Structures of dNTP Intermediate States
during DNA Polymerase Active Site Assembly
Bret D. Freudenthal,1William A. Beard,1and Samuel H. Wilson1,*
1Laboratory of Structural Biology, National Institute of Environmental Health Sciences, National Institutes of Health, P.O. Box 12233,
Research Triangle Park, NC 27709-2233, USA
DNA polymerase and substrate conformational
changes are essential for high-fidelity DNA syn-
thesis. Structures of DNA polymerase (pol) b in
complex with DNA show the enzyme in an ‘‘open’’
conformation. Subsequent to binding the nucleotide,
the polymerase ‘‘closes’’ around the nascent base
pair with two metals positioned for chemistry. How-
ever, structures of substrate/active site intermedi-
ates prior to closure are lacking. By destabilizing
the closed complex, we determined unique ternary
complex structures of pol b with correct and incor-
rect incoming nucleotides bound to the open confor-
mation. These structures reveal that Watson-Crick
hydrogen bonding is assessed upon initial complex
formation. Importantly, nucleotide-bound states rep-
resenting intermediate metal coordination states
occur with active site assembly. The correct, but
not incorrect, nucleotide maintains Watson-Crick
hydrogen bonds during interconversion of these
states. These structures indicate that the triphos-
phate of the incoming nucleotide undergoes re-
arrangement prior to closure, providing an opportu-
nity to deter misinsertion and increase fidelity.
DNA polymerases, and their substrates, undergo conforma-
tional changes upon complex formation. These conformational
changes are believed to hasten or deter right and wrong deoxy-
nucleoside triphosphate (dNTP) incorporation, respectively (Tsai
and Johnson, 2006) and have been referred to as ‘‘induced fit.’’
the incoming dNTP-binding pocket is empty, the enzyme is in an
‘‘open’’ conformation, whereas ternary complex structures with
correct incoming nucleotides show that the polymerase has
‘‘closed’’ around the nascent base pair (Doublie ´ et al., 1999;
Sawaya et al., 1997). DNA polymerases catalyze nucleotidyl
transfer through a two-metal (i.e., Mg2+) mechanism (Steitz,
1999). The crystallographic structure of a substrate complex of
DNA polymerase (pol) b showing two octahedral-coordinated
Mg2+ions in the polymerase active site poised for chemistry
provides compelling evidence for this mechanism (Batra et al.,
2006). Although many structures of DNA polymerases in open
description of the events prior to closure or during active site
assembly is lacking. This is mainly because events during the
open to closed transition or conformational adjustments in the
open conformation upon dNTP binding have been difficult to
determine. Yet, computational studies have highlighted the
importance of intermediate steps during closure and active site
assembly (Radhakrishnan et al., 2006).
The polymerase domain of pol b is composed of three subdo-
mains involved in DNA binding, catalysis, and nucleotide
binding. These are referred to as the D-, C-, and N-subdomains,
respectively (Beard et al., 2006). Pol b also has an accessory
8 kDa lyase domain required in base excision repair. The
hallmark of the open to closed transition in pol b is rotation of
the N-subdomain, positioning a helix N adjacent to the nascent
base pair (Figure 1A). Additionally, there are adjustments in
the positions of the templating nucleotide and side chains in
the C- and N-subdomains critical for active site assembly. These
include (1) Arg283 moving to hydrogen bond to the template
strand in the minor groove; (2) acidic residues in the C-subdo-
main (Asp190, Asp192, and Asp256) repositioning to coordinate
two magnesium ions (Mgcand Mgn); and (3) Asn279 and Arg183
altering their position to hydrogen bond with the base and
triphosphate of the incoming nucleotide, respectively. A result
of the closure is the reorganization of the active site optimizing
the geometry of catalytic atoms necessary for catalysis (Fig-
ure 1B). These changes, deduced from the open and closed
structures, highlight the significance of conformational adjust-
ments of the substrates and polymerase but do not provide
insight into possible selective pressures during nucleotide
discrimination that could occur prior to polymerase closure.
Most nucleoside triphosphates are coordinated to Mg2+in
several coordination states and diastereomers involving non-
bridging oxygens on the b- and g-phosphates (Cohn and
on the a-phosphate has also been reported by Bock (1980).
