Unexpected Role for Helicobacter pylori DNA
Polymerase I As a Source of Genetic Variability
Marı ´a-Victoria Garcı ´a-Ortı ´z1., Ste ´phanie Marsin1., Mercedes E. Arana2, Didier Gasparutto3, Raphae ¨l
Gue ´rois4,5, Thomas A. Kunkel2, J. Pablo Radicella1*
1CEA, Institut de Radiobiologie Cellulaire et Mole ´culaire, UMR 217 CNRS/CEA, Fontenay aux Roses, France, 2Laboratory of Molecular Genetics and Laboratory of
Structural Biology, National Institute of Environmental Health Science, National Institutes of Health, Research Triangle Park, North Carolina, United States of America,
3CEA, Institut Nanosciences et Cryoge ´nie, Grenoble, France, 4CEA, iBiTecS, Gif sur Yvette, France, 5CNRS, URA 2096, Gif sur Yvette, France
Helicobacter pylori, a human pathogen infecting about half of the world population, is characterised by its large intraspecies
variability. Its genome plasticity has been invoked as the basis for its high adaptation capacity. Consistent with its small
genome, H. pylori possesses only two bona fide DNA polymerases, Pol I and the replicative Pol III, lacking homologues of
translesion synthesis DNA polymerases. Bacterial DNA polymerases I are implicated both in normal DNA replication and in
DNA repair. We report that H. pylori DNA Pol I 59- 39 exonuclease domain is essential for viability, probably through its
involvement in DNA replication. We show here that, despite the fact that it also plays crucial roles in DNA repair, Pol I
contributes to genomic instability. Indeed, strains defective in the DNA polymerase activity of the protein, although
sensitive to genotoxic agents, display reduced mutation frequencies. Conversely, overexpression of Pol I leads to a
hypermutator phenotype. Although the purified protein displays an intrinsic fidelity during replication of undamaged DNA,
it lacks a proofreading activity, allowing it to efficiently elongate mismatched primers and perform mutagenic translesion
synthesis. In agreement with this finding, we show that the spontaneous mutator phenotype of a strain deficient in the
removal of oxidised pyrimidines from the genome is in part dependent on the presence of an active DNA Pol I. This study
provides evidence for an unexpected role of DNA polymerase I in generating genomic plasticity.
Citation: Garcı ´a-Ortı ´z M-V, Marsin S, Arana ME, Gasparutto D, Gue ´rois R, et al. (2011) Unexpected Role for Helicobacter pylori DNA Polymerase I As a Source of
Genetic Variability. PLoS Genet 7(6): e1002152. doi:10.1371/journal.pgen.1002152
Editor: Ivan Matic, Universite ´ Paris V, INSERM U571, France
Received March 3, 2011; Accepted May 9, 2011; Published June 23, 2011
This is an open-access article, free of all copyright, and may be freely reproduced, distributed, transmitted, modified, built upon, or otherwise used by anyone for
any lawful purpose. The work is made available under the Creative Commons CC0 public domain dedication.
Funding: This work was supported in part by the Commissariat a ` l’Energie Atomique, a grant from Agence Nationale de la Recherche (ANR-09-BLAN-0271-01) to
JPR and RG, and by Project Z01 ES065070 to TAK from the Division of Intramural Research of the National Institutes of Health, National Institute of Environmental
Health Sciences. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.
Competing Interests: The authors have declared that no competing interests exist.
* E-mail: firstname.lastname@example.org
. These authors contributed equally to this work.
Phenotypic selection from a pool of genetic variants present in
their population allows prokaryotes to successfully adapt to specific
niches and changing environments. The gram-negative bacterium
Helicobacter pylori is one of the most successful human pathogens.
Indeed, it colonises the stomach mucosa of about half the human
population, triggering pathologies that span asymptomatic chronic
gastritis, peptic ulcers and cancer . The study of natural isolates
suggests that the genetic diversity of H. pylori exceeds that recorded
in all other bacterial species studied. Moreover, it is now clear that
even in the course of infection of a single individual, H. pylori
strains display high rates of allelic variation [2,3]. This enhanced
ability to change and the consequent advantage of counting upon
a large pool of variants from which to select the most-fit
combinations have been proposed to facilitate adaptation within
a host as well as colonisation of new hosts [4,5]. Therefore, besides
its clinical importance, the amazing genetic variability of H. pylori
makes it an excellent model for the analysis of the molecular
mechanisms underlying microbial phenotype evolution.
At the origin of the allelic variability are nucleotide changes
that can arise from replication errors either spontaneous or
induced by DNA lesions. H. pylori displays a high rate of
mutations, accounted not only by the lack of mismatch repair
system [6,7] but also by the exposure to genotoxic stresses during
infection leading to the formation of DNA lesions [8,9]. While
replicative DNA polymerases are highly accurate and efficient in
replicating undamaged DNA, the presence of abasic sites or
modified bases will often impede the progression of the
replication fork. It is now well established that in most organisms
DNA polymerases exist that are capable of substituting for the
replicative polymerase and facilitate translesion synthesis (TLS)
allowing the replication machinery to overcome the blockage. In
many cases, TLS is associated with the acquisition of heritable
mutations induced by the incorporation by the TLS polymerase
of an incorrect nucleotide opposite the lesion .
In agreement with the low functional redundancy in its DNA
repair pathways, analysis of the H. pylori genome sequences
predicts the presence of only two putative DNA polymerases.
Indeed, six genes code for the replicative polymerase subunits,
while one gene, HP1470 in the reference strain 26695, codes for a
putative DNA Polymerase I. E. coli Pol I was the first DNA
polymerase discovered and is the most abundant one . The Pol
I bacterial DNA polymerases are multifunctional proteins. In most
PLoS Genetics | www.plosgenetics.org1June 2011 | Volume 7 | Issue 6 | e1002152
bacterial species Pol I presents two distinct functional domains,
a 59 - 39 exonuclease N-terminal domain and a larger C-terminal
domain (Klenow fragment) harbouring the polymerase and the
associated proofreading 39 – 59 exonuclease catalytic sites .
The 59-39 exonuclease activity allows the removal of the RNA
primers of the Okazaki fragments during DNA replication .
The gap-filling capacity of Pol I not only participates in the
replication of the lagging strand but also in DNA excision repair
and in recombination.
