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Published online 30 May 2018 Nucleic Acids Research, 2018, Vol. 46, No. 12 5875–5885
doi: 10.1093/nar/gky402
NAR Breakthrough Article
Pyrophosphate hydrolysis is an intrinsic and critical
step of the DNA synthesis reaction
Jithesh Kottur and Deepak T. Nair*
Regional Centre for Biotechnology, NCR Biotech Science Cluster, 3rd Milestone, Faridabad-Gurgaon Expressway,
Faridabad 121 001, India
Received February 26, 2018; Revised April 20, 2018; Editorial Decision April 24, 2018; Accepted May 15, 2018
ABSTRACT
DNA synthesis by DNA polymerases (dPols) is cen-
tral to duplication and maintenance of the genome
in all living organisms. dPols catalyze the formation
of a phosphodiester bond between the incoming de-
oxynucleoside triphosphate and the terminal primer
nucleotide with the release of a pyrophosphate (PPi)
group. It is believed that formation of the phospho-
diester bond is an endergonic reaction and PPi has
to be hydrolyzed by accompanying pyrophosphatase
enzymes to ensure that the free energy change of
the DNA synthesis reaction is negative and it can
proceed in the forward direction. The fact that DNA
synthesis proceeds
in vitro
in the absence of py-
rophosphatases represents a long-standing conun-
drum regarding the thermodynamics of the DNA syn-
thesis reaction. Using time-resolved crystallography,
we show that hydrolysis of PPi is an intrinsic and crit-
ical step of the DNA synthesis reaction catalyzed by
dPols. The hydrolysis of PPi occurs after the forma-
tion of the phosphodiester bond and ensures that
the DNA synthesis reaction is energetically favor-
able without the need for additional enzymes. Also,
we observe that DNA synthesis is a two Mg2+ ion
assisted stepwise associative SN2 reaction. Overall,
this study provides deep temporal insight regard-
ing the primary enzymatic reaction responsible for
genome duplication.
INTRODUCTION
In all living organisms, deoxyribonucleic acid (DNA) is syn-
thesized by DNA polymerases (dPols), and these enzymes
play a central role in genome duplication. Also, dPols are
essential for many applications in research and biotechnol-
ogy such as DNA sequencing, gene cloning, and Polymerase
Chain Reaction-based diagnostic kits. In both natural and
articial settings, dPols catalyze the template-directed ad-
dition of deoxynucleoside triphosphates (dNTPs) to the
primer strand (1–5). Mg2+ ions play a critical role in the
polymerization reaction, and dPols extends the primer in 5
to 3direction. The formation of a phosphodiester bond (pB
bond) between the ␣-phosphate of the incoming dNTP and
the 3- hydroxyl group of the terminal primer nucleotide rep-
resents the primary chemical reaction catalyzed by dPols.
This reaction also involves breakage of the bond between
the ␣-phosphate, and the bridging oxygen (␣O bond) be-
tween the ␣-and- phosphates resulting in the release of
the pyrophosphate (PPi) moiety as a by-product of the syn-
thesis reaction.
The reaction involving incorporation of a nucleotide into
the primer through the formation of the phosphodiester
bond along with the release of PPi is expected to have a
low free energy change (1,6,7). To ensure that the reaction
moves in the forward direction, it is believed that the PPi
moiety is cleaved by an accompanying pyrophosphatase en-
zyme to render a large negative free energy change (∼7
kcal/mol) to the DNA synthesis reaction (1,8–10). How-
ever, DNA synthesis can proceed smoothly in vitro in the ab-
sence of any pyrophosphatase enzyme, and there is no sat-
isfactory explanation available for this long-standing ther-
modynamic conundrum.
