ArticlePDF Available

Abstract and Figures

DNA synthesis by DNA polymerases (dPols) is central to duplication and maintenance of the genome in all living organisms. dPols catalyze the formation of a phosphodiester bond between the incoming deoxynucleoside triphosphate and the terminal primer nucleotide with the release of a pyrophosphate (PPi) group. It is believed that formation of the phosphodiester 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 pyrophosphatases represents a long-standing conundrum regarding the thermodynamics of the DNA synthesis reaction. Using time-resolved crystallography, we show that hydrolysis of PPi is an intrinsic and critical step of the DNA synthesis reaction catalyzed by dPols. The hydrolysis of PPi occurs after the formation of the phosphodiester bond and ensures that the DNA synthesis reaction is energetically favorable 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 regarding the primary enzymatic reaction responsible for genome duplication.
Content may be subject to copyright.
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
articial 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
The Author(s) 2018. Published by Oxford University Press on behalf of Nucleic Acids Research.
This is an Open Access article distributed under the terms of the Creative Commons Attribution Non-Commercial License
(http://creativecommons.org/licenses/by-nc/4.0/), which permits non-commercial re-use, distribution, and reproduction in any medium, provided the original work
is properly cited. For commercial re-use, please contact journals.permissions@oup.com
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 purication and primer extension assays
PolIV was puried 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 37C (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 4C (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 renement
For the crystals prepared with dTTP, X-ray diffraction data
were collected at the BM14 beamline of ESRF. Data were
processed using iMosm 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 renement in PHENIX, and FoFc
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 FoFc) 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
rened 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 iMosm and Scala. Structure solution and renement
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 renement 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 rened 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
rened 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 modied nucleotides
The modied 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-
ied) were added to a nal concentration of 10 nM, 50 nM
and 2 M, respectively. The reaction mixture was incubated
for 30 min at 37C, 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 37C 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 4C and then frozen. X-ray
diffraction data were collected from the frozen crystals, and
the data collection and renement 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 150to 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 150to 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 FoFcmaps 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 (FoFc)
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 R49A 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 conrm the importance of breakdown of the PPi in the
completion of the polymerization reaction, we conducted
primer extension assays using the modied 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 modication 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 modied 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, modication 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 -modied dTMPnPP. The -modied 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 -modied dGMPnPP opposite template dC. The -modied 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 FoFcmaps 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 conguration for cleavage
and thus prevent incorporation of the wrong nucleotide. It
has been shown that the average Gfor 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 modication 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 afnity 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 rened 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.
Conict of interest statement. None declared.
REFERENCES
1. Watson,J.D., Baker,T.A., Bell,S.P., Gann,A., Levine,M. and
Losick,R. (2013) Molecular Biology of the Gene. 7th edn. Pearson,
(Chapter 3 and Chapter 9).
2. Bessman,M.J., Kornberg,A., Lehman,I.R. and Simms,E.S. (1956)
Enzymic synthesis of deoxyribonucleic acid. Biochim. Biophys. Acta,
21, 197–198.
3. Berdis,A.J. (2009) Mechanisms of DNA polymerases. Chem. Rev.,
109, 2862–2879.
4. Johansson,E. and Dixon,N. (2013) Replicative DNA polymerases.
Cold Spring Harb. Perspect. Biol.,5. a012799
5. Aschenbrenner,J. and Marx,A. (2017) DNA polymerases and
biotechnological applications. Curr. Opin. Biotechnol.,48, 187–195.
6. Peller,L. (1976) On the free-energy changes in the synthesis and
degradation of nucleic acids. Biochemistry,15, 141–146.
7. Peller,L. (1966) Thermodynamic factors in the synthesis of
two-stranded nucleic acids. Proc. Natl. Acad. Sci. U.S.A.,55,
1025–1031.
8. Lapenta,F., Monton Silva,A., Brandimarti,R., Lanzi,M.,
Gratani,F.L., Vellosillo Gonzalez,P., Perticarari,S. and
Hochkoeppler,A. (2016) Escherichia coli DnaE polymerase couples
pyrophosphatase activity to DNA replication. PLoS One,11,
e0152915.
9. Burke,C.R. and Luptak,A. (2018) DNA synthesis from diphosphate
substrates by DNA polymerases. Proc. Natl. Acad. Sci. U.S.A.,115,
980–985.
10. Aravind,L. and Koonin,E.V. (1998) Phosphoesterase domains
associated with DNA polymerases of diverse origins. Nucleic Acids
Res.,26, 3746–3752.
11. Steitz,T.A. (1999) DNA polymerases: structural diversity and
common mechanisms. J. Biol. Chem.,274, 17395–17398.
12. Nair,D.T., Johnson,R.E., Prakash,L., Prakash,S. and Aggarwal,A.K.
(2005) Human DNA polymerase iota incorporates dCTP opposite
template G via a G.C + Hoogsteen base pair. Structure,13,
1569–1577.
13. Nair,D.T., Johnson,R.E., Prakash,L., Prakash,S. and Aggarwal,A.K.
(2005) Rev1 employs a novel mechanism of DNA synthesis using a
protein template. Science,309, 2219–2222.
14. Swan,M.K., Johnson,R.E., Prakash,L., Prakash,S. and
Aggarwal,A.K. (2009) Structural basis of high-delity DNA synthesis
by yeast DNA polymerase delta. Nat. Struct. Mol. Biol.,16, 979–986.
