Como la ADN polimerasa cataliza la repricacion y repara con gran fidelidad

REVIEWS
How DNA polymerases catalyse
replication and repair with contrasting
fidelity
Wen-Jin Wu1, Wei Yang2 and Ming-Daw Tsai1,3
Abstract | DNA polymerases were named for their function of catalysing DNA replication, a process
that is necessary for growth and propagation of life. DNA involving Watson–Crick base-pairing can
be synthesized with high fidelity, the structural and mechanistic origins of which have been
investigated for many decades. Despite this, new chemical insights continue to be uncovered,
including recent findings that may explain newly discovered functions for many DNA polymerases
in DNA repair and mutation. Some of these reactions involve non-Watson–Crick base-pairing. In
addition, certain DNA polymerases have been engineered for a wide variety of applications in
biotechnology and biomedicine. This Review describes the molecular basis for the diverse and
contrasting functions of different DNA polymerases, providing an up‑to‑date understanding of
how these tasks are accomplished and the means by which we can benefit from them.
Institute of Biological
Chemistry, Academia Sinica,
128 Academia Road Sec. 2,
Nankang, Taipei 115, Taiwan.
2
Laboratory of Molecular
Biology, National Institute of
Diabetes and Digestive and
Kidney Diseases, National
Institutes of Health, Bethesda,
Maryland 20892, USA.
3
Institute of Biochemical
Sciences, National Taiwan
University, Taipei 106, Taiwan.
1
Correspondence to M.-D.T.
[email protected]
doi:10.1038/s41570-017-0068
Published online 6 Sep 2017
The first known DNA polymerase, DNA polymerase
I (Pol I), was isolated from Escherichia coli 1,2 and was
shown to faithfully copy template DNA sequences3. This
discovery, made by Arthur Kornberg and co‑workers2 in
1958, stimulated decades of intensive research in iden‑
tifying new polymerases and studying their chemical
mechanisms and biological functions. Scientists have
comprehensively catalogued DNA polymerases from all
three kingdoms of life, with the enzymes being classified
into six major families (A, B, C, D, X and Y) according to
their sequence homology4.
DNA polymerases are known to most chemists for
their role in catalysing DNA replication with high accu‑
racy. Less well known is the function that these enzymes
have in DNA repair, with some polymerases, perhaps
counter-intuitively, even effecting DNA mutations,
some of which are important for life processes5,6. As a
consequence, the fidelity (BOX 1) of deoxyribonucleoside
triphosphate (dNTP) incorporation is highly depend‑
ent on the polymerase catalyst and varies from being
very high (107–108 for replicative polymerases)7 to very
low (close to 2 for mutagenic polymerases)8 (FIG. 1a).
For example, incorporation of a correct Watson–Crick
(W–C) base pair (hereafter referred to as a ‘match’)
mediated by Pol β is governed by Kd,app and kpol (refer
to BOX 1 for definitions), the values for which fall in
the ranges of 10–100 μM and 10–25 s−1, respectively.
For incorporation of an incorrect base pair (hereafter
referred to as ‘mismatch’), Kd,app values increase by a
factor of ~20 (weaker binding) and kpol values decrease
by a factor of ~103–104 (slower reaction). Taken together,
these thermodynamic and kinetic parameters for the
match and mismatch situations result in Pol β fidelity
on the order of 103–105 (REF. 9).
It has been known for some time that there is a degree
of structural variation between polymerases10 (BOX 2). In
this Review, we delineate how these small differences in
polymerase structure enable the enzymes to perform
diverse biological functions, each of which may require
a very different fidelity. This variation is perhaps surpris‑
ing given that polymerases operate through very similar
chemical mechanisms.
Early studies focused on high-fidelity replicative poly‑
merases from bacteria or yeast; however, the mammalian
Pol β, a main player in DNA repair, is the most wellcharacterized polymerase. Although Pol β is not a rep‑
licative polymerase, its fidelity is comparable to that of
replicative polymerases without exonuclease proof‑
reading activity (FIG. 1a), and its kinetic and structural
properties are very similar to replicative polymerases. A
useful understanding of DNA chemistry can be gained by
studying the catalytic cycle (FIG. 1b) and conformational
changes (FIG. 1c) of Pol β11, a system that also serves as a
point of comparison for other enzymes. When in its apo
form (that is, free of substrate), Pol β adopts an extended
conformation (I), which undergoes significant structural
change on binding to DNA, after which it assumes an
open conformation (II). A ternary complex (III) is formed
once the enzyme binds to metal-bound dNTP (MdNTP),
with conformational closure of the N subdomain of Pol β
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Box 1 | Fidelity and error frequency
The fidelity (accuracy of DNA synthesis) of a DNA polymerase (pol) has been defined as
[(kpol/Kd,app)c + (kpol/Kd,app)i]/(kpol/Kd,app)i (REF. 230) or simply (kpol/Kd,app)c/(kpol/Kd,app)i, with the
subscripts c and i referring to the incorporation of correct and incorrect bases,
respectively231,115. Here, kpol is the pseudo-first-order catalytic rate constant, and Kd,app is
the apparent dissociation constant for the dissociation of dNTP (deoxyribonucleoside
triphosphate) from the polymerase–DNA–MdNTP ternary complex, as determined from
pre-steady-state kinetic measurements. Fidelity can also be reported as (kcat/Km)c/(kcat/Km)i
(REF. 232), for which steady-state kinetic measurements allow determination of kcat and
Km, the first-order rate constant (the turnover number) and Michaelis constant,
respectively. Both methods produce quantitatively similar values233. The inverse of
fidelity has been referred to as ‘error frequency’ (REF. 113). MdNTP, metal-bound dNTP.
resulting in the third distinct arrangement, the closed
conformation (IV). It is here that the enzyme effects
dNTP incorporation with concomitant generation of
metal-bound pyrophosphate (MPPi). This product state
(V) undergoes conformational change (V → VI) to
release MPPi (VI →VII), after which the DNA substrate
undergoes translocation and further elongation.
Structural data reported for Pol β in its I12,13, II14,15,
III 16, IV 14,17–21, V 19,20 and VII 14,19,22 states have been
summarized11, and a structure of VI has just been dis‑
closed23. Multiple structures exist for certain states, with
data collection having been performed under slightly
different conditions. Although these results are highly
informative regarding the molecular mechanism by
which DNA polymerases operate, we are continually
deriving important new insights by characterizing the
possible transition state (TS) and intermediate struc‑
tures for each reaction, and by considering how and why
the behaviour of some polymerases deviates from the
established mechanistic paradigms.
This Review first summarizes the chemistry that is
involved in polymerase-catalysed match incorporation,
the mechanism of which is likely to be common to most
polymerases. This discussion is followed by a description
of the mismatch incorporation mechanism, a process
that may proceed in a different manner for each poly‑
merase. With these two aspects covered, we then address
how the conserved structural and mechanistic features
of polymerases facilitate different biological functions,
including repair and bypass of damaged DNA. These
specific functions lend themselves to various applica‑
tions, and we conclude by describing how engineering
polymerases allows these to be realized.
Incorporation of Watson–Crick pairs
Direct monitoring of phosphodiester bond formation.
The formation and cleavage of chemical bonds can be very
rapid and dynamic processes. As a consequence, they are
difficult to observe directly, although a recent report on
femtosecond X‑ray scattering may pave the way for pro‑
gress in this area24. For enzymatic reactions, including the
nucleotidyl transfer reaction catalysed by polymerases, the
active site geometry in the TS can only be inferred from
biochemical and computational analyses25–29. A recent
breakthrough in monitoring phosphodiester bond for‑
mation was achieved using a revised time-resolved (also
referred to as time-lapse) X‑ray crystallography method30.
In this case, it is possible to initiate nucleotidyl transfer
by immersing a non-reactive ground-state crystal of the
Pol η–DNA–CadATP ternary complex in 1 mM aqueous
Mg2+. The incorporation of the 3ʹ‑dA can thus be followed
at atomic resolution because the reaction proceeds much
more slowly in crystallo than it would in solution. At each
time point, the reaction was quenched by freezing the
crystals at 77 K, after which X‑ray diffraction data were
collected. Structural snapshots along the time course for
the phosphodiester bond formation can thus be obtained
(FIG. 2a–c); this approach has also been applied to match
and mismatch incorporation by Pol β19.
Three metal ions are essential for nucleotidyl transfer.