Accordingly, the conformation and coordination of the incoming
dNTP in the active site must undergo rearrangements to achieve
the catalytically active a,b,g-tridentate coordination with the
correct stereospecificity observed in the closed ternary complex
structure poised for chemistry (Figure 1B) (Batra et al., 2006).
Because both the polymerase and triphosphate of the incoming
nucleotide contribute metal ligands, the polymerase active site
is expected to influence metal coordination in an attempt to
modulate active site geometry to promote or deter catalysis.
Accordingly, active site metal coordination should be sensitive
to the open and closed state of the active site as well as nascent
base pair complementarity.
Structure 20, 1829–1837, November 7, 2012 ª2012 Elsevier Ltd All rights reserved 1829
To examine the intermediate nucleotide-binding states in
molecular detail, we used a mutant form of pol b to trap unique
correct and incorrect dNTP/metal-binding states and deter-
states and conformations of the correct incoming nucleotide
during active site assembly. The structures also show that
complementary base pairing can occur in the absence of metal
binding, and that metal binding induces changes in metal coor-
dination to the triphosphate moiety with concomitant changes
in dNTP/protein interactions during active site assembly. An
additional intermediate structure with an incorrect incoming
nucleotide provides insights into molecular strategies to deter
Open R283K Pol b Ternary Complex
Because nucleotide binding to the open pol b binary DNA
complex results in a closed ternary substrate complex, it has
been difficult to isolate intermediates during ternary complex
formation for crystallographic characterization. Here, to alter
the equilibrium between open and closed conformations of
pol b, we utilized a lysine substitution at Arg283, which is located
?20 A˚from the active site. Because Arg283 interacts with the
template strand and other protein residues in the closed (Fig-
ure 1B) but not open conformation, loss of these interactions is
expected to destabilize the closed conformation. Because
a closed polymerase complex is necessary for efficient DNA
synthesis, a moderate reduction in catalytic efficiency with the
mutant enzyme was expected (Table 1), but this is much less
than the loss in efficiency observed with a less conservative
substitution, such as an alanine (Beard et al., 1996).
We determined three ternary complex structures of the R283K
enzyme using a single-nucleotide gapped DNA substrate and
soaking in a divalent metal ion and nonhydrolyzable incoming
dNTP analog, dCMP(CF2)PP, that could base pair with the tem-
plating guanine base (Table 2). Nucleotide analogs were chosen
to prevent catalysis in the crystal because the alternate
approach utilizing a dideoxy-terminated primer lacks a critical
metal ligand (i.e., O30). These analogs have been previously
shown to be excellent nucleotide mimics with similar metal-
binding affinities (Blackburn et al., 1984) and active site coordi-
nation that is indistinguishable from that observed with the
natural nucleotides (Batra et al., 2012; Chamberlain et al.,
2012; Upton et al., 2009).
The R283K ternary complex structures differ in the number of
active site metals and the global polymerase conformation. In all
three of these structures, the incoming cytosine base Watson-
Crick hydrogen bonds with the templating guanine. Comparing
the wild-type pol b DNA binary open complex structure with
two of the mutant ternary complexes that either lack a metal or
include a single-metal reveals that the mutant polymerase is in
the open conformation with rmsds of only 0.18 and 0.38 A˚over
all 326 Ca, respectively (see Figure S1A available online). In
contrast, comparing the wild-type pol b closed ternary complex
structure and the R283K mutant with two active site metals indi-
cates that the mutant enzyme has closed (rmsd of only 0.35 A˚
over 326 Ca atoms (Figure S1B). These small rmsds between
the mutant and wild-type proteins indicate that the lysine substi-
tution at Arg283 does not have a significant impact on the overall
architecture of the protein and is only impacting the equilibrium
between the open and closed state of the polymerase.
Alternative Correct Nucleotide-Binding State
Crystals of the R283K metal-free open ternary complex,
obtained in the presence of 30 mM MgCl2, diffracted to 1.8 A˚
Figure 1. Structural Overview of Wild-type Pol b
(A) Structural overlay of the open binary pol b/DNA complex (3ISB) and the
closed ternary pol b/DNA/dNTP complex (2FMS). The portions of pol b that
undergo a structural change during the open to closed transition are shown in
salmon (open) and yellow (closed). The incoming nucleotide and templating
base are shown in yellow with the Mg2+ions in red. The DNA backbone of the
upstream duplex is represented as an orange ribbon.
