Published online 21 November 2007 Nucleic Acids Research, 2008, Vol. 36, No. 3 705–711
Uracil recognition by replicative DNA polymerases
is limited to the archaea, not occurring with
bacteria and eukarya
Josephine Wardle1, Peter M. J. Burgers2, Isaac K. O. Cann3, Kate Darley4,
Pauline Heslop1, Erik Johansson5, Li-Jung Lin3, Peter McGlynn6, Jonathan Sanvoisin4,
Carrie M. Stith2and Bernard A. Connolly1,*
1Institute for Cell and Molecular Biosciences (ICaMB), University of Newcastle, Newcastle upon Tyne NE2 4HH,
UK,2Department of Biochemistry and Molecular Biophysics, Washington University School of Medicine,
Saint Louis, MO 63110,3Department of Animal Sciences, University of Illinois at Urbana-Champaign, Urbana,
IL 61801, USA,4Replizyme Ltd, York Science Park, York YO10 5DQ, UK,5Department of Medical Biochemistry
and Biophysics, Umea ˚ University, SE-901 87 Umea ˚, Sweden and6Institute of Medical Sciences, University of
Aberdeen, Aberdeen AB25 2ZD, UK
Received September 27, 2007; Revised October 26, 2007; Accepted October 27, 2007
Family B DNA polymerases from archaea such as
Pyrococcus furiosus, which live at temperatures
»1008C, specifically recognize uracil in DNA tem-
plates and stall replication in response to this base.
Here it is demonstrated that interaction with uracil is
not restricted to hyperthermophilic archaea and that
the polymerase from mesophilic Methanosarcina
acetivorans shows identical behaviour. The family
B DNA polymerases replicate the genomes of
archaea, one of the three fundamental domains
of life. This publication further shows that the
DNA replicating polymerases from the other two
domains, bacteria (polymerase III) and eukaryotes
(polymerases d and e for nuclear DNA and polymer-
ase c for mitochondrial) are also unable to recognize
uracil. Uracil occurs in DNA as a result of deamina-
tion of cytosine, either in G:C base-pairs or, more
for example, during replication. The resulting G:U
mis-pairs/single stranded uracils are promutagenic
and, unless repaired, give rise to G:C to A:T transi-
tions in 50% of the progeny. The confinement of
uracil recognition to polymerases of the archaeal
domain is discussed in terms of the DNA repair
pathways necessary for the elimination of uracil.
Family B DNA polymerases from the archaea, for
example the enzyme from Pyrococcus furiosus (Pfu-Pol)
commonly used in the PCR, strongly bind to template-
strand uracil and stall polymerization in response to this
base (1). The ability to recognize uracil arises from a
specialized binding pocket in the amino-terminal domain
of the polymerase (2), which interacts tightly with uracil in
single-stranded DNA (3). Deamination of cytosine con-
verts G:C base-pairs to pro-mutagenic G:U mismatches,
replication of which results in 50% of the progeny
containing a G:C!A:T transition mutation (4). Repair
of G:U mis-pairs is usually initiated by uracil–DNA–
glycosylases (UDGases), enzymes that remove uracil from
DNA by glycosidic bond hydrolysis. In double stranded
DNA, UDGase action initiates a base excision repair
pathway that, ultimately, restores the G:C base-pair (5–7).
Polymerase mediated uracil-induced stalling of replication
is probably the first step of an additional DNA repair
pathway that prevents copying of the G:U mismatch and
permanent fixation of a transition mutation; replication
might therefore offer the final opportunity for mutation
avoidance. The pathways that follow stalling probably
involve recombinational daughter-strand ‘gap’ processes,
often used to repair and restart stalled replication forks
(8,9). Thus far uracil recognition has only been observed
for hyperthermophilic archaeal DNA polymerases and
may be an adaptation to high temperatures, expected
*To whom correspondence should be addressed. Tel: +44 191 222 7371; Fax: +44 191 222 7424; Email: email@example.com
? 2007 The Author(s)
This is an Open Access article distributed under the terms of the Creative Commons Attribution Non-Commercial License (http://creativecommons.org/licenses/
by-nc/2.0/uk/) which permits unrestricted non-commercial use, distribution, and reproduction in any medium, provided the original work is properly cited.
to promote cytosine deamination (10), at which many
archaea live. In this article a mesophilic archaeal Family-B
DNA polymerase has been investigated to elucidate
whether uracil recognition is strictly limited to hyperther-
mophiles. In addition archaea constitute one of the three
fundamental domains of life (11,12), the other two being
bacteria and eukarya. This article also investigates
whether the polymerases responsible for the replication
of DNA in these two domains are able to recognize uracil.