Analysis of the crystal structures of numerous polymer‑
ases led many to believe that each of these enzymes oper‑
ates by a catalytic mechanism that involves two metal
ions10,31 (FIG. 2d). In this mechanism, one site (metal B)
binds to all three phosphate groups of dNTP, two of the
three active site carboxylate groups, as well as a H2O
molecule or a backbone carbonyl (Met14 in the case
of Pol η)32. Thus, metal B has a dual role: to correctly
position the triphosphate and to stabilize the negative
charge that accrues on the oxygen atoms of the PPi leav‑
ing group. Another divalent cation (metal A) binds to all
three carboxylate groups, the α‑phosphate and another
H2O ligand, activating the 3ʹ‑OH moiety of the upstream
primer. Deprotonation of this now acidic 3ʹ‑OH group
(probably by water 27,30 or by the Asp256 carboxylate in
Pol β33) affords a nucleophilic alkoxide that attacks Pα
when the two atoms are collinear with the oxygen atom
that bridges the Pα and Pβ atoms (in accordance with an
SN2‑type in‑line mechanism)10,11,14,18,19,31–34.
Despite the general acceptance of the above mecha‑
nism, recent work has shown that a third metal ion (metal
C) must enter the active site to induce phosphodiester
bond formation (as highlighted in FIG. 2a). The binding
site for metal C can be accurately located by replacing
Mg 2+ with Mn2+, a metal ion that also catalyses DNA syn‑
thesis and is readily detected by X‑ray diffraction even at
low occupancy (FIG. 2b). Phosphoryl transfer takes place
only when the enzyme–substrate complex captures
the third divalent cation through thermal activation35.
Considering the structures of the reaction intermediates
(FIG. 2c), the conversion of conformation 2 to 3 involves
an Arg61 side chain rotating upwards and away from the
third metal binding site, such that metal C can enter the
active site. This metal cation binds to the PPi group and
one of the oxygen atoms of the departing α‑phosphate,
thereby stabilizing the reaction intermediate. These find‑
ings form the basis of a newly proposed three-metal-ion
mechanism for Pol η, in which the third metal ion initi‑
ates the reaction by breaking the existing phosphodiester
bond in dNTP and driving the nucleotidyl transfer 35,36
(FIG. 2e). The native 3ʹ‑OH must be well aligned with the
substrate and the three metal ions for deprotonation
to occur. The catalytic role of a third metal ion is also
supported by quantum mechanics/molecular mechan‑
ics (QM/MM) calculations, which confirm that metal
C can stabilize the negative charge of the PPi product
during DNA replication catalysed by Pol η37.
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A third metal ion has also been identified in timelapse studies of match incorporation by Pol β, although
no snapshot yet exists of the three metal sites being occu‑
pied at the same time as the phosphodiester bond forms.
Instead, the third metal was observed only in the prod‑
uct state (hence, it is referred to as the product metal) in
which the Mg 2+ ion at site A had already been replaced
by Na+ (REF. 19). As is discussed later, metal C has been
observed concomitant with phosphodiester bond forma‑
tion in translesion syntheses mediated by Pol β38,39. Thus,
it is likely that the third divalent metal ion is also present
in the Pol β mechanism for match incorporation. These
A−, B−, C− pols
a
A+, B+, C+ pols
Fidelity
108
107
Y pols
X pols
106
105
104
103
Pol β
b
Conf. Δ
MdNTP
DNAn
DNAn
DNAn
MdNTP
10
Pol λ
Conf. Δ
Chemistry
DNAn
MdNTP
102
DNAn + 1
MPPi
1
ASFV Pol X
− MPPi
− DNAn + 1
DNAn + 1
MPPi
DNAn + 1
VI
VII
Translocation
I
II
III
IV
V
Intermediate
c
Apo Pol β
(extended, form I)
Pol β–DNA
Binary complex
(open, form II)
Pol β–DNA–MgddCTP
Ternary complex
(closed, form IV)
Lyase domain
D (fingers)
C (palm)
N (thumb)
αN
Figure 1 | Polymerase fidelity. The fidelities of DNA polymerases vary greatly, despite
| Chemistry
their mechanisms and structures being largely similar. a | The Nature
range ofReviews
fidelity in
DNA
polymerases is broad. The + and − signs for polymerases in the A-, B-, and C-families
denote the presence and absence, respectively, of the proofreading exonuclease
activity7. The polymerases with very high fidelity feature this domain, whereas the
lower-fidelity polymerases do not. The dashed line extending past 107 denotes that
polymerase fidelity may exceed this value, although confirmation of this is difficult owing
to the limited accuracy associated with biochemical quantification of very low error
rates. b | A simplified catalytic cycle for DNA synthesis mediated by a polymerase11. The
intermediates are denoted using Roman numerals, with M designating a metal ion, usually
in a divalent state. Apo polymerases typically bind to DNA first, followed by the incoming
dNTP (deoxyribonucleoside triphosphate), usually as the metal complex MdNTP. After the
enzyme closes around the substrates, nucleotidyl transfer — referred to as the ‘chemical’
step — results in elongation of DNAn to give DNAn + 1. A more detailed scheme is provided
in FIG. 4c. c | X-Ray crystal structures of forms I (Protein Databank identifier: 1BPD), II (PDB
ID: 1BPX) and IV (PDB ID: 1BPY) of DNA polymerase β (Pol β), the conformations that are
involved in catalysis, with subdomains labelled in both the original notation (with one’s
right hand facing the page) and Wilson’s function-based nomenclature (the DCN system
as outlined in BOX 2). The αN helix of the N subdomain is shown in dark green to illustrate
closure of the N subdomain upon formation of the nascent base pair (grey region). DNA is
shown in yellow. ASFV, African swine fever virus; Conf. Δ, conformational change; ddCTP,
2ʹ,3ʹ‑dideoxy-CTP; MPPi, metal-bound pyrophosphate.
experimental findings have been further probed and
extended through QM/MM calculations that focused
on the specific functions of the third metal40,41.
Of the four structures proposed to be involved in
the key nucleotidyl transfer reaction (FIG. 2c), structure 3
may represent the most important TS or near‑TS form.
Structures 1 and 2 correspond to substrate complex IV in
FIG. 1b, whereas structure 4 corresponds to the product
complex V. The capture of the intermediate with metal B
alone (structure 1) lends support to the early structural
and kinetic studies with Cr(iii)dNTP in the absence of
Mg 2+; these works showed that binding of Cr(iii)dNTP
to Pol β is sufficient to induce the closure of the N sub­
domain of the polymerase20,42. In addition, the Kd value
of the Mg2+ ion (0.5–1 mM) was substantially higher than
that of MgdNTP (30–50 μM)43–45, implying the existence
of a low-affinity metal site. On the basis of the results
from titrations of Pol η with metal ions in crystallo,
Gao and Yang have unequivocally shown that site C is
the one with low affinity for metal ions35,36.
The most common metal ion involved in cataly‑
sis mediated by polymerases is Mg 2+, and it is likely
that Mg2+ is the preferred metal at each binding site.
However, Mn2+ has also been suggested as a natural
metal ion that may be important for some polymerase
functions. The Mn2+ ion has been observed for Pol λ46,47
and Pol μ48–51 in the X‑family, and has been implicated
as an unnatural ‘mutator’ for other polymerases, which
is discussed in the section below on non-W–C incor‑
poration. By contrast, polymerase-bound Ca2+ does not
catalyse the reaction but does result in the formation of
polymerase–DNA–CadNTP ternary complexes that are
inert, such that they are amenable to crystallographic
characterization19,30,35,39,52. The effects that different metal
ions have on the kinetics and fidelity of polymerases
have been reviewed53, but the chemistry governing why
certain polymerases use particular metal ions at specific
sites remains to be elucidated.
A common intramolecular hydrogen bond in the
incoming dNTP. The structures and conformations of
nucleo­bases can also play important roles in the catalysis
of DNA polymerases (FIG. 3). For example, the intraand intermolecular hydrogen-bonding motifs involv‑
ing nucleo­b ases can provide clues regarding the
fidelities of the polymerase enzymes that house these
substrates. Analysis of crystal structures of DNA poly­
merase–DNA–dNTP and RNA polymerase–RNA–
rNTP ternary complexes (where rNTP denotes a
ribonucleoside triphosphate) revealed the existence
of a common intramolecular hydrogen bond between
the 3ʹ‑OH and the β‑phosphate of the incoming dNTP
or rNTP37 (FIG. 3a). This interaction has been sug‑
gested to promote deprotonation of the primer 3ʹ‑OH
by facilitating PP i departure. 2ʹ,3ʹ‑Dideoxy-NTP
(ddNTP) can also be incorporated into DNA by poly‑
merases and is commonly used as a chain terminator
in DNA sequencing. That ddNTP is unable to form
this intramolecular hydrogen bond may contribute to
the catalytic efficiency of the enzyme being reduced
by a factor of >100 when processing this substrate54.
