Download DNA polymerase going in reverse: Requirement for transient metal

Lalith Pereraa,1, Bret D. Freudenthala, William A. Bearda, David D. Shocka, Lee G. Pedersena,b, and Samuel H. Wilsona
a
Laboratory of Genome Integrity and Structural Biology, National Institute of Environmental Health Sciences, National Institute of Health, Research Triangle
Park, NC 27709-2233; and bDepartment of Chemistry, CB 3290, University of North Carolina, Chapel Hill, NC 27599
Edited by Suse Broyde, NYU, New York, NY, and accepted by the Editorial Board August 14, 2015 (received for review June 9, 2015)
DNA polymerases facilitate faithful insertion of nucleotides, a
central reaction occurring during DNA replication and repair. DNA
synthesis (forward reaction) is “balanced,” as dictated by the
chemical equilibrium by the reverse reaction of pyrophosphorolysis.
Two closely spaced divalent metal ions (catalytic and nucleotide-binding metals) provide the scaffold for these reactions. The catalytic
metal lowers the pKa of O3′ of the growing primer terminus, and
the nucleotide-binding metal facilitates substrate binding. Recent
time-lapse crystallographic studies of DNA polymerases have identified an additional metal ion (product metal) associated with pyrophosphate formation, leading to the suggestion of its possible
involvement in the reverse reaction. Here, we establish a rationale
for a role of the product metal using quantum mechanical/molecular
mechanical calculations of the reverse reaction in the confines of the
DNA polymerase β active site. Additionally, site-directed mutagenesis
identifies essential residues and metal-binding sites necessary for
pyrophosphorolysis. The results indicate that the catalytic metal site
must be occupied by a magnesium ion for pyrophosphorolysis to
occur. Critically, the product metal site is occupied by a magnesium
ion early in the pyrophosphorolysis reaction path but must be removed later. The proposed dynamic nature of the active site metal
ions is consistent with crystallographic structures. The transition barrier for pyrophosphorolysis was estimated to be significantly higher
than that for the forward reaction, consistent with kinetic activity
measurements of the respective reactions. These observations provide a framework to understand how ions and active site changes
could modulate the internal chemical equilibrium of a reaction that is
central to genome stability.
DNA polymerase
Although the biological implications are significant, a detailed
chemical description of the reverse reaction is currently lacking.
DNA polymerase (pol) β has served as a model eukaryotic
DNA polymerase for computational, structural, mechanistic, kinetic, and biological studies (10–12). Based on the structure of
DNA bound to the exonuclease site of Escherichia coli DNA polymerase I, a general two-metal ion mechanism for the nucleotidyl
transferase (nuclease and DNA synthesis) reaction was proposed
25 y ago (13). High-resolution crystallographic structures of a
precatalytic ternary complex of pol β are consistent with the twometal ion mechanism (14, 15). The active site metal ions are referred to as the catalytic (Mc) and nucleotide (Mn) metals. The
metal ions coordinate the primer terminus nucleophilic oxygen
(i.e., O3′), oxygens of each phosphate of the incoming nucleotide,
and active site aspartate residues Asp190, Asp192, and Asp256
(Fig. 1). The catalytic metal ion lowers the pKa of the primer
terminus, whereas the nucleotide metal ion balances the developing negative charge during chemistry.
Recent time-lapse crystallography snapshots of postcatalytic
events (i.e., with products) revealed that a Na+ ion replaces
Mg2+ at the catalytic metal ion site and that a new Mg2+-binding
site (Mp) associated with the nascent products (Fig. 2, extended
DNA and PPi) forms transiently (16). The product-associated metal
ion has also been observed in structures of pol η (17) and pol β with
alternate substrates (18, 19). It was proposed that the product
metal ion may play a role in modulating charge distribution
Significance
| pyrophosphorolysis | QM/MM
DNA polymerases use a general two-metal ion mechanism for
DNA synthesis. Recent time-lapse crystallographic studies identified additional adjunct metal ions in the polymerase active site.
One of these ions correlates with appearance of pyrophosphate
and was proposed to be involved in pyrophosphorolysis (reverse
reaction of DNA synthesis). Because DNA polymerases can use
pyrophosphorolysis to remove chain-terminating nucleotides
during chemotherapies, a better understanding of this reaction
is warranted. Through mutagenesis, pyrophosphorolysis measurements, and computational analysis, we examine the role of
metal ions in the reverse reaction. The results indicate that the
product-associated metal ion facilitates pyrophosphorolysis
during the early stages of the reaction but deters the reaction at
later stages, suggesting dynamic behavior that can modulate the
internal chemical equilibrium.