(B) Close-up of the closed ternary pol b active site. Key amino acids, tem-
plating base, and incoming nucleotide are shown in yellow, and important
interactions are indicated (dashed lines). Mgcand Mgnrepresent the catalytic
and nucleotide-binding magnesium ions, respectively.
Table 1. Steady-State Kinetic Parameters for Insertion Opposite
a Templating Guanine in a Single-Nucleotide Gapped Substrate
Wild-type dCTP 48 (2)0.24 (0.05)200 (40)
dATP0.52 (0.05)155 (32)0.0030 (0.0008)
R283KdCTP34 (1)4.6 (1.0)7.4 (1.0)
dATP0.064 (0.006) 225 (26)0.00030 (0.00004)
The results represent the mean (SEM) of at least two independent deter-
DNA Polymerase Intermediate dNTP-Binding States
1830 Structure 20, 1829–1837, November 7, 2012 ª2012 Elsevier Ltd All rights reserved
(Table 2). The incoming nucleotide is in the active site but has
adopted a conformation where the triphosphate moiety is in an
elongated or extended orientation without a bound metal
(Figures2Aand2B).Importantly, thebaseof theincomingnucle-
otide is paired with the templating base in a ‘‘buckled’’ confor-
mation. In this position the O30of the deoxyribose is within
hydrogen-bonding distance to enzyme side chains (Asn279
and Asp276; Figure 2B). These interactions are in sharp contrast
to interactions in the wild-type pol b closed ternary complex,
where Asn279 hydrogen bonds with the minor groove edge of
the base of the incoming nucleotide, Asp276 stacks with the
base of the incoming nucleotide and hydrogen bonds with
Arg40 of the 8 kDa domain, and O30hydrogen bonds with a
nonbridging oxygen on Pb of the triphosphate (Figure 1B). In
this extended orientation the triphosphate is stabilized by
water molecules (Figure S2) and residues Ser180, Gly189,
Arg183, and Arg149 (Figures 2B and S2). These observations
indicate that complementary base pairing could be sampled
upon initial nucleotide binding to the open binary DNA/enzyme
In the absence of metals, active site polymerase side chains
neutralize the charge on the triphosphate. Thus, it appears that
the polymerase can effectively compete with the nucleotide-
bound metal during initial complex formation, suggesting that
the enzyme plays a role in directing proper metal coordination.
This metal-free ternary complex was also observed at higher
MgCl2concentrations (200 mM). Finally, a metal-free state was
unambiguously characterized in the presence of manganese in
a different crystal form (see below).
Alternative Correct Nucleotide Single-Metal-Binding
Structures of the closed complex of wild-type pol b often have
a single Mg2+situated in the nucleotide-binding site (Batra et al.,
2006). Likewise, kinetic and structural characterizations of
exchange-inert metal/nucleotide complexes indicate that pol b
can close in response to metal/nucleotide binding (Arndt et al.,
2001). To trap a one-metal ternary intermediate complex struc-
ture, it was necessary to alter the phosphodiester backbone
at the templating base. To accomplish this, we positioned
Table 2. Data Collection and Refinement Statistics
a, b, c (A˚)54.3, 79.2, 54.854.4, 78.9, 55.155.1, 77.6, 54.954.3, 79.3, 54.655.0, 79.4, 102.1
a, b, g (?)
90, 105.5, 9090, 106.6, 9090, 114.2, 9090, 105.5, 9090, 96.9, 90
4.7 (22.8)5.6 (37.9) 9.8 (59.9)4.9 (36.7)13.6 (70.3)
I/sI35.6 (3.0)26.3 (2.8)15 (2.3)30.3 (2.3)11.1 (2.7)
Completeness (%)99.0 (90.8)99.6 (98.1)98.2 (99.0)99.7 (97.2)99.7 (100)
Redundancy5.2 (1.7)5.0 (3.1)5.7 (3.6)4.9 (2.2)5.9 (5.8)
No. of reflections39,766 28,80219,883 37,97774,621
Molecules per asymmetric unit11112
No. of atoms
32, 44c/41, 32c/–, 28c
Bond lengths (A˚) 0.0080.0060.0070.0080.010
Bond angles (?)1.0271.2050.9811.0121.100
RCSB ID code 4F5N4F5O 4F5Q4F5P4F5R
aHighest-resolution shell is shown in parentheses.
bRefers to the active site metal ions.
cRefers to the open metal-free and closed two-metal pol b molecules (A and B), respectively.
