Nucleic Acids Research, 2009, Vol. 37, No. 9Published online 12 March 2009
The efficiency and fidelity of 8-oxo-guanine
bypass by DNA polymerases d and g
Scott D. McCulloch1, Robert J. Kokoska1, Parie Garg2, Peter M. Burgers2
and Thomas A. Kunkel1,*
1Laboratory of Molecular Genetics and Laboratory of Structural Biology, National Institute of Environmental
Health Sciences Research, Triangle Park, NC 27709 and2Department of Biochemistry and Molecular Biophysics,
Washington University School of Medicine, St Louis, MO 63110, USA
Received November 19, 2008; Revised January 22, 2009; Accepted February 4, 2009
A DNA lesion created
8-oxoG can mispair with adenine during DNA syn-
thesis, it is of interest to understand the efficiency
and fidelity of 8-oxoG bypass by DNA polymerases.
We quantify bypass parameters for two DNA poly-
merases implicated in 8-oxoG bypass, Pols d and g.
Yeast Pol d and yeast Pol g both bypass 8-oxoG and
misincorporate adenine during bypass. However,
yeast Pol g is 10-fold more efficient than Pol d, and
following bypass Pol g switches to less processive
synthesis, similar to that observed during bypass
of a cis-syn thymine-thymine dimer. Moreover,
yeast Pol g is at least 10-fold more accurate than
yeast Pol d during 8-oxoG bypass. These differences
are maintained in the presence of the accessory
proteins RFC, PCNA and RPA and are consistent
with the established role of Pol g in suppressing
ogg1-dependent mutagenesis in yeast. Surprisingly
different results are obtained with human and
mouse Pol g. Both mammalian enzymes bypass
8-oxoG efficiently, but they do so less processively,
without a switch point and with much lower fidel-
ity than yeast Pol g. The fact that yeast and mam-
malian Pol g have intrinsically different catalytic
properties has potential biological implications.
by oxidative stress
One of the most common lesions resulting from oxidative
stress is 7,8-dihydro-8-oxo-guanine (8-oxoG) [(1) and
contains only one extra oxygen atom, yet presents a
major problem for a cell because during DNA synthesis
it can pair with either correct deoxycytidine (dC) or incor-
rect deoxyadenine (dA). NMR and X-ray crystallographic
studies with either a dC?8-oxoG or dA?8-oxoG basepair
shows that neither lesion causes a serious distortion of
the overall helical structure of the DNA (2?5). This is
because the dC?8-oxoG pair exists in the standard anti-
anti conformation, while the dA?8-oxoG mispair exists in
an anti?syn conformation, leading to base pairing along
the Hoogsteen edge of the incoming dA. The dA?8-oxoG
mispair can result in a G?C ! T?A transversion, a sub-
stitution linked to somatic cancers (6). To avoid the
adverse consequences of 8-oxoG?dA mispairs, cells
devote a number of enzymes to processing 8-oxoG (7).
The base excision repair (BER) pathway recognizes and
removes 8-oxoG from the dC?8-oxoG pair using the
MutM/OGG1 glycosylase in E. coli and human cells,
respectively. The dA base from the dA?8-oxoG mispair
is removed by the MutY/MYH glycosylase (E. coli/
human). In addition, MutT/MTH1 (E. coli/human) can
hydrolyze the oxidized precursor 8-oxoGTP to its mono-
phosphate form (8-oxoGMP), thereby preventing incor-
poration opposite either dA or dC in the template.
Lastly, the DNA mismatch repair system has been
shown to recognize 8-oxoG mispairs (8).
DNA repair systems are not perfect, such that template
8-oxoG is sometimes encountered by DNA polymerases
during replication and gap-filling synthesis associated
with DNA repair. Given the tendency for mispairing
by DNA polymerases with associated increase in muta-
tions, many studies have investigated the activities of
specific DNA polymerases when they encounter template
8-oxoG. In an early report, human Pols a and b, calf
thymus Pol d and E. coli Pol I were all able to incorporate
both dC and dA opposite 8-oxoG, in ratios varying from
*To whom correspondence should be addressed. Tel: +1 919 541 2644; Fax: +1 919 541 7613; Email: email@example.com
Scott D. McCulloch, Department of Environmental and Molecular Toxicology, North Carolina State University, Campus Box 7633, Raleigh,
NC 27695, USA
Robert J. Kokoska, U.S. Army Research Office, PO Box 12211, Research Triangle Park, NC, USA
? Published by Oxford University Press 2009
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.