D
NA polymerases are responsible for high-fidelity DNA
synthesis during replication and repair of the genome (1).
Although there are at least 17 human DNA polymerases, they all
use a general nucleotidyl transferase DNA synthesis reaction.
This reaction requires deoxynucleoside triphosphates (dNTPs),
divalent metal ions, and DNA substrate with a primer 3′-OH
annealed to a coding template strand. An inline nucleophilic
attack of the primer 3′-oxyanion on Pα of the incoming dNTP
results in products with DNA extended by one nucleotide (i.e.,
dNMP) and pyrophosphate (PPi). If the enzyme does not release
PPi, pyrophosphorolysis (reverse reaction) can generate dNTP
and a DNA strand that is one nucleotide shorter (Fig. 1) (2).
Although the forward DNA synthesis reaction is preferred, the
pyrophosphorolysis reaction can be biologically significant. Because
DNA polymerases are an attractive chemotherapeutic target, chainterminating nucleoside drugs are often used in a strategy of blocking
DNA synthesis (3–5). However, drug resistance to chain-terminating agents is influenced by the ability of a stalled DNA polymerase
to remove chain-terminating nucleotides through pyrophosphorolysis (6–8). Additionally, pyrophosphorolysis has been reported to
remove misinserted nucleotides opposite specific DNA lesions as a
proofreading activity (9). Accordingly, a better understanding of the
mechanism of this reaction will hasten drug design and intervention.
www.pnas.org/cgi/doi/10.1073/pnas.1511207112
Author contributions: L.P., B.D.F., W.A.B., L.G.P., and S.H.W. designed research; L.P., W.A.B.,
and D.D.S. performed research; L.P., W.A.B., and D.D.S. analyzed data; and L.P., B.D.F., W.A.B.,
L.G.P., and S.H.W. wrote the paper.
The authors declare no conflict of interest.
This article is a PNAS Direct Submission. S.B. is a guest editor invited by the Editorial
Board.
1
To whom correspondence should be addressed. Email: [email protected].
This article contains supporting information online at www.pnas.org/lookup/suppl/doi:10.
1073/pnas.1511207112/-/DCSupplemental.
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DNA polymerase going in reverse: Requirement for
transient metal ions revealed through
computational analysis
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Fig. 1. Proposed two-metal reaction schemes for the forward reaction of
nucleotide incorporation and the reverse reaction of pyrophosphorolysis by
pol β. The conserved active site carboxylates that coordinate two magnesium
ions (green spheres) are shown. The metal nearest Asp256 (D256) is the
catalytic metal, and the metal that coordinates Asp190 (D190) and Asp192
(D192) is the nucleotide-binding metal. Two water molecules coordinating
the metal ions are also shown (small red spheres).
during the reverse reaction (16). To probe this possibility, we
used a quantum mechanical/molecular mechanical (QM/MM)
computational approach (20, 21) that has become an instrumental tool in mechanistic studies on enzyme catalysis. The results
provide evidence that transient metal ions mediate charge distribution within the polymerase active site during chemistry that can
alter the internal chemical equilibrium between the forward and
reverse reactions.
Results
QM/MM Calculations. The energy profile and transition state determined previously for the forward DNA synthesis reaction (22–
24) were used here as a frame of reference, where the energy at
the transition state corresponded to ∼18 kcal/mol and the transition state was at an O3′–Pα distance of ∼2.2 Å. With this in
mind, we initially examined the energy profile of the reverse
reaction for the NacMgnMgp system based on the product
complex observed immediately following catalysis (Fig. 3, Left)
(16). As the distance between the PPi nucleophilic oxygen and Pα
was decreased, the energy profile resembled that of the forward
reaction while these atoms were >2.3 Å apart. However, as this
distance was further decreased, the energy for the reverse reaction became unrealistic during what would traditionally be the
transition state. In the final calculation at a Pα–Oβ distance of
1.7 Å, the three reactive atoms O3′–Pα–Oβ were in positions
similar to those for the transition state of the forward reaction (Fig.
3, Right) (22). However, the energy at this point (∼60 kcal/mol) was
unreasonably high, and the profile indicated the system was essentially locked with no possible products. The product metal ion/
oxygen coordination was maintained by the system throughout the
entire energy profile calculations. The presence of the product
metal ion appeared to block the reaction at Pα–Oβ distances
expected of the transition state.