The absence of other predicted DNA polymerases in H. pylori, in
particular of those capable of TLS, raises several questions
regarding the distribution of roles between the two DNA
polymerases during replication and repair. To investigate these
issues we characterised the protein coded by H. pylori polA gene and
showed that it is able to bypass blocking lesions. Based on our
genetic results we conclude that H. pylori DNA polymerase I, albeit
its important role in cell viability and DNA repair, contributes to
mutagenesis during normal chromosome replication and therefore
to the plasticity of the genome.
HP1470 codes for a bona fide DNA polymerase I devoid
of proofreading activity
The inferred absence of specialised TLS polymerases coded by
the H. pylori genome [14,15] prompted us to study the
characteristics of the putative DNA polymerase I, one of the two
predicted DNA polymerases of this pathogen. The protein
sequence deduced for HP1470 suggested that the protein is a
bona fide DNA Polymerase I orthologue. However, closer
inspection of the amino acid sequence shows that even though
the overall structure of the 39 – 59 exonuclease domain is likely to
be preserved, at least three conserved residues - Asp355, Asp424and
Asp501in E. coli DNA polymerase I - involved in metal binding
and essential for the exonuclease catalytic activity [16–18] are
missing (Figure S1). In order to verify the activities of the H. pylori
DNA Polymerase I (herein Pol I), the protein was expressed in E.
coli and purified to apparent homogeneity (Text S1 and Figure S2).
As expected, Pol I displayed DNA–dependent polymerase
(Figure 1A) and 59 – 39 exonuclease (Figure 1B) activities. In the
polymerisation assays (Figure 1A), extensions beyond the expected
full-length product could be detected. This has previously been
shown to be characteristic of some DNA polymerases lacking a
proofreading activity . To verify the prediction of a lack of 39-
59 exonuclease activity, we then tested Pol I mismatch editing
capacity. In conditions in which the E. coli Klenow fragment
efficiently removed a mispaired base from the primer 39-end, Pol
I showed no exonuclease activity on substrates with different
39-mismatches (Figure 1C). Moreover, with the possible exception
of a G:A after which only one or two bases were incorporated, Pol
I was able to extend primers with various mismatches at their 39-
end (Figure 1D).
An essential role for the 59 – 39 exonuclease domain
In order to explore the role of Pol I in vivo, we generated H. pylori
strains deficient in this protein. The constructs used to disrupt the
gene were designed to replace the 2 kb central region of the gene
with an antibiotic resistance cassette, leaving only 300 bp of the
gene at each extremity. Interestingly, very few clones were
obtained and in all cases analyzed, the cassette was inserted
downstream of the expected site. Sequencing of five independent
insertions showed that the first kilobase of the coding sequence was
always preserved (Figure S3), potentially allowing the 59 – 39
exonuclease to be expressed. To rule out a sequence context bias
for the insertion, we performed transformations with the same
disruption cassette in a strain carrying an extra copy of the polA
gene at the ureA locus. In this case the number of clones recovered
Figure 1. Pol I enzymatic activities. A. Primer extension. Increasing
concentrations (0.1; 1 and 5 nM) of Pol I were used. B. 59 – 39
exonuclease. Concentrations of Pol I used were 10, 50, 100, 150 and
200 nM. C. 39 – 59 exonuclease on mismatched primers. Concentrations
of E. coli Klenow fragment and Pol I were 25 and 100 nM. D. Mismatch
extension. 5 nM Pol I was used. Substrates in A, C and D consisted in a
34-mer template oligonucleotide paired to 18-mer primers as described
on top of each gel (Table S1). For B, the duplex was formed by a 62-mer
(XV82) paired to a 31-mer (XV101) oligonucleotide (Table S1). In all
cases representative gels of at least three independent experiments are
Helicobacter pylori is the main cause of ulcers and gastric
cancers. One the characteristics of this bacterial species is
that it displays an amazing capacity to change its genetic
information. This genetic variability provides H. pylori with
an adaptation potential that allows it to successfully
colonise the stomach of about half the human population.
Here we identified a surprising source of genomic
plasticity in an enzyme also involved in the maintenance
of DNA integrity. Indeed, we show that DNA polymerase I,
one of the only two DNA polymerases that are found in H.
pylori, although essential for DNA replication and repair,
contributes to mutagenesis due to its biochemical
Mutagenesis by H. pylori DNA Pol I
PLoS Genetics | www.plosgenetics.org2June 2011 | Volume 7 | Issue 6 | e1002152
was several orders of magnitude higher compared to those
obtained from transformation of the wild type strain. Analysis of
22 independent insertions showed that 4 were in the ectopic gene
and 18 in the hp1470 locus. Interestingly, in all cases the insertion
resulted in the expected product, a deletion starting 300 bp from
the initiation codon, leading to the truncation of two thirds of the
59 – 39 exonuclease domain. Taken together, these observations
strongly suggest that the 59 – 39 exonuclease activity coded by the
N-terminal domain of Pol I is essential for viability.
Pol I DNA polymerase activity is required for DNA repair
but promotes mutagenesis
We then assessed the capacity of strains defective in DNA
polymerase activity of Pol I (polA) to survive to various genotoxic
treatments. The polA strains used correspond to those strains where
the 39 – 59 exonuclease and polymerase domains are replaced by
an antibiotic resistance cassette, leaving an intact 59 – 39 exonu-
clease domain, shown above to be essential. polA mutants are
extremely sensitive to agents such as ionising radiation, UV light,
hydrogen peroxide and the alkylating agent methyl-methanesul-
fonate (MMS) (Figure 2A–2D), all inducing different types of DNA
damage. Interestingly, when polA was disrupted in a strain
deficient in AP-endonuclease activity (xth)  there was an
additive effect on the sensitivity to MMS, suggesting that Pol I
participates in another pathway besides base excision repair.
These results underscore the crucial role of Pol I in various DNA
To better characterise the in vivo functions of the protein, we
tested the effect of the deficiency in Pol I on H. pylori spontaneous
mutagenesis. The rate of base-pair substitutions was determined
by monitoring the appearance of rifampicin-resistant (Rifr)
colonies  (Figure 3A). Surprisingly, even though DNA
polymerases I are involved in excision repair, inactivation of the
polymerase activity of Pol I not only failed to increase base-pair
mutation rates but resulted in a modest but significant hypo-
mutator phenotype. Moreover, overexpression of Pol I driven by
the strong ureA promoter resulted in a hyper-mutator phenotype
thus indicating that Pol I in vivo generates base-substitutions.