MATERIALS AND METHODS
The purification of the family B DNA polymerase from
P. furiosus (13), Saccharomyces cerevisiae DNA poly-
merases e (14) and d (15) and the family B DNA poly-
merase from Methanosarcina acetivorans (16) have been
described. The Escherichia coli PolIII holoenzyme used
in this publication was PolIII?, which contains the a, e, y,
w, c, d, d’, g and t sub-units but lacks the b clamp (17),
prepared as described (18). The two sub-unit human
mitochondrial DNA polymerase g was purified from
human embryonic kidney cells expressing the SV40 large-
T antigen (HEK293T cells) (19) containing two plasmids
that encode the catalytic and accessory sub-units of the
polymerase (20). The plasmids, pcDNA3.1(-)/Myc-His A
(geneticin antibiotic marker for the catalytic sub-unit,
hygromycin for the accessory) (Invitrogen) place the
coding sequences in-frame with a Myc-His tag (19).
Growth and harvesting of the HEK293T overproducing
cells (growth media containing 2mg/ml geneticin and
100ml/ml hygromycin) has been described (19). Cells were
suspended in ice cold (all subsequent purification steps
were performed at 48C) 20mM sodium phosphate pH 7.4,
10mM NaCl, 20mM imidazole, 250mM sucrose, com-
plete protease inhibitor (Roche), lysed using a French
press and clarified by centrifugation at 1000g for 10min.
DNase I (10mg/ml final concentration and Triton X-100
(1% v/v final concentration) were added to the lysate and,
after incubation for30min, the solution was centrifuged
at 1000g for30min. The crude cell extract was applied
to a 1ml His-Trap column (Amersham-Pharmacia), which
was washed with 20mM sodium phosphate, pH 7.4,
10mM NaCl, 20mM imidazole, 0.1% (v/v) Triton-X100.
The polymerase was eluted using a gradient to 500mM
imidazole (total gradient volume 90ml); appropriate
fractions (monitored by SDS-PAGE) were pooled and
dialysed against 10mM Tris, pH 7.5, 10mM NaCl,
0.1% (v/v) Triton X-100. The volume was reduced to
one-fifth of the original (VivaSpin column, 10kDa cut-off;
Sartorius, Epsom, UK) and the concentrate applied to a
1ml Hep-Trap column (Amersham-Pharmacia). Follow-
ing washing the polymerase was eluted using a gradient to
2M NaCl (gradient volume 60ml); appropriate fractions
(monitored by SDS-PAGE) were pooled and dialysed
against 100mM Tris, pH 7.5, 50mM NaCl, 5mM MgCl2,
5mM dithiothreitol, 30% (v/v) glycerol. Bovine serum
albumin was added (0.5mg/ml final concentration) and
the samples rapidly frozen using liquid nitrogen and
stored at ?808C. Saccharomyces cerevisiae PCNA, RPA
and RFC were purified as described (21,22).
Sequences were aligned using protein blast (http://
Primer-template extension assays
A 16-mer primer (50-GCAGTCCTAGACGCAG-30) and
32-mer template (50-CATCCG(T/U)GG(T/U)GCTATCC
TGCGTCTAGGACTGC-30) were used; annealing pro-
duces a duplex region of 16 base pairs with a single
stranded template-strand extension of 16 bases. A single
uracil is located at one of the two positions indicated
(thymine for controls), which places uracil either seven
or ten bases ahead of the primer-template junction.
