DNA polymerase minor groove interactions
modulate mutagenic bypass of a templating
Bret D. Freudenthal, William A. Beard and Samuel H. Wilson*
Laboratory of Structural Biology, National Institute of Environmental Health Sciences, NIH, PO Box 12233,
Research Triangle Park, NC 27709-2233, USA
Received September 5, 2012; Revised November 5, 2012; Accepted November 7, 2012
A major base lesion resulting from oxidative stress is
8-oxo-7,8-dihydro-20-deoxyguanosine (8-oxoG) that
has ambiguous coding potential. Error-free DNA syn-
thesis involves 8-oxoG adopting an anti-conform-
ation to base pair with cytosine whereas mutagenic
bypass involves 8-oxoG adopting a syn-conformation
to base pair with adenine. Left unrepaired the syn-8-
oxoG/dAMP base pair results in a G–C to T–A
transversion. During base excision repair of this
mispair, DNA polymerase (pol) b is confronted with
gap filling opposite 8-oxoG. To determine how pol b
discriminates between anti- and syn-8-oxoG, we
introduced a point mutation (R283K) to alter insertion
specificity. Kinetic studies demonstrate that this sub-
stitution results in an increased fidelity opposite
8-oxoG. Structural studies with R283K pol b show
that the binary DNA complex has 8-oxoG in equilib-
rium between anti- and syn-forms. Ternary com-
plexes with incoming dCTP resemble the wild-type
enzyme, with templating anti-8-oxoG base pairing
with incoming cytosine. In contrast to wild-type pol
b, the ternary complex of the R283K mutant with an
incoming dATP-analogue and templating 8-oxoG re-
sembles a G–A mismatched structure with 8-oxoG
adopting an anti-conformation. These results dem-
onstrate that the incoming nucleotide is unable to
induce a syn-8-oxoG conformation without minor
groove DNA polymerase interactions that influence
templating (anti-/syn-equilibrium) of 8-oxoG while
Reactive oxygen species (ROS) are generated during
normal metabolic processes and their levels increase in
response to environmental stress. Exposure of DNA to
ROS results in oxidative DNA damage and the generation
of multiple types of DNA lesions. Left unrepaired these
lesions contribute to mutagenesis, cancer and human
disease (1). One of the most abundant lesions caused by
oxidative DNA damage in both the DNA and nucleotide
pools is 8-oxo-7,8-dihydro-20-deoxyguanosine (8-oxoG).
At physiological pH, 8-oxoG has a carbonyl at C8 and
is protonated at N7 (2). These modifications to guanine
lead to additional hydrogen bonding interactions at the
Hoogsteen edge and promote rotation about its glycosidic
bond thereby encouraging both anti- and syn-confor-
mations. This rotation about the glycosidic bond to the
syn-conformation is further facilitated by a backbone
clash between the adducted oxygen (O8) and the
sugar–phosphate backbone in the anti-8-oxoG conform-
The coding potential of 8-oxoG is dictated by the anti- or
syn-conformation of the modified base. Similar to unmodi-
fied deoxyguanine, anti-8-oxoG base pairs with cytosine
through traditional Watson–Crick hydrogen bonding inter-
actions forming a non-mutagenic DNA lesion. In contrast,
the mutagenic syn-8-oxoG conformation is able to base
pair with adenine through its Hoogsteen edge. Kinetic
studies have shown that DNA polymerases insert adenine
opposite 8-oxoG frequently and with enhanced catalytic
compared with guanine, the catalytic efficiency of adenine
insertion opposite 8-oxoG increases almost three orders of
magnitude. If left unrepaired, the pro-mutagenic syn-
8oxoG-dA base pair results in a G–C to T–A transversion
mutation following DNA replication (5). This highlights
the importance of the dual-coding properties of 8-oxoG
being either error free or mutagenic when it is in the anti-
or syn-conformations, respectively.
The prevalence of oxidative stress and subsequent mu-
tagenic properties of 8-oxoG has resulted in an elegant
cellular defense mechanism against 8-oxoG (4). This
lesion is removed from DNA by a glycosylase that
*To whom correspondence should be addressed. Tel: +1 919 541 4701; Fax: +1 919 541 4724; Email: email@example.com
Nucleic Acids Research, 2013, Vol. 41, No. 3Published online 24 December 2012
Published by Oxford University Press 2012.