Because these results indicated the NacMgnMgp system does
not yield an energetically realistic path for pyrophosphorolysis,
we next determined whether an Mg2+ in the catalytic metal ionbinding site, as observed in certain crystallographic structures,
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could favorably influence the energy profile of the reverse reaction. Therefore, the Na+ in the catalytic metal ion site was
substituted with an Mg2+ in preparing the MgcMgnMgp system
(Fig. 4, Left), and the calculations repeated. The configuration
with the lowest energy for this system was found at the Pα–Oβ
distance of 3.0 Å, and this was selected to be the starting point
for this system (Fig. 4, gray structure). All magnesium ions were
octahedrally coordinated in the starting configuration of the
QM component. In this configuration, the distance between the
magnesium ions at the catalytic and nucleotide metal sites was
3.62 Å, and the product metal was 4.99 Å distant from the
nucleotide metal.
As with the first calculation, the energy profile indicated a
catalytically unrealistic reaction path (Fig. 4, Left). When the
Pα–Oβ distance was 1.7 Å, the energy of the system (∼50 kcal/mol)
was far too high to be relevant; the final structure (Fig. 4, colored
structure) was similar to that for the NacMgnMgp system (Fig. 3).
Interestingly, the energy in the vicinity of a possible transition
state for the MgcMgnMgp system was lowered by the presence of
the Mgc, indicating the possible requirement of Mg2+ in the
catalytic metal ion position. Thus, both of these systems were
inappropriate for mediating the reverse reaction, in part because
the product metal ion appeared to be blocking the reaction late
in these reaction paths.
A two-metal ion mechanism was originally proposed in the
nucleotidyl transferase process (13). To examine whether Mg2+
at these two metal ion-binding sites (catalytic and nucleotide)
might be sufficient for pyrophosphorolysis, we modified the potentially reactive core so as to prepare an MgcMgn system by
removing Mgp and replacing it with a water molecule. After
constrained and then unconstrained molecular dynamics (MD)
to achieve equilibrium, a QM/MM initial system was defined
from the final optimized configuration of the classical trajectory.
Several additional water molecules (six) that coordinated the
pyrophosphate and the bridging phosphate were included in the
QM component (Fig. 5, structure representation). In the optimized QM/MM reference system, the Pα–Oβ distance was 3.6 Å.
This was the largest separation for these atoms for all systems
studied. In the other systems for which the product metal was
present, this distance was 0.7 Å shorter. The two magnesium ions
Fig. 2. The product metal-binding site. The product metal (Mgp) coordinates the products of the forward reaction (i.e., nonbridging oxygens on
the incorporated dCMP and PPi). A sodium ion (Nac) occupies the catalytic
metal ion site in this product ternary complex structure (PDB ID code 4KLG).
Metal-coordinating water molecules are shown as small red spheres.
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Fig. 3. QM/MM calculation for the NacMgnMgp system. Energy profile for the system that mimics an intermediate crystallographic substrate/product complex
is shown in the Left panel. The calculated energy is plotted against the Pα–Oβ distance as the reaction coordinate (i.e., the oxygen closest on PPi to the primer
terminal phosphate). The approximate ground, transition, and products states (GS, TS, and PS) of the reverse reaction are indicated. The starting (gray) and
final (color) structures in the QM system are shown in the Right panel. Hydrogens are not shown for clarity, and the dotted line represents a pseudobond
between Pα and Oβ in the final configuration.
were separated by 3.56 Å and maintained octahedral coordination, as in the two systems described above.
The energy profiles for the MgcMgn system is shown in Fig. 5.
Although there were subtle differences in the energy along the
reaction path, the overall features of the energy profile with this
system (Fig. 5, Left) were similar to those observed in the other
two systems. Importantly, the reverse reaction was not observed,
thereby indicating that the mechanism of the reverse reaction is
not simply the same as the forward reaction.
For the three systems described thus far, an instructive comparison of the energy profiles is provided in Figs. 4 and 5. When
Mg2+ occupies the product metal ion site, the energy barrier in
the early part of the reaction path (Pα–Oβ distances from 2.9 to
2.4 Å) is 8–10 kcal/mol lower than for the two-metal ion system
(Fig. 5, Left Inset). Thus, the results for the early portion of these
profiles suggested a beneficial effect of Mg2+ in the product
metal ion site during the initial attack of the PPi oxygen on the
primer terminus phosphate (i.e., Pα). However, it becomes in-
hibitory when these atoms approach 2.3 Å. A similar inhibition
phenomenon occurs when Na+ occupies the catalytic metal site.