7:1 to 1:200 (9). All these polymerases more efficiently
extended the 8-oxoG?dA mispair than the 8-oxoG?dC
pair. More recent studies have reported ratios of dC:dA
insertion opposite 8-oxoG of 2:1 for Pol b (10) and T7
DNA polymerase (11,12), 3:1 for E. coli Pol I (13), 1:1
for E. coli Pol II (13), 1:14 for HIV RT (11), 1:9 for
Bacillus stearothermophilus Pol I (14), 20:1 for RB69
DNA polymerase (15), 3:1 for calf thymus Pol d (16),
20:1 for Saccharomyces cerevisiae Pol Z (17), between 70
and 90:1 for Sulfolobus solfataricus DNA polymerase 4
(Dpo4) (18,19) and between 1:1 and 1:3 for human Pol k
(20,21). Most of these studies involved kinetic analysis of
insertion of a single nucleotide opposite 8-oxoG, and in
some cases, the ability to add a correct nucleotide onto
dC?8-oxoG pairs. These studies clearly demonstrate that
relative to unmodified template guanine, 8-oxoG reduces
thenucleotide selectivityofDNApolymerases, buttovary-
ingdegrees.The structuralbasisforthis reducedselectivity,
and for polymerase-specific variations in the ratio of
dC:dA insertion opposite 8-oxoG, has been investigated
in several studies showing that both base pairs can in
fact exist within the active site of a polymerase, but in dif-
ferent conformations and with different polymerase-
specific interactions (12,14,18,19,22,23). Interestingly, one
such study shows that the dA?8-oxoG anti?syn mispair
can mimic the geometry of a correct base pair and thereby
escape efficient proofreading by the 30exonuclease activity
of the replicative T7 DNA polymerase (9,12).
The present study examines the consequences of
attempts by Pols d and Z to bypass a template 8-oxoG.
The choice of these two particular polymerases with this
lesion was motivated by biological evidence clearly indi-
cating that Pol Z has an important role in suppressing
mutagenesis induced by sunlight (24–30), which not only
generates photodimers but also lesions due to oxidative
stress (31–34). Moreover, S. cerevisiae Pol Z also has a
role in suppressing mutagenesis in cells defective in remov-
ing 8-oxoG due to a defect in the Ogg1 glycosylase
(17,35,36). These data imply that in a manner akin to
the prevailing model for Pol Z-dependent bypass of UV
photoproducts (37,38), when a major replicative poly-
merase like Pol d encounters 8-oxoG, it pauses, thereby
initiating a switch to allow Pol Z to bypass this lesion. We
have been investigating this model based on the specific
hypothesis that polymerase switching during translesion
synthesis (TLS) occurs during transitions from preferred
to disfavored use of damaged primer-templates and
that the polymerase used for each successive nucleotide
incorporated is the one whose properties result in the
highest efficiency. Here, we test this hypothesis by investi-
gating the properties of 8-oxoG bypass by Pols d and Z
using assays designed to quantify the efficiency and the
fidelity of TLS (39–42).
MATERIALS AND METHODS
Materials and reagents
All bacterial strains, plasmids, bacteriophage and other
materials for the assays performed were from previously
described sources (42,43). DNA modification and restric-
tion enzymes were purchased from New England Biolabs
(Ipswich, MA), and oligonucleotides were purchased from
Oligos Etc., Inc. (Wilsonville, OR). Streptavidin was
purchased from Roche Applied Science (Indianapolis,
IN), and dNTPs were purchased from Amersham Bio-
sciences (Piscataway, NJ).
Bypass assays used substrates with 70-mer templates
GCAGAAATTCACTGG; 70Bio-Go: 50-biotin-ATGAC
moiety. The position of the 8-oxoG residue is indicated
by‘x’.Primer strand oligonucleotides
AAG) with a 50-biotin moiety. Bypass efficiency assay
substrates used the ‘blocking’ primer BP14 and the
‘labeled’ primer LP16 that was labeled at the 50end
Substrates were prepared by mixing template oligonucleo-
tide (70Bio-G/70Bio-Go/70Bio-Go2) with 1.2M equiva-
lents each of BP14 and LP16 in 50mM Tris–Cl (pH 7.5)
and 1X SSC, followed by incubation at 758C for 5min,
then cooling to 258C over 2h, protected from light.
Substrates used in the bypass fidelity assay were pre-
pared similarly using only the unlabeled 30Fid primer
bypass assays, a 102-mer template (V9/V9AP1: 50-biotin-
CCCTCTCCCTTCCCTT29-biotin) with both 50and 30
biotin moieties was used, with the position indicated by
‘x’ containing either an undamaged G or a synthetic AP
site (tetrahydrofuran) residue. The primer used (C12-4:
with the region in italics. AP site bypass substrates were
prepared as described above using a 1.2? molar excess of
template to 50 32P end-labeled primer. All bypass efficiency
experiments with human and mouse Pol Z used a 45-mer
template (45TTAG/45TTAGo: 50-CCAGCTCGGTACC
that contained either an undamaged G or 8-oxoG at
the position indicated by ‘x’ with 50 32P-labeled primer
prepared as described earlier. Bypass fidelity reactions
using the 45-mer template were prepared with unlabeled
LBP-24 primer. Templates with different sequence con-
texts indicated in the text, tables and figure legends are
limited to the region in italics in the above 45-mer
Nucleic Acids Research,2009, Vol.37, No. 92831
Proteinisolation and purification
S. cerevisiae polymerase Z was purified as described
previously (44) using a plasmid kindly provided by
Dr. Zhigang Wang (University of Kentucky). Two
separate preparations gave similar results. S. cerevisiae
PCNA, RFC, RPA and three subunit polymerase d,
both exonuclease proficient (dexo+) and deficient forms
(dexo?) were purified as previously described (45–48).
