Copyright ? 2010 by the Genetics Society of America
Participation of DNA Polymerase z in Replication of Undamaged DNA in
Matthew R. Northam, Heather A. Robinson,1Olga V. Kochenova2and Polina V. Shcherbakova3
Eppley Institute for Research in Cancer and Allied Diseases, University of Nebraska Medical Center, Omaha, Nebraska 68198
Manuscript received July 17, 2009
Accepted for publication October 12, 2009
Translesion synthesis DNA polymerases contribute to DNA damage tolerance by mediating replication of
damaged templates. Due to the low fidelity of these enzymes, lesion bypass is often mutagenic. We have
previously shown that, in Saccharomyces cerevisiae, the contribution of the error-prone DNA polymerase
z (Polz) to replication and mutagenesis is greatly enhanced if the normal replisome is defective due
to mutations in replication genes. Here we present evidence that this defective-replisome-induced
mutagenesis (DRIM) results from the participation of Polz in the copying of undamaged DNA rather
than from mutagenic lesion bypass. First, DRIM is not elevated in strains that have a high level of
endogenous DNA lesions due to defects in nucleotide excision repair or base excision repair pathways.
Second, DRIM remains unchanged when the level of endogenous oxidative DNA damage is decreased by
using anaerobic growth conditions. Third, analysis of the spectrum of mutations occurring during DRIM
reveals the characteristic error signature seen during replication of undamaged DNA by Polz in vitro. These
results extend earlier findings in Escherichia coli indicating that Y-family DNA polymerases can contribute to
the copying of undamaged DNA. We also show that exposure of wild-type yeast cells to the replication
inhibitor hydroxyurea causes a Polz-dependent increase in mutagenesis. This suggests that DRIM re-
presents a response to replication impediment per se rather than to specific defects in the replisome
that block DNA synthesis by replicative enzymes
(Prakash et al. 2005). TLS polymerases are present in
all domains of life and include eukaryotic polymerase z
(Polz), Polh, Poli, Polk, and Rev1. Because of the lower
selectivity of their active sites, TLS polymerases often
introduce errors when bypassing lesions or copying un-
2006). In yeast and human cells, DNA synthesis by Polz
is responsible for nearly all mutagenesis induced
by exogenous genotoxicants (Waters et al. 2009). Polz
has been isolated from Saccharomyces cerevisiae as a com-
plex of two subunits encoded by the REV3 and REV7
et al. 2001). At the same time, Polz is very efficient in
extending primers containing a mismatched terminal
nucleotide (Johnson et al. 2000; Guo et al. 2001;
RANSLESION synthesis (TLS) DNA polymerases
have the ability to bypass lesions in template DNA
Haracska et al. 2001; Simhadri et al. 2002). In
accordance with these properties, the main function of
Polz in TLS is proposed to be the extension from nu-
cleotides incorporated opposite DNA lesions by other
and Fuchs 2002; Prakash and Prakash 2002). The
by the Rev1 protein (Acharya et al. 2006). Rev1 is a
deoxycytidyl transferase involved in multiple protein–
protein interactions with other DNA polymerases, and
its essential function in TLS is thought to be structural
of Polz is also stimulated by proliferating cell nuclear
antigen(PCNA), the DNA polymerase processivityfactor
(Garg et al. 2005; Northam et al. 2006).
In addition to the mutagenesis induced by environ-
mental agents, Polz is required for the vast majority of
mutations provoked by endogenous DNA damage. For
rate caused by defects in nucleotide excision repair
(NER) (Roche et al. 1994; Harfe and Jinks-Robertson
2000), base excision repair (BER) (Xiao et al. 2001),
post-replicative DNA repair (Roche et al. 1995;
Broomfield et al. 1998; Xiao et al. 1999), homologous
recombination (Roche et al. 1995; Harfe and Jinks-
Robertson 2000), the overproduction of 3-methylade-
nine DNA glycosylase (Glassner et al. 1998), and the
1Present address: Department of Biological Sciences, Box 2116, Sam
Houston State University, Huntsville, TX 77431-2116.
Universitetskaya emb. 7/9, St. Petersburg, 199034, Russia.
3Corresponding author: University of Nebraska Medical Center, 986805
Nebraska Medical Center, Omaha, NE 68198.
Genetics 184: 27–42 (January 2010)
expression of altered uracil-DNA glycosylases that re-
move undamaged cytosines and thymines in a BER-
increase the level of endogenous replication-blocking
lesions, and the Polz-dependent mutagenesis likely
reflects the function of Polz in the error-prone bypass
of these lesions.
There are also multiple reports documenting Polz-
dependent mutagenesis in situations in which cells are
not expected to accumulate excessive DNA damage.
50–70% of spontaneous mutations in wild-type S.
cerevisiae strains (Cassier et al. 1980; Quah et al. 1980).
Polz also contributes to an increased mutation rate
associated with high levels of transcription (Datta and
Jinks-Robertson 1995) or double-strand break repair
(Holbeck and Strathern 1997). In addition, we and
others have shown that Polz participation in replication
and mutagenesis is promoted by a variety of replication
machinery defects (Shcherbakova et al. 1996; Pavlov
et al. 2001b; Kai and Wang 2003; Northam et al. 2006).
For example, defects in S. cereviseae replicative poly-
merases Pold and Pole that are thought to affect rep-
lication fork progression or the replisome integrity lead
to a mutator phenotype. Eighty to 90% of spontane-
ous mutations in these strains are mediated by Polz
(Shcherbakova et al. 1996; Pavlov et al. 2001b;
Northam et al. 2006). It is not known whether Polz-
dependent mutagenesis in the absence of abnormally
high levels of DNA damage results from error-prone
copying of undamaged DNA or from Polz-mediated
bypass of endogenous lesions present at physiological
levels. The Polz error rate during the copying of
undamaged DNA in vitro is several orders of magnitude
et al. 2006), so its contribution to the genome replica-
tion would be expected to result in increased mutagen-
esis. In Escherichia coli, the high spontaneous mutation
rate in strains with constitutive expression of the SOS
system has been shown to result from the copying of
undamaged DNA by error-prone DNA polymerases
anism mediating the recruitment of Polz in response to
replication defects shares some features with the mech-
anism of polymerase switching during TLS, such as the
requirement for monoubiquitination of PCNA and for
the physical interaction of PCNA with Polz (Northam
et al. 2006). It is possible that the defective replisome
resulting in more need for the error-prone TLS. In this
study, we addressedwhether defective-replisome-induced
mutagenesis (DRIM) reflects the mutagenic bypass of
endogenous damage or error-prone copying of the
undamaged DNA by Polz. The results argue against the
endogenous damage as a key mediator of the mutagenic
response and thus provide evidence for Polz participa-
tion in the copying of undamaged DNA.