Based on structural and biochemical studies, two distinct
mechanisms have been proposed for the synthesis of the pB
bond by dPols. In the classical two-metal mechanism, three
conserved acidic residues in the palm subdomain bind two
Mg2+ ions (11). Structures of dPols in complex with DNA
and dNTP showed that one Mg2+ (Metal B) coordinates
two active-site acidic residues and the triphosphate moiety
of the incoming nucleotide (12–17). The other Mg2+ (Metal
A) coordinates all three active-site acidic residues and 3OH
of the primer terminus (13,18,19) and is thought to facilitate
the formation of the attacking oxyanion by lowering the
*To whom correspondence should be addressed. Tel: +91 124 2848844; Email: deepak@rcb.res.in
C
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5876 Nucleic Acids Research, 2018, Vol. 46, No. 12
pKaof the primer terminus 3O–. The in-line nucleophilic
attack by the 3O– oxyanion on the ␣-phosphate should
lead to the formation of a penta-coordinated bipyramidal
transition state. The metal B may stabilize the developing
negative charge on the triphosphate moiety during the nu-
cleophilic attack. According to this model, the reaction is
hypothesized to be a concerted SN2-type reaction wherein
there are simultaneous formation and breakage of the pB
and ␣O bonds, respectively.
In the recent past, the appearance of an additional diva-
lent metal ion (Metal C) which is distinct from the catalytic
(Metal A) and nucleotide (Metal B) metal ions has been re-
ported (19–21). The metal ion (Metal C) coordinates the
non-bridging oxygens on the phosphates of what previously
were ␣-and-phosphates of the incoming nucleotide and is
predicted to assist breakage of the ␣O bond. According to
this model, the binding of metal C is the rate-limiting step
of the DNA polymerase reaction (21,22). Both the classi-
cal two-metal-ion and the new three-metal-ion mechanisms
propose a concerted SN2-type reaction scheme. However,
based on theoretical analysis, it has been suggested ear-
lier that DNA synthesis by dPols may follow either a con-
certed, stepwise associative or a stepwise dissociative reac-
tion scheme (23,24).
To elucidate the different stages of the DNA synthesis
reaction, we have employed DNA polymerase IV (PolIV)
from Escherichia coli. PolIV is a member of the Y-family
of dPols and capable of template-dependent dNTP incor-
poration (18,25,26). We have conducted time-resolved crys-
tallography on DNA synthesis by PolIV and obtained pe-
riodic snapshots of the reaction. Our studies show that hy-
drolysis of the pyrophosphate moiety is an inherent part of
the DNA synthesis reaction catalyzed by dPols. This step
ensures that the nucleotide incorporation reaction is ener-
getically favorable without the need for accompanying py-
rophosphatases. Also, we observed that the DNA synthesis
is a two Mg2+ ion assisted stepwise associative SN2 reaction
wherein the formation of the pB bond and dissolution of
the ␣O bond occur sequentially. Overall, the study provides
insight regarding the mechanism utilized by dPols to syn-
thesize DNA during DNA replication.
MATERIALS AND METHODS
Protein purication and primer extension assays
PolIV was puried as mentioned before (18,27). Primer ex-
tension assays were carried out at different pH and incu-
bation times to unearth conditions wherein the nucleotide
addition reaction is considerably slowed down. The primer
extension assays were carried out with the following DNA
substrate:
1. Template A
3-GCATGAGCATCCGTAATCACACTGGTCGACAAGTCCATC
CGTGCCATCCT-5
5-XCGTACTCGTAGGCAT-3
The concentrations of protein, DNA and incoming nu-
cleotide used were 100 nM, 100 nM and 25 M, respectively
and the reaction products were processed as mentioned pre-
viously (28). It was seen that at pH 5.2, the rate of reaction
was reduced by >60-fold at 37◦C (Supplementary Figure
S1).
In crystallo DNA synthesis
An 18mer self-annealing oligonucleotide was used for crys-
tallization and has the following sequence: 5TCTAGGGT
CCTAGGACCC 3. The ternary complexes were reconsti-
tuted by mixing PolIV (0.3 mM) with dsDNA (0.36 mM)
followed by addition of 5 mM dTTP (GE Healthcare) or 5
mM dTMPPnP (Jena Biosciences). Crystallization was car-
ried out in the absence of metal ion and crystals were ob-
tained in 0.1 M acetate (pH 5.2) and 5–12% MPD. The in
crystallo reaction was initiated by transferring crystals to
a cryosolution containing 0.1 M acetate (pH 5.2) and 30%
MPD with 5 mM MgCl2. The crystals were incubated for
different time periods at 4◦C (to further reduce the rate of
the nucleotide incorporation reaction) and then ash frozen
in liquid nitrogen to stop the reaction. Subsequently, X-
ray diffraction data were collected from the frozen crystals.