Mouse and human Pol Z were expressed and purified as
described previously (49).
Bypass efficiency reactions using the 70-mer template
substrate and S. cerevisiae proteins were performed as
previously described (39,41,50). All reactions contained
40mM Tris–Cl (pH 7.8), 75mM NaCl, 5mM MgCl2,
1mM ATP, 2mM DTT and 100mg/ml BSA. Reactions
(30ml) contained 1 pmol of DNA substrate that was first
incubated with 10 pmol of streptavidin. When present,
PCNA trimer, RFC complex and RPA were added in
1.2-fold excess compared to DNA substrate. Reactions
with Pol d contained 25mM of each of the four dNTP,
while reactions with Pol Z contained 100mM each. All
components except polymerase were mixed on ice and
then incubated at 308C for 2min. Polymerase (5fmol)
was added to initiate the reaction, and 6ml samples were
removed at the indicated times and mixed with 12ml for-
mamide loading buffer (95% deionized formamide,
25mM EDTA, 0.1% bromophenol blue, 0.1% xylene
cyanol). Products were heated to 958C for 5min
and separated through a 12% denaturing polyacrylamide
gel. Dried gels were visualized and quantified using
a Molecular Dynamics Typhoon 9400 imager and
ImageQuant software. Calculation of termination prob-
ability, bypass amount and relative bypass efficiency
Reactions with human and mouse Pol Z using 45-mer
described (41,51), using 4pmol of substrate and either
10fmol human Pol Z or 16fmol mouse Pol Z. In all reac-
tions used to calculate bypass efficiency parameters, short
incubation times and high DNA to polymerase ratios were
used. The conditions used were chosen to assure that
termination probabilities remained with time, thereby
empirically demonstrating that the majority of product
chains result from a single cycle of synthesis (39).
The assay was performed as previously described (39,42).
Seventy-mer template substrates containing a single
primer (30Fid) were blocked with streptavidin, while
streptavidin complexes blocking the ends. Reaction
conditions were the same as described earlier with the
following exceptions: S. cerevisiae Pol d experiments
contained 8pmol of substrate and 4pmol of polymerase;
S. cerevisiae Pol Z experiments contained 2pmol of
S. cerevisiae proteins were incubated at 308C for 30min.
did not havebiotin–
Reactions with mouse and human Pol Z contained 4 pmol
substrate and 0.8pmol enzyme and were incubated at
378C for 20min. Recovery of the synthesized strand,
annealing to gapped M13 DNA molecules, hybridization
efficiency controls, transfection into E. coli and determi-
nation of error rates and spectra were performed as pre-
viously described (39,42).
The efficiency of 8-oxoGbypass by yeast Pol d
First, we examined the ability of three-subunit yeast Pol d
to bypass 8-oxoG (Figure 1). In comparison to bypass of
undamaged guanine in the same sequence context (lanes
b–d, in Figure 1B and C), 8-oxoG inhibited polymeriza-
tion by wild type (i.e., exo+) Pol d (Figure 1B, lanes i–k)
and exo?Pol d (Figure 1C, lanes i–k). When band inten-
sities were quantified and used to calculate bypass efficien-
cies (Table 1), wild type and exo–Pol d were found to
bypass 8-oxoG with 15% and 31% efficiency, respectively,
compared to bypass of undamaged G in the same
sequence context. This reduced bypass efficiency is due
mainly to reduced extension following incorporation
opposite 8-oxoG (Figure 1B, compare band intensities
at n+3 in lanes d and k). We also performed reactions
with a template of the same sequence but with the 8-oxoG
located four bases downstream, i.e., in a different sequence
context. In this case, Pol dexo+bypassed 8-oxoG with an
efficiency that was 25% of that observed with undamaged
G in the same context (Table 1). Thus in two different
contexts, 8-oxoG substantially reduces processive bypass
synthesis by Pol d. The product chains generated during
8-oxoG bypass by Pol dexo+(Figure 1B) and Pol dexo?
(Figure 1C) were similar, and the bypass efficiency
of Pol dexo–was only slightly higher than the bypass
efficiency of Pol dexo+, indicating that the intrinsic 30
exonuclease activity of Pol d has limited influence on
bypass of 8-oxoG.
Addition of RFC, PCNA and RPA stimulated the
processivity of both forms of Pol d (e.g. Figure 1B
and C, see full-length 70-mer products in lanes g and n).