15. Nair,D.T., Johnson,R.E., Prakash,S., Prakash,L. and Aggarwal,A.K.
(2004) Replication by human DNA polymerase-iota occurs by
Hoogsteen base-pairing. Nature,430, 377–380.
16. Silverstein,T.D., Johnson,R.E., Jain,R., Prakash,L., Prakash,S. and
Aggarwal,A.K. (2010) Structural basis for the suppression of skin
cancers by DNA polymerase eta. Nature,465, 1039–1043.
17. Zahn,K.E., Averill,A.M., Aller,P., Wood,R.D. and Doublie,S. (2015)
Human DNA polymerase theta grasps the primer terminus to
mediate DNA repair. Nat. Struct. Mol. Biol.,22, 304–311.
Nucleic Acids Research, 2018, Vol. 46, No. 12 5885
18. Sharma,A., Kottur,J., Narayanan,N. and Nair,D.T. (2013) A
strategically located serine residue is critical for the mutator activity
of DNA polymerase IV from Escherichia coli. Nucleic Acids Res.,41,
5104–5114.
19. Nakamura,T., Zhao,Y., Yamagata,Y., Hua,Y.J. and Yang,W. (2012)
Watching DNA polymerase eta make a phosphodiester bond. Nature,
487, 196–201.
20. Freudenthal,B.D., Beard,W.A., Shock,D.D. and Wilson,S.H. (2013)
Observing a DNA polymerase choose right from wrong. Cell,154,
157–168.
21. Gao,Y. and Yang,W. (2016) Capture of a third Mg2+ is essential for
catalyzing DNA synthesis. Science,352, 1334–1337.
22. Yang,W., Weng,P.J. and Gao,Y. (2016) A new paradigm of DNA
synthesis: three-metal-ion catalysis. Cell Biosci.,6, 51.
23. Kamerlin,S.C., Sharma,P.K., Prasad,R.B. and Warshel,A. (2013)
Why nature really chose phosphate. Q. Rev. Biophys.,46, 1–132.
24. Ram Prasad,B. and Warshel,A. (2011) Prechemistry versus
preorganization in DNA replication delity. Proteins,79, 2900–2919.
25. Kottur,J. and Nair,D.T. (2016) Reactive oxygen species play an
important role in the bactericidal activity of quinolone antibiotics.
Angew. Chem. Int. Ed. Engl.,55, 2397–2400.
26. Kottur,J., Sharma,A., Gore,K.R., Narayanan,N., Samanta,B.,
Pradeepkumar,P.I. and Nair,D.T. (2015) Unique structural features
in DNA polymerase IV enable efcient bypass of the N2 adduct
induced by the nitrofurazone antibiotic. Structure,23, 56–67.
27. Sharma,A. and Nair,D.T. (2011) Cloning, expression, purication,
crystallization and preliminary crystallographic analysis of MsDpo4:
a Y-family DNA polymerase from Mycobacterium smegmatis. Acta
Crystallogr. Sect. F Struct. Biol. Cryst. Commun.,67, 812–816.
28. Sharma,A. and Nair,D.T. (2012) MsDpo4-a DinB homolog from
mycobacterium smegmatis-Is an Error-Prone DNA polymerase that
can promote G:T and T:G mismatches. J. Nucleic Acids,2012,
285481.
29. Leslie,A.W. and Powell,H. (2007) In: Read,R and Sussman,J (eds).
Evolving Methods for Macromolecular Crystallography. Springer,
Netherlands, Vol. 245, pp. 41–51.
30. Battye,T.G., Kontogiannis,L., Johnson,O., Powell,H.R. and
Leslie,A.G. (2011) iMOSFLM: a new graphical interface for
diffraction-image processing with MOSFLM. Acta Crystallogr. D
Biol. Crystallogr.,67, 271–281.
31. McCoy,A.J., Grosse-Kunstleve,R.W., Adams,P.D., Winn,M.D.,
Storoni,L. C. and Read,R. J. (2007) Phase crystallographic software.
J. Appl. Crystallogr.,40, 658–674.
32. Adams,P.D., Afonine,P.V., Bunkoczi,G., Chen,V.B., Davis,I.W.,
Echols,N., Headd,J.J., Hung,L.W., Kapral,G.J.,
Grosse-Kunstleve,R.W. et al. (2010) PHENIX: a comprehensive
Python-based system for macromolecular structure solution. Acta
Crystallogr. D Biol. Crystallogr.,66, 213–221.
33. Kabsch,W. (2010) Xds. Acta Crystallogr. D Biol. Crystallogr.,66,
125–132.
34. Evans,P.R. and Murshudov,G.N. (2013) How good are my data and
what is the resolution? Acta Crystallogr. D Biol. Crystallogr.,69,
1204–1214.
35. Evans,P.R. (2011) An introduction to data reduction: space-group
determination, scaling and intensity statistics. Acta Crystallogr. D
Biol. Crystallogr.,67, 282–292.
36. Evans,P. (2006) Scaling and assessment of data quality. Acta
Crystallogr. D Biol. Crystallogr.,62, 72–82.
37. Emsley,P. and Cowtan,K. (2004) Coot: model building tools for
molecular graphics. Acta Crystallogr. D Biol. Crystallogr.,60,
2126–2132.