MATERIALS AND METHODS
S. cerevisiae strains: Thewild-typehaploidstrainE134(MATa
and the construction of its pol3-Y708A, rev3D and pol3-Y708A
rev3D derivatives have been described (Shcherbakova and
Kunkel 1999; Northam et al. 2006). The RAD14 and APN2
genes were disrupted in E134 and in the pol3-Y708A mutant by
a selectable hygB (rad14) or kanMX (apn2) cassette as de-
scribed (Wach et al. 1994). The APN1 gene was disrupted
by transformation with a fragment from plasmid pSCP108
(Popoff et al. 1990) containing the hisGTURA3ThisG cassette
followed by selection for the loss of the URA3 marker on
medium containing 5-fluoroorotic acid (FOA; Boeke et al.
1984). The disruptions were confirmed by PCR and by
sensitivity of the mutants to UV irradiation (rad14) or
hydrogen peroxide (apn1 apn2). The quintuple DNA glyco-
sylase mutant DGD39 (MATa leu2-3,112 trp1-289 his7-2 ura3-52
lys1-1 ung1D ntg1D ntg2DTkanMX6 ogg1DTURA3 mag1DT
hphMX4) and the isogenic wild-type strain FF18733 (Kozmin
et al. 2005) were kindly provided by E. Sage (Institut Curie,
Orsay, France). A spontaneous ura3 mutant of DGD39 was
medium. The wild-type chromosomal POL3 and POL2 genes
of FF18733 and the Ura?derivative of DGD39 were then
replaced with the pol3-Y708A and pol2-1 alleles as described
(Shcherbakova et al. 1996; Pavlov et al. 2001b). The strain
Dl(-2)l-7B-YUNI300 (MATa ade2-1 lys2DGG2899-2900 trp1-289
his7-2 leu2DTkanMX ura3D) and its ogg1D mutant (Pavlov
et al. 2002) were obtained from Y. I. Pavlov (University of
Nebraska Medical Center).
Measurement of the spontaneous mutation rate and
hydroxyurea-induced mutant frequency: To measure the rate
of spontaneous mutation, at least nine 7- to 9-ml cultures were
started for each strain from single colonies and grown to the
stationary phase in liquid yeast extract peptone dextrose
medium supplemented with 60 mg/liter adenine and 60
mg/liter uracil (YPDAU). Cells were plated after appropriate
dilutions onto synthetic complete medium containing
l-canavanine (60 mg/liter) and lacking arginine (SC 1 CAN)
for Canrmutant count and onto synthetic complete (SC)
medium for viable count. Yeast extract peptone dextrose and
SC media were prepared as described elsewhere (Rose et al.
1990). Canrmutant frequency was calculated by dividing the
Canrmutant count by the viable cell count. Mutation rate was
calculated from mutant frequency by using the Drake equa-
described (Dixon and Massey 1969). Hydroxyurea (HU)-
but the cultures were started from ?104-cells inoculum rather
than from single colonies and grown in YPDAU medium
containing the indicated concentration of HU. The median
frequency of Canrmutants was used to compare HU-induced
mutagenesis in different strains. To measure the rate of
spontaneous mutation under anaerobic conditions, the cul-
tures were grown in a GasPak anaerobic chamber (BD) prior
to plating on SC 1 CAN and SC media for the mutant and
viable count. Anaerobic conditions were monitored using
GasPak dry anaerobic indicator strips (BD).
Mutational spectra determination: Independent colonies
of the pol3-Y708A and pol3-Y708A rev3D derivatives of E134
were streaked on YPDAU plates, grown for 2 days at 30?, and
replica-plated onto SC 1 CAN medium to select for can1
mutants. One Canrcolony was picked from each patch, and
the CAN1 gene was amplified by PCR and sequenced as
described (Kozmin et al. 2003). The sequencing covered the
28 M. R. Northam et al.
entire open reading frame with the exception of nucleotides
A genetic system for the study of DRIM: Weobserved
previously that many mutations affecting the replisome
components result in a Polz-dependent spontaneous
et al. 2001b; Northam et al. 2006). Here we use two
mutations, pol3-Y708A and pol2-1, that confer the stron-
gest Polz-dependent increase in mutagenesis. The pol3-
Y708A mutation (Pavlov et al. 2001b) results in a single
amino acid change in the polymerase active site of Pold
(Swan et al. 2009a). The pol2-1 mutation is an insertion
of the URA3 gene in the middle of the coding region of
the POL2 gene encoding the catalytic subunit of Pole.
The insertion results in a loss of interaction of the
catalytic subunit with the other subunits of the Pole
holoenzyme (Morrison et al. 1990). DNA replication is
impaired in both mutants as manifested by their slow
growth and, in the case of pol3-Y708A, sensitivity to HU.
Our previous studies support a model in which the
defect in the replication fork progression in these
mutants provides a signal for constitutive monoubiqui-
tination of PCNA and the recruitment of low-fidelity
polymerases, particularly Polz, for DNA replication
(Northam et al. 2006). Those studies indicated that
Y708A andpol2-1strains requirethepresenceofPolz.In
this work, we use this clearly detectable Polz-dependent
mutator phenotype as a measure of Polz involvement in
chromosomal DNA replication.