DNA Polymerase Intermediate dNTP-Binding States
Structure 20, 1829–1837, November 7, 2012 ª2012 Elsevier Ltd All rights reserved 1831
8-oxodeoxyguanine (8-oxoG) in the templating base position
(?3-fold) on dCTP insertion efficiency (Miller et al., 2000) but
requires repositioning of the template backbone of the modified
guanine (Batra et al., 2012; Krahn et al., 2003). The structure of
the binary DNA complex of pol b with 8-oxoG as the templating
base indicates that the DNA backbone of the templating nucleo-
tide assumes alternate positions (Batra et al., 2012). As a result,
the lesion backbone with the common anti-glycosidic conforma-
tion is shifted downstream trapping an intermediate state (Fig-
ure S3). This permitted us to obtain an R283K complex with a
single magnesium ion bound to the incoming nucleotide triphos-
phate, by soaking in the presence of 200 mM MgCl2(Figure 2D).
The R283K open ternary complex one-metal structure reveals
the incoming nucleotide in an intermediate conformation and
position (Figure 2C). The nucleotide-associated metal is coordi-
nated by the triphosphate and active site residues Asp190 and
Asp192 (Figure 2D); the metal coordinates a nonbridging oxygen
on Pg directlyand anonbridging oxygenon Paindirectly through
a water molecule (Figure 2D). In comparison to the metal-free
structure, the hydrogen bond between Asn279 and O30of the
incoming nucleotide is lost, and the incoming nucleotide base
has moved toward a more planar orientation in its Watson-Crick
hydrogen bonding with the templating base (Figures 2C and 3A).
The triphosphate of the incoming nucleotide also undergoes
significant reorganization upon metal binding: Pg now makes
a water-mediated hydrogen bond with Arg149, and Arg183 is
within hydrogen-bonding distance of a nonbridging oxygen on
Pb(Figures 2Dand3A);additionally, Pg isstabilized byhydrogen
bonds with Ser180 and Gly189.
Closed R283K Pol b Ternary Complex
MnCl2for MgCl2, which had been used previously to promote
polymerase closure (Batra et al., 2008; Beard et al., 2009). The
R283K ternary complex structure is in a closed conformation,
and the metals exhibit classical octahedral geometry in the
active site (Figure S4). The lysine at position 283 fails to
make a hydrogen bond in the DNA minor groove, highlighting
a key difference between the mutant and wild-type enzymes,
providing an explanation for the decreased stability of the closed
complex. Comparison of the one-metal and two-metal struc-
tures shows that the incoming nucleotide has undergone
reorganization upon binding the second metal (Figure 3B). The
incoming nucleotide triphosphate now coordinates both metal
ions, and the incoming nucleotide has moved into a planar
geometry with the templating nucleotide, while maintaining
Watson-Crick hydrogen bonding (Figure S4).
Figure 2. Metal-Free and One-Metal Ternary DNA Pol b Structures in the Open Conformation
The metal-free and one-metal structures are tan and cyan, respectively. A 2Fo-Fcelectron density map of the incoming nucleotide in the metal-free (A) and one-
metal (C) pol b structures contoured at 1.2s is shown. The protein is omitted for clarity. The base pairing between the templating base and incoming nucleotide is
highlighted (dashed lines). A close-up of the active site for the metal-free (B) and one-metal (D) pol b structures. Key protein side chains are shown in stick
representation, whereas the nucleotide Mg2+ion and bridging water molecules are shown as red and blue balls, respectively. See also Figures S1, S2, and S3.
DNA Polymerase Intermediate dNTP-Binding States
1832 Structure 20, 1829–1837, November 7, 2012 ª2012 Elsevier Ltd All rights reserved
Closed/Open Pol b Ternary Complex Structure
200mMMnCl2, wefound a crystal form with two pol b molecules
per asymmetric unit (Table 2): one molecule is in an open poly-
merase conformation, whereas the other molecule is closed
(Figure S5). Although a nucleotide is bound in both cases, anom-
nese ions in the closed ternary complex, whereas there is a lack
of anomalous density in the open ternary complex (Figure 4). The
conformations of the bound nucleotides are nearly identical to
those described above.