38. Kolodiazhnyi,O.I. and Kolodiazhna,A. O. (2017) Nucleophilic
substitution at phosphorus: stereochemistry and mechanisms.
Tetrahedron: Assymetry,28, 24.
39. Westheimer,F.H. (1987) Why nature chose phosphates. Science,235,
1173–1178.
40. Da,L.T., Wang,D. and Huang,X. (2012) Dynamics of pyrophosphate
ion release and its coupled trigger loop motion from closed to open
state in RNA polymerase II. J. Am. Chem. Soc.,134, 2399–2406.
41. Genna,V., Gaspari,R., Dal Peraro,M. and De Vivo,M. (2016)
Cooperative motion of a key positively charged residue and metal
ions for DNA replication catalyzed by human DNA Polymerase-eta.
Nucleic Acids Res.,44, 2827–2836.
42. Forterre,P. (2002) The origin of DNA genomes and DNA replication
proteins. Curr. Opin. Microbiol.,5, 525–532.
43. Cheetham,G.M., Jeruzalmi,D. and Steitz,T.A. (1998) Transcription
regulation, initiation, and “DNA scrunching” by T7 RNA
polymerase. Cold Spring Harb. Symp. Quant. Biol.,63, 263–267.
44. Yao,N.Y. and O’Donnell,M.E. (2016) Evolution of replication
machines. Crit. Rev. Biochem. Mol. Biol.,51, 135–149.
45. Fazakerley,G.V., Sowers,L.C., Eritja,R., Kaplan,B.E. and
Goodman,M.F. (1987) Structural and dynamic properties of a
bromouracil-adenine base pair in DNA studied by proton NMR. J.
Biomol. Struct. Dyn.,5, 639–650.
46. M.Davies,J., J.Poole,R. and Sanders,D. (1993) The computed free
energy change of hydrolysis of inorganic pyrophosphate and ATP:
apparent signicance. for inorganic-pyrophosphate-driven reactions
of intermediary metabolism. Biochim. Biophys. Acta (BBA) -
Bioenergetics,1141, 29–36.
47. Minetti,C.A., Remeta,D.P., Miller,H., Gelfand,C.A., Plum,G.E.,
Grollman,A.P. and Breslauer,K.J. (2003) The thermodynamics of
template-directed DNA synthesis: base insertion and extension
enthalpies. Proc. Natl. Acad. Sci. U.S.A.,100, 14719–14724.
48. Pandey,M., Patel,S.S. and Gabriel,A. (2008) Kinetic pathway of
pyrophosphorolysis by a retrotransposon reverse transcriptase. PLoS
One,3, e1389.
49. Shock,D.D., Freudenthal,B.D., Beard,W.A. and Wilson,S.H. (2017)
Modulating the DNA polymerase beta reaction equilibrium to dissect
the reverse reaction. Nat. Chem. Biol.,13, 1074–1080.
50. Rozovskaya,T., Tarussova,N., Minassian,S., Atrazhev,A.,
Kukhanova,M., Krayevsky,A., Chidgeavadze,Z. and
Beabealashvilli,R. (1989) Pyrophosphate analogues in
pyrophosphorolysis reaction catalyzed by DNA polymerases. FEBS
Lett.,247, 289–292.
51. Martynov,B.I., Shirokova,E.A., Jasko,M.V., Victorova,L.S. and
Krayevsky,A.A. (1997) Effect of triphosphate modications in
2-deoxynucleoside 5-triphosphates on their specicity towards
various DNA polymerases. FEBS Lett.,410, 423–427.
52. Sucato,C.A., Upton,T.G., Kashemirov,B.A., Batra,V.K.,
Martinek,V., Xiang,Y., Beard,W.A., Pedersen,L.C., Wilson,S.H.,
McKenna,C.E. et al. (2007) Modifying the beta,gamma
leaving-group bridging oxygen alters nucleotide incorporation
efciency, delity, and the catalytic mechanism of DNA polymerase
beta. Biochemistry,46, 461–471.
53. Sucato,C.A., Upton,T.G., Kashemirov,B.A., Osuna,J., Oertell,K.,
Beard,W.A., Wilson,S.H., Florian,J., Warshel,A., McKenna,C.E.
et al. (2008) DNA polymerase beta delity: halomethylene-modied
leaving groups in pre-steady-state kinetic analysis reveal differences at
the chemical transition state. Biochemistry,47, 870–879.
54. Lecomte,P., Doubleday,O.P. and Radman,M. (1986) Evidence for an
intermediate in DNA synthesis involving pyrophosphate exchange. A
possible role in delity. J. Mol. Biol.,189, 643–652.
55. Vaisman,A., Ling,H., Woodgate,R. and Yang,W. (2005) Fidelity of
Dpo4: effect of metal ions, nucleotide selection and
pyrophosphorolysis. EMBO J.,24, 2957–2967.
56. Olson,A.C., Patro,J.N., Urban,M. and Kuchta,R.D. (2013) The
energetic difference between synthesis of correct and incorrect base
pairs accounts for highly accurate DNA replication. J. Am. Chem.
Soc.,135, 1205–1208.
57. Arzumanov,A.A., Semizarov,D.G., Victorova,L.S., Dyatkina,N.B.
and Krayevsky,A.A. (1996) Gamma-phosphate-substituted
2-deoxynucleoside 5-triphosphates as substrates for DNA
polymerases. J. Biol. Chem.,271, 24389–24394.