DRIM is not affected by an increased level of
endogenous DNA damage: We reasoned that if the
bypass of endogenous damage was a major source of
DRIM, the mutator phenotype of replication-deficient
strains would be stronger when the level of endogenous
damage is elevated. To achieve this, we inactivated the
two major damage repair pathways, NER and BER, in
potentially contribute to DRIM are unknown. There-
fore, we studied the effects of both repair pathways to
by NER and BER could be responsible for the increased
mutagenesis. A similar approach has been used pre-
viously to study the role of endogenous DNA lesions in
et al. 1997). We first inactivated the NER pathway, which
removes a variety of helix-distorting DNA lesions, by
disrupting the RAD14 gene. The Rad14 protein is
involved in damage recognition during NER (Guzder
et al. 1993). The deletion of the RAD14 gene results in a
weak, but clearly measurable, spontaneous mutator
phenotype (Figure 1A, Table S1), consistent with pre-
vious reports (Bertrand et al. 1998; Harfe and Jinks-
Robertson 2000; Scheller et al. 2000; Yu et al. 2003;
Guo et al. 2005). This indicates that the rad14 strain
indeed accrues potentially mutagenic DNA damage.
The mutator effect of the double pol3-Y708A rad14
mutation is equal to the sum of the mutator effects of
the single mutations (Figure 1A, Table S1), suggesting
thatthespontaneousmutations arise throughdifferent,
nonoverlapping pathways in the replication- and repair-
deficient strains. We therefore concluded that the
spontaneous damage that can be repaired by NER plays
no significant role in DRIM.
Next we investigated whether the abasic sites (AP
sites), which are known to present an obstacle for the
replicative DNA polymerases, contribute to DRIM. We
simultaneously disrupted the two genes that encode the
apurinic endonucleases essential for the abasic site
repair, APN1 and APN2, in the pol3-Y708A strain. As
reported earlier (Johnson et al. 1998; Bennett 1999;
Xiao et al. 2001), the double apn1 apn2 mutation itself
caused increased spontaneous mutagenesis (Figure 1B,
Table S1), consistent with the expected mutagenicity of
in the case of rad14, the interaction of the apn1 apn2
double mutation with the pol3-Y708A mutation was
strictly additive (Figure 1B, Table S1). This indicates
that mutagenesis resulting from the AP site bypass and
DRIM represent distinct, nonoverlapping pathways.
Thus, the polymerase stalling at abasic sites does not
appear to make a substantial contribution to DRIM.
In addition to the AP sites, the BER pathway repairs
a variety of other lesions, such as those produced by
reactive oxygen species, alkylation, or deamination. To
determine if any of these lesions are responsible for
yeast DNA glycosylases (Ogg1, Ntg1, Ntg2, Ung1, and
Mag1). The glycosylases are responsible for the recog-
nition and removal of damaged bases, so the quintuple
mutant is expected to be defective in BER of all lesions
in the rate of spontaneous mutation to canavanine
resistance (Canr), which is likely a consequence of the
mutagenicbypass of unrepaired base damage (Figure 1,
C and D; Table S2). When the pol3-Y708A or pol2-1
mutations were introduced into the glycosylase-defi-
cient strain, the resulting spontaneous mutation rate
was close to that expected in the case of the additive
interaction of the BER and DNA polymerase defects
(Figure 1, C and D; Table S2). A small (?20–30%) but
reproducible increase in the mutation rate value in the
pol3-Y708A BER?and pol2-1 BER?over the expected
additive value was observed. This indicates that a minor
fraction of DRIM could be mediated by DNA damage
bypass, at least in the situation in which the damage is
a major source of DRIM, a multiplicative interaction of
the mutator effects would have been observed (a 110-
and 270-fold increase over the wild-type mutation rate
for the pol3-Y708A BER?and pol2-1 BER?strains, re-
DNA Polymerase z Copies Undamaged DNA 29
spectively). The actual mutator effects of the pol3-Y708A
respectively, which is far lower than the mutator effects
expected for the multiplicative interaction. Overall, the
data suggest that the bypass of lesions that are normally
a subject of BER does not make a major contribution
Several studies indicated that NER and BER have
overlapping specificity in respect to the spectrum of
lesions repaired (Swanson et al. 1999; Torres-Ramos
et al. 2000; Gellon et al. 2001). If the bypass of lesions
that could be efficiently repaired by both NER and BER
were a major source of DRIM, the experiments shown
in Figure 1, A–D, would fail to detect this. Therefore, we
also measured DRIM in strains defective simultaneously
in NER and BER. Similar to the results obtained with
strains defective in a single repair pathway, the interac-
tion of the mutator effects caused by the pol3-Y708A
allele and the double NER?BER?deficiency was nearly
additive (Figure 1, E and F; Table S3; Table S4). This
further supports our conclusion that DRIM is unrelated
to the bypass of endogenously generated lesions, in-
cluding the ones that could be processed by both NER
DRIM is not affected by a reduced level of oxidative
DNA damage: Reactive oxygen species are produced in
cells during normal aerobic metabolism. Oxidative
damage to DNA is thought to account for the majority
of endogenously generated DNA lesions. These lesions
are primarily repaired by BER (D’Errico et al. 2008).
Figure 1.—Effect of DNA repair deficiencies
on the spontaneous mutator phenotype of pol3-
Y708A and pol2-1 mutants. (A) Interaction of
the mutator effects of pol3-Y708A and rad14D
alleles. (B) Interaction of the mutator effects of
pol3-Y708A and the double apn1D apn2D muta-
tion. (C and D) Interaction of the mutator effects
of pol3-Y708A and pol2-1, respectively, with the ef-
fect of quintuple DNA glycosylase mutation
ogg1D ntg1D ntg2D ung1D mag1D. (E and F) Inter-
action of the mutator effects of pol3-Y708A and
the double NER?BER?deficiencies, apn1D
apn2D rad14D and ogg1D ntg1D ntg2D ung1D
mag1D rad14D, respectively. The DNA repair mu-
tations designated as NER?and BER?are indi-
cated in ovals. Rate of Canrmutation relative to
the wild type is shown. The data are from Table
S1 (A and B), Table S2 (C and D), Table S3 (E),
and Table S4 (F) and are medians and 95% con-
fidence intervals for at least nine cultures. Rates
expected in the case of additivity were calculated
as a sum of the mutation rates in the strains with
the corresponding single defects. This approach
may slightly overestimate the expected additive
rate value (by a maximum of 9%, 7%, 4.8%,
and 2.6% for the experiments in A–D, respec-
tively) because some of the mutations occurring
spontaneously in the wild-type strain could still
occur in the replication and repair mutants
and thus would be counted twice in this calcula-
30M. R. Northam et al.
