnaX4 forms DnaX3dd’. EMBO J 19: 6536–
6545.
Pursell ZF, Isoz I, Lundström EB, Johansson E & Kunkel TA
(2007) Yeast DNA polymerase epsilon participates in
leading-strand DNA replication. Science 317: 127–130.
Qui Z & Goodman MF (1997) The Escherichia coli polB locus
is identical to dinA, the structural gene for DNA polymerase
II. Characterization of Pol II purified from a polB mutant.
J Biol Chem 272: 8611–8617.
Rangarajan S, Gudmundsson G, Qui Z, Foster PL & Goodman
MF (1997) Escherichia coli DNA polymerase II catalyzes
chromosomal and episomal DNA synthesis in vivo. P Natl
Acad Sci USA 96: 946–951.
Rangarajan S, Woodgate R & Goodman MF (1999) A
phenotype for enigmatic DNA polymerase II: a pivotal role
for pol II in replication restart in UV-irradiated Escherichia
coli. P Natl Acad Sci USA 94: 9224–9229.
Reha-Krantz LJ (1995) Learning about DNA polymerase
function by studying antimutator DNA polymerases. Trends
Biochem Sci 20: 136–140.
Reha-Krantz LJ & Nonay RL (1993) Genetic and biochemical
studies of bacteriophage T4 DNA polymerase 3′?5′
exonuclease activity. J Biol Chem 268: 27100–27108.
Reuven NB, Arad G, Maor-Shoshani A & Livneh Z (1999) The
mutagenesis protein UmuC is a DNA polymerase activated
by UmuD’, RecA and SSB and specialized for translesion
synthesis. J Biol Chem 274: 31763–31766.
Reyes-Lamothe R, Sherratt DJ & Leake MC (2010)
Stoichiometry and architecture of active DNA replication
machinery in Escherichia coli. Science 328: 498–501.
Schaaper RM (1993a) Base selection, proofreading and
mismatch repair during DNA replication in Escherichia coli.
J Biol Chem 273: 23762–23775.
Schaaper RM (1993b) The mutational specificity of two
Escherichia coli dnaE antimutator alleles as determined from
lacI mutation spectra. Genetics 134: 1031–1038.
Schaaper RM (1996) Suppressors of Escherichia coli mutT:
antimutators for DNA replication errors. Mutat Res 350:
17–23.
Schaaper RM (1998) Antimutator mutants in bacteriophage T4
and Escherichia coli. Genetics 148: 1579–1585.
Sevastopoulos CG & Glaser DA (1977) Mutator action by
Escherichia coli strains carrying dnaE mutations. P Natl Acad
Sci USA 74: 3947–3950.
Shcherbakova PV & Fijalkowska IJ (2006) Translesion
synthesis DNA polymerases and control of genome stability.
Front Biosci 11: 2496–2517.
FEMS Microbiol Rev 36 (2012) 1105–1121
DNA replication fidelity in Escherichia coli
Shinagawa H, Iwasaki H, Kato T & Nakata A (1988) RecAdependent cleavage of UmuD protein and SOS mutagenesis.
P Natl Acad Sci USA 85: 1806–1810.
Su XC, Jergic S, Keniry MA, Dixon NE & Otting G (2007)
Solution structure of the domain IVa and V of the tau
subunit of the Escherichia coli DNA polymerase III and
interaction with the alpha subunit. Nucleic Acids Res 35:
2825–2832.
Sutton MD (2010) Coordinating DNA polymerase traffic
during high and low fidelity synthesis. Biochim Biophys Acta
1804: 1167–1179.
Sweasy JB, Witkin EM, Sinha N & Roegner-Maniscalco V
(1990) RecA protein of Escherichia coli has a third essential
role in SOS mutator activity. J Bacteriol 172: 3030–3036.
Taft-Benz SA & Schaaper RM (1998) Mutational analysis of
the 3′?5′ proofreading exonuclease of Escherichia coli DNA
polymerase III. Nucleic Acids Res 26: 4005–4011.
Taft-Benz SA & Schaaper RM (2004) The theta subunit of
Escherichia coli DNA polymerase III; a role in stabilizing the
epsilon proofreading subunit. J Bacteriol 186: 2774–2780.
Takano K, Nakabeppu Y, Maki H, Horiuchi T & Sekiguchi M
(1986) Structure and function of dnaQ and mutD5
mutators of Escherichia coli. Mol Gen Genet 205: 9–13.
Takata K & Wood RD (2009) Bypass specialists operate
together. EMBO J 28: 313–314.
Tang M, Shen X, Frank EG, O’Donnell M, Woodgate R &
Goodman MF (1999) UmuD’2C is an error-prone DNA
polymerase, Escherichia coli pol V. P Natl Acad Sci USA 96:
8919–8924.
Tang M, Pham P, Shen X, Taylor JS, O’Donnell M, Woodgate
R & Goodman MF (2000) Roles of E. coli DNA polymerases
IV and V in lesion-targeted and untargeted SOS
mutagenesis. Nature 404: 1014–1018.