The protocol for preparing crystals utilized here is distinct
from that employed in previous time-resolved crystallog-
raphy studies carried out on DNA polymerases and ,
wherein crystals were grown in the presence of Ca2+ rst
and then incubated with high concentrations (∼100 mM)
of Mg2+ or with Mn2+.
Structure determination and crystallographic renement
For the crystals prepared with dTTP, X-ray diffraction data
were collected at the BM14 beamline of ESRF. Data were
processed using iMosm and the SCALA program in CCP4
(29,30). Using PHASER, the structures corresponding to
different time points were determined using the structure
of PolIVdA:dTMPnPP (4IR1) as a search model (18,31). Be-
fore molecular replacement, the primer nucleotide, incom-
ing nucleotide, and metal ions were removed from the coor-
dinate le of the search model. The MR solutions were sub-
jected to three cycles of renement in PHENIX, and Fo–Fc
maps were calculated for the resolution range of 2.15–40 ˚
A
for all datasets and viewed at 3.3for comparison (Figure
2). The electron density maps (omit Fo–Fc) prepared at the
highest resolution possible for each dataset (Supplementary
Figure S5) were nearly identical to the previous ones (Fig-
ure 2) and in agreement with the all the major conclusions
of the analysis. The terminal primer nucleotide and incom-
ing dTTP were positioned at appropriate locations in the
electron density maps using Coot, and the structures were
rened until convergence using PHENIX (32). For the crys-
tal prepared with dTTP and incubated with 50 mM MgCl2
for 30 min (Supplementary Figure S6),thedatawerecol-
lected at the BM14 beamline of ESRF, and were processed
using iMosm and Scala. Structure solution and renement
were carried out using the same protocol used for structures
obtained using 5 mM MgCl2.
For the crystals obtained with dTMPPnP (Figure 5),
diffraction data were collected at the ID29 beamline of
ESRF and data was processed using XDS, Aimless and
Pointless (33–36). Structure solution and renement were
carried out using the same protocol used for the structures
with dTTP.
Nucleic Acids Research, 2018, Vol. 46, No. 12 5877
For all the rened structures, the majority of the residues
were in the favorable regions of the Ramachandran plot
with only 1% of residues in the disallowed regions. All the
rened structures show the presence of two complexes of
PolIV:DNA:dTTP:Mg2+ which, based on the chain IDs of
the components, are called ABC and FGH. The electron
density maps were clearer for the FGH complex, and hence
this complex was selected for analysis. The electron den-
sity maps were prepared in PHENIX and viewed using
Coot (37). The structures were also analyzed using PyMOL
(Schrodinger Inc.), and all the gures were generated using
PyMOL.
Primer extension assay with modied nucleotides
The modied deoxynucleoside triphosphates dTMPnPP,
dTMPPnP, dGMPnPP and dGMPPnP were obtained from
Jena Biosciences. Primer extension assay was conducted
with different dPols (PolIV, Dbh, Dpo4, MsDpo4, Taq,
Klenow exo-, PfPrex, Pol II and M-MuLV RT) and the sub-
strate DNAs utilized were as follows:
Template A
3-GCATGAGCATCCGTAATCACACTGGTCGACAAGTCCATC
CGTGCCATCCT-5
5-XCGTACTCGTAGGCAT-3
Template C
3-GCATGAGCATCCGTACTCACACTGGTCGACAAGTCCATC
CGTGCCATCCT-5
5-XCGTACTCGTAGGCAT-3
where X =56FAM label.
Protein, DNA and incoming nucleotide (normal or mod-
ied) were added to a nal concentration of 10 nM, 50 nM
and 2 M, respectively. The reaction mixture was incubated
for 30 min at 37◦C, and the reaction products were pro-
cessed as mentioned previously (28).