Since the Rifrmutagenesis test is limited to the detection of
specific base substitutions, we used a forward mutation assay to
explore a larger spectrum of genetic alterations. For such purpose,
we determined the rate of mutations in the rdx gene, leading to
resistance to metronidazole (Mtzr) . The results showed a
much more pronounced effect of Pol I on the Mtzrmutation rates
than in the case of Rifr. Indeed, inactivation of polA resulted in a 4-
fold decrease in spontaneous mutations while its overexpression
increased the rate of mutation by 500-fold (Figure 3B). Sequencing
of Mtzrisolates showed that the spectrum of mutations also
changed. Indeed, sequencing of Mtzrisolates showed that the 10-
fold excess of mutants obtained in a wild type with respect to polA
strain was due to 72- and 9-fold increases in the frequency of base
substitutions and one base-pair frameshifts respectively, while the
frequency of larger deletions or insertions was essentially
unmodified (Table 1). The same trend was observed for the Pol
I over-expressing strain where the increase in mutations was
accounted for by the increase in base substitutions and one base
pair frameshifts, with only 1 out of 36 clones analyzed displaying a
change involving more than one base-pair (22 deletion).
Interestingly, in the overproducing strain the enhanced rate of
base pair substitutions could essentially be accounted for by the
increase in transversions. In conclusion, the excess mutations
Figure 2. polA mutants are sensitive to genotoxic agents. Values
correspond to the average of at least 4 independent determinations
and their SD.
Figure 3. Pol I contributes to mutagenesis. A. Rates of
spontaneous mutations to rifampicin resistance. B. Rates of spontane-
ous mutations to metronidazole (Mtz) resistance. Rates and SD were
calculated by the method of the median from N independent cultures
for wild type, polA and PolA++. N (rif)=76, 50 and 11 and N (Mtz)=20,
36 and 22 cultures, respectively.
Mutagenesis by H. pylori DNA Pol I
PLoS Genetics | www.plosgenetics.org3June 2011 | Volume 7 | Issue 6 | e1002152
observed in strains expressing Pol I were essentially base
substitutions or one nucleotide frameshifts. Taken together these
data confirm that Pol I, although important for DNA repair,
contributes to genetic variability mainly through the generation of
single nucleotide polymorphisms.
Pol I has an accurate DNA polymerase activity
The results presented above prompted us to analyse whether,
besides the lack of proofreading, Pol I harboured an intrinsic
error-prone DNA polymerase activity. The fidelity of Pol I was
determined during synthesis to fill a 407-nt single-stranded gap
within a circular duplex M13mp2 DNA substrate. The gap
contains the lacZ a-complementation sequence that serves as the
target for detecting polymerisation errors that are detected as
light blue and colourless plaques among blue plaques resulting
from correct synthesis . The DNA products of gap filling
by Pol I yielded a lacZ mutant frequency of 0.15%. This
frequency is lower than values obtained after gap filling by several
other exonuclease-deficient family A polymerases, including
0.57% Klenow fragment polymerase , 0.75% for Thermus
aquaticus polymerase , 1.6% for exonuclease-deficient T7
polymerase [23,24], and 0.62% for exonuclease-deficient pol
c . Thus H. pylori Pol I is among the most accurate
exonuclease–deficient members of the family A polymerases
when copying an undamaged DNA template in vitro.
Translesion synthesis by Pol I
The apparent contradictionbetweenthe fidelity of the Pol I DNA
polymerase activity on undamaged DNA and its role in the
generation of mutations prompted us to further investigate the
enzymatic characteristics of Pol I. The lack of proofreading activity,
the consequent capacity to elongate from mismatches and the
spectrum of mutations it generates are reminiscent of TLS
polymerases. We directly addressed this possibility by determining
the ability of purified Pol I to bypass DNA lesions present in the
template strand.Among the lesions tested, some, likethe abasic(AP)
site analogue tetrahydrofurane (THF) and thymine glycol (Tg) are
known to impose a blockage to normal DNA replication [26,27]
while others like 8-oxoguanine (8-oxoG) do not, but have a
miscoding potential . In the case of E. coli Klenow fragment,
DNA synthesis is indeed strongly blocked opposite Tg and THF
residues present in the template strand (estimated bypass efficien-
cies: 5 and 11% respectively). Conversely, albeit also partially
blocked, H. pylori Pol I is able to synthesise through these lesions
(Figure 4A) (estimated bypass efficiencies: 61 and 48%, for Tg and
We next examined the single nucleotide insertion profile
promoted by Pol I opposite the various lesions. Consistently with
the described coding capacity of 8-oxoG, Pol I introduces both A
and C opposite the oxidised guanine (Figure 4B). For the other
lesions tested DNA Pol I follows the A-rule, introducing
Table 1. Sequence alterations in metronidazole-resistant mutants.
Wild typepolA PolA++ ++
No. recovered*Rate (1028) No. recovered* Rate (1028) No. recovered*Rate (1028)
1 bp substitutions 19 (65)436 (11)0.6 14 (39)10725
transversions6 (21) 144 (7)0.4 13 (36)9960
transitions13 (44) 292 (4)0.2 1 (3)321
1 bp insertion2 (7)4 16 (29)1.6 15 (42)11491
1 bp deletion6 (21) 144 (7)0.4 6 (17)4596
.1 bp insertion1 (3)2 22 (41)2.20 (0)0
.1 bp deletion1 (3)26 (11) 0.61 (3) 550
*( ): percent of total events.
Figure 4. Translesion synthesis by Pol I. A. Translesion synthesis by
E. coli Klenow fragment (2,5 nM) and Pol I (20 nM) on 34-mer templates
harbouring a single lesion (X) at position 16. The polymerases have to
add two nucleotides before encountering the lesion. oG=8-oxogua-
nine; dHT=di-hydrothymidine; Tg=thymine-glycol and THF=tetra-
hydrofurane, an abasic site analogue. The concentrations used for the
two DNA polymerases were chosen for yielding the same activity on
undamaged DNA. B. Nucleotide selection. Extension reactions directly
on a lesion were carried out for 5 min in the presence of a single dNTP
(0.1 mM) as indicated for each lane.