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This result further underscores the importance of the
hydrogen bond with 3ʹ‑OH, which has been found to
form stereospecifically with the pro‑S oxygen atom of
the β‑phosphate in dNTP or rNTP. In addition, this
hydrogen bond is also present in the structure of the
Pol β–DNA–Cr(iii)dTMPPCP (where dTMPPCP is
2ʹ-deoxy­thymidine 5ʹ-(β,γ-methylene)triphosphate) ter‑
nary complex bearing metal B alone20 (FIG. 3b). By contrast,
the intramolecular hydrogen bond is not observed in the
complexes that feature a dNTP with l‑stereochemistry,
such as the unnatural Pol λ–DNA–l‑dCTP complex 55.
Watson–Crick base-pairing alone is insufficient to
ensure match incorporation. With the exception of
Pol ν56–58 and Pol θ59–61, most enzymes in the A- and
B-families are replicative polymerases that exhibit
fidelities as high as 106–108 (REFS 7,62) (FIG. 1a). 3ʹ‑5ʹ
Exonuclease proofreading increases fidelity by up to
two orders of magnitude7, such that polymerases without
proofreading (as is the case for most of the polymerases
described in this Review) have fidelities no greater
than ~105–106. One longstanding topic of debate (and
one that has been summarized recently 63) is whether
fidelity can be entirely attributed to the difference in
Gibbs free energy changes between base-pairing of
a match and a mismatch. Even when just considering
matches, extensive early work sought to address whether
hydrogen bonding or shape complementarity is more
important 64–68. Recently, the difference in the reaction
free energy between the incorporation of a match and
a mismatch (ΔΔGinc) has been computed by measur‑
ing the kinetics of the forward and reverse reactions
in both cases. These additional data make it clear that
polymerase fidelity does not depend solely on intrinsic
properties of DNA69 and may well be affected by specific
enzyme–substrate interactions. The prevailing evidence
suggests that the dNTP-induced conformational closure
is fast and that the rate-limiting step of nucleotidyl trans‑
fer is probably the chemical step (IV→V in FIG. 1b) for
Pol β11,20,45,70–77, HIV reverse transcriptase 78, human
Box 2 | Domain and subdomain nomenclatures for polymerases
DNA polymerases comprise multiple independently functioning domains. Of most
mechanistic interest is the catalytic domain, which itself consists of three
interdependent subdomains. The right-hand-based nomenclature — consisting of
fingers, palm and thumb subdomains from the amino (N) to the carboxyl (C) terminus —
is widely used234, and the structures of DNA polymerase β (Pol β) in FIG. 1c are labelled in
this manner. It has also been suggested, based on functional alignment, that the fingers
and thumb subdomains in Pol β should be swapped such that the nomenclature is
left-handed235. The confusion that can result from adopting two forms of nomenclature
for Pol β led to the proposal of function-based (DCN) nomenclature for the three
subdomains of Pol β. From the N to the C terminus, these are D (duplex DNA binding), C
(catalytic) and N (nascent base-pair binding). In this Review, we use the right-hand based
structural alignment for all polymerases and include the DCN nomenclature for Pol β.
In addition to the catalytic domain, polymerases often comprise additional domains
or subdomains that are involved in enzymatic function. For example, Pol β has an 8 kDa
lyase domain at its N terminus (FIG. 1c), whereas many replicative polymerases feature
exonuclease domains for editing at either their N or C termini, and Y‑family
polymerases contain an additional little finger subdomain after the thumb subdomain
at their C termini. Furthermore, some polymerases contain multiple regulatory domains
that are important for their in vivo functions.
Pol η35,36, P2 DNA polymerase IV from Sulfolobus solfa‑
taricus79, KlenTaq1 (REF. 80) and bacteriophage T7 poly‑
merase81; thus, it seems that fidelity arises from extensive
enzyme–substrate interactions and W–C base-pairing in
the TS of the chemical step. This becomes more evident
when comparing these results to the contrasting situation
in which non-W–C incorporation takes place.
Incorporation of non-Watson–Crick pairs
Spontaneous errors in Watson–Crick pairing. Watson
and Crick82,83 observed that deviation from W–C pairing
can arise when tautomeric forms of the bases are present.
For example, a dA–dCTP mismatch, in which A under‑
goes keto–enol-like tautomerization to a structure with
an exocyclic imine, mimics the shape of a W–C base pair
(FIG. 3c). Indeed, this mismatch can be incorporated in
DNA by the high-fidelity Bacillus stearothermophilus
Pol I large fragment84. The incorporation of an ionized
dG–dTTP mismatch has been observed in the reac‑
tion catalysed by avian myeloblastosis virus reverse
transcriptase; the efficiency of dTTP misincorporation
increased as the pH was increased from 6.5 to 9.5 (REF. 85).
The crystal structure of a human Pol λ variant bound
to a DNA substrate with dGTP opposite to a template T
(dT–dGTP) has been solved, confirming that the bases,
despite being mismatched, are nevertheless arranged in
a W–C‑like geometry 86 (FIG. 3d). The pH dependence of
the misincorporation is consistent with the presence of
an ionized base pair. Relaxation dispersion NMR spec‑
troscopy enabled observation, in free duplex DNA, of
transient Hoogsteen pairings87–89 — another form of nonW–C base-pairing that can form with DNA polymerases
as shown in later examples. Departures from W–C pair‑
ing also occur with RNA. Such mismatches — referred
to as wobble pairs — have been observed in dynamic
equilibrium with trace amounts of short-lived W–C‑like
enol tautomers or ionized bases90.
It is important to note that the above deviations from
W–C‑like pairing can arise owing to additional factors:
use of the mutator metal ion Mn2+ for dA–dCTP incor‑
poration and engineering of a polymerase active site for
dT–dGTP. As we describe below, both approaches have
been used in conjunction with other polymerases to
facilitate mismatch formation.
Mn 2+ facilitates mismatched ternary complex
formation of Pol β. The presence of Mn2+ at a polymerase
active site has been observed to enhance the efficiency of
DNA synthesis, although this is often at the expense of
fidelity. Thus, Mn2+ is a mutator metal ion for some poly­
merases19,91–99, probably because it has, relative to Mg2+,
relaxed coordination requirements and greater tolerance
of substrate misalignment94. An example is Pol β, which
lacks the common exonuclease 3ʹ‑5ʹ proofreading domain
and is the smallest eukaryotic polymerase (39 kDa, with
335 residues) as well as the main polymerase responsi‑
ble for base excision repair (BER)11. Owing to the rela‑
tively high fidelity of Pol β, it was difficult to obtain the
structures of mismatched ternary complexes unless Mn2+
was used21. Thus, time-resolved X‑ray crystallography
studies were conducted on ternary complexes that
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a
40 s
T (3′)
80 s
Mg
MgC2+
2+
C
MgB2+
MgA2+
D115
E116
D13 M14
b
30 s
60 s
90 s
New
bond
WatN
MnA2+
MnC2+
MnC2+
M14
D13
c
MgA2+
MgC2+
1
3
2.1 Å
MgB2+
DNAn + 1
MgB2+
O
O
e
4
R61
O
O
−
O
2+
MB
O
Pγ
O
−
O
O
O
O
O
MgB2+
−
O
O
Reaction coordinate
O
+
Pα
O−
H2O
MgA2+
MA2+
O
O
MC2+
−
H O
PPi
Ea, uncat
Figure 2 | Mechanism of phosphodiester bond formation. The
mechanism of phosphodiester bond formation catalysed by DNA
polymerase η (Pol η) involves three metal ions. a | The DNA synthesis
reaction can be followed in crystallo by immersing crystals of the Pol η–
DNA–CadATP ternary complex in a 1 mM solution of MgCl2. Omit maps
(4.0σ; shown in green mesh) — corresponding to the difference between
the observed structure factors (at time intervals between 40 and 230 s)
and the calculated structure factor for the system at 40 s
(Fo(40 – 230 s) − Fc(40 s)) — are superimposed on to structures of the
reaction intermediates. Arrows indicate regions where there is a local
increase in electron density, and the third Mg2+ (MgC2+) cation is circled.