Extension assays (20ml final volume) contained 2.5nM
buffer (archaeal and E. coli polymerases; 20mM Tris–HCl
(pH 8.8), 10mM KCl, 10mM (NH4)2SO4, 5mM MgCl2,
0.1% Triton X-100, 0.1mg/ml bovine serum albumin:
eukaryotic polymerases; 40mM Tris–HCl (pH 7.8),
75mM NaCl, 8mM magnesium acetate, 1mM dithio-
threitol, 0.2mg/ml bovine serum albumin) to which was
added polymerase (1mM final concentration) to initiate
the reaction. Polymerizations were carried out at 308C
for mesophilic DNA polymerases, 728C for thermophilic
DNA polymerases for times of between 0.5 and 30min.
Reactions were stopped by addition of a 2-fold excess of
EDTA (over Mg2+) and 40% formamide and products
detected using denaturing polyacrylamide (15%) gel
electrophoresis followed by phosphorimaging. Some
reactions with yeast Pol d and e were carried out in
the presence of additional replisome components. Here the
primer-template was pre-incubated with 1mM PCNA,
5mM RFC 1mM RPA, 1mM UDGase inhibitor (UGI),
0.5mM ATP, 5mM dNTPs and 8mM MgCl2(other buffer
components as above) for 30s. Reaction was initiated
by adding polymerase at levels that varied between 0.04
at the 50-terminus
32P), 500mM of each dNTP, polymerase specific
Binding of Mac-Pol touracil-containing DNA
The affinity of Mac-Pol for uracil in DNA was determined
using fluorescence anisotropy with hexachlorofluorescein
(hex)-labelled oligodeoxynucleotides (3,23). Assays were
carried out in 1ml volumes containing 6nM Hex-GCC
(pH 7.5), 100mM NaCl and 1mM EDTA and Mac-Pol
(0–160nM) was added until saturation was observed.
Data fitting to give KDhas been described (3,23).
in 10mM Hepes
Amino acid sequence alignment
The N-terminal domain of the family B DNA polymerase
from P. furiosus (Pfu-Pol) contains a pocket responsible
for specific binding of uracil (2), important amino acids of
which are shown in Figure 1. Y7 and P36/Y37 constitute
Nucleic Acids Research, 2008, Vol. 36, No. 3
a lid and base respectively and amino acids 90–97 and
111–116 form the two sides of the pocket. R119 and D123
are involved in a complex hydrogen bonding network,
interlinking many of the pocket amino acids including
Y37. The peptide backbone of Y37 makes hydrogen
bonds to uracil and, hence, Y37 is a particularly important
residue (2). These key uracil-recognizing amino acids are
retained with Mac-Pol, a family B DNA polymerase from
the mesophilic archaeon M. acetivorans (24), as shown in
Figure 1. In general the amino acids shown in Figure 1 are
very well conserved among all euryarchaeal family B
DNA polymerases with no obvious distinction between
thermophilic and mesophilic representatives (Supplemen-
tary Material). The eukaryotic replicative polymerases e
and d are members of the family-B DNA polymerase
family and show amino acid sequence homology with
archaeal enzymes. Elements of the uracil-binding pocket
are apparent for both yeast polymerases (and the relevant
enzymes from a number of eukaryotes, Supplementary
Information), as shown by alignment with Pfu-Pol in
Figure 1. However, not all the key amino acids are
retained and the homology is clearly less striking than
between Pfu-Pol and Mac-Pol. There is no indication of
a residue that could correspond to Y7, although a single
aromatic amino acid, serving as a pocket lid, would be
difficult to identify from sequence alignments as it might
arise from a different region of the eukaryotic polymerase.
The PY pocket base is perfectly conserved with Pol e and
reasonably retained, especially the Y, with Pol d. Similarly
segments corresponding to the pocket walls (amino acids
90–97 and 111–116 in Pfu-Pol) are apparent for both Pols
d and e and particularly noticeable is the near complete
conservation of the equivalents of R119 and D123, both
amino acid identity and spacing being retained. With Pfu-
Pol V93 is a key amino acid for uracil recognition,
mutation to V93Q abolishing interaction (2). With
eukaryotic polymerases this position is invariably occu-
pied with an aliphatic hydrophobic amino acid.