Mutagenesis by H. pylori DNA Pol I
PLoS Genetics | www.plosgenetics.org4June 2011 | Volume 7 | Issue 6 | e1002152
preferentially adenine opposite the lesion. In the case of the abasic
site analogue addition of G opposite THF is also observed
To confirm that the Pol I-dependent in vivo mutagenesis could
be at least partly related to the presence of DNA lesions, we
analysed the effect of Pol I on the spontaneous mutagenesis in
strains lacking Nth, the DNA glycosylase responsible for the
removal of oxidised pyrimidines from DNA. As previously
reported , an nth mutant has a 4-fold higher mutation rate
than its parental strain (Table 2). Inactivation of polA in an nth
background resulted in partial reduction of the mutator phenotype
induced by the lack of Nth, strongly suggesting that a fraction of
unrepaired Nth substrate lesions are normally bypassed by the
DNA Pol I. This result is consistent with a contribution of Pol I to
mutagenesis through TLS.
The success of H. pylori in colonising a large fraction of the
human population has been attributed to its adaptation capacity
based, in turn, on the genetic diversity of the species. At the basis
of the remarkable genetic variation found in H. pylori are
nucleotide polymorphisms that can be rapidly propagated by
recombination between strains . In vivo generation of allelic
diversity is driven by mutation rates significantly higher than those
of most other bacteria . Several mechanisms have been
suggested as contributing to the high mutation frequency of this
pathogen, starting with the lack of homologues of many DNA
repair genes known to be involved in maintaining the genetic
stability in other bacterial species . Among the most remarkable
absences is probably that of a mismatch repair system [6,7]
capable of removing incorrect bases introduced during replication.
The work presented here unveiled another mechanism
contributing to H. pylori high mutation rates. We showed that H.
pylori strains deficient in DNA Pol I polymerase activity have
reduced mutation rates indicating that DNA polymerase I actively
participates in generating allelic diversity. This constitutes a
surprising role for a protein associated with DNA repair and
replication in all the studied bacterial models. The sensitivity of
polA strains to various genotoxic agents confirms that H. pylori Pol I
is involved in various DNA repair pathways such as recombination
and base excision repair. However, their hypomutator phenotype
is in contrast with the 7- to 10-fold-higher spontaneous mutation
frequency in E. coli Pol I-deficient strains . Our results,
together with the absence of DNA polymerases other than DNA
Pol I and Pol III, suggest that the polA gene in this species has been
selected for coding a DNA Pol I capable of fulfilling extra functions
allowing increased mutation rates. Indeed, we showed that DNA
Pol I can not only extend mismatched primers, but also bypass
DNA lesions that would normally block the replicative polymerase
as well as DNA Pol I homologues from other bacteria. How can
those characteristics contribute to increase mutagenesis?
Spontaneous or induced DNA damage is constantly generated
in the genome . In most organisms TLS polymerases allow
replication across damaged DNA avoiding the blockage of the
replication machinery. In E. coli, the SOS response includes the
expression of TLS polymerases that allow survival in such
situations at the expense of induced mutations. In spite of the
lack of evidence for an SOS response system, it is now clear that H.
pylori takes advantage of stress-induced DNA damage to mutate
[8,9]. Such a response necessitates a DNA polymerase capable of
performing mutagenic TLS . Unlike what was shown in B.
subtilis, where Pol I, also lacking the proofreading function, acts in
concert with TLS polymerases PolY1 and PolY2 to bypass lesions
, the biochemical activities of H. pylori Pol I unveiled in this
work would be sufficient to accomplish the task. The hypo-
mutator phenotype of the polA strains and the lack of specialised
TLS polymerases are consistent with this view. So is the increased
proportion of transversions among base substitutions found in the
Pol I overproducing strain with respect to the wild-type (13/14
versus 6/19), as expected for the bypass of non-coding lesions. The
partial dependence on a functional DNA Pol I of the increased
mutagenesis of an nth strain provides further support for this
hypothesis. The oxidative stress generated by infection-induced
inflammation, the acidic medium of the stomach together with the
limited set of H. pylori DNA glycosylases involved in base excision
repair [20,33] favour the formation and persistence of stress-
induced damage in DNA, including modified bases and abasic
sites [9,34]. Therefore, it is likely that beyond its functions in DNA
repair, DNA Pol I plays an important role in the survival of the
bacteria during infection by allowing the replication of damaged
DNA and, concomitantly, by contributing to generate allelic
diversity in response to the stress.
From the enzymatic point of view, H. pylori polymerase I
combines two antagonistic properties not usually found within the
same enzyme. Although it exhibits high accuracy on undamaged
DNA, it is able to efficiently bypass several types of lesions and can
extend mismatched primers. How can such a plasticity be
understood in the context of a single enzyme? Regarding other
Pol I homologues, accuracy was shown to be exquisitely controlled
through a closing mechanism of the fingers domain involving a
tight packing between the active site residues and the nucleotide to
be inserted. Residues in the so-called O-helix were shown to
actively disfavour misincorporation [35,36]. Mutation Y766S
within the O-helix of E. coli Klenow polymerase led to a more
open active site and favoured lesion bypass at the expense of
fidelity [37,38]. Similarly, the more open active site of TLS
polymerases, such as that of yeast Polg or the archaeal Dpo4, is a
major determinant to account for their ability to accommodate
bulky lesions [39,40]. As a first hypothesis, we thought that H.
pylori Pol I active site might have a special open structure
particularly tolerant to mispairs insertions. However, mapping the
conservation of the sequences of both Taq and H. pylori Pol I at
the structure of Taq polymerase showed that both sequences are
strictly conserved all along the active site groove (Figure S4). In
particular, residues of the O-helix involved in the steric-gate
mechanism are identical. Consequently, both the high accuracy of
PolA on undamaged DNA and the conserved nature of the active
site support that, unlike other TLS polymerases, H. pylori Pol I
permissiveness is not due to a more open cavity. Consistently we
have not been able to detect a significant mutagenesis induced by
UV (data not shown). One might suppose that subtle dynamical
properties of the enzyme allow accommodation of small lesions
but not necessarily bulky lesions. A large body of evidence suggests
that this is the case for other members of the Pol A family,
including E. coli Klenow fragment. Indeed, these polymerases were
Table 2. Rates (61028) of spontaneous mutation to Rifr.