Phosphate and pyrophosphate assay
To quantitate the level of phosphates and pyrophosphates
generated during the DNA synthesis reaction, primer ex-
tension assays were carried out initially, as mentioned pre-
viously. The following DNA substrate was utilized:
Template A
3-GCATGAGCATCCGTAATCACACTGGTCGACAAGTCCATC
CGTGCCATCCT-5
5-GTACTCGTAGGCAT-3
The reaction mixture included 100 nM of PolIV, 15 M
DNA duplex and 2 mM of all four dNTPs. After incuba-
tion at 37◦C for 1 h, the reaction was terminated by adding
EDTA solution to a nal concentration of 20 mM. The
amount of phosphate in the reaction mix was determined
using the Phosphate Assay Kit (Sigma-Aldrich) wherein the
sensor reagent is a proprietary formulation of the Malachite
green dye. 100 l of this reagent was added to 50 lofthe
reaction mix followed by incubation at room temperature
for 30 min, and subsequently, absorbance was recorded at
630 nm on a Spectramax M5 plate reader (Molecular De-
vices). The amount of pyrophosphate was determined using
the Pyrophosphate assay kit (MAK168, Sigma) wherein the
sensor reagent is a uorogenic PPi sensor. 50 l of the re-
action mix was added to 50 l of sensor reagent followed
by 30 min of incubation at room temperature. After in-
cubation, the uorescence intensity was measured (ex =
320 nm and em =456 nm) on a Spectramax M5 plate
reader (Molecular Devices). To enable accurate quantita-
tion, initially standard curves were plotted for both Pi and
PPi. All measurements were done in triplicate and with ap-
propriate controls.
RESULTS
Entry of Mg2+ ions triggers conformational changes
Primer extension assays conducted under different reaction
conditions shows that the rate of the polymerization reac-
tion was slowed down considerably at a pH of 5.2 (Sup-
plementary Figure S1). Crystals of the PolIV:DNA:dTTP
complex were grown at this pH in the absence of Mg2+ ions.
The crystals were soaked in Mg2+ for 0, 1, 3, 5, 7, 10, 15, 20,
25, 30, 35, 40, 50 and 60 min at 4◦C and then frozen. X-ray
diffraction data were collected from the frozen crystals, and
the data collection and renement statistics are displayed in
Supplementary Table S1.
Alignment of the ground state structure (0 min), where no
metal ions are present, with the complex obtained 1 min af-
ter addition of the Mg2+ ions, showed only marginal differ-
ences. The incoming nucleotide, therefore, attains the con-
formation compatible with productive catalysis in the ab-
sence of the co-factor ion (Supplementary Figure S2). The
density for both the metal ions appeared 1min after the ad-
dition of Mg2+ ions and the activation of the 3-hydroxyl
group was observed immediately after the metal ions occu-
pied their respective locations. The sugar pucker of the ter-
minal nucleotide of the primer changed from C2-endo to
C3-endo (Supplementary Figure S3). Also, the incoming
nucleotide moved towards the 3OH of the primer termi-
nus. As a result of these changes, the distance between the
3O– of the terminal primer nucleotide and the ␣-phosphate
of the incoming dTTP reduced from 4.6 to 3.5 ˚
A(Fig-
ure 1). Furthermore, the angle formed by the 3O–, the ␣-
phosphate and the bridging oxygen between ␣-and- phos-
phates increased from 150◦to ∼170◦(Figure 1). The confor-
mational changes triggered by binding of Mg2+ ions, there-
fore facilitate the in-line nucleophilic attack by the 3O– of
the terminal primer nucleotide on the ␣-phosphate of the
incoming dTTP.
Phosphodiester bond formation is a stepwise associative SN2
reaction
From 5min onwards, electron density developed gradually
between the 3-end of the primer and ␣-phosphate of the
incoming dTTP. By 10 min, the pB bond was completely
formed resulting in the formation a pentavalent intermedi-
ate. In this intermediate, the ␣-phosphate is simultaneously
bonded to the 3O– of the primer terminus, the bridging
oxygen bonded with the -phosphate, the bridging oxygen
bonded with 5carbon of the deoxyribose sugar and two
oxygen atoms (Supplementary Figure S4). After 10 min, we
observed that there is a gradual decrease in the density of the
5878 Nucleic Acids Research, 2018, Vol. 46, No. 12
Figure 1. Conformational changes triggered by the Mg2+ ions. The terminal primer nucleotide and the incoming dTTP are displayed here in stick rep-
resentation and coloured by element. Upon entry of two Mg2+ ions, the terminal primer nucleotide and the incoming dTTP undergo conformational
changes such that the distance between the 3O– and the ␣-phosphate decreases from 4.6 ˚
A(0min)to3.5 ˚
A (3 min) and the angle between the 3O–, the
-phosphate and the bridging oxygen between the ␣- and - phosphates increases from 150◦to 170◦. The conformational changes, therefore, facilitate the
in-line nucleophilic attack by the 3O– on the ␣-phosphate.