The apparent delayed binding of MgC2+ after product formation is due to
the low electron density of Mg2+ and low occupancy of MgC2+ at 80 s.
b | In crystallo catalysis with Pol η also proceeds in 10 mM MnCl2. The
Fo − Fc omit map for the new phosphodiester bond (blue mesh), the third
Mn2+ cation (MnC2+; blue mesh) and the water molecule WatN (pink
O−
β
nucleobase
nucleobase
MgC
−
−
P
2+
Ea, cat
MgB2+
MA2+
O
O
O
O
H2O
−
O
Pα
−
O
WatN
3.2 Å
Å
4.2
O
.3 Å
2.3 Å 2
MgA
H O
MgC2+
2.1 Å
R61
MgA2+
O
B
R61
2+
R61
nucleobase
4
MgC2+
1
nucleobase
O
3
2
dATP
d
MgA2+
2
Thermal motion
Uncatalysed reaction
Catalysed reaction
DNAn
New
bond
New
bond
MnB2+
D115
E116
600 s
MnC2+
dATP
Energy
230 s
dATP
3′O
S113
140 s
O
O
P
O−
2+
−
−
O
O
MB
β
O
−
O
Pγ
O
O−
O
mesh; WatN is hydrogen bonded to the nucleophilic
3ʹ‑OH) are
Nature Reviews
| Chemistry
contoured at 3σ and superimposed on to the structures at each time
point. c | The reaction coordinate of the DNA synthesis catalysed by
Pol η highlights the roles of the three metal ions, the third of which
promotes phosphoryl transfer to the nucleophilic 3ʹ‑OH by overcoming
the energy barrier to the product state. d | The previously accepted
two-metal-ion mechanism involves deprotonation of 3ʹ‑OH (where :B
is a general base) and nucleophilic attack at Pβ. e | The newly proposed
three-metal-ion mechanism involves metal-induced polarization of the
phosphate and attack of 3ʹ‑OH on Pβ, the latter being a strong
electrophile on account of its binding to MC2+. In parts a–c, hydrogen
atoms have been omitted for clarity. Ea, activation energy. Part a is
adapted with permission from REF. 30, Macmillan Publishers Limited.
Parts b and c are adapted with permission from REF. 35, AAAS. Parts d
and e are adapted with permission from REF. 36, under the Creative
Commons License CC BY 4.0.
NATURE REVIEWS | CHEMISTRY
VOLUME 1 | ARTICLE NUMBER 0068 | 5
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a
β
α
γ
b
γ
β
MgB2+
α
CrB3+
MgA2+
H
H
c
Anti
Anti
H
N
N
N
A* N
R
N
N
H
N
Anti
O
O
Anti
T* N
–
H
N
N
G
N
R
N
N
R
H
O
N
H
W–C-like dT–dGTP
e
O
H
H
N
N
G
Anti
Anti
R
O
W–C-like dA–dCTP
d
C
N
H
O
N
N
N
N
H
N
G
N
R
R
N
H
N
H
Distorted dG–dGTP
H
H
N
Anti
Anti
C
O
H
N
N
R
R
N
H
O
N
H
dC–dGTP
f
N
G
N
N
H
Anti
Syn
N
G
N
R
N
H
N
O
Hoogsteen dG–dGTP
N
H
G
O
N
N
H
N
H
N
N
R
H
Figure 3 | Nucleobases in various ionization states and/or Nature
conformations.
Reviews | DNA
Chemistry
polymerase (Pol)–DNA–MdNTP ternary complexes (where MdNTP is a metal-bound
dNTP) can feature diverse substrates in varied conformations. a | When MgdUMPNPP is
in the Pol β–DNA–MgdUMPNPP ternary complex (Protein Databank identifier: 2FMS), it
forms an intramolecular hydrogen bond (green dashed line) between 3ʹ‑OH and the
pro‑S oxygen of Pβ. The use of this N‑containing and non-hydrolysable analogue of dUTP
enables the intermediate to be characterized. b | The structure of Cr(iii)dTMPPCP in the
Pol β–DNA–Cr(iii)dTMPPCP ternary complex (PDB ID: 1HUO). dTMPPCP is a
non-hydrolysable dTTP analogue in which the O atom that bridges Pβ and Pγ is replaced
with a CH2 group. The B‑site Cr3+ ion is shown in grey. c | The structure of a Watson–Crick
(W–C)‑like dA–dCTP mismatch accommodated by the Bacillus stearothermophilus DNA
polymerase I large fragment (PDB ID: 3PX6) features an adenine tautomer with an
exocyclic imine (A*). d | A W–C‑like dT–dGTP mismatch bound to a Pol λ variant
(PDB ID: 3PML), featuring a deprotonated thymine ring (T*). e | The top panel shows
a dG–dGTP mismatch (PDB ID: 4FK4) and the bottom panel shows a dC–dGTP match
(PDB ID: 4FJH), accommodated by an RB69 L415A/L561A/S565G/Y567A quadruple
mutant. f | A dG–dGTP mismatch with an anti–syn Hoogsteen base pair in ASFV Pol X
(PDB ID: 2M2W). The incoming syn dGTP uses its Hoogsteen face (see FIG. 6a) to bind to
the template G. Hydrogen atoms have been omitted for clarity. dNTP, deoxyribonucleoside triphosphate; dTMPPCP, 2ʹ-deoxythymidine-5ʹ-(β,γ-methylene)triphosphate;
dUMPNPP, 2ʹ‑deoxyuridine‑5ʹ-(α,β-imido)triphosphate.
featured either Mg 2+ or Mn2+ ions19, which enabled com‑
parison between near‑TS intermediate structures (with
mixtures of bound reactants and products) for match
(with Mg 2+) and mismatch (with Mn2+) incorporation
catalysed by Pol β. As shown in the case of dG–dCTP
(FIG. 4a), the intermediate structure observed in the
matched ternary complex of Pol β is similar to that of
Pol η (FIG. 2b), except that metal C was undetectable at
this time point. The complex for the dG–dATP mismatch
(FIG. 4b) also lacks metal C, but its features provide insight
into the structural basis of fidelity: the mismatched
base-pairing is significantly distorted from planarity, and
the distance between the substrate Pα and the product
3ʹ‑OP is longer in the mismatched relative to the matched
structure. This distortion leads to a large increase in the
activation energy that is required for the chemical step of
the mismatch (IV→V, red trace, FIG. 4c), which should be
responsible for the substantially reduced rate of mismatch
relative to match incorporation.
Enlarging the dNTP binding pocket converts the
high-fidelity RB69 polymerase to a low-fidelity
polymerase. One may expect a decrease in fidelity if
the constraints on a polymerase active site are made
less stringent. In this regard, the nascent dNTP binding
pocket of a high-fidelity replicative polymerase from
bacteriophage RB69 was modified by replacing four
bulky amino acid residues with smaller ones to afford
the quadruple mutant L415A/L561A/S565G/Y567A100.
Pre-steady-state kinetic analyses of the mutantcatalysed reaction indicated that its fidelity is lowered by
a factor of 103–106 (REF. 101). Consequently, this mutant
can form stable ground-state ternary complexes with
all 12 mismatches in the presence of the (catalytically
inactive) Ca2+ ion, with the resulting structures featuring
distorted base-pairing at the active site (the structures of
mismatched dG–dGTP and matched dC–dGTP can be
compared in FIG. 3e).
Some low-fidelity polymerases use an enzyme side chain
to select a specific dNTP. The African swine fever virus
(ASFV) Pol X, at 174 residues, is a very small polymerase
that does not feature the lyase domain and duplex DNA
binding subdomain that are present in its mammalian
homologue Pol β, which is twice as large102. In terms of
mismatch incorporation, ASFV Pol X is perhaps the most
extreme case as it catalyses the formation of a dG–dGTP
(G–G) mismatch in addition to the four W–C matches8.
Structures of the free protein have been determined using
solution NMR spectroscopy 103,104, as have those of the
ASFV Pol X–MgdGTP binary complex and ASFV Pol
X–MgdGTP–DNA ternary complex, which indicate that
ASFV Pol X can use either gapped DNA or MgdNTP as
the first substrate105,106. ASFV Pol X can bind to MgdGTP
in a syn configuration in the absence of DNA and form a
dG–dGTP mismatch with an anti–syn Hoogsteen basepair conformation (FIG. 3f). The His115 residue is key to
the catalytic incorporation of dG–dGTP mismatches
(FIG. 5a) , which has recently been confirmed inde‑
pendently by crystallography 107. A double hairpin DNA
with two GAA stem loops was used in the NMR studies
6 | ARTICLE NUMBER 0068 | VOLUME 1
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to acquire high quality NMR spectra106, whereas a natural
one-nucleotide gap DNA was used in the crystallographic
study. The latter analysis located a unique binding pocket
for the 5ʹ‑phosphate group of the downstream primer 107,
which affects dG–dGTP mismatch formation107,108. In the
case of Pol β11 and Pol λ109, this binding is strengthened
by the presence, in the lyase domain, of three cationic
residues that are missing in Pol X.