StrainMutation rate ± SD
Mutagenesis by H. pylori DNA Pol I
PLoS Genetics | www.plosgenetics.org5June 2011 | Volume 7 | Issue 6 | e1002152
shown to be able to incorporate a nucleotide opposite AP sites and
products of cytosine or thymine oxidation (Tg, urea, uracyl-glycol
and others) although not always to elongate from it [19,26,41–44].
Moreover, inactivation of the proofreading activity of Klenow
allows the bypass of most of these lesions [19,43,45–47]. Inter-
festingly, two higher-eukaryote members of the family lacking a
39-59 exonuclease domain, DNA polymerases h and n, have been
shown to be proficient for bypassing Tg and abasic sites [48–50].
Taking into account the strong structural conservation predicted
for the active sites of H. pylori Pol I and the other members of the
family (Figure S4), the work cited above supports the notion that
the loss of proofreading activity can account for the capacity of Pol
I to bypass AP sites and non-bulky damaged bases. In the case of
AP sites this activity will contribute to mutagenesis either by
incorporating in three out of four events the wrong nucleotide or
by inducing frameshifts .
Further support for a role of TLS by Pol I in H. pylori
mutagenesis, comes from the results showing that inactivation of
Pol I partially complements the mutator phenotype of an nth strain
(Table 2). H. pylori Nth is the only DNA glycosylase in this
organism capable of removing oxidised pyrimidines from DNA
. Many of the products of thymine and cytosine oxidation are
pre-mutagenic lesions. In particular, oxidised derivatives from
cytosine as 59-hydroxycytosine, uracyl-glycol and 59-hydroxyur-
acyl [46,51–54] but also from thymine [46,51–54] have been
shown to be bypassed by proofreading-deficient DNA polymerases
and to be mutagenic [55,56].
Besides TLS, another, non-exclusive, mechanism can be
invoked for the role of H. pylori Pol I in mutagenesis. The essential
character of the 59 - 39 exonuclease domain of H. pylori Pol I
strongly suggest that this activity is required for Okazaki fragment
processing, even in the absence of the other protein activities.
Recently, elegant genetic experiments established that E. coli Pol
I proofreading activity plays a crucial role in chromosomal
replication fidelity . The model put forward by the authors
proposes that Pol I performs 1–2% of lagging strand synthesis.
They show that inactivation of the 39 - 59 exonuclease activity
leads to a mutator phenotype with a strong bias towards lagging
strand mutations. The mutator phenotype observed in polA strains
even in the absence of all three TLS polymerases is also consistent
with a proposed role for E. coli Pol I proofreading activity during
replication . In the case of H. pylori, despite accurate
polymerase activity of Pol I, its lack of proofreading capacity
could contribute to mutagenesis during Okazaki fragment
processing. In the absence of Pol I DNA polymerase activity, the
replicative polymerase is the only candidate to perform lagging
strand synthesis. Because of its high fidelity, lower mutation rates
are expected. Conversely, over-expression of Pol I can lead to a
more extensive processing of Okazaki fragments, therefore
increasing the fraction of lagging strand synthesis performed by
this enzyme and leading to a higher level of replication error rates.
In conclusion, independently of the relative contributions of Pol
I to TLS and lagging strand synthesis, the results presented here
strongly support the hypothesis by which in H. pylori the loss of
proofreading activity of this DNA polymerase has been selected for
increasing genome plasticity.
Materials and Methods
H. pylori strains and growth conditions
All H. pylori strains used were in the 26695 genetic background
. To generate gene-specific mutants, the corresponding
open-reading frame (ORF) cloned into pILL570 was disrupted,
leaving 59 and 39 ends (300 bp) of the gene, by a nonpolar
kanamycin- (Km), apramycin- (Apr) or chloramphenicol (Cm)
resistance cassette [59,60]. To generate the Pol I over-expressing
strain, the HP1470 ORF was inserted into pADC vector,
downstream of the ureA promoter, as described . Plasmids
were introduced into H. pylori strains by natural transformation
and recombinants were selected after 3 to 5 days of growth on
either 20 mg/ml Km, 12.5 mg/ml Apr or 8 mg/ml Cm. Allelic
replacement was verified by PCR. As described in the Results
section, the polA mutants used correspond, unless specified, to the
replacement of the equivalent of the Klenow fragment by the
resistance cassette, leaving the 59 to 39 exonuclease domain
intact. Double mutants were obtained by plasmid or genomic
DNA transformation of single mutant or by mixing two mutant
strains together before plating the mix on double selection. H.
pylori cultures were grown at 37uC under a microaerobic
atmosphere on BAB, blood agar base medium supplemented
with an antibiotic mix and 10% defibrillated horse blood.
Sensitivity and mutagenesis assays
For all experiments, H. pylori strains were initially grown for
24 hr on plates with BAB medium. UV, MMS and gamma
irradiation sensitivity assays were performed as described [33,62].
For chemical oxidative stress treatment, H pylori (OD600=1) cell
suspensions were incubated with different concentrations of
hydrogen peroxide (100, 200 and 300 mM). Cells were washed
10 min later, diluted with peptone broth and plated on BAB
plates. Survival was determined as the number of cells forming
colonies on plates after a given treatment divided by the number of
colonies from non-treated cells. Assays to determine spontaneous
mutation rates were performed as described .
All assays were performed at 37uC for 30 min in 20 ml reactions
containing 10 mM Tris-HCl (pH 7.9), 50 mM NaCl, 10 mM
MgCl2, 1 mM Dithiothreitol, 2.5 nM DNA substrate (see Text S1
and Table S1) and variable concentrations of Pol I (specified in the
figure legends). 0.1 mM of either all four dNTPs or each dNTP
individually was included in the reactions except for the
exonuclease activity assays. When required as a control, Klenow
fragment DNA polymerase (Roche) was used. Reactions were
stopped by adding loading buffer (10 mM EDTA, 95% (v/v)
formamide, 0.03% (w/v) bromophenol blue, 0.03% (w/v) xylene
cyanol) and subjected to electrophoresis in 8 M urea-containing
20% polyacrylamide gels. Gels were visualised and quantified
using a Molecular Dynamics PhosphorImager. According to
Koskoska et al. , bypass probabilities were calculated as the
proportion of DNA synthesis products extended beyond the lesion.