The fact that we did not detect a significant effect of
BER on DRIM (Figure 1) suggests that oxidative lesions
are unlikely to play a major role in this mutagenesis
pathway. To provide additional evidence for this, we
strain would beaffectedby anaerobicgrowthconditions
To demonstrate that the level of mutagenic endogenous
damage was indeed reduced in our anaerobic experi-
ments, we measured the spontaneous mutation rate in
ogg1 mutants obtained in three different genetic back-
grounds. The spontaneous mutations in ogg1 strains
result mainly from the accumulation of unrepaired 8-
oxoguanine residues in DNA. The mutator effect of the
anaerobic growth (Figure 2; Table S5). We therefore
concluded that the spontaneous oxidative DNA damage
plays no significant role in DRIM.
The spectrum of spontaneous mutations in the pol3-
Y708A strain shows a characteristic Polz signature: To
gain further insight into the mechanism of DRIM, we
compared the spectrum of spontaneous mutations
generated in the pol3-Y708A strain in the presence and
in the absence of Polz (Table 1, Figure 3 and Figure 4).
We analyzed 178 and 214 independent Canrmutants of
the pol3-Y708A and pol3-Y708A rev3D strains, res-
pectively. The pol3-Y708A spectrum was composed pre-
dominantly of base substitutions (70%) followed by
complex mutations (13%). The latter represented
multiple changes within a stretch of no more than six
nucleotides (Figure 5B). We define these as complex
mutations type I to distinguish them from other types of
Polz?derivative of pol3-Y708A (see below). The other
mutations detected in the pol3-Y708A strain were small
all sequenced mutations) and larger deletions (8%). All
larger deletions affected sequences flanked by short
(three to eight nucleotides) direct repeats (Figure 3).
Deletion of REV3 in the pol3-Y708A strain decreased
50 3 10?8, Northam et al. 2006). The spectrum of
mutations in the rev3D derivative of pol3-Y708A is shown
in Table 1 and Figure 4. To provide an informative
comparison of the pol3-Y708A and pol3-Y708A rev3D
mutational spectra, we calculated the rates of each
individual type of mutation in the two strains as de-
rate in the rev3D derivative was largely due to a large
decrease in base substitution mutations, particularly
GC / CG transversions. The GC / CG changes con-
stituted nearly half of all base substitution mutations in
transversions were found in the pol3-Y708A rev3D strain,
these events upon the inactivation of Polz. Decreases in
the rate of other base substitutions were also observed.
In addition, the rate of type I complex mutations was
five complex changes were found in the pol3-Y708A
rev3D strain, four of them were different from the type I
complex mutations and involved DNA stretches longer
than six nucleotides. We define these larger changes as
complex mutations type II (see Figure 4 for a detailed
description of these mutations).
be specifically attributed to Polz by subtracting the
background mutation rate seen in the absence of Polz
(pol3-Y708A rev3D) from the mutation rate seen in the
presence of Polz (pol3-Y708A) (Table 1). The Polz-
dependent mutations were almost exclusively base sub-
stitutions and complex mutations. A few small deletions
or insertions, mostly ?1 frameshifts, were also appar-
ently generated by Polz. The rates shown in the last
column of Table 1 imply that ?75% of Polz-dependent
mutations are base substitutions, ?16% are complex
mutations, and ?7% are small deletions or insertions.
Interestingly, the rate of large deletions was not signif-
icantly different in the pol3-Y708A and pol3-Y708A rev3D
strains. This indicates that these deletions are not
generated by Polz, but are likely a result of faulty rep-
lication by the defective Pold itself. Mutations in the
POL3 gene have been previously reported to increase
the rate of deletions between short direct repeats (von
Borstel et al. 1993; Tran et al. 1995). The length of the
Polz1and Polz?strains (three to eight nucleotides,
Figures 3 and 4). Interestingly, however, a substantial
difference was seen in the size of the deletions (Figure
waslargerthan40nucleotides,sequenceslonger than 40
nucleotides (up to 646) were deleted in ?42% of all
deletion cases in the Polz?strain. This represents a
Figure 2.—Effect of anaerobic growth conditions on the
spontaneous mutator phenotype of pol3-Y708A (A) and ogg1D
(B) strains. The bar graphs show the rate of Canrmutation in
E134 strain and its pol3-Y708A derivative and in the Dl(-2)l-
7B-YUNI300 strain and its ogg1D derivative. All data are from
Table S5 and are medians and 95% confidence intervals for at
least nine cultures.
DNA Polymerase z Copies Undamaged DNA 31
fivefold increase in the rate of large deletions upon Polz
inactivation, suggesting that Polz could play a role in
suppressing the formation of larger deletions.
HU treatment induces a Polz-dependent mutagenic
response: DRIM has been observed in a variety of DNA
replication mutants, including those with defects in
Pola, Pold, and Pole (Northam et al. 2006). This ob-
servation led to a hypothesis that DRIM is a response to
the impediment of the replication fork progression
rather than to the specific DNA polymerase defects. To
test this, we determined whether treatment of the wild-
type cells with HU would induce Polz-dependent muta-
genesis. HU is known to trigger replication stalling due
to the inhibition of dNTP synthesis while causing no
damage to the DNA or replication proteins. HU is also
known to induce the PCNA monoubiquitylation at
Lys164 (Kannouche et al. 2004; Davies et al. 2008),
and the role of this modification in the recruitment of
specialized DNA polymerases during TLS and DRIM is
well established (Hoege et al. 2002; Northam et al.