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generates a gap in the DNA following removal of the
damaged base and subsequent initiation of base excision
repair (BER). DNA polymerase (pol) b is required to fill
the single-nucleotide-gapped DNA intermediate. In the
repair of the 8oxoG-C base pair, OGG1 glycosylase
removes 8-oxoG resulting in a gapped DNA substrate
with a templating cytosine that will be filled with high
fidelity by pol b. When 8-oxoG escapes repair, there is a
high probability that replication will result in adenine
insertion. Thus, the cell also codes for a glycosylase,
MYH, which removes adenine paired with 8-oxoG and
initiates BER. In this situation, pol b encounters 8-oxoG
as the templating nucleotide and will insert dCMP or
dAMP opposite 8-oxoG resulting in error-free or muta-
genic gap filling, respectively. Kinetic studies with a
templating 8-oxoG have shown that pol b only incorpor-
ates dCTP over dATP by ?2-fold (6). This poor 8-oxoG
fidelity results in futile mutagenic ‘repair’ following inser-
tion of dATP opposite 8-oxoG. Accordingly, mutagenic
repair would be a cellular burden during times of elevated
emphasize the importance of BER and pol b in modu-
lating the repair of oxidative DNA damage and lesion
Currently, pol b is the only wild-type DNA polymerase
that has been structurally characterized with a templating
8-oxoG base paired with either an incoming dCTP or
dATP (7,8). Overall these structures indicate that the
8-oxoG lesion paired with dCTP or dATP is well tolerated
in the polymerase active site. The structure with an
incoming dCTP paired with anti-8-oxoG shows the
greatest structural change at the phosphate backbone of
the anti-8-oxoG, shifting 2.9A˚ and rotating 200?to ac-
(Figure 1A). This movement is a result of a steric clash
between O8 and the phosphate backbone. Recently, the
structure of pol b with a templating syn-8-oxoG
Hoogsteen base pairing with dATP was determined and
indicates that it is similar to that with an incoming dCTP
(7). With the syn-8-oxoG structure, however, Arg283 is
observed to stabilize the syn-conformation through a
hydrogen bond to O8 in the DNA minor groove
(Figure 1B). With guanine or anti-8-oxoG as the
templating base, Arg283 interacts with the templating nu-
cleotide upstream of the coding base. Importantly, Arg283
only interacts with the templating strand after nucleotide
binding that induces subsequent polymerase subdomain
Combining rational site-directed mutagenesis with
kinetic and structural approaches, we are able to identify
DNA minor groove interactions that modulate the coding
potential of 8-oxoG. These results provide mechanistic
insights into substrate specificity, lesion bypass and the
role that the polymerase and incoming nucleotide have
on the anti-/syn-equilibrium of 8-oxoG. In addition to
the mechanistic information obtained, we have also
gained insight concerning the impact the incoming nucleo-
tide has on the conformation of the templating 8-oxoG
and the point at which the 8-oxoG switches between the
syn- or anti-conformations.
stress. These examples
MATERIALS AND METHODS
Human R283K pol b protein was overexpressed in
Escherichia coli and purified as previously described (9).
Binary complex crystals with a templating 8-oxoG in a 1-
nt-gapped DNA were grown in a solution containing
50mM imidazole, pH 7.5, 17% PEG3350 and 350mM
sodium acetate, as previously described (7,10). The
binary complex crystals were transferred to a cryosolution
containing 12% ethylene glycol, 50mM imidazole, pH 7.5,
non-hydrolyzable dNTP analogues (dCMP(CF2)PP or
dAMP(CH2)PP) and 200mM MnCl2. This resulted in for-
mation of the ternary closed complex. Data for the binary
complex crystal structure were determined using the same
cryosolution described above, but lacking the non-
dAMP(CH2)PP was purchased from Jena Biosciences.
Data were collected at 100K on a SATURN92 CCD
detector system mounted on a MiraMax-007HF rotating
anode generator. Data were processed and scaled using
the HKL2000 software package (11). Initial models were
determined using molecular replacement with the previ-
mismatch (3C2M) structure of pol b as a reference (12).
All Rfree flags were taken from the starting model.
Refinement was performed using PHENIX and model
building using Coot (13,14). The figures were prepared
in PyMOL (Schro ¨ dinger LLC). Ramachandran analysis
determined 100% of non-glycine residues lie in the
allowed regions and at least 98% in favoured regions.
The R283K pol b 8-oxoG binary structure was refined
with the templating 8-oxoG base in alternate conform-
ations. After refinement, the occupancies were determined
to be 0.5 for each conformation.
A 34-mer oligonucleotide DNA substrate containing a
single-nucleotide gap with a templating guanine was
created aspreviously described
guanine or 8-oxoG in the coding position (15). Steady-
state kinetic parameters for single-nucleotide gap filling
reactions were determined as previously described (16).
syn-conformation of a templating 8-oxoG, we have
utilized a combination of site-directed mutagenesis,
insights for 8-oxoG lesion bypass during BER. Upon
N forms a hydrogen bond with the oxygen of the nucleo-
tide sugar (O40) upstream of the templating base,
stabilizing the closed state of the polymerase. When
8-oxoG serves as the templating (coding) nucleotide,
Arg283 is also within hydrogen bonding distance of the
adducted oxygen (O8) in the closed conformation
(Figure 1B). These observations indicate that Arg283
has two roles during pol b catalysis when an 8-oxoG is
in the templating position, stabilizing both the closed
polymerase state and the syn-conformation of 8-oxoG.
Nucleic Acids Research,2013, Vol.41, No. 31849
These roles make Arg283 an ideal target for studying the
mechanism of 8-oxoG lesion bypass.