Fig. 4 indicates that additional energy is required, as the Pα–Oβ
distance is reduced below 2.4 Å when Na+, a weak Lewis acid, is
in the catalytic metal ion-binding site. From this comparison, we
deduce that Mg2+, a stronger Lewis acid, is required in the catalytic site. A product Mg2+ ion lowers the activation energy in
the early part of the reaction path but must be displaced midpath
in order for the reaction to proceed beyond the transition state
(∼2.4 Å). It appears that the “strong” coordination (strict octahedral geometry and ligand distances) exhibited by Mg2+ in the
product metal site as the reaction approaches the transition site
region does not permit the reaction to proceed, thereby precluding Mg2+ in the catalytic site facilitating product departure.
Pol β Pyrophosphorolysis Solution Assay. In light of the results
described so far, we decided to confirm that pol β is capable of
catalyzing the reverse reaction. A qualitative assay to measure
Fig. 4. QM/MM calculation for the MgcMgnMgp system. This is similar to Fig. 3, except with magnesium in the catalytic metal site and reoptimizing the
system at the initial MD level before starting QM/MM optimizations. The energy profile (Left) of the NacMgnMgp system is also shown here for comparison
(blue triangles). The starting (gray) and final (color) structures in the QM system are shown in the Right panel.
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Fig. 5. QM/MM calculation for the MgcMgn system. This system was created after exchanging the positions of Mgp in the Mgc–Mgn–Mgp system with that of
the oxygen of a remote water molecule and reoptimizing the system in the beginning at the MD level before starting QM/MM optimizations. The energy
profile (Left) of the MgcMgnMgp (red diamonds) is also shown for comparison. In the Inset, the early gain in energy for the three-metal system is emphasized.
The starting (gray) and final (color) structures in the QM system are shown in the Right panel.
pyrophosphorolysis activity in solution with nicked DNA as
substrate demonstrated that the wild-type enzyme can remove
3′-nucleotides from the substrate in a short period (Fig. 6). In a
single-turnover assay system, where the enzyme concentration exceeded the substrate concentration, removal of the 3′-nucleotide
occurred at a rate of 0.03/s. This rate of the reverse reaction is over
two orders of magnitude less than that of the forward reaction (i.e.,
the insertion of a single nucleotide into a gapped substrate) (25).
A crystallographic structure of the ternary substrate precatalytic
complex indicated that the catalytic metal site was empty for the
alanine mutant of Asp256 (24). We found that the reverse reaction
was also lost with this mutant (Fig. 6), suggesting that there is a
stringent requirement for the catalytic metal ion during pyrophosphorolysis. Additionally, because Arg183 interacts directly with the
PPi product and stabilizes it in the active site (16), the effect of loss
of this interaction was experimentally determined for the reverse
reaction. As expected, the results indicated that loss of the Arg183
interaction severely limits the reverse reaction (Fig. 6).
Assuming that the experimentally determined rate measured
for the forward and reverse reactions represent chemistry, the
experimental free energy difference (3.6 kcal/mol) is similar to
the calculated difference (4 kcal/mol). However, because the
computational uncertainty is similar to the calculated difference,
the chemical equilibrium constant could be significantly different
from the simple ratio of the observed forward and reverse rate
constants (26–30).
QM/MM System to Probe a Transient Product Metal. Time-lapse
X-ray crystallographic structural snapshots had suggested that
the occupancy of the product metal site is transient. The product
metal is not observed until a significant population of PPi is
formed and dissociates before PPi dissociation (16). To better
understand these crystallographic results, we needed to develop
a QM component that could reflect the transient nature of this
metal. However, it is problematic to recreate transient occupation of the product metal site with an optimum number of QM
atoms selected; the expansion of the QM system size to permit
movement of the product metal would be computationally prohibitive. Therefore, we chose a novel approach to mimic the
transient nature of the product metal. We reduced the rigidity
imposed by the Mg2+ ion at the product metal site by substituting
it with Na+. This substitution of Na+ allows an extended coordination distance and prevents the restrictive nature imposed
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by magnesium to preserve its coordination sphere. In addition,
because we computationally observed a beneficial effect of Mg2+
at the catalytic metal site, we switched the catalytic site sodium to
a magnesium ion. Thus, the original NacMgnMgp system became
MgcMgnNap. After equilibration, this system has a relaxed metal
ion–oxygen coordination sphere at the product site that reflects
the transient nature of a metal ion at this site. In the equilibrated
system, Na+ has oxygen coordination distances that are longer
(∼2.4 Å) than those for Mg2+ (∼2.1 Å) (31).
First, to test the idea of using this new system in a full QM/MM Q:11
calculation, we reoptimized the original NacMgnMgp geometry at
the Pα–Oβ distance of 1.7 Å for the new MgcMgnNap system and
found that the system spontaneously converted to the product state
for the reverse reaction (Fig. 7, green triangle). This indicated that
the new system was not trapped in a dead end complex and was
capable of yielding products. Next, an unconstrained optimization
Fig. 6. Pyrophosphorolysis. The reverse reaction (pyrophosphorolysis) was
assayed under single-turnover conditions (E>>DNA) using a 5′-[32P] nicked
DNA substrate with wild-type (WT) pol β or D256A and R183A mutants.