In addition, the accessory proteins increased the percent-
age of product chains reflecting bypass of G and 8-oxoG
by several-fold (compare values in first four columns
and top two lines of Table 1). However, the degree of
stimulation by accessory proteins was similar for both
the undamaged G and 8-oxoG templates, such that the
accessory proteins did not selectively increase the relative
8-oxoG bypass efficiency for either exo+or exo–Pol d
(third line in Table 1). To further confirm that the
accessory proteins are active under the reaction conditions
used here, we performed reactions using a second
undamaged template, in this case one that yields lower
termination probabilities after the first two incorporations
(Figure 2). The processivity of Pol d is somewhat higher
on this template (compare Figure 1B and C, lanes b–d
with Figure 2B, lanes b–e), and the ability of RPA, RFC
and PCNA to increase processivity is clearly seen
(Figure 2B, lanes f–i). We also performed experiments
with 2-fold excess of Pol d over primer-template, in this
Nucleic Acids Research, 2009, Vol. 37,No. 9
2 4 6 2 4 6
2 4 6 2 4 6
a b c d e f gh i j k l m n
2 4 6 2 4 6
2 4 6 2 4 6
a b c d e f gh i j k l m ng’ n’
3' CCG GATTGGG 5'
Figure 1. Inhibition of S. cerevisiae Pol d DNA synthesis by 8-oxoG. (A) Schematic diagram of the substrate used in TLS assays. The template is a
70-mer, the biotinylated primer is a 14-mer and the radio-labeled primer is a 16-mer. (B) Twelve percent dPAGE image of reactions containing a
200:1 substrate:enzyme (S:E) ratio of exonuclease proficient Pol d (Pol dexo+) and either undamaged (G) or damaged (8-oxoG) templates with
blocked ends (see ‘Materials and Methods’ section), without and with the accessory proteins RPA, RFC and PCNA. The template sequence is
given to the left of the image and the number of nucleotide incorporation (+1, +2, etc.) is given on the right. The +40 incorporation represents
synthesis to the end of the template. The position of the 8-oxoG residue is indicated with a star (?). Lanes g0and n0are overexposures of
lanes g and n, respectively. (C) Gel image of reactions containing a 200:1 S:E ratio of exonuclease deficient Pol d (Pol dexo?). Details are the
same as in (B).
Table 1. Bypass efficiency of 8-oxoG by pols d and Z
Relative bypass (%)
Relative bypass (%)
aEfficiency measurements made using 70-mer template with streptavidin blocked ends (see ‘Materials and Methods’ section).
bEfficiency measurements made using 45-mer template (see ‘Materials and Methods’ section).
cBypass efficiency measured in three other template sequence contexts was 30... CGoATC...=220%; 30-... CAGoTC...=180%; 30-...CAGoTT
dExperiments depicted in Figures 1 and 2 were quantified and the bypass amount on each template calculated as previously described (39,41).
Relative bypass is the amount of 8-oxoG bypass compared to undamaged DNA. Switch is defined as a change from lower to higher termination
probability when comparing values at the 8-oxoG and next undamaged base.
Nucleic Acids Research,2009, Vol.37, No. 92833
case with both an undamaged template and a template
containing a synthetic abasic site (tetrahydrofuran).
These experiments more closely approximate the many
published TLS studies that use high-enzyme concen-
trations and permit multiple cycles of polymerization,
evidenced here by extension of nearly all the starting
DNA substrate (Figure 2C). Under these reaction con-
ditions, the accessory proteins clearly stimulate formation
of full-length products, both with undamaged template
(Figure 2C, lanes compare lanes b to c and lanes h to i)
and with the lesion-containing template (Figure 2C,
compare lanes e to f and lanes k to l). The stimulation
of bypass of a synthetic abasic site is consistent with
earlier observations with yeast and calf thymus Pol d
While bypass of 8-oxoG is somewhat problematic for
yeast Pol d, yeast Pol Z bypasses the lesion with ease
(Figure 3B, lanes l–n). In two sequence contexts examined
(one not shown), bypass values are higher for the lesion
than for the corresponding undamaged G, giving relative
bypass efficiencies of 130–250% (Table 1). Also, as
previously reported for bypass of a cis-syn TT dimer by
yeast (40,50) and human Pol Z (41), yeast Pol Z has a
lower probability of terminating processive synthesis
after insertion opposite 8-oxoG (12%, Figure 4A, black
bar) as compared to undamaged G (29%, Figure 4A, gray
bar). This lower termination opposite 8-oxoG corresponds
to a higher probability of adding the next base, i.e. after
insertion opposite 8-oxoG, yeast Pol Z extends the
8-oxoG containing terminus even more efficiently than it
extends the undamaged terminus. Yeast Pol Z then inserts
nucleotides opposite the next three undamaged bases less
efficiently compared to the equivalent positions in the
undamaged template. In other words, after incorporation
opposite 8-oxoG and the next undamaged template
nucleotide (i.e. after bypass), synthesis by yeast Pol Z