R283K pol b incorporation opposite 8-oxoG
syn-conformation of 8-oxoG, we replaced arginine with
lysine generating R283K pol b. We chose to utilize the
conservative lysine substitution because it has only a
moderate impact on polymerase insertion efficiency with
(Figure 2A and Supplementary Figure S1). In comparison,
previous studies have shown that alanine substitution
results in a dramatic loss (>30000-fold) in catalytic effi-
ciency, likely due to the failure of the polymerase to
undergo closure (17). Steady-state kinetic studies where
MgCl2 was substituted with MnCl2 as a divalent
cofactor were also undertaken (Figure 2). Manganese
was necessary for crystallographic characterization of
the closed substrate complex of the R283K mutant
enzyme (see below). This ensured that the solution
Importantly, although the catalytic efficiencies with
MnCl2are greater than that observed with MgCl2, the
specificities (i.e. discrimination) are similar with these
alternate divalent metals (Figure 2A and Supplementary
Figure 2A shows a discrimination plot with the catalytic
efficiencies for dCTP and dATP incorporation opposite
analysethe impact thatArg283hason the
guanine (G) or 8-oxoG (8oG) by wild-type and R283K
pol b with MnCl2. In this type of plot, the magnitude of
discrimination or fidelity is represented by the distance
between the log of the catalytic efficiencies of the alternate
substrates. Opposite a non-damaged guanine, both the
wild-type and R283K pol b have similar catalytic
efficiencies, preferentially inserting dCTP by 3000- and
7000-fold, respectively. The similar efficiencies for dATP
insertion opposite guanine suggest that wild-type and
complexes in the presence of an incorrect nucleotide. In
contrast, dCTP and dATP incorporation opposite 8-oxoG
by wild-type and R283K is very different (Figure 2A).
Wild-type pol b incorporates both dCTP and dATP
opposite 8-oxoG with nearly the same efficiency. This
results from the enhanced incorporation efficiency of
dATP opposite 8-oxoG because of the Hoogsteen
hydrogen bonding interactions between the incoming
dATP and syn-8-oxoG (Figure 1A). In contrast, the cata-
lytic efficiency of R283K is reduced opposite 8-oxoG.
With an incoming dATP, R283K fails to show the dra-
matically enhanced efficiency opposite 8-oxoG observed
with wild-type enzyme (highlighted by a black arrow in
Figure 2A). This suggests that it is unable to form a stable
Hoogsteen base pair with 8-oxoG.
The specificity for insertion of dCTP and dATP
opposite 8-oxoG for select members from different DNA
polymerase families is illustrated in Figure 2B. This inser-
tion ratio provides insight into the templating base and
preferential incorporation of either dATP or dCTP
opposite 8-oxoG. Wild-type pol b has an insertion ratio
of ?1 suggesting that the templating 8-oxoG is stable in
either the anti- or syn-conformation. In comparison, the
Bacillus fragment (A-family) preferentially incorporates
dATP opposite 8-oxoG (dCTP/dATP=0.11), suggesting
a preferential syn-conformation of 8-oxoG in the active
site (18), whereas RB69 DNA polymerase (B-family) pref-
erentially incorporates dCTP (dCTP/dATP=20), sug-
gesting an anti-conformation of 8-oxoG is preferred in
the nascent base pair binding pocket (19). For pol b, the
single lysine substitution at Arg283 results in a mutant
enzyme that is less error prone (dCTP/dATP=15), pref-
erentially incorporating dCTP opposite 8-oxoG.
Structural characterization of the R283K pol b binary
complex with templating 8-oxoG
Structural studies with the open binary DNA complex of
wild-type enzyme with a templating 8-oxoG indicate that
8-oxoG can adopt both the anti- and syn-conformations
(7). To determine whether a templating 8-oxoG can adopt
multiple conformations in the binary complex of the
R283K mutant enzyme, we determined the crystallo-
graphic structure to 1.95A˚ (Table 1). Figure 3A shows
an overlay of the binary R283K pol b complex and the
wild-type binary complex (PDB code 3ISB; root mean
square deviation (RMSD) of 0.17A˚over 307 Ca atoms).
The low RMSD indicates that the R283K mutation does
not have an appreciable impact on the open conformation
of the protein and inspection of the active site indicates no
Figure 1. Structural overview of wild-type pol b with templating
8-oxoG in the active site. (A) Overlay of the templating anti-
G:dCMPCPP (PDB ID 2FMP), anti-8-oxoG:dCMP(CF2)PP (PDB ID
3RJI) and syn-8-oxoG:dAMPCPP (PDB ID 3RJF) in the pol b active
site are shown in yellow, salmon and purple, respectively. The position
of the templating phosphate backbone is indicated for each conform-
ation. (B) Structure of the closed ternary pol b complex with a
templating syn-8-oxoG Hoogsteen base pairing with dAMPCPP
(PDB ID 3RJF) (7). The primer terminus (O30) is indicated. Key
hydrogen bonding interactions are shown as black dashes.