Alternative Incorrect Nucleotide-Binding States
The alternative nucleotide-binding states discussed above
provide insight into how the polymerase binds the correct nucle-
otide, but the stabilization of the incoming nucleotide through
templating base interactions would likely not occur in the case
of a mismatched nucleotide. To probe the incorrect nucleo-
tide-binding states, we obtained a structure of an R283K ternary
complex with an incoming nucleotide creating a mispair in the
active site (dAMPCPP-dG). The fidelity of the mutant enzyme
for insertion of dAMP opposite guanine is similar to that of
wild-type enzyme (Table 1). Numerous attempts to obtain a
metal-free mismatch structure were unsuccessful, likely due to
the absence of base pairing that would stabilize this complex.
However, the crystal of a one-metal mismatch ternary complex
in the open conformation (Table 2) diffracted to 1.85 A˚. The
density corresponding to the sugar and base moieties of the
incoming nucleotide was diffuse, but the triphosphate was
clearly visible (Figure 5A). In contrast to the matched incoming
nucleotide structures, the incoming nucleotide fails to form
a stable interaction with the templating base but is likely near
a helix N.
ture shows that upon polymerase closure the incoming nucleo-
tide is sandwiched into the active site causing distortion and
a shift of the templating base upstream along with O30displace-
ment (Figure 5B) (Batra et al., 2008). The triphosphate moiety in
this one-metal mismatch structure isapproaching the position of
the triphosphate in the closed two-metal mismatch structure
without the benefit of the hydrogen bond with Arg183 or under-
going significant rearrangement upon polymerase closure and
binding of the second metal ion. The precarious position of the
triphosphate, the poor density for the sugar and base moieties,
the scarcity of direct protein interactions, and the open poly-
merase conformation provide an impetus for an incorrect nucle-
otide to rapidly diffuse from the active site, consistent with its
poor binding affinity.
Open/Closed Polymerase Conformations
Previous kinetic (Bakhtina et al., 2005; Zhong et al., 1998) and
structural (Arndt et al., 2001; Batra et al., 2006) characterization
of wild-type pol b indicates that correct nucleotide binding
induces conformational changes that result in an active site
poised forchemistry.The largestconformational changeisrepo-
sitioning of the N-subdomain to close around the nascent base
pair and is accompanied by subtle protein and substrate adjust-
ments. Thus, trapping nucleotide-binding events prior to sub-
domain closure has been difficult to study by crystallography.
To circumvent this issue and capture intermediate nucleotide-
binding states, we utilized a polymerase mutant where Arg283
was replaced with lysine. This arginine residue is within
Figure 3. Comparison of the Intermediate Nucleotide-Binding
States during Metal Binding
The location of the active site aspartate residues, incoming nucleotide, and
templating base (tn) are shown for each structure. The metal-free, one-metal,
and two-metal structures are shown in tan, cyan, and green, respectively. The
two-metal pol b structure are shown in green (see also Figure S4).
(A) Overlay of the metal-free and one-metal pol b structures.
(B) Overlay of the one-metal and two-metal pol b structures.
(C) Overlay of the metal-free, one-metal, and two-metal pol b structures.
(D) A mechanistic model inferred from the three pol b structures with possible
polymerase checkpoints indicated under each step.
DNA Polymerase Intermediate dNTP-Binding States
Structure 20, 1829–1837, November 7, 2012 ª2012 Elsevier Ltd All rights reserved 1833
hydrogen-bonding distance to substrate DNA and other poly-
merase residues in the closed, but not open, conformation.
Although this residue is ?20 A˚from the active site, alanine
substitution results in a low-fidelity mutant with poor activity
(Ahn et al., 1997; Beard et al., 1996). The conservative lysine
substitution retains significant activity and nucleotide discrimi-
nation but exhibits a lower activity relative to wild-type enzyme
as expected for an enzyme where the equilibrium between the
open and closed conformation has been altered (Table 1). For
to subdomain closing and show that Watson-Crick hydrogen
bonding is assessed upon complex formation prior to subdo-
The equilibrium between the open and closed state is ex-
pected to be sensitive to the binding of ligands (e.g., correct/
incorrect nucleotide and catalytic/nucleotide metals) (Kirby
et al., 2012). Arg283 of the N-subdomain indirectly interacts
with Asp192 that contributes metal ligands for both active site
metals through a series of hydrogen bonds that are different in
the alternate states (Sawaya et al., 1997). Another key residue
in this signaling cascade is Glu295 that has been associated
with some human gastric carcinomas when changed to lysine
(Iwanaga et al., 1999). The E295K mutant exhibits poor activity
(Lang et al., 2007) and the inability to form a closed ternary
complex (Kirby et al., 2012), highlighting the critical nature of
the open and closed complexes for polymerase function.