58. Arion,D. and Parniak,M.A. (1999) HIV resistance to zidovudine: the
role of pyrophosphorolysis. Drug Resist. Updat.,2, 91–95.
59. Urban,S., Fischer,K.P. and Tyrrell,D.L. (2001) Efcient
pyrophosphorolysis by a hepatitis B virus polymerase may be a
primer-unblocking mechanism. Proc. Natl. Acad. Sci. U.S.A.,98,
4984–4989.

Supplementary resource (1)

... Time-lapse crystallography yields snapshots of catalytic events as a DNA polymerase transitions through the reaction cycle of nucleotide insertion from nucleotide binding to product formation and PP i release [19][20][21][22][23][24][25][26] . The approach routinely reveals transient intermediate states and conformational transitions during DNA synthesis. ...
... Matched ground state. Time-lapse crystallography of X-family pols β 19,20,[25][26][27] and μ 22 , as well as Y-family polymerase η 21,24 , involved growing polymerase-DNA binary complex crystals, and soaking these crystals in the presence of dNTP and Ca 2+ to generate pol-DNA-dNTP ternary ground state (Ca 2+ -GS) complex crystals [19][20][21][22][23][24][25][26][27] . The addition of Ca 2+ allows dNTP binding but does not support catalysis of nucleotide insertion in crystallo. ...
... The product metal has been observed to coordinate product oxygens of the in crystallo DNA synthesis reactions of X-family pols β 19,20,48,49,51 and μ 22 , as well as Y-family pols η 21,24 and E. coli Pol4 (DinB) 23 . In these enzymes, the product metal is released after bond formation prior to PP i release from the active site. ...
Article
Full-text available
Efficient and accurate DNA synthesis is enabled by DNA polymerase fidelity checkpoints that promote insertion of the right instead of wrong nucleotide. Erroneous X-family polymerase (pol) λ nucleotide insertion leads to genomic instability in double strand break and base-excision repair. Here, time-lapse crystallography captures intermediate catalytic states of pol λ undergoing right and wrong natural nucleotide insertion. The revealed nucleotide sensing mechanism responds to base pair geometry through active site deformation to regulate global polymerase-substrate complex alignment in support of distinct optimal (right) or suboptimal (wrong) reaction pathways. An induced fit during wrong but not right insertion, and associated metal, substrate, side chain and pyrophosphate reaction dynamics modulated nucleotide insertion. A third active site metal hastened right but not wrong insertion and was not essential for DNA synthesis. The previously hidden fidelity checkpoints uncovered reveal fundamental strategies of polymerase DNA repair synthesis in genomic instability. DNA polymerase (pol) λ performs DNA synthesis in base excision and double strand break repair. How pol λ accomplishes nucleotide insertion that can lead to mutagenesis and genomic instability was unclear. Here the authors employ time-lapse crystallography to reveal hidden polymerase checkpoints that enable right and wrong natural nucleotide insertion by pol λ.
... It is supposed to stabilize the charge and structure of the pentacovalent transition state and assist the leaving of the pyrophosphate, after formation of the new phosphodiester bond [78]. Furthermore, time-resolved crystallography using DNA pol IV (Y-family) revealed the intrinsic pyrophosphate hydrolysis by the DNA pol, ensuring an energetically favourable reaction [80]. Utilizing β-γ-non hydrolysable dNTPs, this associated pyrophosphate hydrolysis was also proven to be essential for DNA polymerization of the A-and B-family [80]. ...
... Furthermore, time-resolved crystallography using DNA pol IV (Y-family) revealed the intrinsic pyrophosphate hydrolysis by the DNA pol, ensuring an energetically favourable reaction [80]. Utilizing β-γ-non hydrolysable dNTPs, this associated pyrophosphate hydrolysis was also proven to be essential for DNA polymerization of the A-and B-family [80]. ...
... (2) Time resolved crystallography of E. coli DNA pol IV (Y-family) showed no third metal ion in the C-site, during and after bond formation [80]. Only by using elevated amounts of MgCl2 (50 mM for 30 min), the C site was occupied by a Mg 2+ ion. ...
... 1 Furthermore, hydrolysis of PP i represents an intrinsic and critical step of DNA synthesis. 11 In contrast to PPase, all other known phosphoesterase enzymes catalyze the hydrolysis of ester bonds in organic P compounds through a unidirectional (disequilibrium) reaction. 12 Therefore, both equilibrium and disequilibrium oxygen isotope signatures are proxies for assessing the presence of biological activity in various ecosystems. ...
... This result is consistent with the base-catalyzed hydrolytic reaction in PP i as a concerted SN2-type reaction with simultaneous formation and breakage of the P−O bonds. 11 As expected, the order of formation of heavier isotopologues at the expense of lighter isotopologues in the P 2 16 O 7 and H 2 18 O (i.e., P i derived from PP i ) was inverse to that of the P 18 O 4 and H 2 16 O system (Figure 3a−f). ...
... While the forward reaction has been studied extensively by Szostak and Richert from the perspective of prebiotically plausible oligonucleotide formation and lately even translation, [38][39][40][41][42][43][44][45][46][47] the hydrolysis of phosphodiesters and pyrophosphates has received somewhat less attention. [48,49] In this context, we report the design, synthesis, and selfassembly, of ribose-and ribonucleotide-based amphiphiles, composed of the polar ribomonophosphate or ribonucleotide head group and a nonpolar alkyl tail group. Nine different compounds with unique properties were successfully synthesized and characterized. ...