Reactions were initiated with MgCl2/PPi and stopped after 1, 3, 5, or 10 min.
The products were separated on a 16% denaturing gel as outlined in Materials and Methods. The first lane (P) represents the full-length primer (n),
whereas the shorter products (n–x) represent continued pyrophosphorolysis
reactions generating additional products. Loss of pyrophosphorolysis activity
by removing a catalytic metal-binding ligand through D256A mutation indicates that the catalytic metal is essential for the reverse reaction. Likewise,
removing a hydrogen bond with PPi through R183A substitution dramatically decreases pyrophosphorolysis.
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Fig. 7. Influence of Nap late in the reaction coordinate. The Nac of the NacMgnMgp system was replaced with a magnesium ion and Mgp with a sodium ion (i.e.,
swapping the two ions occupied at catalytic and product metal ion sites as shown in the Right panel) in the final conformation (Pα–Oβ = 1.7 Å) shown in Fig. 3. Relaxing
the resultant system using a QM/MM reoptimization yielded a triphosphate product conformation (green triangle).
of the original NacMgnMgp system in its geometry at a Pα–Oβ distance of 1.7 Å spontaneously resulted in a state similar to that of the
starting point of the reaction path (SI Appendix, Fig. S1, open triangle at a Pα–Oβ distance of 2.9 Å). Thus, because a product state
could be observed in the MgcMgnNap system, we extended the
QM/MM examination to all geometries in the energy profile of
the MgcMgnNap system.
The energy profile for the MgcMgnNap system is shown in Fig. 8
(green circles). When the catalytic metal site is occupied by Mg2+
and the product metal site by Na+, a complete reaction profile with
a broad transition state (2.0–2.2 Å) is observed, with an activation
barrier of ∼22 kcal/mol. Fig. 8 also provides comparison of the twometal (MgcMgn) and three-metal (MgcMgnMgp) ion systems for the
reverse reaction with that of a system in which a third metal
(a sodium that mimics a transient metal) occupies the product metal
site. A metal ion in the product metal site substantially lowers the
energy in the initial phase of the reaction path. Importantly, the
transient nature of the product metal ion (i.e., replacement with a
sodium ion) facilitates the product state by producing a catalytically
competent reaction path. Nevertheless, the energy barrier at the
transition state of the reverse reaction is higher than that reported
for the forward reaction (22–24) (i.e., ∼22 vs. ∼18 kcal/mol). In
addition, a weak coordination for the water molecules solvating Na+
is observed (Fig. 8) in comparison with that of the Mg2+ at the
product metal ion site.
Charge Calculations and the Mechanism of Transient Metal Effects
on the Reverse Reaction. The QM component in these studies
consisted of 113 atoms. A substantial number of atoms in this
quantum subsystem are buried. It is generally recognized that
Fig. 8. QM/MM calculation for the MgcMgnNap system. All QM/MM optimized configurations in Fig. 3 were subjected to the swapping of the metal ions
occupied at catalytic and product metal-binding sites to create this system. The energy profile (Left) for the reoptimized structures for the MgcMgnNap system
(green circles) is compared with the MgcMgnMgp and MgcMgn systems (red diamonds and black squares, respectively). The Inset shows the energy profiles for
the transition region. A structural state (Right) that resembles the expected triphosphate product (in color) is compared with an early configuration in the
reaction coordinates (gray).
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charge calculations using electrostatic potential fitting schemes,
such as Merz–Singh–Kollman (32), Chelp (33), and ChelpG (34),
can become unreliable for buried charges (35). Therefore, we
selected an atomic charge method that yields a stable charge
distribution for atoms. The CM5 charge model (35), an extension
of Hirshfeld population analysis proposed by the Truhlar group
and that is adapted to accommodate buried atoms properly, was
used for calculating QM component charges. We subsequently
performed a QM cluster calculation on the QM atoms (including
protons at the pseudoatom positions in the boundaries) in the
presence of the charge distribution of the MM atoms, so as to
preserve the surrounding QM/MM environment that significantly influences the QM component. The resultant electrostatic
charges obtained from the CM5 charge scheme are tabulated (SI
Appendix, Table S1 A–D; the atom identities are given in SI
Appendix, Table S1E). For clarity, we present the atomic charge
differences (current – initial value) at each selected step.