Bypass efficiencies were then calculated for each enzyme and each
substrate as the ratio of the bypass probability of a specific
damaged base with respect to that of an undamaged nucleotide in
the same position.
M13mp2 fidelity assay
The assay was performed as described previously . Gap-
filling DNA synthesis was performed in a reaction mixture (25 ml)
containing 50 mM Tris (pH 6.8), 50 mM NaCl, 10 mM MgCl2,
1 mM DTT, 0.2 mM each of dNTP and 0.2 nM of gapped
M13mp2 DNA substrate. Reactions were initiated by adding Pol
I, incubated at 37uC for 30 min, and terminated by adding EDTA
to 20 mM. When DNA products were analyzed by agarose gel
electrophoresis , the majority of the gapped molecules were
filled to completion. However, a minority of DNA products
migrated as if synthesis had paused at the palindrome just
upstream of the open reading frame of the LacZ gene. In this
Mutagenesis by H. pylori DNA Pol I
PLoS Genetics | www.plosgenetics.org6June 2011 | Volume 7 | Issue 6 | e1002152
minority population, only about 75% of the template used to score
errors had been copied. As a consequence, the lacZ mutant
frequency observed for the ensemble reaction products may
slightly underestimate the error rate of H. pylori Pol I.
(Escherichia coli) and of polymerase A (Helicobacter pylori) homologs
from various bacterial species spanning the region of the 39-59
exonuclease domain. The four positions involved in chelating the
divalent metals (as shown in Figure 1) are highlighted by red
triangles on top of the alignment. The aligned species were
selected to sample species with or without the consensus site
required for metal-binding in the 39-59 exonucleolytic site (name
written in black or red, respectively). In every clade of the bacterial
classification, polymerases lacking these residues can be found.
Among e proteobacteria, Helicobacter hepaticus is the closest species
to Helicobacter pylori with an a priori functional 39-59 exonucleolytic
site. Among c proteobacteria, some species among the Pseudomonas
clade do not contain the consensus site. Among the species tested,
all b proteobacteria were found with a conserved functional site.
Conversely, among the Firmicutes or Actinobacteria tested, none
of them have the correct consensus site.
Multiple sequence alignment of polymerase I
was purified to near homogeneity as judged by analysis on 10%
SDS polyacrylamide gel electrophoresis and staining with
Pol I protein. H. pylori Pol I overexpressed in E. coli
transformed with the plasmid carrying the polA ORF in which its
central part was replaced by an antibiotic-resistance cassette
(ATBR), leaving 300 base pairs (bp) of the ORF at each end.
Sequence analyses indicated that all the antibiotic-resistant
recombinants recovered had the ATBRcassette inserted 998 bp
downstream the initiation codon. The integration was likely to
have occurred through a recombination between 7 bp repeats. (A)
Disruption of the polA gene. Wild type strains were
Expected recombination event. (B) Actual event in which the final
product would allow the expression of the first 330 amino acids of
the protein where resides the 59 – 39 exonuclease activity.
polymerase (3LWM) with its various domains highlighted in yellow
(39-59 exonuclease), red (palm), green (thumb) and cyan (fingers).
DNA complexed to the polymerase is colored in purple (B) Surface
representation of the Taq polymerase domain in the same
orientation as in panel A. The surface is coloured with respect
to the conservation between the sequences of the Taq and that of
H. pylori PolA. Red color indicates the invariant positions while
colors ranging from orange to pale yellow report residues with
decreasing similarity. (C) A section of the surface representation
highlights the O-helix residues engulfing the nucleotides as
identical between both Taq and H. pylori PolA polymerase.
(A) Ribbon representation of a structure of the Taq
DNA substrates preparation and Pol I expression and
Sequence of DNA oligonucleotides used for preparing
We thank Drs. Katarzyna Bebenek, Danielle Watt, and Esma Bentchikou
for thoughtful comments on the manuscript and Dr. Luis Blanco for his
advice and for providing MVGO with facilities for performing some of the
biochemical assays. We also thank Dr. Robert Fuchs for encouraging us to
explore how TLS is performed in H. pylori.
Conceived and designed the experiments: M-VG-O SM TAK JPR.
Performed the experiments: M-VG-O SM MEA. Analyzed the data:
M-VG-O SM MEA RG TAK JPR. Contributed reagents/materials/
analysis tools: DG RG. Wrote the paper: M-VG-O SM RG JPR.
Analysis of sequences and structural models: RG.
1. Suerbaum S, Michetti P (2002) Helicobacter pylori infection. N Engl J Med 347:
2. Falush D, Kraft C, Taylor NS, Correa P, Fox JG, et al. (2001) Recombination
and mutation during long-term gastric colonization by Helicobacter pylori:
estimates of clock rates, recombination size, and minimal age. Proc Natl Acad
Sci U S A 98: 15056–15061.
3. Israel DA, Salama N, Krishna U, Rieger UM, Atherton JC, et al. (2001)
Helicobacter pylori genetic diversity within the gastric niche of a single human
host. Proc Natl Acad Sci U S A 98: 14625–14630.
4. Kang J, Blaser MJ (2006) Bacterial populations as perfect gases: genomic
integrity and diversification tensions in Helicobacter pylori. Nat Rev Microbiol
5. Tenaillon O, Toupance B, Le Nagard H, Taddei F, Godelle B (1999) Mutators,
population size, adaptive landscape and the adaptation of asexual populations of
bacteria. Genetics 152: 485–493.
6. Bjorkholm B, Sjolund M, Falk PG, Berg OG, Engstrand L, et al. (2001)
Mutation frequency and biological cost of antibiotic resistance in Helicobacter
pylori. Proc Natl Acad Sci U S A 98: 14607–14612.
7. Pinto AV, Mathieu A, Marsin S, Veaute X, Ielpi L, et al. (2005) Suppression of
homologous and homeologous recombination by the bacterial MutS2 protein.