... It is known that pyrophosphatase can break the diphosphate bond efficiently under mild conditions. [49,77] A 365 μm solution of X 15 ppX 15 was incubated at 37°C in a 0.5 mm HEPES buffer (pH = 7.5) with the enzyme for 3 days. To our surprise, X 15 ppX 15 was not hydrolyzed with this specific enzyme (Table S1). ...
Article
Full-text available
The aqueous self-assembly of amphiphiles into aggregates such as micelles and vesicles has been widely investigated over the past decades with applications ranging from materials science to drug delivery. The combination of characteristic properties of nucleic acids and amphiphiles is of substantial interest to mimic biological self-organization and compartmentalization. Herein, we present ribose- and ribonucleotide-based amphiphiles and investigate their self-assembly as well as their fundamental reactivity. We found that various types of aggregates are formed, ranging in size from nanometers to micrometers and all amphiphiles exhibit aggregation-induced emission (AIE) in solution as well as in the solid state. We also observed that the addition of 1-ethyl-3-(3-dimethylaminopropyl)-carbodiimide (EDC) leads to rapid and selective dimerization of the amphiphiles into pyrophosphates, which decreases the critical aggregation concentration (CAC) by a factor of 25 when compared to the monomers. Since the propensity for amphiphile dimerization is correlated with their tendency to self-assemble, our results may be relevant for the formation of rudimentary compartments under prebiotic conditions.
... The reason DNA pols catalyze DNA synthesis irreversibly in vivo is because the pyrophosphate product is continuously degraded by cellular pyrophosphatases and dNTP substrates are continuously resupplied. However, evidence also exists that E. coli pol IV and S. cerevisiea Rev1 are capable of directly hydrolyzing pyrophosphate, making the polymerization reaction irreversible (Kottur and Nair, 2018;Weaver et al., 2020). The classic TS theory postulates that the TS is a saddle point on the energy landscape with the spatial gradient being zero, i.e., the highest energy point on the reaction trejactory but also the lowest energy point in all other directions with a fixed chemical composition. ...
Article
Full-text available
Almost all DNA polymerases (pols) exhibit bell-shaped activity curves as a function of both pH and Mg2+ concentration. The pol activity is reduced when the pH deviates from the optimal value. When the pH is too low the concentration of a deprotonated general base (namely, the attacking 3'-hydroxyl of the 3' terminal residue of the primer strand) is reduced exponentially. When the pH is too high the concentration of a protonated general acid (i.e., the leaving pyrophosphate group) is reduced. Similarly, the pol activity also decreases when the concentration of the divalent metal ions deviates from its optimal value: when it is too low, the binding of the two catalytic divalent metal ions required for the full activity is incomplete, and when it is too high a third divalent metal ion binds to pyrophosphate, keeping it in the replication complex longer and serving as a substrate for pyrophosphorylysis within the complex. Currently, there is a controversy about the role of the third metal ion which we will address in this review.
... For instance, when we do PCR reactions in test tubes, we never add any pyrophosphatase to remove PPi. Or, as a recent study reported, PPi hydrolysis may be an intrinsic step of the DNA synthesis reaction catalyzed by DNA polymerases [49]. Nonetheless, neither explanation can explain the discrepancy in cell cycle alteration between IPP1 suppression in yeasts and PFK2 depletion in Toxoplasma. ...
Article
Full-text available
Many biosynthetic pathways produce pyrophosphate (PPi) as a by-product, which is cytotoxic if accumulated at high levels. Pyrophosphatases play pivotal roles in PPi detoxification by converting PPi to inorganic phosphate. A number of apicomplexan parasites, including Toxoplasma gondii and Cryptosporidium parvum, express a PPi-dependent phosphofructokinase (PPi-PFK) that consumes PPi to power the phosphorylation of fructose-6-phosphate. However, the physiological roles of PPi-PFKs in these organisms are not known. Here, we report that Toxoplasma expresses both ATP- and PPi-dependent phosphofructokinases in the cytoplasm. Nonetheless, only PPi-PFK was indispensable for parasite growth, whereas the deletion of ATP-PFK did not affect parasite proliferation or virulence. The conditional depletion of PPi-PFK completely arrested parasite growth, but it did not affect the ATP level and only modestly reduced the flux of central carbon metabolism. However, PPi-PFK depletion caused a significant increase in cellular PPi and decreased the rates of nascent protein synthesis. The expression of a cytosolic pyrophosphatase in the PPi-PFK depletion mutant reduced its PPi level and increased the protein synthesis rate, therefore partially rescuing its growth. These results suggest that PPi-PFK has a major role in maintaining pyrophosphate homeostasis in T. gondii. This role may allow PPi-PFK to fine-tune the balance of catabolism and anabolism and maximize the utilization efficiency for carbon nutrients derived from host cells, increasing the success of parasitism. Moreover, PPi-PFK is essential for parasite propagation and virulence in vivo but it is not present in human hosts, making it a potential drug target to combat toxoplasmosis.