Escherichia coli PolIII is a member of the Pol C family
(17) and amino acid homology between the a sub-unit
(the polypeptide that possess the polymerase activity)
and Pol B family members is limited to key regions that
contribute to the active sites (polymerase and proof-
reading exonuclease) and are involved in dNTP recogni-
tion (25,26). The a sub-unit of PolIII does not contain
a sequence with any similarity to the uracil-recognition
region of archaeal family B polymerases and crystal
structures of two representatives (27,28) show no evidence
for the presence of a uracil-binding pocket. The poly-
merase sub-unit of mitochondrial Pol g belongs to the
A family (20), again polymerases with only limited
homology to the B group (25,26) and no hint of sequence
elements matching the uracil-binding region of Pfu-Pol.
No crystal structures are yet available for Pol g, but other
family A members of known structure e.g. E. coli Klenow
fragment (29) and Taq-Pol (30) show no semblance of
a uracil-binding pocket. Thus amino acid alignments
suggest that the mesophilic Mac-Pol should recognize
uracil, eukaryotic Pols d and e may interact with the base
and provide no evidence for this function with bacterial
PolIII and mitochondrial Pol g.
Primer-template extension assays
Hyperthermophilic archaeal DNA polymerases stall repli-
cation on encountering uracil, most easily assayed
using template strands with a single uracil in defined
positions (1–3). This assay has been extended to the study
of a number of other replicating polymerases and Figure 2
shows that primer extension catalysed by Mac-Pol is
halted by the presence of uracil. Figure 2 also shows data
obtained with Pfu-Pol; it is apparent that the stalling
position is the same for both the hyperthermophilic
and the mesophilic polymerase i.e. four bases prior to
encounter of uracil. In contrast, Figure 2 demonstrates
that the replicating polymerases from yeast, S. cerevisiae
DNA polymerases e (Sce-Pol e) and d (Sce-Pol d) are able
to replicate past uracil, giving full length rather than
truncated products. The results shown in Figure 2, for the
two yeast polymerases, were obtained using an excess
of PCNA, RFC and RPA, replisome components that
increase polymerase processivity and accuracy (15,31,32).
However, an additional experiment using Sce-Pol d
without added replisome components also resulted in
read through of uracil, giving identical results to those
shown in Figure 2 (data not shown). Thus PCNA, RFC
and RPA are without influence in respect to uracil
recognition. Yeast Pol d unusually contains NY at
positions 36/37, other eukaryotic Pol ds have PY or HY
here (supplementary). However, the yeast enzyme is not
an unusual outlier as identical results (not shown) were
seen with human Pol d. In Figure 2 the replicating DNA
polymerase from E. coli (E. coli Pol III?) has been tested,
as with the eukaryotic enzymes the presence of uracil did
not result in cessation of replication and complete read
through was observed. Finally Figure 2 evaluates human
Pol g, the two sub-unit enzyme responsible for mitochon-
drial DNA replication (20). This polymerase has relatively
low activity, only extending a small fraction of the primer;
nevertheless, it is apparent that stalling of replication
in response to template strand uracil does not take place.
Binding ofmesophilic archaealpolymerase
Primer-template extension assays are qualitative in nature,
more accurate data can be obtained using fluorescence
anisotropy to measure the affinity of the polymerase
for uracil-containing DNA (3,24). Performing such an
7 36/ 90 – 97 111–116 119 123
Pfu-Pol: Y PY PQDVPTIR EYD-IPF R D
Mac-Pol: Y PY PKDVPEIR ESD-ILF R D
Yeast Pol : P NY PHMVNKLR TYDNIAY R D
Yeast Pol : T PY SNQLFEAR EYD-VPY R D
Figure 1. The amino acids that form the uracil-binding pocket of
Pfu-Pol (2) are shown (numbers above refer to the positions of amino
acids in the Pfu-Pol sequence). An alignment is shown for the corre-
sponding amino acids in Mac-Pol and yeast Pols d and e. Homology is
excellent between Pfu-Poland Mac-Pol and partial between Pfu-Pol and
the two yeast enzymes. A more comprehensive table showing more
species is given in the Supplementary Material.