2006). In the experiment shown in Figure 7, we mea-
sured the frequency of the Canrmutation in yeast
cultures grown in the presence of varying concentra-
tions of HU. A concentration of 200 mm causes a nearly
complete cell-cycle arrest in most strains, so we used a
range of smaller HU doses that do not interfere dra-
situation in the replicative polymerase mutants that are
able to replicate their DNA and divide. While HU was
clearly mutagenic in the wild-type strain, the mutant
frequency was reduced up to 15-fold in the rev3 strain
lacking Polz. This indicates that the phenomenon of
Spontaneous can1 mutations in the pol3-Y708A strain and its rev3D derivative
Rate of Polz-dependent
GC / AT
AT / GC
GC / TA
GC / CG
AT / CG
AT / TA
Large rearrangements ($8 nucleotides)
Deletions between short direct repeats
Complex mutations type I (#6 nucleotides)d
Complex mutations type II ($7 nucleotides)d
300 21450 250
aTwo can1 mutants of the pol3-Y708A strain carried double base substitutions. The mutations were separated by 93 and 476
nucleotides in these mutants. These were counted as four individual base substitutions.
bRate for each type of mutation was calculated as follows: MRi¼ (Mi/MT) 3 MR, where Miis the number of mutations of the
particular type, MTis the total number of mutations, and MR is the rate of Canrmutation in the corresponding strain determined
by fluctuation analysis, 3 3 10?6for the pol3-Y708A and 5 3 10?7for the pol3-Y708A rev3D strain (Northam et al. 2006).
cRate of Polz-dependent mutations was calculated by subtracting the rate for the pol3-Y708A rev3D strain from the rate for the
dComplex mutations type I are defined as replacements of one to six adjacent nucleotides with a different sequence no more
than six nucleotides long. Complex mutations type II are defined as replacements where the original sequence or the new se-
quence or both are longer than six nucleotides.
32 M. R. Northam et al.
Figure 3.—Spectrum of spontaneous can1 mutations in the pol3-Y708A strain. The coding sequence of the CAN1 gene is shown.
Letters and triangles above the sequence indicate base substitutions and single base deletions, respectively. Letters with a ‘‘1’’
symbol indicate single base insertions. Red boxes show complex mutations. Deletions of 2–5 nucleotides are designated by a line
above the sequence with a number of deleted nucleotides shown above the line. For larger deletions, short direct repeats flanking
the deleted region are shown by colored arrows with a deletion identification number inside the arrow. The green box indicates
a 4-nucleotide insertion within an 11-nucleotide region.
DNA Polymerase z Copies Undamaged DNA33
Figure 4.—Spectrum of spontaneous can1 mutations in the pol3-Y708A rev3D strain. Dashed colored lines below the CAN1 se-
quence show multi-base deletions between imperfect direct repeats. The corresponding imperfect repeats are indicated by the
same-color shaded boxes. The non-identical nucleotides within the repeats are in yellow. A deleted region not flanked by short
repeats is shown by a purple dashed line below the CAN1 sequence. A 2-nucleotide addition is shown by a line above the CAN1
34M. R. Northam et al.
Polz-dependent mutagenesis is not a unique feature of
the replicative polymerase mutants that we study, but
rather represents a general response to replication fork
stalling. Additional, Polz-independent pathways appar-
ently contribute to the HU-induced mutagenesis as well
because the rev3 strain still showed an elevated fre-
quency of Canrmutation in the presence of HU,
particularly at higher HU doses (Figure 7; Table S6).
In this study, we provide several lines of evidence that
DRIM is unrelated to the role of Polz in mutagenic TLS
and represents Polz-dependent error-prone copying of
undamaged DNA. First, epistatic analysis of mutation
rates in DNA replication and repair mutants indicated
that mutagenesis induced by endogenous lesions and
DRIM represent separate, nonoverlapping pathways.
Second, DRIM remains unchanged when spontaneous
oxidative damage is eliminated. Third, the spectrum of
mutations occurring during DRIM shows similarities to
the error specificity of purified Polz on undamaged
DNA in vitro. This demonstrates that Polz can be re-
cruited to perform DNA synthesis on undamaged DNA
templates. This could potentially involve processive
error-prone DNA synthesis by Polz, extension of mis-
matches generated by the defective replicative DNA
mutagenic response with HU illustrates that DRIM does
not require the presence of a defective replicative DNA
polymerase that could generate mismatches due to re-
laxed nucleotide selectivity. Thus, the studies of DRIM
a variety of situations where the fork progression is
The data presented in Table 1 and Figure 5 indicate
that two types of events, complex mutations and GC /
CG transversions, can be considered the hallmarks of
the Polz mutational signature in the pol3-Y708A strain.
Importantly, these two types of mutations were also the
unique features of the spectrum of errors generated by
purified yeast Polz during replication of undamaged
DNA in vitro (Zhong et al. 2006). The G?G and C?C
mispairs were among the three most frequent errors
made by Polz in vitro. This predicts that a high rate of
GC / CG transversion would result if Polz were to
contribute to DNA synthesis in vivo. The purified Polz
created complex mutations at an average frequency
of 12%, which is similar to the frequency of complex
in the pol3-Y708A strain (15%). The overall representa-
sequence with the inserted nucleotides above the line. A 22-nucleotide duplication is indicated by a line above the sequence. All
other symbols are as in Figure 3.
DNA Polymerase z Copies Undamaged DNA 35
in vitro and in vivo spectra, including the predominance
of base substitutions, the same proportion of complex
mutations, and a low frequency of frameshifts. The only
major difference between the two spectra was the very
Polz and the relatively low rate of the corresponding
be caused by differences in the reporter genes used,
contribution of other replication proteins, and/or
correction of these mispairs by the DNA mismatch
There is a remarkable similarity between the types of
DNA template in vitro and in the pol3-Y708A strain
in vivo. In both cases, these are multiple changes within
no more than six or seven bases, and about half are
7-nucleotide stretches are typically two base substitu-
tions or a base substitution and a single base deletion/
addition separated by several unchanged nucleotides
(Figure 5B and Zhong et al. 2006). Incontrast, complex
much lower frequency during Polz-dependent TLS.