Neither the high efficiency of 8-oxoG bypass by yeast
Pol Z (Table 1) nor the switch to less processive synthesis
(Figure 4A and B) is strongly influenced by the presence
ab c defghi
2 4 6 8 26 804
5 5 0 5 5
550 5 5
5 ’ -C C T T T G C G A AT T C - -T2 5- -G C G G C T C C C
5 ’ -C C T T T G C G A AT T C - - T2 5- -G C G G C T C C C
5 ’ -C C T T T G C G A AT T C - -T2 5- - G C G G C T C C C
3´ CCCTCG GGCGT 5'
Figure 2. Stimulation of Pol d activity by accessory proteins. (A) Schematic diagram of the substrate used in TLS assays. The template is a 102-mer
with biotin at both the five and three ends. The radio-labeled primer is a 26-mer. (B) Time course of reactions of Pol dexo?using undamaged template
that does not have sequence specific pause sites. Reactions are confirmed to be under single-interaction conditions (see ‘Materials and methods’
section). The template sequence is given to the left of the image and the number of nucleotide incorporation (+1, +2, etc.) is given on the right. The
+46 incorporation represents synthesis to the end of the template. (C) Gel images of reaction products created using a S:E ratio of 1:2 (polymerase
excess) with 102-mer templates containing either undamaged G or tetrahydrofuran (an AP site mimic) at the fifth incorporation position (?). These
images show that in the presence of the accessory proteins, stimulation of AP site bypass occurs, similar to a previous report (53). Details are the
same as in (B). Panels (B) and (C) confirm that the accessory proteins are active under the conditions used.
Nucleic Acids Research, 2009, Vol. 37,No. 9
of the accessory proteins. As reported earlier at a higher
enzyme to substrate ratio (53), there is clear stimulation of
Pol Z by RPA, PCNA and RFC (Figure 3B, compare
amount of product with 6 or more incorporations
in lanes b–d with e–g and in lanes l–n with o–q). The
stimulation is similar with the undamaged and damaged
templates. The results were somewhat different with
human and mouse Pol Z. Both polymerases are less
processive than yeast Pol Z [compare termination prob-
abilities in Figure 4A and C and reference (50)]. Despite
this lower processivity, both mammalian polymerases
readily bypass 8-oxoG, with no detectable block to syn-
thesis (Figure 3C and D). In fact, they do so with relative
bypass efficiencies consistently higher than for the unda-
maged template (150–220%, Table 1 and its legend).
These values in excess of 100% are largely due to a slightly
higher efficiency of insertion opposite the lesion compared
to the equivalent undamaged bases. However, after the
8-oxoG was copied, the mammalian enzymes failed to
(Figure 3C and D, Figure 4C and data not shown). This
differs from yeast Pol Z and from the behavior of human
and mouse Pol Z when copying a TT dimer (41,51).
Fidelity of 8-oxoGbypass
Next, we measured the error rates of Pol dexo+, Pol dexo?
from yeast and Pol Z from yeast, human and mouse when
bypassing an 8-oxoG in the presence of all four dNTPs.
Enough enzyme and time were provided to allow complete
synthesis to the end of the template. The newly synthesized
strand was recovered and hybridized to gapped M13mp2
DNA, which was introduced into E. coli cells that were
then plated to score M13 plaque color phenotype (39,42).
The undamaged G or 8-oxoG is within a TAG codon
in the lacZa gene in M13mp2 DNA. Correct incorpora-
tion of dC results in M13 plaques that are faint blue
(due to slight read through of the nonsense codon),
whereas stable misincorporation of dA or dG opposite the
G or 8-oxoG results in dark blue plaques. Sequencing
M13 DNA from dark blue plaques identifies the substitu-
tion and allows calculation of error rates.
In this assay, Pol dexo+and Pol dexo?have much lower
fidelity when bypassing 8-oxoG than when copying the
undamaged template (Table 2). This is reflected in the
dramatically higher dark blue plaque frequencies when
copying the damaged template (23–30%) as compared to
Human Pol η
Mouse Pol η
Yeast Pol η
a b c d e f g
kl m n o p q
2 4 6 2 4 60
2 46 2 4 60
5´-T C G G T A C C G G G T T A G C C
3' CCG GATTGGG 5'
3' CCG GATTGGG 5'
Figure 3. High-efficiency bypass of 8-oxoG by Pol Z. (A) Schematic diagram of the substrates used in TLS assays. For yeast Pol Z the substrate is as
described in Figure 1. For human and mouse Pol Z, the template is a 45-mer and the radio-labeled primer is a 24-mer. (B) Reactions were performed
as described in Figure 1 legend using a 200:1 S:E ratio of S. cerevisiae Pol Z, either in the absence of any accessory proteins (lanes b–d and l–n), or
with RPA, RFC and PCNA (lanes e–g and o–q). The template sequence is given to the left of the image and the number of nucleotide incorporation
is given on the right. The position of the 8-oxoG residue is indicated with a star (?). (C and D) Bypass efficiency reactions were performed using a
400:1 S:E ratio of human Pol Z and a 250:1 S:E ratio of mouse Pol Z.