␣O bond (Figure 2, Supplementary Figure S5). The analy-
sis of the omit Fo–Fcmaps showed that the pB bond for-
mation and ␣O bond dissolution happen sequentially and
not concomitantly. The DNA synthesis reaction, therefore,
follows the stepwise associative SN2 scheme (23). Further,
the DNA synthesis reaction may also represent an exam-
ple of the alternate two-step mechanistic model of SN2(P5)
reactions proposed recently by Kolodiazhnyi and Kolodi-
azhna (38). In this alternate model, the pentavalent inter-
mediate exists in a lower-energy state between two higher-
energy transition states corresponding to, in this case, the
formation of pB bond and dissolution of the ␣O bond.
Unlike postulated by the classical two-metal-ion mecha-
nism, the DNA synthesis reaction does not follow a con-
certed SN2 reaction scheme as the pB bond formed rst fol-
lowed by dissolution of the ␣O bond (Movie S1) (11). Addi-
tionally, there was no electron density present in the appro-
priate location for a third Mg2+ (Metal C) ion in the maps as
observed in the case of DNA polymerases and . Hence,
our studies show that two Mg2+ ions are adequate to enable
synthesis of the pB bond (Figure 2& Supplementary Fig-
Nucleic Acids Research, 2018, Vol. 46, No. 12 5879
Figure 2. Electron density maps depicting the sequence of events during phosphodiester (pB) bond formation: Omit electron density maps (Fo–Fc)
calculated to a resolution of 2.15 ˚
A are displayed at a contour of 3.3. These maps show that the electron density corresponding to formation of pB appears
rst (5, 7 and 10 m) followed by dissolution of ␣O (15, 20 and 25m) and subsequent hydrolysis of the PPi moiety to phosphate ions (30, 35, 40, 50 and
60 m). The terminal primer nucleotide and the incoming dTTP are displayed here in stick representation and colored by element. Na+and Mg2+ ions are
shown as purple and green spheres respectively.
ure S5). This observation gave rise to the possibility that the
third metal ion observed in earlier studies may be due to the
use of Mn2+ or high concentrations of Mg2+. The electron
density maps for a crystal incubated with 50 mM MgCl2
for 30 min showed the presence of density corresponding to
the third Mg2+ (Metal C) ion (Supplementary Figure S6).
Hence, the third metal ion may play no actual part in DNA
synthesis.
Also, the time-resolved experiment clearly shows that a
pentavalent intermediate is formed and decomposes gradu-
ally during the DNA synthesis reaction (Figure 2& Supple-
mentary Figure S5). The selection of the multivalent phos-
phorous element to form the backbone of genetic material
may in part be due to the need to form this transient pen-
tavalent intermediate during formation of the DNA poly-
mer (39).
Pyrophosphate hydrolysis is critical for DNA synthesis
The products of the nucleotide incorporation reaction are
the extended primer strand which has increased in length by
one nucleotide, and the PPi moiety from the dNTP. Surpris-
ingly, we observed an additional step in the reaction wherein
there is hydrolysis of the PPi moiety to generate two individ-
ual phosphate ions- Pi and ␥Pi corresponding to the -
and ␥-phosphate of dTTP, respectively (Figure 2&Movie
S1). The Arg49 residue involved in stabilizing the PPi in the
active site moved away after breakdown of PPi (Supplemen-
tary Figure S7). Consistent with the observed importance of
5880 Nucleic Acids Research, 2018, Vol. 46, No. 12
the R49 residue, the R49→A mutant protein lost the abil-
ity to catalyze the DNA synthesis reaction (Supplementary
Figure S8). After hydrolysis of the PPi moiety, ␥Pi diffused
out rst, and the two metal ions were retained in the com-
plex. The mechanism of PPi release by dPols has been the
subject of scrutiny in the past, and our study shows that this
happens through the hydrolysis of PPi into free phosphates
(40,41).
