Nucleic Acids Research, 2008, Vol. 36, No. 7Published online 14 February 2008
Eukaryotic Y-family polymerases bypass a
3-methyl-2’-deoxyadenosine analog in vitro
and methyl methanesulfonate-induced DNA
damage in vivo
Brian S. Plosky1, Ekaterina G. Frank1, David A. Berry2, Graham P. Vennall2,
John P. McDonald1and Roger Woodgate1,*
1Laboratory of Genomic Integrity, National Institute of Child Health and Human Development, National Institutes of
Health, Bethesda, MD 20892-3371 and2Berry & Associates, Inc., 2434 Bishop Circle East, Dexter, MI 48130, USA
Received November 9, 2007; Revised January 26, 2008; Accepted January 29, 2008
N3-methyl-adenine (3MeA) is the major cytotoxic
lesion formed in DNA by SN2 methylating agents.
cellular replicases because the N3-methyl group
hinders interactions between the polymerase and
the minor groove of DNA. However, this hypothesis
has yet to be rigorously proven, as 3MeA is
intrinsically unstable and is converted to an abasic
site, which itself is a blocking lesion. To circumvent
these problems, we have chemically synthesized a
3-deaza analog of 3MeA (3dMeA) as a stable
phosphoramidite and have incorporated the analog
into synthetic oligonucleotides that have been used
in vitro as templates for DNA replication. As
expected, the 3dMeA lesion blocked both human
DNA polymerases a and d. In contrast, human
polymerases g, ı and i, as well as Saccharomyces
cerevisiae polg were able to bypass the lesion, albeit
with varying efficiencies and accuracy. To confirm
the physiological relevance of our findings, we show
that in S. cerevisiae lacking Mag1-dependent 3MeA
repair, polg (Rad30) contributes to the survival of
cells exposed to methyl methanesulfonate (MMS)
and in the absence of Mag1, Rad30 and Rev3,
human polymerases g, ı and i are capable of
restoring MMS-resistance to the normally MMS-
DNA is subject to a variety of chemical modifications that
alter its structure. Such alterations can block basic cellular
functions such as transcription and/or replication and can
lead to cell death, mutagenesis and cancer in higher
eukaryotes. One such modification is DNA methylation,
which can be caused by endogenous chemicals, products
of metabolism, environmental exposure or treatment with
several cancer chemotherapeutics. Not surprisingly, cells
have developed several evolutionarily conserved mechan-
isms for repairing or tolerating this type of DNA damage,
including base excision repair (BER), nucleotide excision
repair (NER), recombination and translesion DNA
synthesis (TLS) (1).
Methylating agents primarily react with exocylic nitro-
gen or oxygen atoms on purines and pyrmidines, with the
reaction mechanism (SN1 or SN2) determining the relative
ratio of oxygen to nitrogen modifications (2). The major
products in DNA exposed to SN2 methylating agents are
N7-methylguanine and N3-methyladenine (3MeA), while
there is very little methylation of oxygen atoms on the
bases or the sugar phosphate backbone. 3MeA accounts
for ?20% of the base damage formed by SN2 methylating
agents (2) and is considered to be the major cytotoxic
lesion produced by such chemicals, based on the fact that
bacterial and viral DNA polymerases are blocked before
adenine residues but not guanine, on templates treated
with either SN1 or SN2 methylating agents (3).
3MeA is primarily removed by BER, although NER
appears to provide an important back-up mechanism in
the absence of BER in eukaryotes (4–7). Mouse embryo-
nic fibroblasts (MEFs) lacking Aag, the DNA glycosylase
that normally removes 3MeA from DNA, are sensitive to
methyl methanesulfonate (MMS) and the compound
methyl lexitropsin, which preferentially methylates N3 of
adenine (8). Indeed, Aag?/? cells become arrested in S
phase longer than their wild-type counterparts treated
with either methylating agent, suggesting that the unre-
paired 3MeA residues are a block to replication in vivo.
*To whom correspondence should be addressed. Tel: +1 301 217 4040; Fax: +1 301 217 5815; Email: firstname.lastname@example.org
? 2008 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.
However, it has been extremely difficult to prove that
3MeA blocks replication directly, as the half-life of 3MeA
in vitro is estimated to be between 12 and 24h (9), thereby
precluding biochemical analysis. Furthermore, assuming
3MeA has a similar, or even faster decay in vivo, it seems
likely that by the time the MMS-treated Aag?/? cells
arrest in S phase, a significant portion of the 3MeA
residues would be converted to replication-blocking abasic
sites. The fact that the arrested cells eventually complete
S phase (8) suggests that the replication-block is either
removed by another repair mechanism, or that specialized
DNA polymerases are able to bypass the damaged site.
Several eukaryotic DNA polymerases are capable of
performing TLS. Perhaps the best-characterized eukar-
yotic TLS polymerases are polz, a B-family polymerase
(10,11), and polZ, poli, polk and Rev1, all of which are
Y-family polymerases (12). Based upon structural studies,
the Y-family polymerases appear to be good candidates
to facilitate TLS of 3MeA, since unlike high-fidelity
replicative polymerases, they do not make the same
contacts with N3 of adenine in the minor groove of
duplex DNA (13).