Nucleic Acids Research,2008, Vol. 36,No. 3707
experiment with Mac-Pol (Figure 3) gave a KDof 9.7nM
for binding to a single-stranded 22-mer containing a
centrally located uracil. As shown in Figure 3 an identical,
within the error limits of the assay, KDof 8.5nM was
seen with Pfu-Pol (previously we also obtained a value
of 8nM (2,3) with Pfu-Pol). Thus the binding affinity of
thermophilic and mesophilic archaeal polymerase for
uracil appears to be the same.
The results presented in this publication confirm that
family-B DNA polymerases from thermophilic archaea
cease DNA polymerization in response to template strand
uracil and this property is shared by the polymerase from
the mesophilic archaeon M. acetivorans (25). In contrast
DNA polymerases e and d from S. cerevisiae, E.coli DNA
polymerase III and human mitochondrial polymerase g
all read-through uracil. Thus, at least as assessed with
purified proteins, only the archaeal domain appears
to possess a polymerase able to bind uracil tightly and
subsequently stall replication. Several factors may be
responsible for the differences between archaea and
Have appropriatepolymerases been compared?
If uracil-induced stalling serves to protect from the conse-
quences of copying G:U mismatches, it is important to
compare polymerases responsible for genome replication,
rather than playing, for example, a specialized role in
DNA repair. PolIII has been unequivocally assigned
as the replicating polymerase in E.coli (17) and with
eukaryotes Pol e appears to copy the leading strand and
Pol d the lagging (31,33) and, between them, these two
polymerases are responsible for nuclear DNA replication.
Similarly Pol g has been established as the enzyme
responsible for eukaryotic mitochondrial DNA replication
(20). With the archaea the polymerase responsible for
DNA replication awaits absolute confirmation; therefore
it remains a possibility that the family-B polymerase is a
specialized enzyme dedicated to uracil repair. However,
the family-B enzymes are by far the most likely candidates
for DNA replication, being the only polymerases present
in all archaea (34–36) and clearly related to the eukaryotic
replicative polymerases d and e. An unusual hetero-
dimeric polymerase (classified as family D) has been
identified in euryarchaea (37) and both the family B and
family D polymerases are essential for euryarchaeal
survival (38). Based on the biochemical properties of the
family B and D polymerases from Pyrococcus abysii it was
suggested that B copies the leading strand and D the
lagging (39). Recently, the same group has proposed
an alternative scenario; Pol D initially elongating RNA
primers before a switch to Pol B directed synthesis (40),
with Pol B elongating almost all the leading strand and an
undefined fraction of the lagging. Crenarchaea lack Pol D,
instead multiple forms of Pol B are found and are assumed
P T U7 U10 P T U7 U10
T U7 P T U7 P T U7 P
P T U7 U10 P T U7 U10
E. Coli PolIII*Pfu-Pol
P EP T U7 U10 P T U7 U10
- - -
following primer-template was used:
The templates contain a single uracil (thymine in controls) either 7 or
10 bases ahead of the primer-template junction. The polymerases
under investigation are shown above each panel. Individual gel lanes
are labelled: P (primer); polymerase not added, serves as marker for
migration of unextended 16-mer primer. EP (extended primer, only
used with Pol g), chemically synthesized 32-mer corresponding to fully
extended primer, serves as marker for full extension. T (thymine),
control template lacking uracil. U7/U10; templates containing uracil
either 7 or 10 bases ahead of primer-template junction. The archaeal
polymerases Mac-Pol and Pfu-Pol fully extend the primer when the
template lacks uracil. In contrast, the presence of uracil in the template
strand leads to truncated products due to uracil-induced stalling
of polymerization. All other polymerase (yeast Pols d and e, E. coli
PolIII?and mitochondrial Pol g) fully extend the primer regardless of
whether uracil is present or not in the template.