1850Nucleic Acids Research, 2013,Vol.41, No. 3
localized structural perturbations when compared with
Examination of the 8-oxoG in the templating position
indicates that 8-oxoG has an elevated B-factor (48A˚2)
relative to the rest of the DNA (28A˚2). The high
B-factor correlates with the globular density for this
templating base. As shown in Figure 3B, 8-oxoG can be
modelled into an omit map in either the anti- or
syn-conformations and likely adopts multiple orientations
in the active site when pol b is in an open conformation.
The templating 8-oxoG was initially modelled and refined
in either the syn- or anti-conformation (Supplementary
Table 1. Data collection and refinement statistics
R283K pol b 8-oxoG R283K pol b 8-oxoG:dCMP(CF2)PPR283K pol b 8-oxoG:dAMPCPP
a, b, c (A˚)
?, ?, ? (?)
Bond lengths (A˚)
Bond angles (?)
RCSB ID code
P21 P21 P21
aHighest resolution shell is shown in parentheses.
Figure 2. Steady-state kinetic analysis of wild-type and R283K pol b. (A) A discrimination plot of 1-nt gap filling by wild-type and R283K pol b
opposite non-damaged guanine (G) and 8-oxoG (8oG) with dCTP or dATP in the presence of MnCl2. The dCTP and dATP incorporation is shown
with a green and red bar, respectively. The black arrow highlights the impact of the R283K mutation on dATP incorporation opposite 8-oxoG.
(B) Table of select DNA polymerases (polymerase family) that have been characterized kinetically for incorporation opposite 8-oxoG (6,18,19,20–32).
The dCTP/dATP ratio shows the preference for incorporation of dCTP relative to dATP. A value of 1 indicates no preference of incorporation,
a value >1 indicates a preference for dCTP and a value <1 indicates a preference for dATP incorporation. The likely preferred glycosidic
conformation of the templating 8-oxoG is indicated.
Nucleic Acids Research,2013, Vol.41, No. 31851
Figure S2). However, omit map analysis of either confor-
mer indicated the existence of multiple conformations.
In addition, thephosphodiester
templating nucleotide can be modelled into two stable
conformations. These observations are consistent with
the templating 8-oxoG in the R283K pol b open binary
conformation being in equilibrium between the anti- and
syn-conformations prior to binding the incoming nucleo-
tide and undergoing polymerase subdomain closure.
Structure of R283K ternary 8-oxoG complexes
To determine the crystallographic structure of the ternary
R283K pol b protein with a templating 8-oxoG, we
utilized non-hydrolyzable dNTP analogues to prevent ca-
talysis while maintaining the catalytically active primer
terminus. Hydrolysis of the incoming nucleotide by an
activated water molecule that infiltrates the active site
has been observed in the absence of a primer O30(i.e.
dideoxy-terminated primer sugar) (8). Importantly, these
analogues are excellent mimics of dNTPs having similar
metal affinities and coordination properties as native nu-
cleotides (33). Since Arg283 is involved in stabilizing the
closed conformation of pol b, it was not surprising that in
the presence of MgCl2the mutant enzyme remained in an
open conformation (see below). This is consistent with the
moderate loss of catalytic efficiency (Figure 2A and
Supplementary Figure S1). As demonstrated previously
(34), we utilized MnCl2to promote polymerase closure
to overcome the weak ability of R283K pol b to form a
Crystals of the R283K ternary complex with a
templating 8-oxoG (anti-conformation) base pairing with
an incoming dCTP diffracted to 2.2A˚. This ternary
complex is closed with a-helix N forming one face of the
nascent base pair binding pocket (Figure 4). A superpos-
ition with the analogous wild-type complex indicates that
the mutant structure is similar to that of the wild-type
complex with an RMSD of 0.40A˚(305 Ca) (Figure 4B).
In contrast to arginine at residue 283, Lys283 fails to form
a hydrogen bond with the oxygen (O40) of the sugar ring
upstream of 8-oxoG. The incoming dCTP forms Watson–
Crick hydrogen bonds with 8-oxoG, while the two Mn2+
ions are coordinated with octahedral geometry that
include oxygens from the primer terminus (O30), nucleo-
side triphosphate and active site aspartates (Asp190, 192
and 256). As observed in the wild-type ternary complex
(7,8), 8-oxoG inthe R283K
anti-conformation and the phosphodiester backbone is
displaced by the adducted oxygen (O8). The position of
backbone) is nearly identical in both the wild-type and
mutant ternary complexes (Figure 4B).
To provide molecular insight into the kinetic observa-
tion of the reduced dATP incorporation opposite 8-oxoG
by the R283K mutant (Figure 2A), we solved the crystal-
lographic structure of the R283K pol b ternary complex
with an incoming dATP analogue and templating 8-oxoG
to 2.0A˚(Table 1). Surprisingly, the structure shows that
the R283K mutant failed to undergo complete closure and
is trapped in an intermediate conformation (Figure 5).