A major obstacle that has to date prevented the study of
3MeA TLS in vitro has been the inherent instability of the
3MeA lesion. To circumvent these problems, we have
synthesized a stable 3-deaza analog of the nucleoside
3-methyl-20-deoxyadenosine that can be incorporated into
synthetic oligonucleotides as 3-deaza-3-methyladenine
(3dMeA). Here, we show that human replicative poly-
merases pola and pold are blocked by 3dMeA, while
human and Saccharomyces cerevisiae Y-family poly-
merases are capable of bypassing the modified base
in vitro. In agreement with our in vitro observations, we
also demonstrate that human DNA polymerases Z, i and
k have the ability to restore MMS-resistance to a
normally MMS-sensitive mag1D rad30D rev3D strain
of S. cerevisiae.
MATERIALS AND METHODS
Ethenoadenosine phosphoramidite was purchased from
Glen Research (Sterling, VA, USA). All oligonucleotides
used for in vitro replication and PCR assays, were
synthesized by Lofstrand Labs Limited (Gaithersburg,
MD, USA) and gel purified prior to use. Ethenoadenine
and 3dMeA bases were incorporated into oligonucleotides
using ultra-mild synthesis conditions.
Human pold (14), GST-poli (15), His-polZ (16) and
S. cerevisiae polz (GST-Rev3/Rev7) (17),were purified as
previously described. Human pola was purchased from
S. cerevisiae polZ and Rev1 protein were purchased
from Enzymax (Lexington, KY, USA). Mouse Aag was
purchased from Trevigen (Gaithersburg, MD, USA).
USA). Human polk,
Synthesis of the3-deaza-3-methyl-dA-phosphoramidite
A detailed protocol outlining the chemical synthesis of the
3-deaza-3-methyl-dA-phosphoramidite is available online
as Supplementary Data.
Invitro Aagexcision assay
To measure DNA glycosylase activity on various sub-
strates, 50-[32P] 29mer, 50-GCT CGT CAG ACG ATT
TAG AGT CTG CAG TG-30
ethenoadenine or 3dMeA underlined and in bold font),
was annealed to its complementary strand. Double-
stranded DNA of 0.4pmol was treated with 3U of
mAag or mock treated for 1h at 378C. NaOH was added
to a final concentration of 100mM along with 10mM
Tris, 1mM EDTA (final) and the samples were incubated
at 378C to cleave any resulting abasic sites. Samples were
resolved on a 15% gel (8-M urea) and visualized with a
Molecular Dynamics phosphorimager and ImageQuant
(with the adenine,
In vitro replication assays were performed using the 29mer
oligonucleotide 50-GCT CGT CAG ACG ATT TAG
AGT CTG CAG TG-30as a template (with the location of
the undamaged adenine, or 3dMeA underlined and in
bold font). For most experiments described herein, this
template was annealed to a [32P]-labeled 16mer primer
with the following sequence; 50-CAC TGC AGA CTC
TAA A -30. For the extension assays reported in Table 3,
the [32P]-labeled primer was a 17mer with the sequence;
50-CAC TGC AGA CTC TAA AX -30, where X is either
A, or T. Primer-template DNAs were prepared by
annealing the 50[32P]-labeled primer to the unlabeled
template DNA at a molar ratio of 1:1.5. Standard 10-ml
reactions contained 40mM Tris–HCl at pH 8.0, 5mM
MgCl2, 100mM of each ultrapure dNTP (Amersham
Pharmacia Biotech, NJ, USA), 10mM DTT, 250mg/ml
BSA, 2.5% glycerol and 10nM primer/template DNA.
The concentration of polymerase added varied and is
given in the legends to figures 3, 4, 5 and 7. After
incubation at 378C (or 308C for yeast enzymes) for 5min,
reactions were terminated by the addition of 10ml of 95%
formamide/10mM EDTA and the samples heated to
1008C for 5min and briefly chilled on ice. Reaction
mixtures (5ml) were resolved on 15% polyacrylamide, 8M
urea gels and analyzed with a Molecular Dynamics
phosphorimager and ImageQuant software.
For steady-state kinetic reactions, each polymerase was
assayed to determine the amount of enzyme and nucleo-
tide that would result in <20% incorporation (18,19):
0.4U/reaction for pola, 1.2nM for human polZ, 1.8nM
for poli, 1.5nM for polk and 1.4nM for S. cerevisiae
polZ. All reactions were performed in 10ml in the standard
reaction buffer described earlier, except those involving
poli, where the concentration of magnesium chloride was
reduced from 5 to 0.25mM. Reactions were initiated by
the addition of the dNTP and lasted for 1.5–5min for the
Nucleic Acids Research, 2008, Vol. 36, No. 72153
correct nucleotides and 5–10min for incorrect nucleotides,
depending on the polymerase. On unmodified templates,
dNTP concentrations ranged from 0.01 to 100mM for the
correct dTTP and from 1 to 500mM for the incorrect
dNTPs. For Y-family polymerases on the 3dMeA contain-
ing template, dTTP concentrations ranged from 0.1mM to
1mM while incorrect dNTPs ranged from 10mM to 1mM
(except for poli reactions where dATP ranged from 2 to
100mM, while dGTP and dCTP ranged from 10 to
300mM). For the data shown in Table 3, dCTP
concentrations varied from 0.2 to 10mM on the unda-
maged template and from 10 to 300mM for 3dMeA-
containing template. For pola with the 3dMeA-containing
template, dATP and dTTP were varied from 0.1 to 1mM.