to pol b. However, it does not exhibit large subdomain reposi-
tioning in response to nucleotide binding (Garcia-Diaz et al.,
2005). It utilizes a template strand-slippage mechanism that
opens and closes the active site upon dNTP binding at the
expense of frameshift fidelity. Interestingly, alanine or lysine
substitution of the arginine residue that corresponds to Arg283
of pol b (i.e., Arg517) results in a mutant enzyme with reduced
frameshift fidelity. Structural characterization of the mutant
binary DNA complexes indicates that the template strand is in
multiple conformations consistent with a reduced ability to
form a closed complex (Bebenek et al., 2008).
Nucleotide Binding and Metal Coordination
DNA polymerases bind substrates in an ordered fashion with
DNA binding first (Tanabe et al., 1979). Subsequently, dNTPs
bind in a template-dependent manner to preserve Watson-Crick
base pairing. In addition to substrates, catalysis requires two
metals that are coordinated by three active site carboxylates
(Steitz, 1999). The catalytic metal (metal A) lowers the pKAof
the 30-OH of the growing primer strand and coordinates active
site carboxylates (Asp190, Asp192, and Asp256; Figure 1). Addi-
tionally, a nucleotide-binding metal (metal B) coordinates three
nonbridging oxygens from each phosphate of the incoming
nucleotide and two active site carboxylates (Asp190 and
Asp192). The nucleotide-associated metal hastens binding of
the incoming nucleotide and assists PPidissociation. The cata-
lytic metal is believed to bind last because metal B facilitates
binding of the incoming nucleotide that would complete the
coordination sphere for metal A. Prior to polymerase binding
the incoming nucleotide is complexed with divalent magnesium.
Magnesium can bind to any of the phosphates forming mono-,
bi-, or tridentate complexes. Additionally, magnesium can
interact with either oxygen on the a- and/or b-phosphates
creating four or two diastereomers for a,b- and b,g-bidentate
complexes, respectively (Eckstein, 1980). A high-resolution
structure of the pol b active site poised for chemistry indicates
that the nucleotide-bound magnesium is coordinated in a triden-
tate fashion interacting with the pro-RPoxygens of the a- and
b-phosphates (Batra et al., 2006). Thus, the polymerase must
either select the correct conformer or influence the coordination
of the triphosphate. Although it is unlikely that the metal affinity
for the analogs influences the trapping of the structural interme-
diates (Blackburn et al., 1984), it remains to be determined
whether steric features might be involved.
Comparing the three nucleotide and metal positions observed
in the matched structures highlights the reorganization that must
Figure 4. Active Site of Each Pol b Molecule in the Two Molecules per Asymmetric Unit Structure
The active site carbons for the closed two-metal (A) and open metal-free (B) pol b molecules are shown in green and tan, respectively. The carbons of the primer
terminus base pair are gray. The Mn2+ionsare shown as red spheres. A Fo-Fcdensitymap for each incoming nucleotide is shown in green and contoured to 2.7s.
An anomalous mapcontoured at5sover theentireasymmetric unitis shown inred forbothactive sites, highlighting thepresence ofMn2+inthe closedactive site
(A) and lack of metal in the open active site (B) soaked with 200 mM MnCl2.The base pairing between the complementary basesis highlighted (dashed lines). Key
protein side chains are shown in stick representation. See also Figure S5.
DNA Polymerase Intermediate dNTP-Binding States
1834 Structure 20, 1829–1837, November 7, 2012 ª2012 Elsevier Ltd All rights reserved
occur within the active site and incoming nucleotide to achieve
optimal geometry (Figures 3A–3C). This reorganization is en-
couraged when base pair complementarity is maintained,
providing the opportunity for nucleoside triphosphate adjust-
ments to attain an optimal catalytic conformation. There is also
significant movement of the templating and incoming nucleotide
bases, as both metals complete their optimal coordination.