The charge variations during the reaction profiles can be used
to examine the redistribution of the electron density during the
course of the reaction (SI Appendix, Fig. S2). In the initial portion of the reaction path (2.9–2.4 Å), the variations of charge
for all four systems were similar. Interestingly, however, in the
MgcMgnNap system, for which the reverse reaction was observed,
a relatively larger charge variation was found for the new primer Q:12
O3′ product (atom 62, variation of about 0.24e for MgcMgnNap
versus 0.09e–0.14e for the other three systems) of the sugar ring.
This oxygen atom became more negative as the transition state
developed and resolved. The result in this case was reminiscent
of the charge variation found for the same oxygen atom during the
forward reaction (24), except in this case the oxygen becomes more
negative instead of less negative (as in the forward reaction) after
the transition state. This charge variation is mediated by the catalytic magnesium ion, explaining the requirement for magnesium at
the catalytic site (Fig. 9). Participation of water molecules in charge
redistribution was significant, as revealed by the relatively larger
charge variation of several of the water molecules (water oxygen
atoms 96 and 99), again especially in the MgcMgnNap case for which
the reverse reaction was observed.
Discussion
It is a basic tenant of the theory of enzyme action that the
enzyme catalyzes both the forward and reverse reaction. Thus,
for forward reactions that do not have a large negative free
energy change, the reverse reaction can become an important
Fig. 9. Reaction path and various metal ion-binding site occupancies during the course of the pyrophosphorolysis reaction. The starting configuration (Top
Right) of the reverse reaction is the product structure of the nucleotide incorporation reaction. A sodium ion is observed in the catalytic metal-binding site. In
the transition state (Top Middle), the catalytic metal site requires a magnesium ion. The conversion of PPi to dNTP is found in the product state of the
pyrophosphorolysis reaction (Top Left). Shown are potential metal ion occupancies at (A) the catalytic metal ion-binding site and (B) the product metal ionbinding site for various segments of the reaction path to facilitate the pyrophosphorolysis.
6 of 9 | www.pnas.org/cgi/doi/10.1073/pnas.1511207112
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in the DNA polymerase catalytic cycle had not occurred to us until
time-lapse crystallographic structural intermediates revealed a new
metal-binding site accompanying product formation was transient in
nature (i.e., not observed in pre- or postcatalytic structures) (16).
Previous work had noted that sodium easily exchanged for magnesium at the catalytic metal-binding site (15, 16). The sodium exchange for the catalytic metal ion is probably facilitated by the loss
of a metal-binding ligand (primer O3′) as DNA is extended by a
nucleotide in the forward reaction. The calculations provide an
additional feature of the catalytic site metal ion exchange; when a
sodium ion occupies the catalytic metal site, the reverse reaction
was blocked. Accordingly, loss of the catalytic magnesium ion after
insertion (forward reaction) commits the reaction forward.
In hindsight, the dynamic nature of the metals is not surprising.
During the course of the chemical reaction, developing negatively
charged ligands must be neutralized in the product state. Thus,
metal–ligand interactions in the ground state are broken during the
course of the reaction, and new product ligands generate new metal
interactions. This appears to be the situation with a metal ion at the
product metal-binding site described here. Because products must
be released for catalytic cycling, the stabilizing influence of the
product metal ion must be removed to permit product dissociation.
Conclusions
We have performed a series of QM/MM calculations and pyrophosphorolysis assays to examine the effect of the product metal ion
on the pyrophosphorolysis reaction catalyzed by pol β. This adjunct
metal was observed near the end of the nucleotide insertion reaction in the recent time-lapse crystallographic structures (16–19).
Our results from QM/MM calculations indicate that the pyrophosphorolysis reaction is dependent on a magnesium ion in the
catalytic metal site. Additionally, a magnesium or sodium ion in the
product site reduces the activation barrier in the initial portion of
the pyrophosphorolysis reaction coordinate. However, continued
occupancy of the product site by a magnesium ion shows a strong
inhibitory effect as the reaction proceeds into the anticipated
transition state region; that is, Pα–Oβ distances are less than 2.4 Å
and must be removed for the pyrophosphorolysis reaction to proceed. The calculated transition state energy barrier for the pyrophosphorolysis reaction is within a range (∼22 kcal/mol) that is
generally consistent with our solution kinetics studies. These observations provide a framework to understand how ions and active
site charge distribution modulate the internal equilibrium of a reaction that is central to genome stability.