Replication products were separated on 15% polyacryl-
amide gels containing 8-M urea and visualized with a
Molecular Dynamics phosphorimager and quantified with
The apparent Vmaxand Kmvalues for each enzyme and
nucleotide were determined from a Hanes–Woolf plot by
linear least-squares fit as described previously (18). The
catalytic efficiency of nucleotide insertion was calculated
as the ration of Vmax/Kmand the frequency of misinsertion
was calculated as (Vmax/Km)incorrect/(Vmax/Km)correct as
described previously (18) using SigmaPlot software
(SPSS, Chicago, USA).
Generationof yeast strains and plasmids
All yeast strains were derived from the W303 background
(20). MAG1 was disrupted by PCR amplification of the
URA3 gene from pRS416 using primers with 40nt of
homology to upstream and downstream of MAG1
(MagUraF, 50-ATG AAA CTA AAA AGG GAG TAT
GAT GAG TTA ATA AAA GCA GCA GAG CAG
ATT GTA CTG AGA GTG C-30and MagUraR, 50-TTA
GGA TTT CAC GAA ATT TTC TTC TGC CTT CAT
CAT GGC AGC GGT ATT TTC TCC TTA CGC-30)
and transformed into C10-15a (W303 RAD5+mata)
(20). Positive disruptants were confirmed by PCR and
MMS sensitivity. The mag1D haploid strain was mated to
C10-10a, in order to obtain the mag1D rad30D double
mutant (BPC1-4d) and a backcrossed mag1D (BPC1-2a)
strain. BPC1-4d (mag1D::URA3 rad30D:HIS3 mata) was
mated with C17-1A (rev3D:HisG-URA3 mata) to obtain
mag1D rev3D (BPC2-8c), rad30D rev3D (BPC2-5a) double
mutants and the mag1D rad30D rev3D (BPC2-13c) triple
mutant. Since MAG1 and REV3 disruptions were both
marked by the URA3 gene, all strains genotypes were
confirmed by PCR for these two genes by triplex PCR
with the following reverse primer for URA3 (URA3_44R;
50-ACT AGG ATG AGT AGC AGC ACG-30) and
forward andreverse primers
(MAG1_95upF; 50-TGG CCA CTG CCC TCT GAT
ATG-30and MAG1_298R; 50-CTT GGC CAC TGA TCT
GTT GAG-30) or REV3 (REV3_355upF; 50-ACC ATT
GTC CAA AGC TGT CGC-30and REV3_223R; 50-ACG
TGG CAC AAT ACT TGA TGC C-30).
Plasmids expressing human and S. cerevisiae Y-family
(Stratagene, La Jolla, CA, USA). POLI was cloned by
digesting p6-1 (21) with NcoI, filling in the overhang with
Klenow fragment, followed by digestion with AvaI and
subsequent cloning into the SmaI site of pESC-LEU to
generate pBP65. POLH was cloned as a NotI–BamHI
fragment from pCDNA-XPV (22) into pESC-LEU
digested with NotI and BglII to generate pPB66. POLK
was cloned into pESC-LEU by first digesting pBP65 with
NcoI, filling the ends with Klenow fragment to blunt end
and subsequently digesting the vector with XmaI. An
EcoRV–XmaI fragment from pHSE2 (a kind gift from
Haruo Ohmori, University of Kyoto, Japan), encoding
POLK was subsequently cloned into the vector to generate
pBP98. Saccharomyces cerevisiae RAD30 was cloned as an
NcoI–PstI fragment from pJM231 into the similarly
digested plasmid, pBP65, to generate pBP82.
MMS toxicity for each genotype was assessed on over-
night cultures. Yeast were harvested and washed twice
with PBS. MMS was diluted to 0.25% in PBS and aliquots
of each strain were removed at selected time intervals,
washed with PBS and diluted for plating on YPAD agar
plates. Colonies were counted after 5 days at 308C. For the
rad30D rev3D) was transformed with pBP65 (expresses
human poli), pBP66 (expresses human polZ), pBP98
(expresses human polk), pBP82 (expresses S. cerevisiae
polZ) or pESC-LEU. Yeast strains were cultured over-
night in complete synthetic raffinose medium lacking
L-leucine. One hour prior to MMS treatment, the cultures
were harvested by centrifugation and transferred to
synthetic galactose medium to induce the expression of
polymerases. Cells were harvested and treated as described
above, except dilutions of each culture were plated on
synthetic galactose agar plates lacking L-leucine.