These changes appear to drive the templating base toward the
template-primer junction and the incoming nucleotide toward
a more planar orientation (Figure 3C).
Although altered coordination and reorganization of the
incoming nucleotide within the active site are expected to be
rapid, these steps can be thermodynamically significant. The
ability to capture thesestates through site-directed mutagenesis
and crystallization suggests that these structures are energeti-
callystable and thatthese alternate transient coordination states
will impact the binding of correct and incorrect nucleotides
(discussed below). The intermediate nucleotide conformations
in the open complex suggest that proper coordination of the
nucleotide-binding metal will not occur until the polymerase
closes. This is consistent with the lack of protein coordination
of the Bacillus DNA polymerase I (Wu and Beese, 2011). This
also suggests that metal ligand exchange events may be
a common strategy utilized by DNA polymerases to encourage
or deter catalytic activation for right and wrong nucleotides,
The structures of the intermediate metal/nucleotide conforma-
tions provide insight into additional opportunities for the poly-
merase to select the correct incoming nucleotide. Although
many studies have attempted to relate polymerase discrimina-
tion to rate-limiting conformational changes, it now appears
minants for substrate selection (Tsai and Johnson, 2006). Thus,
an incorrect nucleotide deters incorporation by inducing an
unfavorable catalytic conformation.
The structures suggest additional steps during nucleotide
binding that could provide checkpoints for substrate selection
that involve metal and nucleotide reorganization prior to poly-
merase closure (Figure 3D). Initial nucleotide binding to the
open polymerase complex involves side chains neutralizing the
negative charge on the triphosphate moiety with the loss of an
improperly coordinated metal (step 1). This allows the poly-
merase to sample base pair complementarity, while providing
an opportunity for the triphosphate to achieve proper divalent
metal coordination. In the one-metal-binding stage (step 2),
the triphosphate undergoes reorganization attempting to find
correct catalytic coordination. When the nucleotide-binding
metal achieves good coordination, the catalytic metal (step 3)
binds and stabilizes the closed polymerase conformation.
Importantly, these states represent alternate conformations
that are expected to interconvert and will depend on the nature
of the specific substrates.
Comparing the matched and mismatched alternative nucleo-
tide-binding states indicates a significant difference in the path-
ways during the polymerase reaction. The ability of the correct
going significant rearrangement, provides a fidelity checkpoint
because the correct nucleotide will be stabilized through
Watson-Crick hydrogen bonding. In comparison the mis-
matched nucleotide is stabilized in the active site through the
triphosphate and lacks the stabilizing interactions with the tem-
plating base, thus reducing its affinity for the polymerase active
site. Additionally, upon polymerase closure the matched nucleo-
tide is rearranged to form a catalytically competent complex,
whereas a mismatched nucleotide is sandwiched into the active
site resulting in movement of the primer terminus away from the
triphosphate (Batra et al., 2008).
a mismatch is easily understood in terms of the expected low
Figure 5. One-Metal dG-dAMPCPP Mismatch Open R283K Pol b Structure
(A) The one-metal mismatch pol b structure is shown in magenta with the Mg2+ion and water molecule shown in red and blue, respectively. Key active site
residues are shown with important interactions highlighted with dashed lines. A 2Fo-Fcelectron density map of the incoming nucleotide contoured at 1.2s
highlights the disordered base and sugar moiety for the incoming nucleotide.
(B) Overlay of the pol b dG-dAMPCPP one-metal open R283K and two-metal closed wild-type enzyme (3C2M) structures. The one-metal structure is in magenta,
and the two-metal structure is shown in purple. The open and closed position of helix N, the active site aspartate residues, and the divalent metal ions are
indicated. Mn2+is shown in purple and corresponds to the two-metal closed mismatch wild-type pol b structure.