During the bypass of site-specific model lesions in
yeast, complex mutations constituted ,3% of all TLS-
associated mutations and typically involved sequence
replacements of no more than 3 adjacent nucleotides
(Gibbs et al. 1995; Gibbs and Lawrence 1995; Gibbs
et al. 2005). Simultaneous changes of .3 nucleotides
were not seen in the spectrum of UV-induced mutations
TLS assay system used by the Z. Wang laboratory,
multiple changes in larger stretches of DNA (up to 12
nucleotides) are seen at a high frequency during the
Zhao et al. 2006). These, however, are seen in rev3
mutant strains as well, so they are apparently not
to represent a characteristic signature of Polz-mediated
bypass of endogenous DNA damage in vivo (Harfe and
Jinks-Robertson 2000). In that study, only mutations
leading to a 11 frameshift were analyzed, and the vast
majority of these mutations occurred in TTT homonu-
cleotide runs. In contrast, among the frameshift-associ-
ated complex mutations found in the pol3-Y708A strain,
the majority did not involve homonucleotide runs,
via a frameshift in a mononucleotide repeat. This
provides an additional indication that mutagenesis
mediated by DNA damage bypass and DRIM occurs via
different mechanisms, supporting the view that DRIM
results from copying of undamaged DNA by Polz.
A recent report suggested that a strong Polz-
dependent increase in mutagenesis could result from
the accumulation of extended stretches of single-
stranded DNA (ssDNA) in yeast, presumably due to
et al. 2008). The accumulation of ssDNA at stalled
replication forks has been suggested to serve as a signal
for the Rad6/Rad18-dependent monoubiquitylation of
PCNA, which, in turn, is required for the recruitment of
TLS polymerases to sites of DNA damage (Davies et al.
2008). We have previously reported that the pol3-Y708A
dependent mutators, show a robust constitutive mono-
ubuquitylation of PCNA, which is required for DRIM
(Northam et al. 2006). This is consistent with the idea
that the replication mutants may have an excessive
amount of ssDNA at the replication forks. However,
of endogenous lesions present in single-stranded re-
gions. First, even a nearly complete replication block,
such as the one resulting from HU treatment, increases
the amount of ssDNA at the replication forks no more
than twofold (Sogo et al. 2002; Davies et al. 2008).
This increase is likely to be even smaller in the rep-
lication mutants, such as pol3-Y708A, which are quite
capable of DNA replication. The mutator effect due to
to be less than twofold. Second, anaerobic growth
conditions, which presumably reduce the damage in
ssDNA, had no effect on DRIM (Figure 2, Table S5).
Third, the spectrum of mutations that was suggested to
result from the Polz-dependent bypass of endogenous
lesions accumulating in ssDNA (Yang et al. 2008) is
drastically different from the mutational signature of
DRIM (Table 1). Particularly, the ssDNA-stimulated mu-
tagenesis showed no complex changes involving more
Figure 5.—GC / CG transversions and com-
plex mutations are the two characteristic features
of spontaneous Polz-dependent mutagenesis in
the pol3-Y708A strain. (A) The spectrum of base
substitutions in the CAN1 gene of the pol3-Y708A
strain. The proportions of individual base substi-
tution were calculated from the data shown in
Table 1. (B) Complex mutations found in the
CAN1 gene of the pol3-Y708A strain. The super-
script numbers indicate nucleotide position.
36M. R. Northam et al.
than two adjacent nucleotides (a frequency of ,3%), a
significantly higher proportion of small indels (21% vs.
rather than transversions among base substitutions.
Taken together, these observations indicate that DRIM
in double- or single-stranded DNA.
It is interesting to compare the mutational signature
of Polz seen in the pol3-Y708A strain with the specificity
a substantial contribution of Polz may be expected. Polz
is responsible for 50–70% of spontaneous mutations in
wild-type yeast strains (Cassier et al. 1980; Quah et al.
1980). Multiple changes within stretches of up to seven
nucleotides were reported to occur at ?10% frequency
in the CAN1 gene (Tishkoff et al. 1997; Ni et al. 1999).
Such changes, however, were not seen by other labora-
tories in spontaneous Canrmutants (Tran et al. 2001;
Huang et al. 2002; Rattray et al. 2002) or in other
reporter systems (Lee et al. 1988; Kang et al. 1992; von
Borstel et al. 1993; Scheller et al. 2000). It is possible
that the contribution of Polz to mutagenesis could vary
depending on the genetic background and/or growth
conditions. Remarkably, a mutational spectrum appar-
ently showing the Polz signature was seen in the strain
carrying another mutant allele of POL3, mut7-1 (von
Borstel et al. 1993). As in the case of pol3-Y708A, the
mut7-1 spectrum shows the predominance of base
substitutions, almost half of which are GC / CG
transversions, a smaller number of frameshifts, and a
sizable proportion of multiple changes within stretches
of up to six nucleotides. This is consistent with our
earlier observation that many mutations in the repli-
some components could lead to the recruitment of Polz
to the primer terminus (Northam et al. 2006).
The high frequency of complex changes during Polz-
dependent DNA synthesis is intriguing. It is clear that
the capacity to make complex mutations lies in Polz
itself, as it generates these mutations at the same
frequency in vitro in the absence of any other proteins
(Zhong et al. 2006). The mechanism could potentially
involve multiple rounds of misinsertion and extension
(Zhong et al. 2006). Most DNA polymerases are very
inefficient in extending primers with even a single
mismatched nucleotide at the 39-end (Pavlov et al.
2006). Polz is a notable exception; it is highly capable of
utilizing primers with a mismatched terminal nucleo-
tide. The ability of Polz to extend primers containing
multiple mismatches, however, was not investigated.