3dMeA is astableanalog of N3-methyladenine
3-Methyl adenosine is unstable in vitro with an estimated
half-life of just 12–24h (23). This short half-life has
therefore limited biochemical or enzymatic studies on the
lesion. To circumvent these problems, we have synthesi-
zed a 3-deaza-3-methyl-20-deoxyadenosine
3-methyl-20-deoxyadenosine. The 3-deaza- analog has the
same overall structure as the naturally occurring adduct
(Figure 1A), but it lacks the positive charge associated
with the N3 atom that normally destabilizes the glycosidic
bond, and is therefore very stable. The analog can be
synthesized as a phosphoramidite (Figure 1B) and can be
incorporated into oligonucleotides by standard chemical
Since 3MeA is excised from DNA by the alkyladenine
DNA glycosylase (Aag) (24), we determined if 3dMeA is
also a substrate for Aag by treating either unmodified
duplex DNA or DNA containing 3dMeA, or ethenoade-
nine (eA) with purified mouse Aag followed by hydroxide
treatment. eA is a well-characterized substrate for Aag
(25) and as noted in Figure 2, is completely excised from
the substrate, as all of the eA oligonucleotide is cleaved
Nucleic Acids Research, 2008, Vol. 36, No. 7
at the resulting abasic site, by hydroxide treatment
(Figure 2). In contrast, the 3dMeA containing DNA
shows relatively little cleaved substrate, and there is no
detectable cleavage product in the unmodified control.
This demonstrates that Aag can excise 3dMeA, but to a
much lesser extent than eA and presumably the naturally
Treatment with NaOH in the absence of Aag confirms
that 3dMeA analog is indeed stable and that even boiling
of the DNA to anneal the lesion containing strand to its
complementary strand, did not result in abasic sites that
could be subsequently hydrolyzed by treatment with
NaOH. We suspect that the 3dMeA analog may not be
because of its stabilized glycosidic bond. Indeed, it has
been proposed that the weakened glycosidic bond of
several Aag substrates may facilitate excision by the
3dMeA is astrongkinetic blockto replicative
polymerases, but isbypassed by Y-family polymerases
To date, there has been no direct evidence of 3MeA
blocking a replicative DNA polymerase. We therefore
compared human pola and pold to polZ, poli, and polk in
the presence of the four standard deoxynucleotides
to determine which enzymes were capable of replicating
a template containing 3dMeA. Under standard reaction
conditions and with an undamaged template, each
polymerases utilizes ?10–20% of the primer (Figure 3A,
left), however, virtually no extension of the primer
annealed to the 3dMeA-containing template was observed
in the presence of pola and pold, indicating that the lesion
is a strong kinetic block to replicative polymerases. By
comparison, both incorporation and bypass of the lesion
was observed in the presence of human polZ, i or k
(Figure 3). Steady-state kinetic analyses revealed that
incorporation of T opposite the 3dMeA lesion only
occurred with an efficiency of 0.15–3% of that opposite
an undamaged A (Table 1). However, when one compares
the catalytic activity (Vmax/Km) of the Y-family enzymes
ability to incorporate opposite the 3dMeA lesion, it is 125-
to 1200-fold more efficient than the incorporation by pola
(Table 1) (full kinetic parameters are supplied as
Very recently, we discovered that the catalytic activity
of poli in vitro is dramatically enhanced in the presence
of low concentrations of Mg2+or Mn2+(27). Indeed,
poli-dependent incorporation opposite the 3dMeA lesion
increased significantly when comparing primer extension
in 0.25mM versus 5mM MgCl2and lesion bypass was
greatly stimulated in the presence of 0.25mM MnCl2
(Figure 4). Similar to studies with other B-family
polymerases (28), low levels of Mn2+also appeared to
Figure 1. Synthesis ofasynthetic 3-deaza-3-methyl-dA phosphoramidite.
(A) Chemical structures of 3-methyl-20-deoxyadenosine and 3-deaza-
3-methyl-20-deoxyadenosine. Replacement of the N3 with carbon
removed the positive charge and helps stabilize the glycosidic bond.
(B) Schematic of the synthesis of the 3-deaza-3-methyl-dA phosphor-
amidite (i) Di-benzoylation of 3-deaza-dA using benzoyl chloride in
(2-cyanoethyl)phosphoramidic chloride and diisopropylethylamine in
Figure 2. 3-deaza-3-methyl adenine is a stable analog of 3MeA. Mouse
alkyladenine glycosylase (mAag) excises both 3-methyladenine (3MeA)
and ethenodeoxyA (eA). 0.4pmol of undamaged, 3dMeA- or eA-
containing DNA was treated with 3U of mAag, or mock treated for
1h at 378C. To hydrolyze the resulting abasic sites, NaOH was added
to a final concentration of 100mM along with 10mM Tris, 1mM
EDTA (final) and the samples were incubated at 378C. Samples were
resolved on a 15% polyacrylamide gel containing 8-M urea. The
nucleotide sequence of the 29mer duplex DNA is shown at the top
of thepanel andtheposition
29mer oligonucleotide and 12mer product are shown on the right
side of the gel.
Nucleic Acids Research, 2008, Vol. 36, No. 7 2155
stimulate human pold’s activity in the primer extension
assays with the undamaged template, as well as enable a
small amount of incorporation and extension beyond the
3dMeA lesion (Figure 4).