... PP i production during DNA synthesis only accounts for 1% of the cell's energy budget (Figure 1), whereby some DNA polymerases can also operate with NDP substrates (Burke and Lupták, 2018). In addition, some polymerases use the irreversible effect of PP i hydrolysis by possession of a pyrophosphatase domain that cleaves PP i as the enzyme moves forward (Kottur and Nair, 2018). PP i generation and hydrolysis render both the reactions of the core and polymerization reactions during replication, transcription and translation irreversible under the physiological conditions of the cell. ...
Article
Full-text available
The possible evolutionary significance of pyrophosphate (PPi) has been discussed since the early 1960s. Lipmann suggested that PPi could have been an ancient currency or a possible environmental source of metabolic energy at origins, while Kornberg proposed that PPi vectorializes metabolism because ubiquitous pyrophosphatases render PPi forming reactions kinetically irreversible. To test those ideas, we investigated the reactions that consume phosphoanhydride bonds among the 402 reactions of the universal biosynthetic core that generates amino acids, nucleotides, and cofactors from H2, CO2, and NH3. We find that 36% of the core’s phosphoanhydride hydrolyzing reactions generate PPi, while no reactions use PPi as an energy currency. The polymerization reactions that generate ~80% of cell mass – protein, RNA, and DNA synthesis – all generate PPi, while none use PPi as an energy source. In typical prokaryotic cells, aminoacyl tRNA synthetases (AARS) underlie ~80% of PPi production. We show that the irreversibility of the AARS reaction is a kinetic, not a thermodynamic effect. The data indicate that PPi is not an ancient energy currency and probably never was. Instead, PPi hydrolysis is an ancient mechanism that imparts irreversibility, as Kornberg suggested, functioning like a ratchet’s pawl to vectorialize the life process toward growth. The two anhydride bonds in nucleoside triphosphates offer ATP-cleaving enzymes an option to impart either thermodynamic control (Pi formation) or kinetic control (PPi formation) upon reactions. This dual capacity explains why nature chose the triphosphate moiety of ATP as biochemistry’s universal energy currency.
Article
Bacillus stearothermophilus large fragment (BST LF ) DNA polymerase is reported, isolated on silica via a fused R5 silica-affinity peptide and used in nucleic acid diagnostics. mCherry (mCh), included in the fusion construct, was shown as an efficient fluorescent label to follow the workflow from gene to diagnostic. The R5 immobilisation on silica from cell lysate was consistent with cooperative R5-specific binding of R5 2 -mCh-FL-BST LF or R5 2 -mCh-H10-BST LF fusion proteins followed by non-specific protein binding (including E. coli native proteins). Higher R5-binding could be achieved in the presence of phosphate, but phosphate residue reduced loop-mediated isothermal amplification (LAMP) performance, possibly blocking sites on the BST LF for binding of β- and γ-phosphates of the dNTPs. Quantitative assessment showed that cations (Mg ²⁺ and Mn ²⁺ ) that complex the PPi product optimised enzyme activity. In malaria testing, the limit of detection depended on Plasmodium species and primer set. For example, 1000 copies of P. knowlesi 18S rRNA could be detected with the P.KNO-LAU primer set with Si-R5 2 -mCh-FL-BST LF , but 10 copies of P. ovale 18S rRNA could be detected with the P.OVA-HAN primer set using the same enzyme. The Si-immobilised BST LF outperformed the commercial enzyme for four of the nine Plasmodium LAMP primer sets tested. Si-R5 2 -mCh-FL-BST LF production was transferred from Cambridge to Accra and set up de novo for a trial with clinical samples. Different detection limits were found, targeting the mitochondrial DNA or the 18S rRNA gene for P. falciparum. The results are discussed in comparison with qPCR and sampling protocol and show that the Si-BST LF polymerase can be optimised to meet the WHO recommended guidelines. Graphical abstract
Article
Full-text available
COVID-19 has exposed stark inequalities between resource-rich and resource-poor countries. International UN- and WHO-led efforts, such as COVAX, have provided SARS-CoV-2 vaccines but half of African countries have less than 2% vaccinated in their population, and only 15 have reached 10% by October 2021, further disadvantaging local economic recovery. Key for this implementation and preventing further mutation and spread is the frequency of voluntary [asymptomatic] testing. It is limited by expensive PCR and LAMP tests, uncomfortable probes deep in the throat or nose, and the availability of hardware to administer in remote locations. There is an urgent need for an inexpensive “end-to-end” system to deliver sensitive and reliable, non-invasive tests in resource-poor and field-test conditions. We introduce a non-invasive saliva-based LAMP colorimetric test kit and a $51 lab-in-a-backpack system that detects as few as 4 viral RNA copies per μL. It consists of eight chemicals, a thermometer, a thermos bottle, two micropipettes and a 1000–4000 rcf electronically operated centrifuge made from recycled computer hard drives (CentriDrive). The centrifuge includes a 3D-printed rotor and a 12 V rechargeable Li-ion battery, and its 12 V standard also allows wiring directly to automobile batteries, to enable field-use of this and other tests in low infrastructure settings. The test takes 90 minutes to process 6 samples and has reagent costs of $3.5 per sample. The non-invasive nature of saliva testing would allow higher penetration of testing and wider adoption of the test across cultures and settings (including refugee camps and disaster zones). The attached graphical procedure would make the test suitable for self-testing at home, performing it in the field, or in mobile testing centers by minimally trained staff.