The role of an enzyme side chain in selecting a spe‑
cific dNTP has also been confirmed in the case of Rev1,
a Y-family polymerase that repairs human DNA. Indeed,
the Rev1–DNA–MgdCTP ternary complex has been
characterized, and the enzyme, initially identified as a
deoxycytidyl transferase110, mediates incorporation of
dCTP opposite to template G with very high specificity.
To a lesser extent, Rev1 can also install dCTP opposite
to an abasic site or an O6‑methylguanine during transle‑
sion DNA synthesis110,111. The origin of the very high
specificity that Rev1 shows for dCTP became evident
after the crystal structure of yeast Rev1 bound with
template G and dCTP112 was determined. The dCTP
substrate does not form a base pair with the template
G, but instead hydrogen bonds to the Arg324 side chain
of Rev1. Furthermore, Rev1 sets aside the template base
and uses Arg324 as a template to form an Arg–dCTP
pair that mimics a DNA base pair (FIG. 5b). Thus, Rev1
uses its protein side chain to dictate the identity of not
only the incoming dNTP, but also the template base112.
Understanding fidelity attenuation with the mediumfidelity Pοl λ. In the X‑family pols, Pol λ exhibits a
fidelity (calculated from the reported error frequencies to
be 30–9,100)113 between those of Pol β (1,700–93,000)114
and ASFV Pol X (1.9–7,700)8. Thorough structure–
function studies have been conducted on Pol λ109, and a
recent report indicates that, similar to ASFV Pol X, Pol λ
possesses high MgdNTP affinity in the absence of DNA52.
Analogous to the stabilizing interaction provided by
b Helix N
a Helix N
Open
Open
Closed
PPi
dG
dCTP
Reactant
Product
Closed
MgB2+
D192
MgA2+
PPi
dATP
MnB2+
D192
dG
D190
MnA2+
D256
D256
c
D190
TS
Gibbs free energy
WT mismatch
I260Q mismatch
WT match
EDn
II
N
EDnN
III
E′DnN
IV
MA2+
E′DnNMA
IV
E′Dn + 1PPiMA
V
− MA2+
E′Dn + 1PPi
V
EDn + 1PPi
VI
− PPi
EDn + 1
VII
Intermediate
Reaction coordinate
Figure 4 | Structural and energetic differences near the transition state of the nucleotidyl transfer
mediated
by Pol β.
Nature Reviews
| Chemistry
a | A near‑TS intermediate structure of a dG–dCTP match (yellow; Protein Databank Identifier: 4KLF) has been determined
from time-resolved X‑ray crystallography by immersing a crystal of the ternary complex Pol β–DNA–CadCTP in MgCl2
(200 mM, 20 s). The αN helix is closed; the open αN helix of the DNA binary complex (cyan; PDB ID: 3ISB) is shown for
comparison. b | Similarly, the near‑TS intermediate structure of a dG–dATP mismatch (magenta; PDB ID: 4KLS) has been
determined from a crystal immersed in MnCl2 (200 mM, 10 min). The αN helix is also closed, although not as fully as that in
part a. The structures depicted in parts a and b feature 1:1 mixtures of bound reactants and products. c | Free energy
diagrams, based on pre-steady-state kinetic analyses, have been proposed for wild-type (WT) and mutant Pol β. The
corresponding intermediates from FIG. 1b (where the roles of metal A and metal B are not dissected) are shown below the
reaction scheme here. E, enzyme in open conformation; Eʹ, enzyme in closed conformation; Dn, DNA; M, metal; N, MdNTP
with MB only; Pol β, DNA polymerase β; PPi, metal-bound pyrophosphate; TS, transition state. Part c is adapted with
permission from REF. 119, American Chemical Society.
NATURE REVIEWS | CHEMISTRY
VOLUME 1 | ARTICLE NUMBER 0068 | 7
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a
dG (anti)
dGTP (syn)
dG (anti)
H115
dGTP (syn)
H115
b
c
dG
ddC
dG
Y505
L431
Mg2+
dCTP
R324
Mg2+
F506
A431
I492
d
Pathway
A
Pol
(ASFV Pol X, Pol λ)
Pathway
B
Watson–Crick base pairing
Pol–DNAn
Pol–dNTP
Pol–DNAn–dNTP
Pol–DNAn + 1–PPi
Pol–DNAn–dNTP
Pol–DNAn + 1–PPi
Mismatch base pairing
Figure 5 | Polymerases overcome Watson–Crick pairing byNature
engaging
in multiple
Reviews
| Chemistry
interactions with substrates. a | Stereo views of the ASFV DNA polymerase X
(Pol X) active site, highlighting the interactions between MgdGTP and His115 in the Pol X–
MgdGTP binary complex (thin lines; Protein Databank identifier: 2M2U), and the Pol X–
DNA–MgdGTP ternary complex (sticks; PDB ID: 2M2W). In each case, His115 helps to keep
the incoming dGTP in a syn conformation. b | Rev1 uses its Arg324 side chain as a DNA
template base to pair up with incoming dCTP, with template G having been set aside
from the DNA helix (PDB ID: 2AQ4). c | The structures of the tyrosine–phenylalanine (YF)
motifs and surrounding regions for the MnMgdCTP complex of Pol λ (dark blue; PDB ID:
5DDY), as well as the MgdCTP complex of the Pol λ L431A mutant (cyan; PDB ID: 5CR0).
The L431A mutation provides space for Ile492 to move away from the Phe506 ring,
allowing Phe506 to orient itself parallel to Tyr505, a conformation that facilitates dNTP
binding. d | The canonical reaction mechanism for polymerase enzymes involves initial
binding of DNA (pathway A; see also FIG. 1b). ASFV Pol X and human Pol λ, and possibly
other lower-fidelity polymerases, may also bind dNTP first (pathway B). ASFV, African
swine fever virus; dNTP, deoxyribonucleoside triphosphate.
His115 in ASFV Pol X, Pol λ makes use of its Tyr505 side
chain to bind to the dNTP substrate through π–π inter‑
actions; mutation of Tyr505 into a smaller Ala reduced
the affinity significantly. Structural analysis suggested that
Pol λ maintains its medium fidelity by binding the sub‑
strate in a well-defined hydrophobic pocket that features
Leu431, Ile492, Tyr505 and Phe506 residues. In support,
it was predicted and subsequently demonstrated that the
L431A mutation enhances MgdNTP pre-binding (FIG. 5c)
and lowers fidelity.
The mechanism of fidelity. Having established the struc‑
tures and mechanisms that are involved in match and
mismatch incorporations, we now address the main
factors that are responsible for the high fidelity of cer‑
tain DNA polymerases. The most extensively studied
effect is the MdNTP-induced conformational change
(closure of the N subdomain or thumb subdomain for
Pol β but the fingers subdomain for other high-fidelity
poly­merases10). The closed conformation has been well
studied in many intermediate structures of polymerase–
DNA–MdNTP complexes. Work on the R61A mutant
of Pol η also indicated that the closed conformation is a
prerequisite for aligning the primer and dNTP such that
a third metal ion can bind30,35,36. A major point of interest
is the role of conformational closure in differentiating
correct and incorrect dNTP. This conformational change
was once believed to be the rate-limiting step and thus
the main fidelity-controlling step115. However, as men‑
tioned above, it is the chemical step that is now recog‑
nized as being rate limiting. Nevertheless, recent studies
have revealed that mismatched MgdNTP can induce
only partial conformational closure or none at all116–118.
Further studies suggested that the conformational clo‑
sure differentiates dNTP by a thermodynamic rather than
a kinetic effect. As becomes evident on considering the
free energy profile for the Pol β catalytic cycle (FIG. 4c), mis‑
match incorporation induces conformational closure at a
rate comparable to that induced by match incorporation
(III → IV, FIG. 4c). However, the correct dNTP can better
stabilize the closed form IV119; this has been supported
by recent single-molecule studies120,121 and is logical given
that the N subdomain (represented by the characteristic
αN helix) is closed in the near‑TS intermediate structures
in reactions that lead to both match (FIG. 4a) and mismatch
incorporation (FIG. 4b). Taken together, the results indicate
that both match and mismatch incorporations (if the latter
does occur) proceed through analogous conformational
trajectories that involve closure of the N subdomain119.