Next, we examined the single nucleotide insertion
profile opposite the 3dMeA lesion promoted by polZ,
poli and polk (Figure 5) and discovered that both polZ
and poli are error-prone, in that they readily misincorpo-
rate A opposite the 3dMeA lesion. Indeed, the ability of
both polymerases to incorporate A opposite 3dMeA is
consistent with the increase in A:T to T:A transversions
observed in vivo in mice exposed to MMS (29). In
contrast, polk appears to be fairly accurate, as it primarily
inserts T opposite 3dMeA. Analysis of the steady-state
kinetics for each enzyme (Table 2) reflects the results
shown in Figure 5. Poli and polZ misinsert A opposite
3dMeA with a frequency of 0.46 and 0.48 relative to
incorporationof the correctbase T,respectively.
In contrast, polk is 10-fold more accurate than either
polZ or poli and misincorporates A opposite 3dMeA with
a frequency of 0.04. Each polymerase appears to have
higher than expected efficiency of inserting C, but this may
simply occur as a consequence of the local sequence
context, since the next 50template base is a G. While at
first glance all three of the Y-family polymerases appear to
be error-prone, they are, in fact, more accurate than pola,
which actually misincorporates A opposite 3dMeA 4-fold
better than T, in the steady-state assays (Table 2).
Finally, we examined the ability of polZ, poli and polk
to extend from a base paired with 3dMeA. The primer
terminus was either a ‘correctly’ paired T:3dMeA, or was
an A:3dMeA mispair (Table 3). Both polZ and polk
extended the correctly paired T:3dMeA primer terminus
relatively well and did so with an efficiency of ?8–10%
of that compared to a normal T:A base-pair (Table 3).
In contrast, poli only extended the T:3dMeA primer with
Figure 3. Ability of human DNA polymerases to bypass 3-deaza-3-
methyl adenine in vitro. Standard reactions contained 100mM all 4 dNTPs
and lasted for 5min at 378C. Reactions contained 0.2U pola, 5nM pold,
3nM polZ, 1.5nM poli and 3nM polk. The nucleotide sequence of the
template DNA is shown on the left-hand side of the gel. The ‘A’ in bold
font is either undamaged (left-hand panel) or 3-deaza-3-methyl adenine
(3dMeA; right-hand panel). As clearly seen, the 3dMeA lesion is a strong
block to replication by human DNA polymerases a and d, but can be
bypassed by human polymerases Z, i and k.
Table 1. Efficiency of insertion of T opposite undamaged A, or 3dMeA by various eukaryotic polymerases
TemplateVmax/Km(mM?1min?1) TemplateVmax/Km(mM?1min?1)Efficiency of insertionb
Efficiency of insertionc
A3.43dMeA 0.0002 5.88?10?5
A13.73 3dMeA 0.025
aHs, Homo sapien; Sc, S. cerevisiae.
bInsertion opposite 3dMeA relative to the insertion opposite an undamaged A.
cVmax/Kmopposite 3dMeA relative to the Vmax/Kmopposite 3dMeA by Hs pola.
dIn the presence of 0.25mM MgCl2.
Figure 4. Ability of human DNA polymerases d and i to bypass
3-deaza-3-methyl adenine in the presence of low Mg2+/Mn2+in vitro.
Standard reactions contained 100mM all 4 dNTPs and lasted for 5min
at 378C. Reactions contained 5nM pold and 4nM poli. The nucleotide
sequence of the template DNA is shown on the left-hand side of the
gel. The ‘A’ in bold font is either undamaged (left-hand panel) or
3-deaza-3-methyl adenine (3dMeA; right-hand panel). Track 1, No
dNTPs; Track 2, 0.25mM MgCl2; Track 3, 5mM MgCl2; Track 4,
0.25mM MnCl2. As clearly seen, the 3dMeA lesion is a strong block to
replication by human DNA polymerases d even in the presence of Mn.
In contrast, poli-dependent incorporation opposite the lesion is
stimulated by 0.25mM MgCl2 and significant bypass is observed in
the presence of 0.25mM MnCl2.
Nucleic Acids Research, 2008, Vol. 36, No. 7
an efficiency of about 4% relative to an undamaged base-
pair. Both poli and polk extended the A:3dMeA mispair
?3- to 4-fold less efficiently than the T:3dMeA base-pair.
In contrast, human polZ actually extended the A:3dMeA
mispair slightly better than the correctly paired T:3dMeA
Saccharomyces cerevisiae polgisimportant intolerating
MMS-induced damage inthe absence ofMAG1
Based on our in vitro findings with the human Y-family
polymerases, we were eager to determine if Y-family
polymerases play a role in tolerating 3MeA in vivo. We
chose to use S. cerevisiae as a model because it has a
limited number of DNA polymerases compared with
higher eukaryotes. Saccharomyces cerevisiae polZ is
encoded by the RAD30 gene, and it is reported that
rad30D strains are somewhat sensitive to MMS (30,31).
However, in the W303 background, a RAD30 disruption
is not sensitive to MMS (Figure 6A). Mag1 is the only
DNA glycosylase that repairs 3MeA in S. cerevisiae and
disruption of the MAG1 gene makes yeast highly sensitive
to methylating agents, such as MMS. In order to
determine if S. cerevisiae polZ is involved in tolerating
lesions normally repaired by Mag1 (i.e. 3MeA), we
generated a rad30D mag1D strain. Interestingly, the
double mutant is more sensitive to MMS than the
mag1D strain, suggesting that polZ may help facilitate
bypass of persisting 3MeA lesions in vivo (Figure 6A).