Preprint
Full-text available
ATP is universally conserved as the principal energy currency in cells, driving metabolism through phosphorylation and condensation reactions. Such deep conservation suggests that ATP arose at an early stage of biochemical evolution. Yet purine synthesis requires six phosphorylation steps linked to ATP hydrolysis. This autocatalytic requirement for ATP to synthesize ATP implies the need for an earlier prebiotic ATP-equivalent, which could drive protometabolism before purine synthesis. Why this early phosphorylating agent was replaced, and specifically with ATP rather than other nucleotide triphosphates, remains a mystery. Here we show that the deep conservation of ATP reflects its prebiotic chemistry in relation to another universally conserved intermediate, acetyl phosphate, which bridges between thioester and phosphate metabolism by linking acetyl CoA to the substrate-level phosphorylation of ADP. We confirm earlier results showing that acetyl phosphate can phosphorylate ADP to ATP at nearly 20 % yield in water in the presence of Fe ³⁺ ions. We then show that Fe ³⁺ and acetyl phosphate are surprisingly favoured: a panel of other prebiotically relevant ions and minerals did not catalyze ADP phosphorylation; nor did a number of other potentially prebiotic phosphorylating agents. Only carbamoyl phosphate showed some modest phosphorylating activity. Critically, we show that acetyl phosphate does not phosphorylate other nucleotide diphosphates or free pyrophosphate in water. The phosphorylation of ADP monomers seems to be favoured by the interaction between the N6 amino group on the adenine ring with Fe ³⁺ coupled to acetyl phosphate. Our findings suggest that the reason ATP is universally conserved across life is that its formation is chemically favoured in aqueous solution under mild prebiotic conditions.
Article
Full-text available
Enzyme catalysis has been studied for over a century. How it actually occurs has not been visualized until recently. By combining in crystallo reaction and X-ray diffraction analysis of reaction intermediates, we have obtained unprecedented atomic details of the DNA synthesis process. Contrary to the established theory that enzyme-substrate complexes and transition states have identical atomic composition and catalysis occurs by the two-metal-ion mechanism, we have discovered that an additional divalent cation has to be captured en route to product formation. Unlike the canonical two metal ions, which are coordinated by DNA polymerases, this third metal ion is free of enzyme coordination. Its location between the α- and β-phosphates of dNTP suggests that the third metal ion may drive the phosphoryltransfer from the leaving group opposite to the 3′-OH nucleophile. Experimental data indicate that binding of the third metal ion may be the rate-limiting step in DNA synthesis and the free energy associated with the metal-ion binding can overcome the activation barrier to the DNA synthesis reaction. Electronic supplementary material The online version of this article (doi:10.1186/s13578-016-0118-2) contains supplementary material, which is available to authorized users.
Article
Full-text available
Trans-lesion synthesis polymerases, like DNA Polymerase-η (Pol-η), are essential for cell survival. Pol-η bypasses ultraviolet-induced DNA damages via a two-metal-ion mechanism that assures DNA strand elongation, with formation of the leaving group pyrophosphate (PPi). Recent structural and kinetics studies have shown that Pol-η function depends on the highly flexible and conserved Arg61 and, intriguingly, on a transient third ion resolved at the catalytic site, as lately observed in other nucleic acid-processing metalloenzymes. How these conserved structural features facilitate DNA replication, however, is still poorly understood. Through extended molecular dynamics and free energy simulations, we unravel a highly cooperative and dynamic mechanism for DNA elongation and repair, which is here described by an equilibrium ensemble of structures that connect the reactants to the products in Pol-η catalysis. We reveal that specific conformations of Arg61 help facilitate the recruitment of the incoming base and favor the proper formation of a pre-reactive complex in Pol-η for efficient DNA editing. Also, we show that a third transient metal ion, which acts concertedly with Arg61, serves as an exit shuttle for the leaving PPi. Finally, we discuss how this effective and cooperative mechanism for DNA repair may be shared by other DNA-repairing polymerases.
Article
The activity of DNA polymerase underlies numerous biotechnologies, cell division, and therapeutics, yet the enzyme remains incompletely understood. We demonstrate that both thermostable and mesophilic DNA polymerases readily utilize deoxyribonucleoside diphosphates (dNDPs) for DNA synthesis and inorganic phosphate for the reverse reaction, that is, phosphorolysis of DNA. For Taq DNA polymerase, the KMs of the dNDP and phosphate substrates are ∼20 and 200 times higher than for dNTP and pyrophosphate, respectively. DNA synthesis from dNDPs is about 17 times slower than from dNTPs, and DNA phosphorolysis about 200 times less efficient than pyrophosphorolysis. Such parameters allow DNA replication without requiring coupled metabolism to sequester the phosphate products, which consequently do not pose a threat to genome stability. This mechanism contrasts with DNA synthesis from dNTPs, which yield high-energy pyrophosphates that have to be hydrolyzed to phosphates to prevent the reverse reaction. Because the last common ancestor was likely a thermophile, dNDPs are plausible substrates for genome replication on early Earth and may represent metabolic intermediates later replaced by the higher-energy triphosphates.
Article
This review is devoted to the stereochemistry of nucleophilic substitution reactions at phosphorus. The study of the reactions of phosphoryl group transfer is important for biological and molecular chemistry. The stereochemistry and mechanisms of SN1(P) monomolecular and SN2(P) bimolecular nucleophilic substitution reactions of organophosphorus compounds are discussed. It has been shown that hydrolysis of many natural phosphates proceeds according to the monomolecular SN1(P) mechanism via the formation of metaphosphate intermediate (PO3⁻). SN2(P) nucleophilic substitution at chiral trivalent or pentavalent phosphorus compounds proceeds via the formation of penta-coordinated transition state or pentacoordinate intermediate.