Tessman ES & Peterson P (1985) Plaque color method for
rapid isolation of novel recA mutants of Escherichia coli
K-12: new classes of protease-constitutive recA mutants.
J Bacteriol 163: 677–687.
Timms AR & Bridges BA (2002) DNA Pol V-dependent
mutator activity in an SOS-induced Escherichia coli strains
with a temperature-sensitive DNA polymerase III. Mutat Res
499: 97–101.
Tuppen HA, Blakely EL, Turnbull DM & Taylor RW (2010)
Mitochondrial DNA mutations and human disease. Biochim
Biophys Acta 1797: 113–128.
Uchida K, Furukohri A, Shinozaki Y, Mori T, Ogawara D,
Kanaya S, Nohmi T, Maki H & Akiyama M (2008)
Overproduction of Escherichia coli DNA polymerase DinB
(Pol IV) inhibits replication fork progression and is lethal.
Mol Microbiol 70: 608–622.
Vandewiele D, Fernandez de Henestrosa AR, Timms AR,
Bridges BA & Woodgate R (2002) Sequence analysis and
phenotypes of five temperature sensitive mutator alleles of
dnaE, encoding modified alpha-catalytic subunits of
Escherichia coli DNA polymerase III holoenzyme. Mutat Res
499: 85–95.
FEMS Microbiol Rev 36 (2012) 1105–1121
1121
Wagner J & Nohmi T (2000) Escherichia coli DNA polymerase
IV mutator activity: genetic requirements and mutational
specificity. J Bacteriol 182: 4587–4595.
Wagner J, Gruz P, Kim SR, Yamada M, Matsui K, Fuchs RPP
& Nohmi T (1999) The dinB encodes a novel E. coli DNA
polymerase, DNA pol IV, involved in mutagenesis. Mol Cell
4: 281–286.
Wagner J, Fujii S, Gruz P, Nohmi T & Fuchs RPP (2000) The
b clamp targets DNA polymerase IV to DNA and strongly
increases its processivity. EMBO Rep 1: 484–488.
Wang F & Yang W (2009) Structural insight into translesion
synthesis by DNA Pol II. Cell 139: 1279–1289.
Wickner S & Hurwitz J (1974) Conversion of oX174 viral
DNA to double-stranded form by purified Escherichia coli
proteins. P Natl Acad Sci USA 71: 4120–4124.
Witkin EM (1974) Thermal enhancement of ultraviolet
mutability in a tif-1 uvrA derivative of Escherichia coli B-r:
evidence that ultraviolet mutagenesis depends upon the
inducible function. P Natl Acad Sci USA 71: 1930–1934.
Witkin EM & Kogoma T (1984) Involvement of the activated
form of RecA protein in SOS mutagenesis and stable DNA
replication in Escherichia coli. P Natl Acad Sci USA 81:
7539–7543.
Witkin EM, McCall JO, Volkert MR & Wermundsen IE (1982)
Constitutive expression of SOS functions and modulation of
mutagenesis resulting from resolution of genetic instability
at or near the recA locus of Escherichia coli. Mol Gen Genet
185: 43–50.
Wolff E, Kim M, Hu K, Yang H & Miller JH (2004)
Polymerase leave fingerprints: analysis of the mutational
spectrum in Escherichia coli rpoB to assess the role of
polymerase IV in spontaneous mutation. J Bacteriol 186:
2900–2905.
Woodgate R & Ennis DG (1991) Levels of chromosomally
encoded Umu proteins and requirements for in vitro UmuD
cleavage. Mol Gen Genet 229: 10–16.
Yao NY & O’Donnell M (2008) Replisome dynamics and use
of DNA trombone loops to bypass replication blocks. Mol
BioSyst 4: 1075–1084.
Yao NY, Georgescu RE, Finkelstein J & O’Donnell ME (2009)
Single-molecule analysis reveals that the lagging strand
increases replisome processivity but slows replication fork
progression. P Natl Acad Sci USA 106: 13236–13241.
Yeiser B, Pepper ED, Goodman MF & Finkel SE (2002) SOSinduced DNA polymerases enhance long-term survival and
evolutionary fitness. P Natl Acad Sci USA 99: 8737–8741.
Zechner EL, Wu C & Marians KJ (1992a) Coordinated leading
and lagging strand synthesis at the Escherichia coli DNA
replication fork: II. Frequency of primer synthesis and
efficiency of primer utilization controls Okazaki fragment
size. J Biol Chem 267: 4045–4053.
Zechner EL, Wu C & Marians KJ (1992b) Coordinated leading
and lagging strand synthesis at the Escherichia coli DNA
replication fork: III. Polymerase-primer interaction governs
primer size. J Biol Chem 267: 4054–4063.
ª 2012 Federation of European Microbiological Societies
Published by Blackwell Publishing Ltd. All rights reserved