Transient Metals and the Reverse Reaction. The calculations indicate
that pyrophosphorolysis requires a temporal order of metal binding
in the catalytic and product metal sites (Fig. 9). Starting with the
crystallographic ternary product structure (Fig. 3, structural image),
magnesium in the product metal site is beneficial as the Pα–Oβ
distance is reduced from 2.9 to 2.4 Å. This is evident by comparing
the energy of the MgcMgn system with that of the MgcMgnMgp
system; the latter system was 8–10 kcal/mol more favorable (Fig. 4).
After this point in the reaction path, the presence of magnesium
in the product metal site was inhibitory. This inhibition arises from
the Mg2+ sterically blocking bond formation in the computational
studies, thus indicating the product metal must be transiently bound
during catalysis. Substituting a sodium ion for the magnesium ion
permits the reaction to proceed given the “loosened” ligand coordination of sodium compared with magnesium. An additional
critical observation is that the reaction can only proceed when the
catalytic metal site contains a magnesium ion. Regarding the catalytic metal site, the crystallographic ternary product structure
contains a sodium ion in this site (Fig. 3, structural image). As
the Pα–Oβ distance is reduced from 2.9 to 2.4 Å, the energy with
either sodium or magnesium in the catalytic site was similar (Fig.
4). After this point, however, magnesium in the catalytic site was
favorable. This was evident when the inhibitory effect of magnesium in the product metal site was removed by substituting with
sodium at this site (Fig. 8). This transient behavior of the active
site metals during the reverse reaction is illustrated in Fig. 9.
Perera et al.
PNAS Early Edition | 7 of 9
PNAS PLUS
Overview. The notion that transient metal ions could be important
Given the positive charge of a metal ion in the context of the
active site, it is likely that a transient metal would have a significant impact on charge distribution during the reaction. In this
way, the impact of the metal ions in the QM/MM systems used
here can be rationalized. For the reverse reaction, the switch
from Mg2+ to Na+ at the product site mimics the transient nature
of this metal. Likewise, Mg2+ at the catalytic site is absolutely
required, indicating that magnesium must rebind to this site for
pyrophosphorolysis to occur. Although the mechanism of the ion
exchange at these two sites originates for different reasons, the
transient feature of active site metals can provide catalytic versatility (i.e., modulate the active site chemical equilibrium) and
may be a general strategy used by other metal-dependent enzymes. Nonetheless, metal ion transitions during the course of a
reaction present a computational challenge and opportunity.
Because many DNA polymerases undergo subdomain motions
that accompany substrate binding, the active site also transitions
from an open, solvent-exposed state to a closed, less solventaccessible state (11). In this context, it should be noted that these
calculations are performed in an environment of the closed polymerase active site and are thus sensitive to the internal chemical
equilibrium at the enzyme active site. As described above, active site
metals can modulate the internal equilibrium in the closed polymerase state. Open/closed enzyme conformational changes would
be expected to also alter the overall equilibrium. For example, rapid
reopening after DNA synthesis that permits PPi dissociation would
“pull” the reaction forward, thereby increasing the observed equilibrium constant toward DNA synthesis.
Because protein dynamics are not considered here, it is important
to acknowledge the technical limitations of the present work. Chung
et al. (36), in their review of the method used here, note the challenging task to carry out stable geometry optimization of a complex
system with an enormous number of degrees of freedom that leads
to a great number of potential conformations. By necessity, our
sampling is limited but, we believe in this case, is adequate for the
current exploration. In addition, the QM/MM methodology that
was developed to treat the essential parts of the enzymatic reaction
by QM can give rise to potential errors due to insufficient size of the
QM system, treatments of the QM and MM boundaries, and
electrostatic interactions between the QM and MM systems. Previously, Ryde and coworkers (37) reported that the mean absolute
errors emanating from systems could be as much as 7 kcal/mol.
Despite these potential errors, the magnitude of which is difficult to
systematically estimate, the current level of methodology is quite
useful for combining with crystallography and experimental measurements when considering mechanistic possibilities.
condition-dependent consideration. Such is the likely case for
the pol β chemical nucleotide insertion (forward) and pyrophosphoroylsis (reverse) reactions. We have previously used
QM/MM methods to complement our experimental structural
studies of the forward chemical nucleotide insertion for pol β
(22–24). Given the substantial evidence that the reverse reaction
occurs and the recent identification of an adjunct third metal
transiently observed during substrate/product interconversion (16),
we embarked on a series of QM/MM simulations that model the
reverse reaction. A logical progression of calculations NacMgnMgp,
MgcMgnMgp, and then MgcMgn did not provide a productive path;
that is, the transition barriers were too large to be feasible. However, when we used the geometries generated by the NacMgnMgp
system to optimize energies and geometries for an MgcMgnNac
system, a reasonable path for pyrophosphorolysis that is energetically feasible was found that also accommodates the transient nature of the product metal.