Previous studies have shown that polz is responsible for
most MMS-induced mutagenesis (32,33), and deletion of
REV3 (encoding the catalytic subunit of polz) further
sensitizes mag1D strains to MMS (Figure 6B). Therefore,
it is possible that both polZ and polz are important for
survival after treatment with MMS in a mag1D back-
ground (Figure 6B). Indeed, the triple mutant is sig-
nificantly more sensitive than either double mutant
(Figure 6B). This suggests that polz and polZ act in
independent repair pathways to tolerate unrepaired base
damage caused by MMS. Similar observations and
conclusions were recently drawn by Johnson et al. (34).
To determine which TLS polymerases in S. cerevisiae
are capable of bypassing unrepaired 3MeA, we assayed
the ability of S. cerevisiae polZ, polz, Rev1 and polz in
conjunction with Rev1 to bypass 3dMeA in vitro
(Figure 7). Similar to human polZ, S. cerevisiae polZ
In contrast, polz exhibits a much weaker ability to
bypass the lesion (Figure 7, left-hand panel). Rev1, a
dCMP transferase that is necessary for the function of
Figure 5. Ability of human DNA polymerases Z, i and k to
(mis)incorporate opposite 3-deaza-3-methyl adenine. Standard reactions
contained 100mM all 4 dNTPs (4), or each nucleotide separately (G, A,
T, C) and lasted for 5min at 378C. Reactions contained 4nM polZ,
6nM poli and 4nM polk. The nucleotide sequence of the template
DNA is shown on the left-hand side of the gel. The ‘A’ in bold font
indicates the location of the 3dMeA lesion.
Table 2. Fidelity of nucleotide insertion of opposite 3dMeA by various
aHs, Homo sapien; Sc, S. cerevisiae.
bIn the presence of 0.25mM MgCl2.
Table 3. Kinetics of single nucleotide extensiona
(3dMeA) by Y-family DNA polymerases
aIncorporation of C opposite undamaged G.
bHs, Homo sapien; Sc, S. cerevisiae.
cIn the presence of 0.25mM MgCl2.
Nucleic Acids Research, 2008, Vol. 36, No. 72157
polz in vivo, also has minimal activity on either the
undamaged A-template, or 3dMeA-containing template,
as well as having little, to no stimulatory effect, on polz’s
ability to bypass the 3dMeA lesion. Rev1 is clearly
catalytically active under our assay conditions, as the
enzyme is able to insert C opposite an undamaged
template G, as well as further stimulate polz activity
(Figure 7, right-hand panel). Our in vitro data, combined
with the MMS sensitivities of the mag1D rad30D and the
mag1D rad30D rev3D strains therefore supports the idea
that polZ replicates past unrepaired 3MeA lesions in the
absence of Mag1.
Human Y-family polymerases rescue theMMS sensitivity
of mag1Drad30D rev3Dstrains of S. cerevisiae
The enhanced MMS sensitivity of the mag1D rad30D
rev3D strain gave us an opportunity to test the ability of
human polZ, poli and polk to bypass alkylation damage
in vivo. When each human polymerase (as well as
S.cerevisiae polZ, as a control), was expressed from a
galactose inducible promoter in the triple mutant, we
discovered that all of the human polymerases rescued the
MMS-sensitivity of the mag1D rad30D rev3D strain, albeit
to varying degrees (Figure 8). Quite remarkably, expres-
sion of human polk confers MMS resistance on the mag1D
rad30D rev3D strain to the same extent as overproducing
S. cerevisiae polZ. Both human polZ and poli also confer
MMS-resistance, but to a lesser degree than human polk
or S. cerevisiae polZ.
In some regard, it is really quite amazing that the
human polymerases are able to confer MMS-resistance in
the heterologous yeast survival assay, given the myriad of
protein interactions that are believed to be required for the
activity of the polymerases in vivo (35). Clearly, most of
these protein–protein interactions must be conserved
throughout evolution for the human polymerases to be
able to function in S. cerevisiae. However, it is unlikely
that these protein–protein interactions occur with the
same efficiency in the heterologous system, and as a result,
it is possible that the ability of human poli to restore
MMS-resistance in S. cerevisiae is compromised by
weakened protein–protein interactions with S. cerevisiae’s
TLS accessory proteins. The same cannot be said of
human polZ’s inability to restore MMS-resistance to the
same extent as S. cerevisiae polZ, as human polZ has
previously been shown to fully complement the UV-
sensitivity attributed to a polZ-deficiency in a rad52D
rad30D S. cerevisiae strain (36).