To conrm the importance of breakdown of the PPi in the
completion of the polymerization reaction, we conducted
primer extension assays using the modied nucleotides
dTMPPnP and dGMPPnP, wherein the bond between the
-and␥-phosphates is non-hydrolyzable. If PPi is the -
nal by-product of the reaction, the polymerases should in-
corporate dTMPPnP and dGMPPnP opposite template dA
and dC respectively, as there is no modication in the bond
between the ␣-and-phosphates. The dPols tested include
PolIV (E. coli), Dpo4 (Sulfolobus solfataricus), Dbh (Sul-
folobus acidocaldarius), MsDpo4 (Mycobacterium smegma-
tis), the Klenow fragment of DNA polymerase I (E. coli),
the polymerase module of the Pfprex protein (Plasmodium
falciparum), DNA polymerase II (E. coli) and M-MuLV RT
(Moloney Murine Leukemia Virus). PolIV, Dpo4, Dbh and
MsDpo4 belong to the Y-family of dPols, the Klenow frag-
ment and Pfprex are representatives of the A-family, DNA
Polymerase II is a member of the B-family, and M-MuLV
RT belongs to the Reverse Transcriptase family. All the
tested dPols failed to incorporate the modied nucleotides
(Figures 3and 4). These studies show that the hydrolysis of
PPi for completion of the synthesis reaction may be a con-
served feature of all dPols.
Biochemical assays that assess the amount of inorganic
phosphate (Pi) and PPi generated during the dNTP incor-
poration reaction showed that the level of Pi produced is
much higher than that of PPi. This observation is in line
with the inference that PPi hydrolysis is critical for the com-
pletion of the DNA synthesis reaction (Figure 5).
X-ray diffraction data were collected from crystals of
PolIVDNA:dTMPPnP complex soaked with Mg2+ for 0min and
60min (Supplementary Table S2). The corresponding elec-
tron density maps showed that on Mg2+ ion entry, the sugar
pucker of the terminal primer nucleotide changes and the
incoming nucleotide moves closer to the terminal primer
nucleotide. The distance between the 3O- of the terminal
primer nucleotide and the ␣-phosphate of dTMPPnP is 3.3
˚
A and the angle formed between these two atoms and the
bridging oxygen between ␣-and- phosphates is 171◦(Sup-
plementary Figure S9). However, the reaction does not pro-
ceed to completion; incorporation does not occur (Figure 6)
and, this observation reinforces the importance of PPi hy-
drolysis.
Temporal sequence of events during the dNTP incorporation
reaction
Based on our studies, the following series of events may oc-
cur during the DNA synthesis reaction catalyzed by PolIV
(Figure 7& Movie S2). Initially, binding of dNTP along
with Mg2+ ions initiates the DNA synthesis reaction in the
DNA polymerase active site. (I) Once all the components re-
quired for the DNA synthesis are assembled in the correct
location and conformation, Mg2+ ion activates the 3-OH of
the primer nucleotide, and this results in the change in the
sugar pucker from C2-endo to C3-endo form, (II-V) phos-
phodiester bond (pB) formation results in the appearance
of a penta-covalent transition state which is followed by ␣O
bond dissolution and release of PPi, (VI) PPi is further hy-
drolyzed to two phosphate ions followed by reorientation
of the Arg49 residue. (VII) ␥Pi diffuses out rst (VIII) both
metal ions and Pi are released followed by DNA translo-
cation so that the newly incorporated nucleotide moves out
of the nucleotide-binding site to make way for the next nu-
cleotide to enter, and a new cycle of DNA synthesis can
start. The identity of the incoming nucleotide will be deter-
mined by the next unpaired template nucleotide. DNA repli-
cation is hypothesized to have existed in DNA viruses even
before the emergence of the Last Universal Common An-
cestor (42). Hence, it is possible that the proposed scheme
may have appeared early in evolution and may be common
for all dPols (43,44).