Alternatively, the complex changes could arise through
a relocation of the primer, copying a short DNA se-
quence elsewhere in the genome, and the realignment
of the primer at the original location. A ‘‘misincorpora-
tion slippage’’ model has been proposed to explain the
generation of complex mutations by Polz during the
endogenous DNA damage bypass (Harfe and Jinks-
Robertson 2000). In this model, misincorporation of a
nucleotide opposite a lesion in the template yields an
the adjacent homopolymeric run, resulting in a base
substitution accompanied by a frameshift. By analogy to
this model, a destabilization of the 39 terminus could
result from the frequent stalling of the defective rep-
licative polymerase in our case, which could provide an
opportunity for a temporary relocation of the primer.
Figure 6.—Size of deletions
between short direct repeats in
the CAN1 gene of the pol3-
Y708A strain and its rev3D deriva-
tive. The exact locations of the
deletion breakpoints are given
in Figures 3 and 4.
DNA Polymerase z Copies Undamaged DNA 37
Both misinsertion–extension and relocation mecha-
nisms could potentially contribute to the generation of
complex mutations. Among 12 complex changes in the
by asequence inthe vicinity of the mutation site (Figure
S1). The others could occur by different mechanisms or
involve primer relocation to a more distant site.
The analysis of the DNA sequence context of Polz-
dependent base substitutions suggests that the contri-
bution of Polz to DNA synthesis is not the same during
class of mutations, GC / CG transversions, the
sequence context was significantly different depending
on whether the ‘‘G’’ or ‘‘C’’ was originally present in the
coding or noncoding strand of the CAN1 gene (Table
S7). In 29 of 30 cases (97%) when ‘‘G’’ was the original
coding strand base, the mutations occurred at sequen-
ces containing between two and eight consecutive G?C
pairs. In contrast, when ‘‘C’’ was the original coding
strand base, the surrounding sequence was generally
A?T-rich, with 17 of 25 mutations (68%) at sites having
A?T pairs both 39 and 59 to the ‘‘C.’’ In fact, 40% of all
coding strand ‘‘C’’substitutions occurred at five distinct
TCAA sites (the mutated ‘‘C’’ is underlined), suggesting
that it may be the preferable sequence context for this
orientation of the G?C pair. The seemingly opposite
context preferences for G / C vs. C / G changes may
result from a combination of several factors: There
could be different sequence context requirements for
the formation of G?G vs. C?C mispairs. There could also
be differences in the frequency at which Polz is
recruited to copy the two opposite strands. Finally, the
sequence context preferences for the same Polz-de-
pendent replication error could be different on the two
DNA strands due to, for example, different composition
of the replicating complexes. Further studies using a
system that would distinguish between the replication
be required to distinguish between these possibilities.
Studies of the mechanism of DNA polymerase switch-
ing during TLS led to two models that are not
necessarily mutually exclusive (reviewed recently in
Waters et al. 2009). According to the first model,
stalling of the replicative polymerase at the site of the
lesion leads to the recruitment of TLS polymerases that
then act in the context of the replication fork to bypass
the lesion. Resumption of the accurate processive
replication requires a switch back to the replicative
DNA polymerase, which becomes possible once the
lesion is bypassed. In the second model, once the fork
encounters a lesion, aquick restart of replication occurs
downstream of the lesion, leaving a gap betweenthe site
of the lesionand the site of the restart. TLS polymerases
then bypass the lesion and, possibly, fill the remaining
gap, thus working outside of the replication fork. In
yeast, the electron microscopy and two-dimensional gel
support for the second idea (Lopes et al. 2006). Similar
to the mechanism of TLS, Polz-dependent DNA synthe-
in the context of the replication fork or as a part of the
post-replicative gap filling process. Errors made during
ongoing DNA replication are expected to be corrected
by the DNA mismatch repair (MMR) system. Indeed, we
previously observed that Polz-dependent base substitu-
tion errors in the pol3-Y708A background are corrected
by MMR. The rate of Polz-dependent base substitutions
increases ?30-fold when MMR is inactivated (Pavlov
etal. 2001b).It, however,cannot beexcludedthaterrors
made during the gap-filling synthesis could be pro-
replicative gaps. In this case, the effect of MMR on Polz-
made during the gap-filling synthesis are not corrected.
Further experiments would be required to distinguish
between these possibilities. Additionally, it is possible
that the replisome defects lead to the accumulation of
double-strand breaks (DSB), and the processing of
these breaks could contribute to the increased Polz-
dependent mutagenesis (Holbeck and Strathern
1997). However, the types of mutations seen during
DSB-repair-associated mutagenesis [a large proportion
of frameshifts and very few GC / CG transversions and
complex mutations (Rattray et al. 2002)] is signifi-
cantly different from the mutational spectrum gener-
ated during Polz-dependent DRIM. This argues against
a major contribution of the DSB repair to DRIM.
The observation that an error-prone TLS polymerase
can contribute to the copying of undamaged DNA is
highly important given the possible devastating effects
of mutagenic DNA synthesis on genome stability. In-
Figure 7.—Mutagenesis induced by HU in the wild-type
strain E134 and its rev3 derivative. The data are from Table
S6 and are median mutant frequencies for at least 11 cultures.
The HU-induced mutant frequency shown on the graph was
calculated by subtracting the mutant frequency in the absence
of HU from the mutant frequency in the HU-treated cultures.
The difference between the two strains is statistically signifi-
cant at all HU doses, as indicated by nonoverlapping 95%
confidence intervals (Table S6).
38M. R. Northam et al.
terestingly, the other yeast TLS polymerase, Polh, is
apparently not able to access the primer terminus in the
is impeded due to the intrinsic replisome defects. Polh
has an extremely low fidelity (Matsuda et al. 2000), so
any contribution of this polymerase to the copying of
undamaged DNA would be expected to be highly
mutagenic. Unlike Polz, Polh did not appreciably
contribute to DRIM (Pavlov et al. 2001b). This suggests
that the participation of Polh in replication is more
strictly regulated. In agreement with this idea, over-
production of Polh did not increase the rate of sponta-
neous mutation in yeast or in human cells (Pavlov et al.