Consistent with this conclusion, some lower-fidelity
poly­merases, including Pol μ51, Pol λ52 and terminal
deoxynucleotidyl transferase (TdT)122,123, exist in a closed
conformation even in their substrate-free forms. The
DinB homologue (Dbh) polymerase from S. solfataricus,
an error-prone enzyme from the Y-family, is also closed
in its apo form, and human Pol η is closed when in the
Pol η–DNA binary complex124. Furthermore, as described
above, several lower-fidelity polymerases are able to bind
with MdNTP before binding DNA, which led to the pro‑
posal of a partially random sequential mechanism (FIG. 5d)
for some polymerases52,106. Different polymerases may
operate through a combination of pathways A (in which
DNA is bound first) and B (in which MdNTP is bound
first) to achieve their specific functions.
As shown in FIG. 4c, the main influence on fidelity,
based mainly on the studies of Pol β, is the nature of the
TS of the nucleotidyl transfer reaction70. This is supported
by kinetic analyses119, as well as by consideration of the
near‑TS structures19. In catalysis mediated by Pol η, bind‑
ing of metal C is the rate-limiting sub-step of the chemi‑
cal step35,36. The near‑TS structures of Pol β complexes of
matched and mismatched substrates differ in many ways,
including the interactions involving active site residues,
metal ions, W–C pairing and DNA binding (FIG. 4a,b).
These differences are reflected in the higher relative
energies of the TS and the intermediates that are close
to the TS in the case of mismatch incorporation (FIG. 4c).
Indeed, small structural perturbations can affect fidelity,
and mismatches may occur more often if the enzyme
relaxes its stringency for correct dNTP incorporation
(for example, by enlarging the active site) or is inherently
8 | ARTICLE NUMBER 0068 | VOLUME 1
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selective for binding a specific dNTP (as described above
for Rev1 and dCTP). In addition, binding of the A‑site
M2+ is a key step in the discrimination against an incor‑
rect incoming dNTP, and Mn2+ is less selective than is
Mg 2+ in this regard34. Similar reasoning can explain why
the mutagenic I260Q variant of Pol β has a lower fidelity
than the native Pol β125, as kinetic119, small angle X‑ray
scattering 117 and recent crystallographic analyses13 all sug‑
gest that the main cause for the lower fidelity of I260Q is
its ability to form a relatively stable mismatched ternary
complex (FIG. 4c). Further elaboration of the mechanisms is
provided in BOX 3.
DNA repair, damage bypass and mutation
Roles of DNA polymerases in DNA damage responses.
DNA can be damaged by exogenous agents such as reac‑
tive oxygen species (ROS), alkylating agents or UV light.
More than 50,000–70,000 DNA sites can be damaged per
cell per day 126–128. If left unrepaired, DNA lesions can lead
to mutations and cancer formation. To defend against
these possibilities, living systems make use of various
DNA repair mechanisms that involve many other pro‑
teins in addition to polymerases. DNA damage and repair
are broad fields of active research5,11,109,128–132, with the
2015 Nobel Prize in Chemistry having been awarded to
Thomas Lindahl, Paul Modrich and Aziz Sancar for their
work on the molecular mechanisms of DNA repair. Our
Box 3 | Kinetic origins of fidelity
The question as to which step is rate limiting for match incorporation, mainly for
high-fidelity DNA polymerases, has been the subject of debate over the past two
decades. This is despite it being known, for four decades now, that in the evolutionary
process, enzymes have evolved to stabilize the transition state of the chemical step
(FIG. 1b, IV → V) in their catalytic cycle, to a point where this energy approaches that
of the transition states associated with the physical steps, such as substrate binding,
product release and conformational changes. At this point, any further stabilization
of the transition state of the chemical step would not have a significant effect on the
overall rate236. Thus, for match incorporation catalysed by DNA polymerases, the
chemical step should be either rate limiting (for less well-evolved enzymes) or very
close to the physical steps (for more evolved enzymes). An important point is that
fidelity is determined by the difference between parameters for match and mismatch
incorporation. As high-fidelity DNA polymerases have not evolved to incorporate
mismatches, the energy of the transition state of the chemical step should be much
higher in the case of mismatches (FIG. 4c, red trace) relative to matches (green trace).
This is evidenced by values for kpol, the pseudo-first-order catalytic rate constant for
incorporation, being a few orders of magnitude lower in the case of mismatches.
Thus, the chemical step (including its sub-steps) is the key step for the origin of
fidelity, although other steps could also contribute partially. Low-fidelity DNA
polymerases may have evolved to facilitate mismatch incorporation depending on
their specific functions, for example, by exploiting greater control of nucleotide
binding. This could also be achieved by artificially engineering the high-fidelity
enzyme to facilitate nucleotide binding or by replacing Mg2+ with Mn2+.
Nonetheless, there are different viewpoints and exceptions, as well as many
computational studies that are largely beyond the scope of this Review. For future
studies, we remind the reader that the ‘thio effect’ (that is, the ratio of the rate of a
phosphate substrate versus a phosphorothioate substrate) — often used in deciphering
whether the conformational closure or the nucleotidyl transfer is rate limiting — is not
a definitive approach because the full thio effect when the chemical step is fully rate
limiting is often unknown and is highly variable70, and the thio effect measured for
enzymatic phosphoryl transfer reactions can vary from 1 to 100,000 (REF. 237). In
addition, the effects of site-specific mutation on kinetics should be examined critically
and interpreted cautiously in both experimental238 and computational studies239.
discussion here largely focuses on human polymerases, as
we express more repair polymerases than do lower spe‑
cies, and our enzymes have been the subjects of recent
discoveries concerning new mechanisms and functions.
When DNA damage occurs, cells usually initiate
specific repair mechanisms before the synthesis phase
(S phase) of the cell cycle, in which DNA is replicated.
These mechanisms, often in conjunction with activation
of checkpoint proteins, are generally referred to as DNA
damage response. Common repair mechanisms include,
but are not limited to, BER, nucleotide excision repair
(NER), ribonucleotide excision repair (RER) and mis‑
match repair 128. The roles of polymerases in these repair
pathways are usually to catalyse ‘re‑synthesis’ after the
damaged nucleobases have been excised through mul‑
tiple steps. BER usually involves the use of Pol β to fill a
single-nucleotide gap, whereas NER involves Pol δ and
possibly Pol κ133, and RER involves mainly Pol δ to fill
longer gaps131. Another role of polymerases in response
to DNA damage is translesion synthesis, which usually
occurs either before the lesion can be repaired by one
of the mechanisms mentioned above or after the lesion
escapes the repair mechanisms. The aim is to try to
insert a correct base to avoid mutation, and, if this is not
possible, a mismatched dNTP is incorporated and then
fixed in the post-replication repair.
New functions of human DNA polymerases. The 17
human DNA polymerases that have been identified to
date belong to the A-, B-, X- and Y-families, with none
being in the C- or D-families. Of the A‑family poly­merases
(mainly replicative), Pol γ functions in high-fidelity DNA
synthesis in mitochondria134,135, the nuclear Pol θ (which
features polymerase and helicase domains) also partici‑
pates in double-strand break repair (DSBR)61,136–138 and the
nuclear Pol ν can perform translesion synthesis58,139. Of
the B‑family polymerases (also replicative), Pol α can use
dNTP to extend an RNA primer before it can be used by
Pol δ or Pol ε4. Pol δ also participates in DNA repair and
binds weakly to the sliding clamp proliferating cell nuclear
antigen during replication. Pol δ dissociates on reach‑
ing a stalled site, leaving the antigen on the DNA140 and
allowing a polymerase capable of translesion synthesis,
such as Pol η, to synthesize past a stalled site such as a
T–T dimer 141. Very recently, Pol δ has also been shown
to participate in alternative telomere maintenance142.
It is generally accepted that the main role of Pol ε is in
leading-strand DNA replication, although it is still under
debate whether Pol ε or Pol δ is the main polymerase in
this function143–145. The X‑family polymerases126,146, includ‑
ing Pol β11,12,17, Pol λ147,148, Pol μ48,51 and TdT122, specialize
in BER, translesion synthesis, somatic hypermutation4
and DSBR or V(D)J recombination (the latter involving
random rearrangement of variable, diverse and joining
DNA regions). The Y‑family polymerases, including
Pol κ, Pol ι, Pol η and Rev1, are mainly responsible for
translesion synthesis of bulky adducts129,149,150. Telomerase,
which belongs to the reverse transcriptase family, is
responsible for replication of the chromosome end151,152.
An exciting newly discovered polymerase is
PrimPol 153,154, which belongs to the archaeal and
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eukaryotic primase superfamily, and is so named because
it can function as both a primase and a polymerase.