Structural studies with wild-type pol b indicate that
during polymerase closure, a moderate structural repos-
itioning occurs at the lyase domain whereas the largest
structural change occurs with the pol b N-subdomain
(i.e. fingers of right-handed DNA polymerases). This re-
positioning results in the nascent base pair sandwiched
Figure 4. R283K pol b ternary complex with a templating anti-8-
oxoG:dCMP(CF2)PP base pair. (A) R283K pol b ternary complex
active site with 8-oxoG in the anti-conformation base pairing with
dCMP(CF2)PP. The key hydrogen bonding interactions are shown as
black dashes. Mn2+and water molecules are shown as red and blue
spheres, respectively. The key active site residues and Lys283 (K283)
are shown in stick format. (B) Structural overlay of the wild-type and
R283K pol b ternary complexes are shown in salmon and cyan, respect-
ively. Mn2+from the R283K pol b structure is shown as red spheres and
the Mg2+from the wild-type pol b structure is shown in green.
Figure 3. R238K pol b binary complex with a templating 8-oxoG in
the active site. (A) Overlay of the wild-type (PDB ID 3ISB) and R283K
(PDB ID 4GXI) pol b binary complex structures in grey and purple,
respectively. (B) A Fo?Fc omit map (green) contoured at 2.5s is
shown for the templating 8-oxoG in the R283K pol b binary structure.
The templating 8-oxoG can be modelled in both the syn- and
anti-conformations with the phosphate backbone in two orientations.
1852Nucleic Acids Research, 2013,Vol.41, No. 3
between the primer terminal base pair and a-helix N. As
shown in Figure 5A, a-helix N of R283K is in an inter-
mediate position between the open and closed states. The
lyase domain has undergone complete closure, indicating
that this partial closure event is likely due to the R283K
mutation in a-helix N. A signalling cascade between the
active site and a-helix N during polymerase closure
involves Asp192, Arg258, Tyr271, Phe272, Arg283,
Glu295 and Tyr296 (35–37). Figure 5B illustrates the
relative position of these key residues for the open (PDB
code 3ISB), intermediate (R283K) and closed (PDB code
2FMS) states of pol b. The aspartate residues involved in
binding the active site metal ions are shown coordinating
the MnCl2ions in the intermediate and closed conform-
ations. In comparison, other key residues for the inter-
mediate R283K ternary complex are in alternative
conformations between the open and closed states.
Lys283 is in an intermediate location due to the loss of
the hydrogen bond in the DNA minor groove. Tyr271 is
positioned in an intermediate conformation and has not
fully moved to its closed position, thus failing to promote
the movement of Phe272 to its ‘closed’ conformation. The
intermediate conformation of Tyr271 is likely stabilized by
a hydrogen bond between its side chain hydroxyl and N2
of 8-oxoG (Figure 6B, discussed below).
In the wild-type pol b syn-8-oxoG:dATP ternary
complex, Arg283 stabilizes both the syn-conformation of
8-oxoG and the closed polymerase complex (7). By
putting a lysine residue at this position, the kinetic
studies suggest that the syn-conformation of 8-oxoG in
the active site was not as common as with wild-type
enzyme, indicated by the decreased dATP insertion
Figure 5. Intermediate conformation of the R283K pol b ternary complex with templating anti-8-oxoG and incoming dAMPCPP. (A) Structural
overlay of the R283K anti-8-oxoG:dAMPCPP ternary complex with the wild-type open binary (PDB ID 3ISB) and closed ternary (PDB ID 2FMS)
pol b complexes shown in green, purple and yellow, respectively. The lyase domain and a-helix N (Helix N) are indicated. The position of a-helix N
is designated as open, intermediate (Int.) or closed for each structure. The incoming nucleotide for the R283K ternary complex is coloured green in
the active site. (B) Key amino acids that are repositioned during subdomain closure are shown for the open (PDB ID 3ISB), intermediate (R283K
8-oxoG:dATP) and closed (PDB ID 2FMS) pol b complexes. Helix N is shown in a ribbon representation and key amino acids are in stick format.
Mn2+and Mg2+are shown as red and yellow spheres, respectively.
Figure 6. R283K pol b ternary complex with a templating anti-8-oxoG and incoming dAMPCPP. The templating anti-8-oxoG, incoming dAMPCPP
and a-helix (Helix) N are shown in green. Mn2+and water molecules are shown as red and blue spheres, respectively. (A) Active site of the R283K
pol b anti-8-oxoG:dAMPCPP with a Fo?Fcmap (green) contoured at 3s. (B) Key active site hydrogen bonding interactions between the templating
anti-8-oxoG and incoming dATP are shown with black dashes. The key amino acids are shown in stick format and the octahedral geometry for each
metal ion is illustrated.
Nucleic Acids Research,2013, Vol.41, No. 31853
efficiency (Figure 2A). Consistent with the kinetic studies,
the structure of the corresponding ternary complex of the
mutant indicates that the dATP analogue fails to base pair
with the templating 8-oxoG. Figure 6A shows an omit
map for the templating anti-8-oxoG and incoming dATP
in the R283K active site. The incoming dATP analogue
shows clear density for the sugar and phosphate moieties,
while the base is more diffuse indicative of multiple pos-
itions and is consistent with previous structures of
mismatches in the confines of the pol b active site (38).