Nucleic Acids Research,2009, Vol.37, No. 92835
the undamaged template (0.05–0.15%). Sequence analysis
followed by error rate calculations was performed for
Pol dexo+and Pol dexo?, in each case in the absence and
in the presence of RFC, PCNA and RPA (Table 3). The
results indicate that 40–50% of bypass of 8-oxoG by Pol d
results in stable misincorporation of dA and/or dG
opposite the lesion. Rates are similar with Pol dexo+and
Pol dexo?, indicating that these damaged mismatches are
not proofread by the 30exonuclease activity of yeast Pol d.
The accessory proteins have little effect on error rates
for dA?8-oxoG mismatches generated by either Pol
dexo+or Pol dexo?. In the presence of the accessory
factors, both forms of Pol d showed a 6-fold decrease in
the rate of 8-oxoG?dG mismatches. This difference is
statistically significant (Fisher’s exact test, P=0.012 and
0.011 for Pol dexo+and Pol dexo?, respectively).
Pol Z lacks intrinsic proofreading activity and, when
copying undamaged DNA, it is much less accurate
[Tables 2 and 3, and refs (49,50,54)] than Pol d [Tables 2
and 3, and ref. (55)]. However, when the fidelity of Pol Z
was measured for bypass of 8-oxoG, yeast Pol Z was
more accurate than yeast Pol d, as reflected in lower
dark blue plaque frequencies (Table 2) and in much
lower error rates for dA?8-oxoG and dG?8-oxoG
mismatches calculated after sequence analysis (Table 3).
The accessory proteins had little if any affect on the
fidelity of 8-oxoG bypass by yeast Pol Z. Error rates
for 8-oxoG bypass by human and mouse Pol Z are
much higher than for yeast Pol Z. Dark blue plaque
frequencies of ?30% were observed (Table 2), and the
error rates for dA misincorporation (44% for mouse,
45% for human) are 17-fold higher than for yeast Pol Z.
Similar and high error rates for 8-oxoG bypass by human
Pol Z were observed in three other sequence contexts
(Table 3, note c).
This study tests the hypothesis that enzymatic switching
during TLS occurs during transitions from preferential to
disfavored use of damaged primer-templates and that the
polymerase or 30-exonuclease used for each successive
nucleotide incorporated is the one whose properties
Termination Probability (%)
Template (3' 5')
Yeast Pol η
Template (3' 5')
Termination Probability (%)
Mouse Pol η
Template (3' 5')
Termination Probability (%)
Yeast Pol η + RPA/RFC/PCNA
Figure 4. Termination probability analysis during 8-oxoG bypass by Pol Z. Gel images of reaction products seen in Figure 3 were quantified using
ImageQuant software, as described previously (39,41). (A) Graph of termination probability (vertical axis, 0–60%) at each of the first seven
incorporations (horizontal axis) for S. cerevisiae Pol Z without accessory proteins. Undamaged template and 8-oxoG template values are indicated
by light gray and black bars, respectively. Error bars are the standard deviation for six measurements (three time points each for two separate
experiments). The switch from low to high termination probability after 8-oxoG has been bypassed is indicated with dashed-line arrows. (B) Graph
of termination probability for S. cerevisiae Pol Z with RPA, RFC and PCNA. Details are the same as in (A). (C) Graph of termination probability
for mouse Pol Z without accessory proteins on a 45-mer template (see ‘Materials and methods’ section). Note that mouse and human Pol Z display
nearly identical properties (41,49,51).
Table 2. Dark blue plaque frequency for G and 8-oxoG bypass
aValues given are the numbers of plaques of each type counted and the
resulting mutation frequencies for G/8-oxoG bypass reactions. Data
shown is from experiments using a 70-mer template with streptavidin-
blocked ends (see ‘Materials and Methods’ section).
bThis data has been previously published (50).
cAdditional experiments using a 45-mer template (see ‘Materials
and Methods’ section) gave dark blue plaque frequencies of 32%
(30-CCGoATC), 29% (30-CCAGoTC) and 32% (30-CCGoATT).