Uniquely, PrimPol can use both dNTP and rNTP sub‑
strates to initiate DNA and RNA synthesis, respectively,
which is in contrast to primase, for which only rNTP
can be used. PrimPol can also function in translesion
synthesis153,154 but is highly error-prone155.
a
H
R
N
N
H 2N
9 8
7
3 4 5
2
6
1
N
H
N
O
Hoogsteen face
Approaches used by DNA polymerases to deal
with the 8‑oxo‑dG lesion and mutation. 8‑Oxo‑
7,8‑dihydroxy‑2ʹ‑deoxyguanosine (8‑oxo‑dG) is an
abundant mutagenic oxidative DNA lesion, occurring
nearly 2,800 times per human cell per day 128. In addi‑
tion, dGTP can also be oxidized to 8‑oxo-dGTP and
misincorporated into DNA by many polymerases, such
as Pol α156, mitochondrial Pol γ157, Pol β38,156,158,159, Pol λ160,
N
Oxidation
N
H 2N
dG
3 4 5
2
6
1
N
H
N
2 3
H
9
8
7
N
O
H
N
H
dATP (anti)
8-oxo-dG dAMP
(anti)
(syn)
MgC2+ MgB2+
NaA+
N
N
4
1
6 5
c
PPi
8-oxo-dGMP
(syn)
R
N
9 8
7
8-oxo-dG (syn)
b
dA
(anti)
O
R
D190
D192
MnC2+
MnB2+
MnA2+
D256
d
8-oxo-dG
(syn)
dCTP
(anti)
8-oxo-dG
(syn)
Mg2+
dATP
(syn)
Mg2+
e
Fingers
Palm
DNA
Little finger
Thumb
Figure 6 | Certain polymerases, by virtue of the size and nature
their active
sites,
Natureof
Reviews
| Chemistry
are active in translesion syntheses. a | 8‑Oxo‑7,8‑dihydroxy‑2ʹ‑deoxyguanosine
(8‑oxo‑dG) uses its Hoogsteen face to pair with an incorrect dATP in syn–anti
conformations. b,c | Structures of dA–8‑oxo-dGTP (anti–syn; Protein Databank
identifier: 4UAY) and 8‑oxo‑dG–dATP (syn–anti; PDB ID: 4RQ8) Hoogsteen base pairs,
respectively, accommodated by DNA polymerase β (Pol β). d | Pol ι uses its exceptionally
narrow active site to force the purine bases of 8‑oxo‑G or dATP to adopt a syn
conformation. The 8‑oxo‑dG–dCTP pair shown on the left (syn–anti; PDB ID: 3Q8P) is
more stable than 8‑oxo‑dG–dATP shown on the right (syn–syn; PDB ID: 3Q8Q), as the
former has one more hydrogen bond (dashed lines). e | Space-filling representations of
the narrow active site of Pol ι (PDB ID: 3Q8P), shown on the left, and the wide active site
of Pol η, shown on the right. The narrow active site of Pol ι forces a template 8‑oxo‑dG to
adopt a more compact syn conformation (yellow spheres) to provide space for incoming
dCTP (wheat-coloured spheres). The wide active site of Pol η accommodates DNA
featuring a bulky cis-syn thymine dimer (yellow spheres), as well as an incoming
dAMPNPP (2ʹ‑deoxyadenosine‑5ʹ-(α,β-imido)triphosphate, wheat-coloured spheres).
HIV‑1 reverse transcriptase156, Bacillus stearothermophilus
Pol I large fragment161 and even telomerase162. Once incor‑
porated, most of the lesions can be repaired by the BER
mechanism, but the sheer abundance of the lesions means
that some will persist into the S phase. As 8‑oxo‑dG can
use its Hoogsteen face to pair with an incorrect dATP
(syn–anti, FIG. 6a) in addition to the W–C‑like match with
dCTP (anti–anti)163, it can generate dG to dT transver‑
sions161 and lead to cancers. Recent studies have elucidated
the reasons for the variation in the response of different
polymerases to the 8‑oxo‑dG lesion.
Pol β has been shown to insert 8‑oxo-dGTP opposite
to a template dA in preference to dC158. The structures
of the intermediates in the Pol β‑catalysed reaction for
8‑oxo-dGTP insertion opposite to a template dA or dC
have been determined using time-lapse crystallography 38.
The dA–8‑oxo-dGMP mismatch forms a good anti–syn
Hoogsteen base pair (FIG. 6b), which induces structural
changes in the active site such that the mismatch, although
compromising the subsequent DNA ligation process159,
cannot be identified as a damage site by Pol β. Timelapse crystallography has also been used to show that
the 8‑oxo‑dG–dAMP mismatch also exists in a syn–anti
(Hoogsteen) conformation39 (FIG. 6c). The corresponding
dC–8‑oxo-dGMP and 8‑oxo‑dG–dCMP complexes from
the two studies both exist in an anti–anti conformation,
as is usually adopted by matched structures. Importantly,
the N subdomain was closed39,159 and the third metal ion
was also observed in the complexes involving 8‑oxo‑dG or
8‑oxo-dGTP described above, although it was missing in
the mismatch complexes of undamaged dNTP19.
In contrast to Pol β, the Y‑family enzyme Pol ι pref‑
erentially incorporates a correct dCTP opposite to an
8‑oxo‑dG lesion, a reaction that defines the unique
biological role of Pol ι in protection against oxidative
stress164. In comparing the four structures of Pol ι with
the 8‑oxo‑dG–dCTP match and the dATP, dGTP, or
dTTP mismatch163, it was found that the exception‑
ally narrow active site of Pol ι forces the purine bases
of template 8‑oxo‑dG and incoming dGTP or dATP
to each adopt a syn conformation. This stereochem‑
istry and an extra hydrogen bond favour the smaller
8‑oxo‑dG–dCTP (syn–anti Hoogsteen) base pair over
the 8‑oxo‑dG–dATP (syn–syn) base pair (FIG. 6d) and
normal W–C (anti–anti) base pairs, leading to correct
dCTP incorporation opposite to the template 8‑oxo‑dG.
The origin of Pol λ fidelity in promoting error‑free
bypass of 8‑oxo‑dG has recently been reported165. Seven
novel crystal structures and kinetic data point to Pol λ
having a flexible active site that can tolerate 8‑oxo‑dG in
either the anti‑ or syn‑conformation, with discrimination
against the pro‑mutagenic syn‑conformation occurring
at the extension step.
Lesion bypass across O6‑methylguanine. The
O6‑methylguanine (O6Me‑dG) moiety is a methylated
DNA lesion that is produced by various alkylating agents.
When left unrepaired, O6Me‑dG causes G to A muta‑
tions, owing to Pol β pairing the methylated base with an
incorrect dTTP much more frequently (~30‑fold) than
with a correct dCTP166. By using the mutator Mn2+ to
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promote formation of an O6Me‑dG–dTTP mismatched
ternary Pol β complex, it has been shown that Pol β
adopts a catalytically competent closed conformation,
with the O6Me‑dG–dTTP pair recognized as a pseudo
W–C base pair 97. By contrast, the enzyme adopts an
open conformation in the O6Me‑dG–dCTP ternary
complex 97,167. These results provide the structural basis
for the carcinogenic O6Me‑dG lesion.
Lesion bypass across an abasic site. Abasic DNA sites
are estimated to occur approximately 10,000 times per
day in each human cell168. These are referred to as being
apurinic or apyrimidinic (AP) owing to the absence of
purine or pyrimidine bases, respectively. Abasic sites
may arise spontaneously or can be induced by chemo­
therapeutics. The chemistry behind the deleterious
effects of AP sites has been reviewed169. Referred to as
the ‘A rule’, polymerases from families A and B are most
likely to install a dATP substrate opposite to an abasic
site170,171, which leads to the transversion mutations that
are found in cancer cells172. It has been shown that the
A‑family polymerase KlenTaq (a Klenow-fragment ana‑
logue of Taq polymerase) follows the A rule by using the
side chain of Tyr671 to bind incoming dATP173. By con‑
trast, Pol β from the X‑family uses its lyase subdomain
to remove the abasic site from DNA following incision
of its 5ʹ‑phosphate, and an irreversible inhibitor of a
2‑phosphato‑1,4‑dioxobutane derivative that mimics
an AP DNA lesion has been shown to shut down the
lyase activity of Pol β (with a half-maximal inhibitory
concentration, IC50, of ~21 μM)174.