2001a; King et al. 2005). An interesting possibility is that
Polz has specifically evolved as a polymerase capable of
not necessarily related to damage in the template DNA.
This would be consistent with the high promiscuity of
Polz for the extension of primer termini that could not
be efficiently used by other DNA polymerases. In this
case, it would be reasonable to expect that the control
over the access of Polz to the primer terminus could
be more relaxed, which would allow it to respond to a
wide variety of situations that slow replication fork
In addition to the pol3-Y708A and pol2-1 strains dis-
cussed in this study, the recruitment of Polz has been
shown to be responsible for a significant proportion
of spontaneous mutations in several other DNA replica-
tion mutants. These include pol3-t mutants with a
temperature-sensitive Pold, dpb3 mutants with the de-
letion of an accessory subunit of Pole, and the pol1-1
As discussed previously, the spectrum of spontaneous
indicative of the involvement of Polz. Although it
appears to be a common mechanism of mutagenesis,
the recruitment of Polz is clearly not the only way by
which a replicative DNA polymerase mutation can pro-
duce a mutator phenotype. In recent years, mutations
were identified that affect the nucleotide selectivity, but
not the catalytic activity of Pola, Pold and Pole (Niimi
et al. 2004; Li et al. 2005; Venkatesan et al. 2006; Nick
mutations are not expected to cause defects in the
replication fork progression that could trigger the re-
cruitment of specialized DNA polymerases. The vast
majority of spontaneous mutations in the correspond-
ing mutants does not require Polz (Suzuki et al. 2009; Y.
I. Pavlov, personal communication) and is thought to
result from inaccurate DNA synthesis by the replicative
DNA polymerases themselves. Similarly, the mutator
phenotype of strains defective in the proofreading
activity of Pold and Pole (Morrison et al. 1991; Simon
et al. 1991) apparently results from the low-fidelity
synthesis by the replicative polymerases (Shcherbakova
et al. 2003; Fortuneetal. 2005) andisnot affected bythe
inactivation of Polz (Shcherbakova et al. 1996). It has
been reported recently that the temperature-sensitive
display a mutator phenotype that is independent of Polz
(Mito et al. 2008). This is in contrast to our earlier
observation that nearly two-thirds of spontaneous muta-
tions in the pol3-t strains require Polz when the cells are
grown at 30? (Northam et al. 2006), the condition at
which pol3-t strains show much more severe growth and
genome stability defects (Gordenin et al. 1991, 1993;
a different strain background as suggested by Mito et al.
(2008), it is likely that the effect of the pol3t mutation on
lead to the recruitment of Polz are not accumulated in
sufficient amounts at 23?, where pol3t strains have no
detectable growth defect. Further studies will be needed
to determine the exact nature and/or level of the
aberrant replication intermediates necessary for the
activation of one or another mutagenesis pathway.
Interestingly, the mutator phenotype of the pol3t
strains observed by Mito et al. (2008) required another
TLS polymerase, Rev1. While the catalytic activity of
Rev1 can operateduring TLS, particularly during abasic
site bypass, the essential role of Rev1 in most forms of
TLS is structural (Waters et al. 2009). Yeast Rev1 and its
mammalian homologs are involved in multiple physical
interactions with other TLS polymerases and the Pol32
subunit of Pold (Acharya et al. 2009; Waters et al.
2009). This led to the idea that Rev1 could provide a
docking site to help exchange different DNA poly-
merases at the replication fork. Binding of Rev1 to Polz
also stimulates the mismatch extension activity of Polz
(Acharya et al. 2006). The TLS function of yeast and
mammalian Rev1, in turn, is regulated by monoubiqui-
tylation of the Lys164 residue of PCNA (Garg and
Burgers 2005; Guo et al. 2006; Wood et al. 2007). Since
we previously established that PCNA ubiquitylation at
Lys164isrequiredfor Polz-dependent DRIM (Northam
et al. 2006), it is plausible that Rev1 could play an
organizing role in this mutagenesis pathway as well.
On the other hand, the structural basis for nucleotide
insertion by Rev1 (Nair et al. 2005; Swan et al. 2009b) is
on undamaged DNA is the insertion of C opposite
responsible for the wide spectrum of base substitutions
and complex mutations observed during DRIM. Experi-
ments are in progress to investigate the role of Rev1 and
other factors involved in TLS, such as Pol32 or cell-cycle
checkpoint proteins (Koren 2007), in Polz-dependent
In addition to Polz-dependent DNA synthesis, the
cells presumably have other ways of tolerating the
replication defects. In fact, the inactivation of Polz in
DNA Polymerase z Copies Undamaged DNA39
growth defect beyond that conferred by the replication
mutation itself (our unpublished data). This suggests
that the replication can proceed in these strains without
the genome-destabilizing mutagenic DNA synthesis by
Polz. Our results suggest, however, that Polz plays an
essential role in preventing larger genome rearrange-
ments in this situation, such as large deletions between
repeated sequences. There is evidence that deletions
between short direct repeats are mediated by DNA
polymerase slippage (Tran et al. 1995). The dramatic
increase in the size of such deletions in the Polz?strain
(Figure 6) is consistent with a hypothesis that longer
stretches of ssDNA could form in the absence of Polz,
which would permit replication slippage over longer
distances. Mammalian cells lacking Polz show an ele-
vated rate of DSB and large chromosomal rearrange-
ments, particularly translocations (Van Sloun et al.
2002; Wittschieben etal.2006). Itwould beinteresting
to determine whether the increase in the size of
deletions in the yeast Polz?cells and the genomic
instability in the mammalian Polz?cells reveal the same
role of Polz in the control of genome stability.
We thank Youri Pavlov for yeast strains and for critically reading the
manuscript, Evelyne Sage and Stas Kozmin for providing DNA
glycosylase mutants, Victoria Liston and Corinn Grabow for technical
rate measurement. This work was supported in part by National
Institutesof Health grants ES011644 and ES015869 and by a Nebraska
Department of Health and Human Services grant LB506 to P.V.S. and
the Blanche Widaman Fellowship to M.R.N.
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