2. Primer-templateextension assays.Inall casesthe
0 150 200 250
Figure 3. Binding titration for Pfu-Pol (black circles) and Mac-Pol
(white circles). The polymerases were added to Hex-GCCCGCGGG
AUATCGGCCCTTA (6nM) and the fluorescence anisotropy mea-
sured (the same number of measurements were carried out for both
polymerases; in some cases the white circles obscure the black). The
titration was carried out three times to give a KDof 8.5?1.3nM for
Pfu-Pol and 9.7?1.6nM for Mac-Pol.
Nucleic Acids Research, 2008, Vol. 36, No. 3
to be solely responsible for DNA replication (34–36).
In conclusion it appears that all the polymerases studied
in this publication serve to replicate the genomes of the
organisms from which they are derived.
Is uracilrecognition aconsequence ofhigh temperature
Prior to this publication read-ahead recognition of uracil
has only been demonstrated with polymerases purified
from archaea that occupy high temperature niches.
The family-B polymerases from the hyperthermophiles
P. furiosus, Pyrococcus woisei and Thermococcus literalis
(1), Sulfolobus solfataricus (41) and Pyrococcus horikishii,
Thermococcus gorgonarius, Sulfurisphaera ohwakuensis,
Aeropyrum pernix and Pyrodictium occultum (unpublished
observations) all stall replication in response to uracil.
Here, for the first time, a family B polymerase from
a mesophilic archaeon, M. acetivorans (optimum growth
temperature 35–408C) (24), has been shown to bind
uracil-containing DNA as tightly as Pfu-Pol and to halt
replication when this base is encountered. Thus recogni-
tion of uracil is not exclusively a property of enzymes
isolated from hyperthermophiles and, although the
bacterial and eukaryotic polymerases were from meso-
philes (E. coli, yeast and humans), differences in uracil
sensing as compared to archaea cannot simply be
Further information may be available by studying PolIII
from hyperthermophilic bacteria such as Aquifex aeolicus
(42), a polymerase very similar in organization to that
from E. coli and anticipated to behave identically.
in habitat temperature.
Are the archaea defective instandard uracil repairenzymes?
Uracil arises in DNA in two ways, polymerase catalysed
de novo incorporation using dUMP (from dUTP) (44,44)
and deamination of cytosine in G:C base-pairs (4). Both
are detrimental to cells; A:U base-pairs, arising from
de novo incorporation, may interfere with DNA binding
proteins and lead to futile cycles of DNA repair (7)
and G:U mis-pairs, the consequence of deamination,
are mutagenic following replication (4–7). All organisms
protect the genome from uracil using dUTPase (enzymes
that hydrolyses dUTP to dUMP and PPi) and UDGase
(enzymes that excise uracil from DNA to initiate base
excision repair). Archaea possess both dUTPase (45) and
UDGase (46,47) and their mutation rates appear similar
to mesophilic bacteria and eukaryotes (48,49). Therefore,
the archaea do not appear deficient in uracil-protecting
enzymes or DNA repair pathways in general and, it is,
therefore, unlikely that the archaea have a polymerase-
based uracil repair pathway because they are lacking in
the standard uracil defences.
Why do onlythe archaea possessareplicating polymerase
that recognizes uracil?
Accurate removal of uracil, or indeed most damaged
bases, from the genome relies on the double stranded
nature of DNA; following excision of the aberrant base,
the complementary strand is used as a template for
re-synthesis of the correct sequence (5–7). Problems arise
when DNA damage is encountered during replication and
under such circumstances polymerization is often halted
and appropriate repair initiated. Pathways include fork
reversal followed by damage removal, template switching
using the copy of the complementary strand to direct
polymerization and recombination with the copying of
a sister chromosome (8,9,50). A key step appears to be the
cessation of DNA replication, which serves as the signal
for initiation of the relevant mutli-step/multi-protein
DNA repair system. Replication linked repair is, there-
fore, often considered in the context of DNA lesions that
halt the replicating polymerase: examples include; single
and double strand breaks, through which the polymerase
cannot copy; DNA crosslinks, which cannot be unwound;
and bulky damaged bases or abasic sites, to which the
polymerase cannot easily match an incoming dNTP.