Nucleic Acids Research, 2009, Vol. 37,No. 9
result in the highest efficiency and the highest fidelity of
bypass. We previously examined this for two lesion-
polymerase combinations that are highly relevant to muta-
genesis and cancer susceptibility, i.e. Pols Z and d bypass
of a cis-syn TT dimer resulting from sunlight exposure
(40,41). Here, we extend the effort to bypass of template
8-oxoG. This lesion results from exposure to sunlight and
other types of environmental stress, both exogenous and
endogenous, and convincing evidence shows that yeast
Pol Z suppresses mutagenesis in yeast strains deficient
the Ogg1 glycosylase that normally removes 8-oxoG
from DNA (17,35,36). Those genetic data are strongly
supported by the biochemical properties of yeast Pols d
and Z reported here. Template 8-oxoG impedes synthesis
by yeast pol d (Figure 1), while yeast pol Z efficiently
incorporates opposite 8-oxoG and the next undamaged
template nucleotide (Figure 3A) to achieve very efficient
bypass (Table 1) and then switches to less processive syn-
thesis (Figure 4A and B). Yeast pol Z stably incorporates
C rather than A opposite 8-oxoG by a factor of 67-fold
(Table 3, error rate of 150?10?4for yeast Pol Z with
accessory proteins). This result is consistent with kinetic
studies of yeast Pol Z (17,56). By comparison, bypass of
8-oxoG by wild type yeast pol d is 33-fold less accurate
(Table 3, error rate of 4900?10?4with accessory pro-
teins), and neither its proofreading activity nor accessory
proteins improve fidelity (Table 3). All these properties
strongly support the proposal (17) that yeast Pol Z,
although clearly not error-free, contributes to bypass of
spontaneously generated 8-oxoG in a manner that sup-
It is well known that 8-oxoG not only arises sponta-
neously but is also generated by exposure to UV radiation.
This and the fact that XPV patients lacking functional Pol
Z have greatly increased susceptibility to sunlight-induced
skin cancer raises the interesting issue of whether mam-
malian Pol Z also bypasses 8-oxoG in a manner that
suppresses mutagenesis. This possibility is supported by
a report (17) that, like yeast Pol Z, human Pol Z also
bypasses 8-oxoG accurately. This led to the suggestion
that, in addition to suppressing sunlight-induced skin
cancer, human Pol Z may also suppress internal cancers
that would otherwise result from mutagenic bypass of
8-oxoG in DNA (17). Although there is no compelling
evidence that XPV patients have increased susceptibility
to internal cancers, a recent study of human cell lines
has concluded that human Pol Z suppresses 8-oxoG-
dependent mutations when plasmid DNA that was
damaged in vitro by methylene blue plus light is replicated
in vivo (57).
On the other hand, we find that the human and mouse
Pol Z error rate for bypass of 8-oxoG approaches 50%
(Table 3). These high error rates are observed in multiple
nucleotide insertion studies (58,59) showing that human
Pol Z inserts dA and dC opposite 8-oxoG with similar
efficiencies and then extends the resulting termini with
similar efficiencies. Importantly, the high error rates we
observe for human and mouse Pol Z differ by only
about 2-fold from the error rates for bypass of 8-oxoG
by bovine Pol d (83%, 5:1 ratio of A to C incorporation)
and human Pol a (99.5%, 200:1A to C incorporation) (9).
These comparisons predict that switching from mamma-
lian Pol d or Pol a to mammalian Pol Z in order to
bypass 8-oxoG would have at most a 2-fold affect on
8-oxoG-dependent mutagenesis. Consistent with this is
a recent demonstration that the frequency of 8-oxoG-
dependent mutagenesis in a gapped plasmid is high, and,
more importantly, it is similar in Pol Z-deficient and Pol
Z-proficient human cells (60). Also relevant here may be
the observation that, unlike for bypass of a TT dimer (41),
we see no evidence for a switch to less processive syn-
thesis after bypass of 8-oxoG by mouse and human
Pol Z (Figure 2B, Table 1). This represents a second
Table 3. Error specificity during G and 8-oxoG bypass
G ! C
G ! T
G ! C
G ! T
aAdditional experiments using a 45-mer template (see ‘Materials and Methods’ section) with slight sequence difference gave error rates of: (30-...
CGoATC...) G!C=610?10?4, G!T=4600?10?4; (30-...CAGoTC...) G!C=<230?10?4, G!T=4500?10?4; (30-...CAGoTT...)
bDNA from dark blue plaques described in Table 2 was sequenced and error rates (?10?4) were calculated as previously described (42). Only changes
at the G/8-oxoG site are given (the TAG stop codon is in italics in the left-most column). Note that G!A changes are not detectable by color
screening as the amber stop codon (TAG) changes instead to an ochre stop codon (TAA) in the lacZa sequence.
cThese sequenced samples are the same as those used in a published report (50). The specific error rates shown here have not previously been
Nucleic Acids Research,2009, Vol.37, No. 92837
difference between the yeast and mammalian Pol Z. Given
structure–function studies of other polymerases (10,12,14,
15,18,19,23), these differences may depend on amino acids
variations at the active sites and/or the little finger
domains of yeast and mammalian pol Z.