Article
DNA polymerases catalyze efficient and high-fidelity DNA synthesis. While this reaction favors nucleotide incorporation, polymerases also catalyze a reverse reaction, pyrophosphorolysis, that removes the DNA primer terminus and generates deoxynucleoside triphosphates. Because pyrophosphorolysis can influence polymerase fidelity and sensitivity to chain-terminating nucleosides, we analyzed pyrophosphorolysis with human DNA polymerase β and found the reaction to be inefficient. The lack of a thio-elemental effect indicated that this reaction was limited by a nonchemical step. Use of a pyrophosphate analog, in which the bridging oxygen is replaced with an imido group (PNP), increased the rate of the reverse reaction and displayed a large thio-elemental effect, indicating that chemistry was now rate determining. Time-lapse crystallography with PNP captured structures consistent with a chemical equilibrium favoring the reverse reaction. These results highlight the importance of the bridging atom between the β- and γ-phosphates of the incoming nucleotide in reaction chemistry, enzyme conformational changes, and overall reaction equilibrium.
Article
A multitude of biotechnological techniques used in basic research as well as in clinical diagnostics on an everyday basis depend on DNA polymerases and their intrinsic capability to replicate DNA strands with astoundingly high fidelity. Applications with fundamental importance to modern molecular biology, including the polymerase chain reaction and DNA sequencing, would not be feasible without the advances made in characterizing these enzymes over the course of the last 60 years. Nonetheless, the still growing application scope of DNA polymerases necessitates the identification of novel enzymes with tailor-made properties. In the recent past, DNA polymerases optimized for diverse PCR and sequencing applications as well as enzymes that accept a variety of unnatural substrates for the synthesis and reverse transcription of modified nucleic acids have been developed.
Article
It is generally assumed that an enzyme-substrate (ES) complex contains all components necessary for catalysis and that conversion to products occurs by rearrangement of atoms, protons, and electrons. However, we find that DNA synthesis does not occur in a fully assembled DNA polymerase–DNA–deoxynucleoside triphosphate complex with two canonical metal ions bound. Using time-resolved x-ray crystallography, we show that the phosphoryltransfer reaction takes place only after the ES complex captures a third divalent cation that is not coordinated by the enzyme. Binding of the third cation is incompatible with the basal ES complex and requires thermal activation of the ES for entry. It is likely that the third cation provides the ultimate boost over the energy barrier to catalysis of DNA synthesis.
Article
DNA Polymerases generate pyrophosphate every time they catalyze a step of DNA elongation. This elongation reaction is generally believed as thermodynamically favoured by the hydrolysis of pyrophosphate, catalyzed by inorganic pyrophosphatases. However, the specific action of inorganic pyrophosphatases coupled to DNA replication in vivo was never demonstrated. Here we show that the Polymerase-Histidinol-Phosphatase (PHP) domain of Escherichia coli DNA Polymerase III α subunit features pyrophosphatase activity. We also show that this activity is inhibited by fluoride, as commonly observed for inorganic pyrophosphatases, and we identified 3 amino acids of the PHP active site. Remarkably, E. coli cells expressing variants of these catalytic residues of α subunit feature aberrant phenotypes, poor viability, and are subject to high mutation frequencies. Our findings indicate that DNA Polymerases can couple DNA elongation and pyrophosphate hydrolysis, providing a mechanism for the control of DNA extension rate, and suggest a promising target for novel antibiotics.
Article
Recent studies posit that reactive oxygen species (ROS) contribute to the cell lethality of bactericidal antibiotics. However, this conjecture has been challenged and remains controversial. To resolve this controversy, we adopted a strategy that involves DNA polymerase IV (PolIV). The nucleotide pool of the cell gets oxidized by ROS and PolIV incorporates the damaged nucleotides (especially 8oxodGTP) into the genome, which results in death of the bacteria. By using a combination of structural and biochemical tools coupled with growth assays, it was shown that selective perturbation of the 8oxodGTP incorporation activity of PolIV results in considerable enhancement of the survival of bacteria in the presence of the norfloxacin antibiotic. Our studies therefore indicate that ROS induced in bacteria by the presence of antibiotics in the environment contribute significantly to cell lethality.
Article
The machines that decode and regulate genetic information require the translation, transcription and replication pathways essential to all living cells. Thus, it might be expected that all cells share the same basic machinery for these pathways that were inherited from the primordial ancestor cell from which they evolved. A clear example of this is found in the translation machinery that converts RNA sequence to protein. The translation process requires numerous structural and catalytic RNAs and proteins, the central factors of which are homologous in all three domains of life, bacteria, archaea and eukarya. Likewise, the central actor in transcription, RNA polymerase, shows homology among the catalytic subunits in bacteria, archaea and eukarya. In contrast, while some “gears” of the genome replication machinery are homologous in all domains of life, most components of the replication machine appear to be unrelated between bacteria and those of archaea and eukarya. This review will compare and contrast the central proteins of the “replisome” machines that duplicate DNA in bacteria, archaea and eukarya, with an eye to understanding the issues surrounding the evolution of the DNA replication apparatus.