Mol Cell 17: 113–120.
8. Kang JM, Iovine NM, Blaser MJ (2006) A paradigm for direct stress-induced
mutation in prokaryotes. FASEB J 20: 2476–2485.
9. O’Rourke EJ, Chevalier C, Pinto AV, Thiberge JM, Ielpi L, et al. (2003)
Pathogen DNA as target for host-generated oxidative stress: role for repair of
bacterial DNA damage in Helicobacter pylori colonization. Proc Natl Acad
Sci U S A 100: 2789–2794.
10. Tippin B, Pham P, Goodman MF (2004) Error-prone replication for better or
worse. Trends Microbiol 12: 288–295.
11. Kornberg A, Baker TA (1992) DNA Replication. New York: Freeman.
12. Joyce CM, Grindley ND (1984) Method for determining whether a gene of
Escherichia coli is essential: application to the polA gene. J Bacteriol 158:
13. Okazaki R, Arisawa M, Sugino A (1971) Slow joining of newly replicated DNA
chains in DNA polymerase I-deficient Escherichia coli mutants. Proc Natl Acad
Sci U S A 68: 2954–2957.
14. Alm RA, Ling LS, Moir DT, King BL, Brown ED, et al. (1999) Genomic-
sequence comparison of two unrelated isolates of the human gastric pathogen
Helicobacter pylori. Nature 397: 176–180.
15. Tomb JF, White O, Kerlavage AR, Clayton RA, Sutton GG, et al. (1997) The
complete genome sequence of the gastric pathogen Helicobacter pylori. Nature
16. Derbyshire V, Freemont PS, Sanderson MR, Beese L, Friedman JM, et al.
(1988) Genetic and crystallographic studies of the 39,59-exonucleolytic site of
DNA polymerase I. Science 240: 199–201.
17. Derbyshire V, Pinsonneault JK, Joyce CM (1995) Structure-function analysis of
39R59-exonuclease of DNA polymerases. Methods Enzymol 262: 363–385.
18. Liu X, Hou J, Liu J (2006) Chlamydial DNA polymerase I can bypass lesions in
vitro. Biochem Biophys Res Commun 345: 1083–1091.
19. Clark JM, Beardsley GP (1989) Template length, sequence context, and 39-59
exonuclease activity modulate replicative bypass of thymine glycol lesions in
vitro. Biochemistry 28: 775–779.
20. Mathieu A, O’Rourke EJ, Radicella JP (2006) Helicobacter pylori genes
involved in avoidance of mutations induced by 8-oxoguanine. J Bacteriol 188:
21. Jeong JY, Mukhopadhyay AK, Akada JK, Dailidiene D, Hoffman PS, et al.
(2001) Roles of FrxA and RdxA nitroreductases of Helicobacter pylori in
susceptibility and resistance to metronidazole. J Bacteriol 183: 5155–5162.
22. Bebenek K, Kunkel TA (1995) Analyzing fidelity of DNA polymerases. Methods
Enzymol 262: 217–232.
Mutagenesis by H. pylori DNA Pol I
PLoS Genetics | www.plosgenetics.org7 June 2011 | Volume 7 | Issue 6 | e1002152
23. Bebenek K, Joyce CM, Fitzgerald MP, Kunkel TA (1990) The fidelity of DNA Download full-text
synthesis catalyzed by derivatives of Escherichia coli DNA polymerase I. J Biol
Chem 265: 13878–13887.
24. Eckert KA, Kunkel TA (1990) High fidelity DNA synthesis by the Thermus
aquaticus DNA polymerase. Nucleic Acids Res 18: 3739–3744.
25. Longley MJ, Nguyen D, Kunkel TA, Copeland WC (2001) The fidelity of
human DNA polymerase gamma with and without exonucleolytic proofreading
and the p55 accessory subunit. J Biol Chem 276: 38555–38562.
26. Ide H, Kow YW, Wallace SS (1985) Thymine glycols and urea residues in M13
DNA constitute replicative blocks in vitro. Nucleic Acids Res 13: 8035–8052.
27. Strauss B, Rabkin S, Sagher D, Moore P (1982) The role of DNA polymerase in
base substitution mutagenesis on non-instructional templates. Biochimie 64:
28. Grollman AP, Moriya M (1993) Mutagenesis by 8-oxoguanine: an enemy
within. Trends Genet 9: 246–249.
29. Suerbaum S, Josenhans C (2007) Helicobacter pylori evolution and phenotypic
diversification in a changing host. Nat Rev Microbiol 5: 441–452.
30. Bates H, Randall SK, Rayssiguier C, Bridges BA, Goodman MF, et al. (1989)
Spontaneous and UV-induced mutations in Escherichia coli K-12 strains with
altered or absent DNA polymerase I. J Bacteriol 171: 2480–2484.
31. Lindahl T (1993) Instability and decay of the primary structure of DNA [see
comments]. Nature 362: 709–715.
32. Duigou S, Ehrlich SD, Noirot P, Noirot-Gros MF (2005) DNA polymerase I acts
in translesion synthesis mediated by the Y-polymerases in Bacillus subtilis. Mol
Microbiol 57: 678–690.
33. O’Rourke EJ, Chevalier C, Boiteux S, Labigne A, Ielpi L, et al. (2000) A novel 3-
methyladenine DNA glycosylase from helicobacter pylori defines a new class
within the endonuclease III family of base excision repair glycosylases. J Biol
Chem 275: 20077–20083.
34. Eutsey R, Wang G, Maier RJ (2007) Role of a MutY DNA glycosylase in
combating oxidative DNA damage in Helicobacter pylori. DNA Repair (Amst)
35. Kiefer JR, Mao C, Braman JC, Beese LS (1998) Visualizing DNA replication in
a catalytically active Bacillus DNA polymerase crystal. Nature 391: 304–307.
36. Li Y, Korolev S, Waksman G (1998) Crystal structures of open and closed forms
of binary and ternary complexes of the large fragment of Thermus aquaticus
DNA polymerase I: structural basis for nucleotide incorporation. EMBO J 17:
37. Bell JB, Eckert KA, Joyce CM, Kunkel TA (1997) Base miscoding and strand
misalignment errors by mutator Klenow polymerases with amino acid
substitutions at tyrosine 766 in the O helix of the fingers subdomain. J Biol
Chem 272: 7345–7351.