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Materials and Methods
Pyrophosphorolysis Solution Assays. Bacterially expressed wild-type human
pol β and mutants (D256A and R183A) were purified as described previously
(38). Reactions were conducted in 50 mM Mes, 25 mM Tris, 25 mM ethanolamine (pH 7.5 adjusted at 37 °C) containing 100 mM KCl, 1 mM DTT, and
100 μg/mL BSA. Pol β (2 μM) was preincubated with nicked DNA (200 nM) for
5 min at 37 °C. The reaction was initiated by manual mixing with a prewarmed solution of 20 mM Mg2+ and 2 mM PPi (1:1, vol/vol) (final enzyme,
DNA, MgCl2, and PPi concentrations were 1 μM, 100 nM, 10 mM, and 1 mM,
respectively). Aliquots (10 μL) were removed at various times and quenched
in an equal volume of 0.3 M EDTA, pH 8.0. Reaction substrates and products
were resolved on 16% denaturing (8 M urea) polyacrylamide gels and visualized by phosphorimagery using a Typhoon 8600 (GE Healthcare) imager
and ImageQuant software.
Computational Procedure for QM/MM Calculations. Initial structures for theoretical calculations were prepared using a recent high-resolution crystallographic structure of the ternary product complex [Protein Data Bank (PDB)
ID code 4KLG] where the catalytic metal site is occupied by a sodium ion and
magnesium ions occupy the nucleotide and product metal sites. MD simulations were carried out in a completely solvated aqueous medium after
hydrogens and neutralizing sodium ions were added. Positions of all crystallographic water molecules were preserved initially. The pyrophosphate
charge was taken to be –3, and the phosphate on the pyrophosphate near
the newly formed DNA primer terminus was assumed to be completely
deprotonated. All MD trajectory calculations were carried out with the
Amber12SB force field using the PMEMD module of Amber.12 (39). Water
molecules were represented by the TIP3P model (40). Long-range interactions
were treated with the particle mesh Ewald method (41). After initial NPT
trajectory at 10K to adjust the density of the system near 1.0 g/cm3, a 20-ns
constant volume/constant temperature (T = 300K) equilibrium simulation with
the sequentially decreasing harmonic constraint force constants (from 50 to
0.1 kcal/mol/nm) applied to the protein, DNA, and metal ions in the crystallographic structure ensures that the system coordinates represent a precatalytic
state. Before QM/MM calculations, we optimized several configurations selected from MD simulations and used the lowest energy system as the starting
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configuration for the reaction path calculation; other configurations are within
3 kcal/mol to this lowest energy conformation. The lowest energy configuration
was used for the reaction path calculation. The system with a magnesium ion at
the active site was prepared from the same coordinates (PDB ID code 4KLG). A
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the MM region. The only reaction coordinate present in the current protocol
was the distance between the phosphorous atom of the phosphate group of
the primer terminus and the nearest oxygen atom of PPi.
ACKNOWLEDGMENTS. This research was supported by Research Projects
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the Intramural Research Program of the National Institute of Health,
National Institute of Environmental Health Sciences and in association with
the National Institutes of Health Grant U19CA105010.
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Q: 7_PNAS allows up to five keywords. You may add 2 keywords. Also, please check the order of your
keywords and approve or reorder them as necessary.
Q: 8_Please provide a new title that does not contain a colon.
Q: 9_PNAS articles should be accessible to a broad scientific audience. As such, please spell out the d in
dNMP.
Q: 10_PNAS discourages claims of priority; is this truly novel? If not, please either (a) replace the term
“novel” with a term such as “previously unidentified” or (b) remove it altogether to avoid the claim
of priority.
AUTHOR QUERIES
AUTHOR PLEASE ANSWER ALL QUERIES
2
Q: 11_PNAS discourages claims of priority; is this truly new? If not, please either (a) replace the term
“new” with a term such as “previously unidentified” or (b) remove it altogether to avoid the claim
of priority. Please replace all instances of “new system” (if needed) throughout the article.
Q: 12_PNAS discourages claims of priority; is this truly new? If not, please either (a) replace the term
“new” with a term such as “previously unidentified” or (b) remove it altogether to avoid the claim
of priority.
Q: 13_Please state the basis (e.g., vol/vol) for all concentrations greater than 1%.
Q: 14_PNAS articles should be accessible to a broad scientific audience. As such, please spell out
PMEMD.
Q: 15_PNAS articles should be accessible to a broad scientific audience. As such, please spell out TIP3P.
Q: 16_PNAS articles should be accessible to a broad scientific audience. As such, please spell out NPT.