DISCUSSION
Our studies reveal that breakdown of the PPi moiety is criti-
cal for completion of the DNA synthesis reaction. The con-
version of PPi to Pi (G=−7kcal/mol) is essential to ren-
der a large overall negative free energy change (G=−6.5
kcal/mol) to the DNA synthesis reaction (45,46). It was pre-
viously believed that dPols act in tandem with pyrophos-
phatase enzymes that cleave the PPi, so that the coupling of
the two reactions provides an overall negative free energy
change (1,8). It is clear from this study that the PPi hydrol-
ysis step is inherently part of the complete dNTP incorpo-
ration reaction catalyzed by dPols and therefore, the DNA
synthesis reaction carried out by dPols is energetically fa-
vorable without the need of any other enzyme activity. The
entropic penalty imposed by nucleotide incorporation is ex-
pected to be substantial since the mobility of the dNTP re-
duces drastically on polymerization (24,47). PPi hydrolysis
may also serve to reduce the effect of reduction in entropy
on the energetics of the DNA synthesis reaction. The en-
ergy released due to the breakdown of PPi may also aid in
translocation of DNA on the polymerase so that the next
unpaired template nucleotide reaches the active site and a
new round of DNA synthesis can begin.
PPi is known to participate in pyrophosphorolysis, which
is the reverse of the polymerization reaction and involves ex-
cision of the terminal nucleotide of the primer strand (48).
Pyrophosphorolysis results in restoration of the dNTP and
the length of the primer reduces by one nucleotide. The hy-
drolysis of the PPi moiety ensures that the probability of the
pyrophosphorolysis reaction is minimal and DNA synthesis
is irreversible.
Recently, a study on DNA polymerase has shown that
utilization of dNTPs that are non-hydrolyzable at the -
␥position leads to the promotion of the pyrophosphorol-
ysis reaction instead of the DNA synthesis reaction (49).
Also, the utilization of a PPi analog, with an imido- linkage
has been shown to promote pyrophosphorolysis (50). More-
over, modication at the -and␥- phosphates of dNTPs
results in a drastic reduction or complete failure of incor-
poration of the incoming nucleotide by different dPols (51–
Nucleic Acids Research, 2018, Vol. 46, No. 12 5881
Figure 3. Primer Extension Assays with template dA and incoming dTTP, dTMPnPP and dTMPPnP. Opposite template dA, different dPols added dTTP
and could not add the ␣-modied dTMPnPP. The -␥modied dTMPPnP was also not incorporated into the primer by these enzymes and this
observation validates the importance of PPi hydrolysis in the DNA synthesis reaction.
5882 Nucleic Acids Research, 2018, Vol. 46, No. 12
Figure 4. Primer Extension Assays with template dC and incoming dGTP, dGMPnPP and dGMPPnP. The tested dPols added dGTP and could not add
the ␣-modied dGMPnPP opposite template dC. The -␥modied dGMPPnP was also not incorporated into the primer and therefore, hydrolysis of
PPi is important for completion of the DNA synthesis reaction.
Nucleic Acids Research, 2018, Vol. 46, No. 12 5883
Figure 5. Comparison of PPi and Pi formation during DNA synthesis.
The displayed graph shows that, during DNA synthesis, the amount of Pi
generated is >5-fold as compared to PPi.
Figure 6. DNA synthesis reaction does not occur in the presence of
dTMPPnP. Omit Fo–Fcmaps are displayed at a contour of 3.3 for the
PolIVDNA:dTMPPnP complex before (A) and 60 min after the addition of
Mg2+ (B). The electron density maps show that although the 3-OH is ac-
tivated, the DNA synthesis reaction does not proceed further. These ob-
servations are in line with the inference that cleavage of the pyrophosphate
moiety is critical for the irreversible completion of the dNTP incorporation
reaction.
53). These observations are in line with the inference that
cleavage of the PPi is critical for completion of the DNA
synthesis. It has been suggested previously that dissociation
of metal A from the polymerase prevents pyrophosphorol-
ysis (21). However, our studies indicate that the hydrolysis
of PPi may be the primary strategy to avoid the reversal of
the synthesis reaction. The breakdown of PPi will, therefore
allow for smooth progression of the replication fork and en-
sure that the cell is not subjected to replication stress.