We have described a novel procedure for the synthesis of
a phosphoramidite that is a stable 3-deaza analog of
3-methyl-20-deoxyadenosine (Figure 1). By using this
analog in replication assays, we provide the most direct
evidence currently available that 3MeA is a significant
block to two of the three main replicases in eukaryotes,
namely pola and pold. Furthermore, we demonstrate that
three human Y-family polymerases (polZ, poli and polk)
are capable of insertion opposite the 3dMeA lesion, as
Figure 6. Survival of S. cerevisiae exposed to MMS. Exponentially
growing strains of S. cerevisiae were exposed to 0.25% MMS for 10, 20
or 30min, washed and subsequently plated on YPAD for 5 days at
308C. (A) Disruption of REV3 makes S. cerevisiae mildly sensitive to
MMS, but disruption of RAD30 has no observable effect on MMS
sensitivity. (B) Disruption of MAG1 sensitizes S. cerevisiae to MMS,
and disruption of RAD30 sensitizes the strain to MMS, indicating that
the Rad30 encoded polZ helps protect S. cerevisiae from lesions that
are normally repaired by Mag1. (C) Disruption of REV3 also sensitizes
a mag1D strain to MMS, and the disruption of both RAD30 and REV3
synergistically enhances the lethality of MMS, indicating that polZ and
polz may operate in separate pathways to repair lesions normally
removed from the genome by Mag1. Three independent isolates were
tested for each strain and standard deviations, which were all below 1
log of survival have been omitted for clarity.
Nucleic Acids Research, 2008, Vol. 36, No. 7
well as extension beyond the modified base (Figures 2–4),
with polk being the most accurate and polZ the most
efficient in vitro (Tables 1 and 2). Similarly, S. cerevisiae
polZ bypassed the 3dMeA lesion with the greatest
efficiency of the Y-family polymerase assayed (Table 1),
whilst polz showed little ability to traverse the lesion
in vitro (Figure 6A). Human polZ, S. cerevisiae polZ and
human polk, all extended bases incorporated opposite
3dMeA with an efficiency of 8–14% relative to an
undamaged primer terminus (Table 3). Human polZ did
not discriminate between a correctly paired, or mispaired
S. cerevisiae polZ both preferred to extend the correctly
paired T:3dMeA primer terminus 3- to 4-fold better then
the A:3dMeA mispair. Poli extended the T:3dMeA base-
pair poorly, but like human polk and S. cerevisiae polZ
preferred the correctly paired primer terminus over the
Our kinetic data on the ability of human pols Z, i and k
to misinsert bases opposite a 3dMeA lesion in vitro does
not agree well with a recent report in which 3MeA was
modeled into the active site of the respective enzymes (34).
Based upon molecular modeling it was hypothesized that
polk and polZ should be able to insert a base opposite the
3MeA lesion equally as well as opposite an undamaged
base. However, while significantly better than human
pola, human polk and polZ inserted a base opposite
3dMeA with an efficiency of ?0.2–2% of that opposite an
undamaged base, suggesting some steric hindrance of the
3dMeA lesion in the active site of the respective Y-family
enzymes. Similarly, it was also hypothesized that poli
should be able to extend a T:3MeA base pair efficiently
(34), but in our hands, this only occurred with an
efficiency of about 4% of that of an undamaged base pair.
To examine the role of Y-family polymerases in
tolerating 3MeA in vivo, we utilized strains of S. cerevisiae
that carried a Rad30 (polZ) deletion. At least in the wild-
type W303 and CL1265-7C backgrounds (data for
CL1265-7C not shown), the absence of polZ did not
appear to render the strain sensitive to MMS (Figure 6A).
Figure 7. Ability of S. cerevisiae DNA polymerases to bypass 3-deaza-3-methyl adenine in vitro. Standard reactions contained 100mM all 4 dNTPs
and lasted for 20min at 308C. Reactions contained 4nM polZ, 6nM polz and 15nM Rev1. The nucleotide sequence of the DNA templates is shown
on the left-hand side of each gel. Left-hand panels: the ‘A’ in bold font is either undamaged adenine or 3-deaza-3-methyl adenine. As can be seen,
polZ bypasses the 3dMeA lesion much more efficiently than polz. Rev1 has negligible activity on either template, and did not appreciably stimulate
the activity of polz on the 3dMeA template. Right-hand panels: both Rev1 and polz are catalytically active, as they are able to incorporate dCMP
opposite undamaged G. Rev1 also stimulates polz in the presence of dCTP alone as well as in the presence of all 4 dNTPs.
Figure 8. Human Y-family polymerases can restore MMS-resistance to
a normally MMS-sensitive rad30D rev3D mag1D strain of S. cerevisiae.
Exponentially growing strains of S. cerevisiae harboring plasmids
expressing human polymerases Z, i and k or S. cerevisiae polZ under
the control of a galactose inducible promoter were induced in complete
synthetic galactose media without leucine. Media was removed, and
cells were washed and exposed to 0.25% MMS for 10, 20 or 30min,
washed and subsequently plated on complete synthetic galactose plates
lacking leucine for 5 days at 308C. Three independent isolates were
tested for each strain and standard deviations, which were all below
1 log of survival, have been omitted for clarity. As clearly seen, human
polZ and i can restore MMS-resistance to the normally MMS-sensitive
strain, but the greatest effect was observed with human polk, which was
as efficient as native S. cerevisiae polZ in restoring MMS-resistance.