Importantly, the templating
anti-conformation with the phosphate backbone shifted
away from the adducted oxygen. In addition, the inability
of the incoming nucleotide to be stabilized with Hoogsteen
hydrogen bonds suggests that the syn-conformation of
8-oxoG is not significantly populated. Figure 6B shows
key hydrogen bonding interactions in the R283K active
site. Lys283 fails to form hydrogen bonding interactions
with 8-oxoG or the sugar of the upstream template nu-
cleotide. The templating 8-oxoG forms hydrogen bonding
interactions with two water molecules that have infiltrated
the active site, likely due to the partially open intermediate
state of a-helix N. These water molecules are also within
hydrogen bonding distance of N6 of the incoming dATP.
The N2 of anti-8-oxoG is hydrogen bonding to the
hydroxyl of Tyr271. The incoming dATP analogue is
within hydrogen bonding distance of Asp276(Og) and
Asn279(Nd2) with N6 and N3 of dATP, respectively.
The triphosphate of the incoming nucleotide participates
in the octahedral coordination of the two active site
MnCl2 ions. Interestingly, the primer terminus is not
coordinating the catalytic metal ion and has shifted
away from the triphosphate permitting a water molecule
to occupy this coordination position.
Overlaying the ternary substrate complexes of wild-type
and mutant enzymes with 8-oxoG/dATP shows significant
differences in the active site with an RMSD of 0.8A˚over
291 Ca atoms. Localized analysis of the active site of the
two proteins (Figure 7A) shows that the R283K ternary
complex is not fully closed in contrast to wild-type
enzyme. The templating 8-oxoG in the R283K pol b
active site has undergone major adjustments shifting
upstream by 3.7A˚ at the sugar moiety. This upstream
template shift results in movement of the primer
terminus away from the catalytically competent orienta-
tion. Overlaying the mismatch structure of wild-type pol b
containing a templating guanine and incoming dATP
analogue with the mutant anti-8oxoG:dATP structure
has an RMSD of 0.34A˚over 277 Ca atoms, highlighting
the similarities between the two structures. Figure 7B
shows that while a-helix N of the R283K structure has
not undergone complete closure, the templating strand,
incoming nucleotide and primer terminus are in a similar
Comparing key amino acid interactions indicates that
Asp276 and Asn279 form similar contacts in the
mismatch and mutant structures (Figure 7C); the major
differences are at residues Lys283, Tyr271 and Phe272.
The lysine at 283 in the mutant fails to interact with N3
of the templating guanine as observed in the wild-type
mismatch structure (38); the loss of this interaction
Figure 7. Structural comparison of R283K anti-8-oxoG:dAMPCPP
and wild-type pol b ternary complexes. Active site aspartate residues
(D190, D192 and D256), residue 283 (K283 or R283), templating
8-oxoG, incoming nucleotide and the primer terminus (O30) are
indicated.(A) Structural overlay
oxoG:dAMPCPP and wild-type syn-8-oxoG:dAMPCPP pol b ternary
complexes are shown in green and purple, respectively. The key move-
ments of the primer terminus and templating strand are highlighted
with red arrows. The templating nucleotide is labelled n and its
upstream neighbour is n?1. (B) Structural overlay of the R283K
anti-8-oxoG:dATP and mismatch wild-type G:dATP pol b (PDB ID
3C2M) ternary complexes are shown in green and brown, respectively.
(C) Key amino acid contacts between the active site residues and the
templating base or incoming nucleotide
oxoG:dATP and mismatch wild-type G:dATP pol b ternary complexes
are shown with green and brown dashes, respectively. The movement of
the phosphate backbone as a result of the adducted oxygen on 8-oxoG
templating base is highlighted with a red arrow.
of theR283K anti-8-
of the R283K anti-8-
1854 Nucleic Acids Research, 2013,Vol.41, No. 3
probably prevents complete closure of the N-subdomain.
As a result of the partial closing of the mutant, Tyr271
and Phe272 are in intermediate positions. The similarities
8oxoG:dATP structures are consistent with the kinetic
characterization (Figure 2A). The incorporation efficiency
of dATP insertion opposite 8-oxoG with the R283K
mutant is similar to that observed for wild-type enzyme
opposite a non-damaged guanine. Thus, the mutant dis-
criminates against dATP insertion opposite 8-oxoG
through a similar conformational strategy as wild-type
enzyme to deter insertion of dATP opposite guanine.
The oxidized purine 8-oxoG is able to adopt either an
anti- or syn-conformation that will promote error-free or
mutagenic replication, respectively. The alternate coding
potential of 8-oxoG is modulated by the glycosidic torsion
Hoogsteen edges are used for base pairing with cytosine
or adenine, respectively. Structural characterization of
duplex DNA containing 8-oxoG indicates that the glyco-
sidic preference of 8-oxoG is determined by its base-
pairing partner, inducing the syn-conformation when
paired with adenine (39,40) and the anti-conformation
when paired with cytosine (41,42). The 8-oxoG nucleoside
favours the syn-conformation due to steric repulsion with
the sugar. In this context, 8-oxodGTP is preferentially
inserted opposite adenine since the anti-conformation is
excluded from the dNTP-binding pocket due to steric
and electrostatic barriers between O8 and Pa (43).