The cleavage of the PPi will contribute towards enforc-
ing delity. The presence of a mispair in the active site will
lead to distortion of the spatial alignment of the PPi group
with respect to the enzyme residues and the Mg2+ ion. As
a result, the rate at which PPi group is broken down will
reduce considerably and thus affect completion of the syn-
thesis reaction (52–55). Although the PPi moiety can poten-
tially move to bind in the correct orientation after the dis-
solution of the ␣O bond, pyrophosphorolysis might happen
before PPi has attained the right conguration for cleavage
and thus prevent incorporation of the wrong nucleotide. It
has been shown that the average G◦for incorporation of
the correct dNTP is −5.2 kcal/mol and that for the addition
of the incorrect dNTP is −0.13 kcal/mol (56). These obser-
vations suggest that the PPi moiety is cleaved only when the
correct dNTP is incorporated and are in line with the in-
ference that cleavage of PPi moiety by dPols will contribute
towards ensuring delity of the DNA synthesis reaction.
The modication of the ␥-phosphate has been shown to
affect the ability of viral reverse transcriptase to incorpo-
rate nucleotides, and hence, it is possible that PPi cleavage
may be an evolutionarily conserved feature of all replica-
tive polymerases (57). Enhancement in pyrophosphoroly-
sis activity has been implicated in the ability of mutant vi-
ral polymerases to remove chain terminating inhibitors that
are used as drugs such as zidovudine (58,59). It is believed
that these mutations enhance the afnity for PPi, and thus
promote the reverse reaction. However, our studies raise the
possibility that these mutations might prevent/reduce cleav-
age of PPi by the enzyme and thus promote pyrophospho-
rolysis.
Overall, we provide concrete experimental evidence that
hydrolysis of the pyrophosphate moiety is an intrinsic and
critical step of the DNA synthesis reaction. Also, DNA syn-
thesis is a stepwise associative SN2 reaction assisted by two
Mg2+ ions. The study brings to light the mechanism of the
fundamental reaction responsible for genome duplication
and the insight obtained from this study may aid the devel-
opment of improved PCR-based diagnostic kits and novel
therapeutic strategies against retroviruses.
DATA AVAILABILITY
The Structure factors and the rened co-ordinates have
been deposited in the PDB with the following codes: 5YUR
(0 min), 5YUS (1 min), 5YUT (3 min), 5YUU (5 min),
5YUV (7 min), 5YUW (10 min), 5YV3 (15 min), 5YUX
(20 min), 5YUY (25 min), 5YV4 (30 min), 5YUZ (35 min),
5YV0 (40 min), 5YV1 (50 min), 5YV2 (60 min), 5YYD
(dTMPPnP: 0 min), 5YYE (dTMPPnP: 60 min) and 5ZLV
(50 mM MgCl2; dTTP: 30 min).
5884 Nucleic Acids Research, 2018, Vol. 46, No. 12
Figure 7. Mechanism of incorporation of dTTP opposite dA by DNA polymerase IV. The different steps associated with the DNA synthesis reaction
catalyzed by PolIV are displayed. The terminal primer nucleotide (dC), the incoming nucleotide (dTTP) and active site residues are shown in stick repre-
sentation and colored according to element. The Mg2+ ions are shown in the form of green spheres.
SUPPLEMENTARY DATA
Supplementary Data are available at NAR Online.
ACKNOWLEDGEMENTS
We thank Minakshi Sharma and Mary K. Johnson for
purifying Pfprex and MsDpo4, respectively; Dr Dinakar
M. Salunke (ICGEB, New Delhi) and Prof. Jayant B.
Udgaonkar (NCBS, Bangalore) for critically reading the
manuscript; Prof. S. Ramaswamy (inStem, Bangalore) for
discussions. We thank the X-ray diffraction facility located
at the Regional Centre for Biotechnology. DTN thanks Dr.
Hassan Belrhali & Dr Babu Manjashetty (BM14 beamline,
ESRF) and Dr Danielle de Sanctis (ID29 beamline, ESRF)
for help with X-ray diffraction data collection.
FUNDING
Regional Centre for Biotechnology; Data collection at the
BM14 beamline of ESRF (Grenoble, France) was sup-
ported by the BM14 project––a collaboration between
DBT, EMBL and ESRF; Data collection at ID29 was facil-
itated by the ESRF Access Program of RCB which is sup-
ported by Department of Biotechnology, Government of
India [BT/INF/22/SP22660/2017]. Funding for open ac-
cess charge: Intramural Funding from Regional Centre for
Biotechnology.
Conict of interest statement. None declared.
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