DNA Polymerase Intermediate dNTP-Binding States
Structure 20, 1829–1837, November 7, 2012 ª2012 Elsevier Ltd All rights reserved 1835
affinity of the wrong nucleotide. Accordingly, if the polymerase
can effectively compete for metal binding, incorrect nucleotides
will be at a disadvantage for binding. In contrast we also were
not able to trap a one-metal complex for a natural G-C base
pair; instead, we relied on a biologically important oxidative
DNA lesion, 8-oxoG (Ames and Gold, 1991). In this situation
the phosphate backbone of the templating 8-oxoG accommo-
dates the normal anti-conformation of the base through a 4 A˚
displacement (Batra et al., 2012; Krahn et al., 2003). Although
this lesion only reduces insertion efficiency 3-fold relative to
guanine, it suggests that the polymerase is sensitive to alter-
ations in the templating pocket and that the one-metal complex
would not accumulate during transition to a closed complex.
The influence of metal binding on reorganization of the incoming
nucleotide identifies a role for the nucleotide-binding metal
outside its role in chemistry. Recently, a structural analysis of
pol h nucleotide insertion identified a third metal occupying
the active site following chemistry (Nakamura et al., 2012).
This metal binds to the DNA and pyrophosphate products
successfully competing with an active site arginine that
hydrogen bonds to the triphosphate moiety of the incoming
nucleotide prior to chemistry. Thus, metals appear to play
important roles in substrate binding and product release by
competing with the polymerase for substrate and product coor-
Human R283K pol b protein was overexpressed in E. coli as previously
described (Beard and Wilson, 1995). Binary complex crystals with either
a templating guanine or 8-oxoG were grown as previously described by Batra
et al. (2006, 2012). The binary complex crystals were transferred to a cryoso-
lution containing 12% ethylene glycol, 50 mM imidazole (pH 8.0), 20%
PEG3350, 90 mM sodium acetate, 5 mM nonhydrolyzable dNTP analogs
(dCMP(CF2)PP or dAMP(CH2)PP), and either MgCl2or MnCl2. This resulted
in formation of the ternary complex. The final metal-free ternary complex
was soaked in 30 mM MgCl2 and also observed in 200 mM MgCl2.
The one-metal-matched and -mismatched ternary complex was soaked
in 200 mM MgCl2. The two-metal-matched ternary complex was soaked in
200 mM MnCl2. The two molecules per asymmetric unit were soaked in
200 mM MnCl2.
Data were collected at 100 K on a SATURN92 CCD detector system
mounted on a MiraMax-007HF rotating anode generator. Data were pro-
cessed and scaled using the HKL2000 software package (Otwinowski and
Minor, 1997). Initial models were determined using molecular replacement
with the previously determined open (3ISB) or closed (2FMS) structure of pol
b as a reference (Beard et al., 2009). All Rfreeflags were taken from the starting
model. Refinement was carried out using PHENIX and model building using
Coot (Adams et al., 2010; Emsley and Cowtan, 2004). The figures were
prepared in PyMOL (Schro ¨dinger). Ramachandran analysis determined that
100% of nonglycine residues lie in the allowed regions and at least 97% in
A 34-mer oligonucleotide DNA substrate containing a single-nucleotide gap
witha templating guanine was created as previously describedwith a templat-
ing guanine in the coding position (Cavanaugh et al., 2010). Steady-state
kinetic parameters for single-nucleotide gap-filling reactions were determined
as previously described (Beard et al., 2004).
The atomic coordinates and structure factors reported in this paper have been
deposited in Protein Data Bank (http://www.pdb.org): 4F5N (matched, zero
metals); 4F5O (matched, one metal); 4F5P (mismatched, one metal); 4F5Q
(matched, two metals); and 4F5R (two molecules/ASU).
Supplemental Information includes five figures and can be found with this
article online at http://dx.doi.org/10.1016/j.str.2012.08.008.
We thank Drs. R.E. London, L.C. Pedersen, and V.K. Batra for critical reading
of the manuscript and Dr. C.E. McKenna for the a,b-difluoromethylene dCTP
analog. This research was supported by Research Project Numbers
Z01-ES050158 and Z01-ES050161 to S.H.W. in the Intramural Research
Health Sciences and was in association with the National Institutes of Health
Grant 1U19CA105010. B.D.F., W.A.B., and S.H.W. designed the research;
B.D.F., W.A.B., and S.H.W. analyzed data; B.D.F. performed research; and
B.D.F., W.A.B., and S.H.W. wrote the paper.
Received: July 17, 2012
Revised: August 8, 2012
Accepted: August 10, 2012
Published online: September 6, 2012
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