While the structural studies provide insight into the
inherent conformations of 8-oxoG when in solution or
duplex DNA, kinetic analysis of polymerase specificity
tabulated in Figure 2B indicates that the polymerase
active site influences the conformation of 8-oxoG during
replication. To date only a select number of polymerases
have been structurally characterized with a templating
8-oxoG in the active site, these include RB69 (B-family)
(19,44,45), Dpo4 (Y-family) (20), pol k (Y-family) (21,22),
pol i (Y-family) (46), pol Z (Y-family) (47), T7 (A-family)
(48) and pol b (X-family) (7,8). Structures of ternary sub-
strate complexes indicate that with an incoming dCTP-
positioned opposite anti-8-oxoG, very little structural
changes are required to accommodate the lesion in the
active site. In the case of pol b, the only appreciable
change is the repositioning of the phosphate backbone
of 8-oxoG to accommodate O8 of the modified guanine
residue (7,8) (Figure 1). In other cases (e.g. RB69 and T7),
the phosphate backbone does not sterically interfere with
O8 due to a 90?bend in the template strand at the active
site permitting the polymerase to modify the conformation
of the sugar–phosphate backbone (19,48). There are
however specific active site contacts with the templating
8-oxoG base. Both T7 (48,49) and Dpo4 DNA polymer-
ases (20) form stabilizing hydrogen bonds with the anti-8-
oxoG at the adducted oxygen (O8). RB69 has a strong
preference for inserting dCTP opposite anti-8-oxoG due
tointeractions with the
discouraging the syn-8-oxoG conformation (44). Altering
these residues enhances 8-oxoG mutagenesis (20,44,49).
Together these studies highlight the impact that the
active site has on conformational selection of the anti-/
syn-conformation of 8-oxoG in both the templating and
incoming nucleotide positions.
Active site contacts regulate mutagenic or error-free
bypass of 8-oxoG
The kinetics of nucleotide incorporation opposite 8-oxoG
by wild-type pol b indicates a lack of discrimination for
dCTP and dATP incorporation (Figure 2). This implies
that in solution the active site easily accommodates both
the anti- and syn-conformations of 8-oxoG and that the
incoming nucleotide may dictate the orientation of the
templating base in the pol b active site. This is also con-
sistent with the open binary pol b structure displaying
8-oxoG in multiple conformations based on diffuse
electron density and high B-factors (Figure 3 and
Supplementary Figure S2). However, structural studies
of pol b with a templating syn-8-oxoG Hoogsteen base
pairing to dATP indicate that Arg283 forms a stabilizing
hydrogen bond with O8 in the closed ternary complex (7).
This contact is unique to syn-8-oxoG and not observed
with non-damaged guanine and anti-8oxoG; in these latter
cases, Arg283 interacts with the nucleotide upstream of
the templating nucleotide. Thus, the pol b active site
8-oxoG. To test whether this contact influences 8-oxoG
specificity, we generated a R283K mutant that resulted in
pol b favouring error-free dCTP incorporation opposite
anti-8oxoG (Figure 2). This indicates that the nucleotide
alone is not able to dictate the orientation of the 8-oxoG
in the active site of pol b and that active site contacts play
a key role in regulating the error-free and mutagenic
bypass of 8-oxoG. Surprisingly, the loss of Arg283 inter-
actions resulted in pol b utilizing a mismatch strategy to
avert misinsertion of dATP opposite 8-oxoG. This
strategy utilizes the formation of a mismatch ternary
complex in which the templating strand is shifted
upstream, repositioning of the primer terminus away
from the catalytic metal, and the subsequent generation
of an apparent abasic site (i.e. the templating pocket lacks
a base) (38). Together these studies infer that the base
pairing of the incoming dATP to the templating 8-oxoG
is not strong enough to maintain or induce the syn-
conformation, and that the closed polymerase active site
has an inherent preference for the anti-conformation that
can only be overcome by Arg283 stabilizing the syn-
conformation of 8-oxoG.
Previous computational studies with wild-type pol b
containing a templating syn-8-oxoG Hoogsteen base
pairing with dATP resulted in the polymerase having a
partially open active conformation (50). This was a con-
sequence of a-helix N failing to undergo complete closure.
In failing to undergo closure, key amino acids that are
involved in facilitating polymerase closure and enzyme ac-
tivation fail to reach the fully closed conformation. These
studies also observed that an anti-8oxoG failed to
hydrogen bond with an incoming dATP and resulted in
Nucleic Acids Research,2013, Vol.41, No. 3 1855
a mismatched ternary complex in the active site. The inter-
mediate state of the N-subdomain results in additional
water molecules penetrating the active site (Figure 6).
This is consistent with other structural studies using
Bacillus fragment Pol I where incomplete subdomain
(i.e. fingers) closure permitted additional water molecules
to penetrate the active site (51).