It is of course possible that the intrinsically low 8-oxoG
bypass fidelity of the catalytic subunit of mammalian
Pol Z that we observe here can be improved. Our data
(Table 1) indicate that RFC, PCNA and RPA may slightly
improve bypass efficiency, but do not improve the 8-oxoG
bypass fidelity of either yeast Pol d or yeast Pol Z
(Table 3). This is consistent with earlier studies showing
that these accessory proteins have at most a 1.5-fold
effect on the fidelity of yeast Pol Z when bypassing a TT
dimer (50) or on the base substitution or indel error rates
of yeast Pol d and yeast Pol Z when copying undamaged
DNA (50,61). Theoretically, the situation could be dif-
ferent for the human proteins, based on the observation
that PCNA and RPA allowed correct insertion of dC
opposite 8-oxoG to proceed 68-fold more efficiently than
incorporation of dA opposite 8-oxoG in single nucleotide
insertion experiments (59). Thus, it will be interesting
to measure the error rate for a complete 8-oxoG bypass
reaction by mammalian Pol Z in the presence of the acces-
sory proteins. Although accessory proteins do not strongly
influence single base error rates by many polymerases,
including yeast Pol Z during 8-oxoG bypass (Table 3),
it remains possible that mammalian accessory proteins
might improve Pol Z fidelity. Other possibilities for
improving the fidelity of 8-oxoG-dependent bypass by
Pol Z include (but are not limited to) correction
of 8-oxoG-containing mismatches by mismatch repair
(8,62) or by proofreading. Either correction process
could suppress mutagenesis during TLS, even for TLS
by a highly inaccurate polymerase, especially if multiple
cycles of polymerization-error correction are permitted.
Relevant here is the finding that 8-oxoG containing
mismatches made by Pol d are not proofread by its intrin-
sic exonuclease activity (Table 3), which is consistent
with studies showing that the large fragment of E. coli
DNA polymerase I (9), T7 DNA Pol (12) and RB69
Pol (15,63) also do not efficiently proofread dA?8-oxoG
mismatches. This has been rationalized by the observation
that an dA?8-oxoG mismatch mimics the geometry of a
correct base pair, especially regarding interactions of
atoms in the DNA minor groove with side chains at
the polymerase active site (12,14). Nonetheless, even
though Pol Z lacks an intrinsic exonuclease activity, it
is theoretically possibly that mismatches made during
Pol Z bypass of some lesions, e.g. 8-oxoG that retains
base coding potential, may be proofread by a separate
exonuclease. Genetic evidence exists for extrinsic proof-
reading of Pol a replication errors by Pol d (64) and
biochemical evidence has been obtained for extrinsic
proofreading of human Pol Z errors during SV40 origin-
dependent replication of undamaged DNA (65) and for
Pols d and e-dependent proofreading of Pol Z misincor-
porations opposite the 30T of a TT dimer (40). For extrin-
sic proofreading to get around the geometric mimicry
mentioned earlier, Pol d and/or Pol e may need to
bind to the mismatched terminus directly via the
exonuclease active site, as already reported for two other
replicative DNA polymerases, T7 DNA pol (66) and
RB69 pol (67).
The capacity of Pol Z to suppress 8-oxoG-dependent
mutagenesis may also depend on when the lesion is
encountered, whether by a replication fork or during
post-replication gap-filling (17). Also relevant may be
the physiological state of the cell when the 8-oxoG is gen-
erated. For example, the error rates for bypass of 8-oxoG
by Pol d (9) and Pol Z (Table 3) were all measured for
reactions containing 100mM dNTPs. This is approxi-
mately the dNTP concentration induced when budding
yeast are exposed to the UV mimetic 4-NQO (68). Thus
comparatively high-dNTP concentrations may be relevant
to bypass of 8-oxoG or other lesions induced by sunlight
or other exogenous environmental stress. On the other
hand, the strongest evidence that Pol Z suppresses
8-oxoG-dependent mutagenesis is for 8-oxoG arising
spontaneously (17). Interestingly, dNTP concentrations
in yeast are normally several-fold lower than in the
induced state (69). Given that the efficiency of bypass of
8-oxoG by yeast Pol e depends on the dNTP concentra-
tion (69), the successful competition among the many
polymerases present in a cell for a lesion may depend
not only on lesion identity, but also on whether the
lesion arose spontaneously or was induced by exogenous
environmental stress. Because the four dNTPs are not
present in equimolar amounts in vivo [ref. (69) and earlier
studies reviewed in refs (70,71)], the nature of the
polymerase competition, as well as the fidelity of
TLS, may vary depending on the lesion, the 50flanking
template sequence and the amount of each dNTP avail-
able for insertion opposite the lesion and subsequent
The authors thank Katarzyna Bebenek and Zachary
Pursell for thoughtful comments on the manuscript.
NIH Grant GM032431 [to P.M.B.]; Intramural Research
Program of the NIH, National Institute of Environmental
Health Sciences [to T.A.K.]. Funding for open access
Institutes of Health, National Institute of Environmental
Conflict of interest statement. None declared.
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