Nucleic Acids Research, 2008, Vol. 36, No. 72159
However, in the S288C background, a mild sensitivity
has been previously reported (37). Since a large number
of genes are known to be important for tolerating MMS in
yeast (31), it is possible that subtle genetic differences
between the W303 and the S288C backgrounds might
account for the discrepancy between our observations and
the data reported by others. Thus, despite the fact that
S. cerevisiae polZ bypasses the 3dMeA lesion in vitro
(Figure 7), it does not appear to play a primary role in
protecting wild-type cells from the cytotoxic effects of
alkylation damage in vivo. Presumably such observations
can be explained by the fact that 3MeA is not only
intrinsically labile, but is efficiently removed from the
genome by the Mag1 glycosylase (38). Interestingly,
S. cerevisiae strains lacking both Mag1 and polZ are
significantly more sensitive to the cytotoxic effects of
MMS than a wild-type strain (Figure 6B). We believe that
such observations reveal an important role for polZ in the
bypass of persisting 3MeA lesions in vivo. It is also possible
that the increased MMS-sensitivity may be partially due to
a requirement for polZ-dependent bypass of abasic sites.
However, previous studies indicate that polZ has limited
ability to traverse an abasic site in vitro (39) and as a
consequence, it is believed that polZ plays only a minor
role in the bypass of abasic site in vivo (40).
While polz showed little ability to bypass the 3dMeA
lesion in vitro, a rev3D strain nevertheless exhibited mild
MMS-sensitivity in vivo (Figure 6A) (41). However, it
should be noted that the strain is proficient for Mag1 and
it is conceivable that the MMS sensitivity is actually due
to an inability to bypass abasic lesions generated through
the actions of the Mag1 glycoslyase, rather than defects in
the bypass of 3MeA (32,33). The idea that polZ and polz
potentially act in two separate pathways to facilitate
bypass of 3MeA and abasic sites, respectively, is
supported by the fact that the mag1D rad30D rev3D
triple mutant is considerably more MMS-sensitive than
either the mag1D rad30D or mag1D rev3D strains
The enhanced MMS sensitivity of the mag1D rad30D
rev3D triple mutant allowed us to assay the role of human
polZ, poli and polk in the tolerance of alkylation damage
in vivo. While both expression of polZ and poli increased
MMS-resistance that was only rivaled by overexpression
of endogenous S. cerevisiae polZ (Figure 8). We believe
that our data reflects how these enzymes might participate
in the tolerance of alkylation damage in higher eukar-
yotes. Indeed, polk appears to be important for survival
after MMS exposure in both polk-deficient MEFs and in
polk-deficient DT40 chicken lymphoblasts (42), and in
both cases, it is assumed that the BER pathway in these
cell lines remains fully functional. Furthermore, the role
for polk/polIV-like polymerases in tolerating cellular
alkylation damage appears to be well conserved through-
out evolution, as it has recently been reported that
Escherichia coli dinB (polIV)-deficient strains are con-
siderably more sensitive to MMS damage than wild-type
The role of polZ in tolerating MMS-induced lesions
in vivo appears less clear. PolZ appears to be important for
budding yeast to tolerate MMS-induced damage, but only
in the absence of BER (Figure 6A), and a similar situation
may arise in human cells. Individuals with the variant
form of Xeroderma Pigmentosum (XP-V) lack functional
polZ, and are susceptible to sunlight-induced skin cancer
and while cells from these individuals are mildly sensitive
to ultraviolet light, they are not sensitive to methylating
agents, such as MMS (44).
The role of poli in the TLS of alkylation damage in
mammals remains enigmatic. The Poli gene in the
129-derived inbred strain of mice has a stop codon in
the second exon, effectively making the mice homozygous
Poli(?/?) (45). Mice and embryonic stem cell lines derived
from 129-derived strains are widely used in the study of
DNA repair and mutagenesis, and appear to have no
obvious sensitivity to methylating agents. However, it is
possible that several ‘knockout’ mice generated using
129-derived embryonic stem cells could be ‘double knock-
outs’ for both Poli and the target gene of interest (46,47).
Of direct importance to our current study, is the fact that
two separate groups generated Aag(?/?) mice and cell
lines from 129-derived embryonic stem cells (29,48).
Interestingly, there were differences between the two
studies in the sensitivity of the mice and MEFS to various
alkylating agents. Elder et al., found that the Aag(?/?)
primary fibroblasts exhibited mild sensitivity to MMS, but
not bischroroethyl nitrosourea or mitomycin C, while
Engelward and colleagues, who generated homozygous
Aag(?/?) cells directly from a 129-derived embryonic
stem line, observed that the MEFS were hypersensitive
to MMS, bischloroethyl nitrosourea and mitomycin
C. Essentially, the cells used by Elder et al. (29), were
Aag(?/?) while those used by Engelward et al. (48), were
likely to have been Aag(?/?), Poli(?/?). A subsequent
study by Sobol and colleagues (49) found that cells
independently derived from Aag(?/?) were not sensitive
to MMS at all. Thus, subtle genotypic strain differences
could readily account for the various phenotypes. As a
consequence, it will be interesting to assay the MMS
sensitivity of congenic C57Bl6-derived mice lacking Aag
and Poli, to determine if poli plays a role in protecting
mammalian cells from alkylation-induced DNA damage.
Supplementary Data are available at NAR Online.
This work was supported by funds from the NICHD/NIH
Intramural Research Program. We thank Alexandra
Vaisman for help preparing the figures and Haruo
Ohmori for kindly providing the polk plasmid, pHSE2.
Funding to pay the Open Access publication charges for
this article was provided by the NICHD/NIH Intramural
Conflict of interest statement. None declared.
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