Stabilization of 8-oxoG during polymerase closure
The diffuse electron density for the templating 8-oxoG in
the binary pol b active site indicates that 8-oxoG is in
equilibrium between anti- and syn-conformations prior
8-oxoG in the closed ternary complex of wild-type pol b
exhibits a single glycosidic conformation indicating that
the polymerase has captured 8-oxoG in a stable anti- or
syn-orientation depending on the incoming nucleotide
(7,8). This raises the question of when does 8-oxoG
form a stable configuration in the polymerase active site,
during or after polymerase closure. Structural analysis of
the wild-type and R283K pol b active site in the closed
ternary complex indicates the templating 8-oxoG is unable
to accommodate glycosidic rotation between the anti- and
syn-configurations after closure. Supplementary Figure S3
indicates that the nascent base pair binding pocket is spa-
tially limited in the closed conformation and would not
permit free rotation around the glycosidic bond of the
templating nucleotide. The inability to stabilize the
syn-8oxoG conformation in the R283K active site results
in a mismatch ternary complex inferring that the polymer-
syn-conformation. For Arg283 to contact the templating
8-oxoG, it must move ?6A˚towards the active site during
polymerase closure. This large translation necessary for
Arg283 to stabilize the syn-8-oxoG indicates the polymer-
ase captures the 8-oxoG conformation during polymerase
closure and the lack of this contact during closure results
in a mismatch ternary complex. This type of mechanism
allows the polymerase to scan the templating base during
subdomain closure and capture either the anti- or
syn-conformation based on the incoming nucleotide
together with the energetically stable conformation of
the templating base.
Based on the work presented here, we propose mechan-
istic steps of 8-oxoG lesion bypass by pol b. First, pol b
binds to single-nucleotide-gapped DNA with a templating
8-oxoG forming an open binary state with the phosphate
backbone and templating 8-oxoG in equilibrium between
both the anti- and syn-conformations. Then, an incoming
nucleotide binds to the open binary complex. In the case
of an incoming dCTP, the equilibrium is shifted to the
Watson–Crick hydrogen bonding interactions and the
movement of the phosphodiester backbone. In the case
of an incoming dATP, the nucleotide likely forms
weaker Hoogsteen hydrogen bonds with the templating
syn-8-oxoG in the open conformation, but does not ap-
preciably shift the 8-oxoG equilibrium towards the
syn-conformation. This is based on the inability of the
incoming dATP to stably form a Hoogsteen base-pairing
and stabilizedby both
interaction in the absence of the Arg283 residue. In
addition, recent studies by our group to structurally char-
acterize nucleotide-binding states to the open conform-
ation of pol b with an 8-oxoG templating base
consistently captured the incoming dCTP base pairing
with the anti-8oxoG in the open conformation, but
incoming dATP (34). Following nucleotide binding, pol
b undergoes closure with an incoming dCTP base
pairing to anti-8oxoG, resulting in a stable ternary
complex poised for insertion. In the case of an incoming
dATP, wild-type pol b captures the syn-conformation
through hydrogen bonding interactions between Arg283
and O8 of 8-oxoG during subdomain closure. This
syn-conformation is further stabilized by the incoming
dATP Hoogsteen base pairing with the templating base
to form the final ternary complex poised for insertion.
The lack of the Arg283 contact during subdomain
closure results in the templating 8-oxoG having a prefer-
ential orientation for the anti-conformation resulting in a
mismatch ternary complex, leading to reduced incorpor-
ation of dATP.
The ability of a polymerase to undergo mutagenic or
error-free lesion bypass has important ramifications for
the replication of the genome. In the case of pol b, the
balance between the anti- and syn-conformations is not
dictated solely by the incoming nucleotide and is partially
controlled by active site residues that contact the
templating base. In this sense, pol b actively promotes
mutagenesis through stabilizing the syn-8oxoG conform-
ations by Arg283 thereby promoting insertion of adenine.
This is similar to the mutagenic interaction that Asn279 of
pol b provides to stabilize the syn-conformation of an
incoming nucleotide 8-oxodGTP while it Hoogsteen base
pairs with adenine (6,43). These residues make wild-type
pol b mutagenic during interaction with these oxidized
substrates since they promote the mutagenic syn-8-oxoG
conformation. Accordingly, mutating either the Arg283 or
Asn279 residue in pol b results in increased specificity for
dCTP incorporation opposite anti-8-oxoG. These results
suggest that it might be possible to reduce the mutagenic
response of cells to oxidative stress by expressing ration-
ally engineered pol b modified at Asn279 and/or Arg283.
4GXI, 4GXJ, 4GXK.
Supplementary Data are available at NAR Online:
Supplementary Figures 1–3.
support from Lars Pedersen and the Collaborative
Crystallography Group at NIEHS.
1856 Nucleic Acids Research, 2013,Vol.41, No. 3
Program of the National Institutes of Health, National
Institute of Environmental Health Sciences and was in
association with the National Institutes of Health
Research Project [Z01-ES050158].
for openaccess charge:
Conflict of interest statement. None declared.
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