Uracil presents a formidable problem for replication-
coupled repair, as it is a near perfect analogue of thymine
and mistaken for this base by most polymerases (7).
In general polymerases, as with the bacterial and eukary-
otic enzymes studied in this publication, do not stop on
encountering uracil; rather they insert adenine opposite
with high efficiency, resulting in irreversible fixing of a
mutation, should the uracil have arisen by deamination
of a G:C base-pair. Recognition of uracil by a specialized
pocket in the archaeal DNA polymerases allows replica-
tion to be halted and presumably facilitates repair by the
same pathways used with ‘intrinsically-stopping’ lesions
such as strand breaks. A more pertinent question to the
one posed as this sub-heading’s title might be, therefore,
‘what happens when uracil is encountered during replica-
tion in bacteria and eukaryotes?’ One possibility, despite
dUTPase and UDGase being present in all three domains
of life, is that uracil defence is more active in bacteria/
eukaryotes, such that uracil is encountered, during
replication, at levels low enough to be tolerated without
cessation of DNA polymerization. Such a scenario seems
unlikely and may be especially problematic with mito-
chondria as, although the organelle contains UDGase
activity, it is subject to a high mutagenic load due to
reactive oxygen species arising from oxidative metabo-
lism (20). An alternative is that UDGase itself might
act a uracil sensor during replication. In eukaryotes, one
particular UDGase (UNG) interacts with PCNA and
RPA and is associated with replication foci, excising uracil
arising from incorporation of dUMP during replication
(51,52). Here, UNG appears to travel with the replication
apparatus and following incorporation of dUMP ‘back
tracks’ to initiate base excision repair of A:U base-pairs
in the newly synthesized double-stranded DNA. UNG
may also have a role in standard base excision repair
of G:U mismatches (53,54) and it has been tentatively
suggested that the enzyme may further act on G:U mis-
matches that have escaped repair prior to replication (6).
Here, though, UNG would have to scan ahead of the
polymerase to generate an abasic site that would then
stall the approaching polymerase, allowing entry into
recombination-based repair pathways. It is not clear how
UNG is organized such that it can fulfil these quite
different roles, in one case tracking backwards and
acting post-replication, in the other reading forward
Nucleic Acids Research,2008, Vol. 36,No. 3709
and acting pre-replication. Interestingly the UDGase from
archaea also interacts with PCNA (55). A final possibility
is that bacteria and eukyarotes contain as yet unidentified
uracil-binding proteins that travel with the replication
apparatus and passively stall DNA synthesis in an
analogous manner to the uracil-binding pocket of archaeal
In summary, this publication indicates that uracil
sensing by replicating polymerases is a property of archaea
(both thermophilic and mesophilic), not shared with
bacteria and eukaryotes. The repair processes used by
archaea following uracil-induced stalling await elucida-
tion, as do the mechanisms by which bacteria and eukary-
otes deal with uracil encountered during replication.
Eukaryotes contain a third family-B DNA polymerase,
Pol z (zeta), which shows homology to the archaeal family
B DNA polymerases but is involved in the by pass of
damaged bases rather than DNA replication (56). Primer-
template extension assays clearly demonstrated that Pol z
does not stall replication on encountering uracil and this
result is included for completion.
Supplementary Data are available at NAR Online.
J.W. is a PhD student supported by Cancer Research UK
(CRUK). B.A.C. was supported by grants from UK
BBSRC and the European Union. P.M.J.B. was sup-
ported, in part, by grant GM 32431 from the National
Institutes of Health. E.J. was supported by the Swedish
Research Council, the Swedish Cancer Society and
Svenska Sma ¨ rtafonden. P.M. was supported by the UK
MRC and the Lister Institute of Preventive Medicine.
I.K.O.C. was supported by National Science Foundation
Grant MCB-023841. Funding to pay the Open Access
publication charges for this article was provided by The
University of Newcastle.
Conflict of interest statement. None declared.
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