38. Lone S, Romano LJ (2003) Mechanistic insights into replication across from
bulky DNA adducts: a mutant polymerase I allows an N-acetyl-2-aminofluorene
adduct to be accommodated during DNA synthesis. Biochemistry 42:
39. Rechkoblit O, Kolbanovskiy A, Malinina L, Geacintov NE, Broyde S, et al.
(2010) Mechanism of error-free and semitargeted mutagenic bypass of an
aromatic amine lesion by Y-family polymerase Dpo4. Nat Struct Mol Biol 17:
40. Washington MT, Prakash L, Prakash S (2001) Yeast DNA polymerase eta
utilizes an induced-fit mechanism of nucleotide incorporation. Cell 107:
41. Ide H, Petrullo LA, Hatahet Z, Wallace SS (1991) Processing of DNA base
damage by DNA polymerases. Dihydrothymine and beta-ureidoisobutyric acid
as models for instructive and noninstructive lesions. J Biol Chem 266:
42. Matray TJ, Haxton KJ, Greenberg MM (1995) The effects of the ring
fragmentation product of thymidine C5-hydrate on phosphodiesterases and
klenow (exo-) fragment. Nucleic Acids Res 23: 4642–4648.
43. Paz-Elizur T, Takeshita M, Livneh Z (1997) Mechanism of bypass synthesis
through an abasic site analog by DNA polymerase I. Biochemistry 36:
44. Sagher D, Strauss B (1983) Insertion of nucleotides opposite apurinic/
apyrimidinic sites in deoxyribonucleic acid during in vitro synthesis: uniqueness
of adenine nucleotides. Biochemistry 22: 4518–4526.
45. Hatahet Z, Zhou M, Reha-Krantz LJ, Ide H, Morrical SW, et al. (1999) In vitro
selection of sequence contexts which enhance bypass of abasic sites and
tetrahydrofuran by T4 DNA polymerase holoenzyme. J Mol Biol 286:
46. Purmal AA, Lampman GW, Bond JP, Hatahet Z, Wallace SS (1998) Enzymatic
processing of uracil glycol, a major oxidative product of DNA cytosine. J Biol
Chem 273: 10026–10035.
47. Shibutani S, Grollman AP (1993) On the mechanism of frameshift (deletion)
mutagenesis in vitro. J Biol Chem 268: 11703–11710.
48. Arana ME, Seki M, Wood RD, Rogozin IB, Kunkel TA (2008) Low-fidelity
DNA synthesis by human DNA polymerase theta. Nucleic Acids Res 36:
49. Arana ME, Takata K, Garcia-Diaz M, Wood RD, Kunkel TA (2007) A unique
error signature for human DNA polymerase nu. DNA Repair (Amst) 6:
50. Seki M, Masutani C, Yang LW, Schuffert A, Iwai S, et al. (2004) High-efficiency
bypass of DNA damage by human DNA polymerase Q. EMBO J 23:
51. Feig DI, Sowers LC, Loeb LA (1994) Reverse chemical mutagenesis:
identification of the mutagenic lesions resulting from reactive oxygen species-
mediated damage to DNA. Proc Natl Acad Sci U S A 91: 6609–6613.
52. Kreutzer DA, Essigmann JM (1998) Oxidized, deaminated cytosines are a
source of CRT transitions in vivo. Proc Natl Acad Sci U S A 95: 3578–3582.
53. Najrana T, Saito Y, Uraki F, Kubo K, Yamamoto K (2000) Spontaneous and
osmium tetroxide-induced mutagenesis in an Escherichia coli strain deficient in
both endonuclease III and endonuclease VIII. Mutagenesis 15: 121–125.
54. Purmal AA, Kow YW, Wallace SS (1994) Major oxidative products of cytosine,
5-hydroxycytosine and 5-hydroxyuracil, exhibit sequence context-dependent
mispairing in vitro. Nucleic Acids Res 22: 72–78.
55. Kamiya H (2003) Mutagenic potentials of damaged nucleic acids produced by
reactive oxygen/nitrogen species: approaches using synthetic oligonucleotides
and nucleotides: survey and summary. Nucleic Acids Res 31: 517–531.
56. Wallace SS (2002) Biological consequences of free radical-damaged DNA bases.
Free Radic Biol Med 33: 1–14.
57. Makiela-Dzbenska K, Jaszczur M, Banach-Orlowska M, Jonczyk P,
Schaaper RM, et al. (2009) Role of Escherichia coli DNA polymerase I in
chromosomal DNA replication fidelity. Mol Microbiol 74: 1114–1127.
58. Tago Y, Imai M, Ihara M, Atofuji H, Nagata Y, et al. (2005) Escherichia coli
mutator (Delta)polA is defective in base mismatch correction: the nature of in
vivo DNA replication errors. J Mol Biol 351: 299–308.
59. Heuermann D, Haas R (1998) A stable shuttle vector system for efficient genetic
complementation of Helicobacter pylori strains by transformation and
conjugation. Mol Gen Genet 257: 519–528.
60. Skouloubris S, Thiberge JM, Labigne A, De Reuse H (1998) The Helicobacter
pylori UreI protein is not involved in urease activity but is essential for bacterial
survival in vivo. Infect Immun 66: 4517–4521.
61. Kang J, Huang S, Blaser MJ (2005) Structural and functional divergence of
MutS2 from bacterial MutS1 and eukaryotic MSH4-MSH5 homologs.
J Bacteriol 187: 3528–3537.
62. Marsin S, Mathieu A, Kortulewski T, Guerois R, Radicella JP (2008) Unveiling
novel RecO distant orthologues involved in homologous recombination. PLoS
Genet 4: e1000146. doi:10.1371/journal.pgen.1000146.
63. Kokoska RJ, McCulloch SD, Kunkel TA (2003) The efficiency and specificity of
apurinic/apyrimidinic site bypass by human DNA polymerase eta and
Sulfolobus solfataricus Dpo4. J Biol Chem 278: 50537–50545.
Mutagenesis by H. pylori DNA Pol I
PLoS Genetics | www.plosgenetics.org8 June 2011 | Volume 7 | Issue 6 | e1002152