The enlarged active site of Pοl η enables bypass of the
bulky T–T dimer. DNA synthesis often stalls when a rep‑
licative polymerase encounters a bulky adduct such as a
cyclobutane–pyrimidine dimer (CPD)175, which forms by
UV‑induced [2+2] cycloadditions between pyrimidine
bases. In many species, including bacteria, fungi, plants
and some mammals (marsupials and the species below),
a T–T dimer can be repaired by photoactivation involving
photolyase176–179. The apo structure of the catalytic core of
yeast (Saccharomyces cerevisiae) Y‑family Pol η features an
active site that is more open than that of replicative poly‑
merases, such that Pol η could potentially accommodate
a bulky DNA lesion, for example, a T–T dimer 180. Crystal
structures have been determined at four time points in the
Pol-η-catalysed DNA synthesis on a substrate featuring
CPDs32. The structures reveal a uniquely enlarged active
site in Pol η, enabling the enzyme to accommodate the
bulky T–T dimer while maintaining excellent stereochem‑
ical control during catalysis. In addition, Pol η acts as a
‘molecular splint’ to stabilize the damaged DNA. A com‑
parison between the narrow active site of Pol ι bound with
an 8‑oxo‑dG lesion and the wide active site of Pol η bound
with a CPD lesion is shown in FIG. 6e. A deficiency in
Pol η can cause UV sensitivity and a variant of the human
syndrome xeroderma pigmentosum181,182.
Misincorporation of rNTP. It has long been recog‑
nized that DNA polymerases can make the mistake
of processing rNTPs instead of dNTPs183,184, which is
NATURE REVIEWS | CHEMISTRY
unsurprising given that the former are present, on aver‑
age, at 30–200‑fold higher cellular concentrations185,186.
Most polymerases use a steric gate183,184,187 that would
clash with the 2ʹ‑OH group of an rNTP188. However, it
has recently been demonstrated that even high-fidelity
replicative polymerases, such as Pol α, Pol δ and Pol ε,
incorporate more than 10,000 rNTPs into the yeast
(S. cerevisiae) nuclear genome in each round of repli‑
cation186, with the estimated value being greater in the
human genome131. Furthermore, the X‑family enzymes
Pol β and, to a lesser extent, Pol λ can also incorpo‑
rate rNTPs opposite to normal bases or 8‑oxo-dG189.
Incorporation of rNTP into DNA, if not repaired, can
cause genomic instability and serious diseases. Like
other forms of damage, living systems have multiple
pathways to repair DNA, with RER being the primary
one in this case131.
DNA polymerases involved in mutagenic functions.
As described above, replicative polymerases minimize
mutations and achieve very high fidelity, whereas repair
polymerases are expressed to fix and bypass life-threat‑
ening DNA damage and mutation. There are also some
polymerases that are involved in mutagenesis, a normal
life process. For example, the low-fidelity ASFV Pol X
has been implicated in a mutagenic BER pathway8,71,104,190,
which could be a survival mechanism for the virus
under stress.
Mutagenesis is an important part of our immune
response. In order to produce specific antibodies to
fight against diverse pathogens or other foreign sub‑
jects, B cells must first generate a diverse repertoire of
B cell receptors, which involves a key randomization step
called V(D)J recombination. Furthermore, activation of
B cell receptors by a foreign antigen triggers somatic
hypermutation, a process by which the immune system
adapts to combat pathogens. Recent studies indicate
that three of the X‑family polymerases, Pol λ, Pol μ and
TdT, participate in V(D)J recombination, whereas two
Y‑family pols, Pol η and Rev1 (and possibly additional
polymerases), are involved in somatic hypermutation.
Although the detailed biochemical mechanisms of the
processes that involve these polymerases are still sub‑
jects of intensive study 6,191, it is intuitively clear that that
these polymerases have characteristically low fidelities,
as described in the preceding section, because their likely
role is to synthesize mismatched DNA.
Applications of DNA polymerases
Polymerases have diverse and essential biological roles,
but further, and more than any other class of enzymes,
they have found varied applications in biotechnology
and health-related industries. The most obvious appli‑
cations are in DNA amplification and manipulation,
including the polymerase chain reaction (PCR) and
its error-prone process. Polymerases are also useful in
DNA and RNA sequencing, as well as in other emerg‑
ing applications that are described below. For brevity, we
introduce only the most sophisticated applications, plac‑
ing emphasis on recent developments that may either
enhance current technologies or spawn new ones.
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DNA amplification and manipulation. Many poly‑
merases have been widely used in applications such as
the amplification of DNA by PCR192,193, site-directed
mutagenesis, error-prone PCR, DNA sequencing, diag‑
nosis by detection of DNA or RNA with methy­lation
(for example, 5ʹ‑methylated cytidine) and aptamer
selection by systematic enrichment of ligands by expo‑
nential amplification194. In particular, the bacteriophage
T7 polymerase has found use in the cloning of genes.
Some of these many applications involve polymerases
incorporating modified dNTPs into growing DNA.
For example, the A‑family KlenTaq as well as B‑family
KOD (Thermococcus kodakarensis polymerase) and
9°N (Thermococcus sp. 9°N-7 polymerase) can process
dNTP analogues with very bulky groups; the struc‑
tural basis for their surprisingly wide substrate scope
is now well known194. The high temperatures required
to denature double-stranded DNA demand that PCR
use only very thermally stable polymerases195. In this
regard, both high-fidelity and error-prone archaeal
DNA polymerases with high thermostability have
been developed. The use of archaeal polymerases in
biotechnology, particularly in different types of PCR,
has recently been reviewed195.
Recent development in the use of polymerases in DNA
sequencing. Certain polymerases can faithfully copy a
DNA strand; thus, it makes sense to use these enzymes
to sequence genes of interest. The approach most widely
adopted is the chain terminator method, which was
introduced by Sanger and co-workers196,197 but has now
been revised and improved in many ways. One strategy
for improving signal detection and throughput makes
use of reversible terminator dNTP analogues that fea‑
ture fluorophore labels198, and this technology is now
being put to commercial use, for example, by Illumina
Cambridge Ltd. However, dNTP bearing a bulky label
may be difficult for a polymerase to process; this prob‑
lem has been addressed by the development of mutants
such as the 9°N penta-mutant of D141A/E143A/L408S/
Y409A/P410V, an enzyme engineered by New England
Biolabs and termed Therminator III. In this 3ʹ‑5ʹ‑exo−
polymerase variant, the bulky amino acids are replaced
by smaller ones, such that bulky dNTP analogues can
be incorporated efficiently. The reversible terminator
approach is now widely used in second-generation
sequencing, but although these methods constitute tre‑
mendous improvements over the Sanger approach, they
are limited in the lengths of DNA that can be read in each
step. Some of these problems can be overcome using the
single-molecule real-time sequencing method developed
by PacBio199,200. This method, unlike second-generation
sequencing, does not require a pause between read steps
to deprotect the 3ʹ‑OR group or to remove the fluoro‑
phore from the base. This new method is now classified
as a third-generation sequencing tool.
A very different strategy is key to fourth-genera‑
tion DNA sequencing technologies such as the Oxford
Nanopore Technologies Nanopore sequencers. These
devices feature a nanopore, formed by the bacteriophage
φ29 DNA packaging motor or other materials201, through
which a single strand of DNA passes. Changes in electri‑
cal current are measured and can be related back to the
sequence of bases that were drawn through the nanopore.
Recently, the nanopore technology has been combined
with sequencing-by‑synthesis, with the resulting nano­
pore-sequencing-by-synthesis methodology report‑
edly having a false positive background detection rate
below 1.2%202. Over the past decade, the next-generation
DNA sequencing technology 200 has reduced the cost of
sequencing genomes by five orders of magnitude203,204; a
person’s whole genome can now be sequenced for as little
as US$999 (REF. 205).
Engineering polymerases to synthesize DNA with
unnatural base pairs. Attempts to expand the genetic
alphabet involve the development of unnatural base
pairs, which include nucleobase shape mimics65, hydro‑
phobic pairs66,67,206–209, metal-containing base pairs210 and
base pairs with altered hydrogen-bonding motifs68,211,212.
An unnatural base pair between 2‑amino‑6-(2‑thienyl)
purine and pyridin‑2‑one can lead to the incorporation
of unnatural amino acids into proteins213. A nucleo‑
tide analogue featuring the unnatural hydrophobic
base 7-(2‑thienyl)imidazo[4,5‑b]pyridine increases the
chemical and structural diversity of DNA in generating
DNA aptamers. Aptamers with this base target proteins
with an affinity that is two orders of magnitude greater
than do aptamers that contain only natural bases214,215.
Remarkably, many DNAs that contain unnatural hydro‑
phobic bases can be replicated by various polymer‑
ases216–218, such that one can even create semi-synthetic
organisms216,219,220.
A directed evolution approach can be used to prepare
polymerase variants with desirable and novel functions,
some of which we now mention221,222. Applications in
mRNA diagnosis, such as pathogen detection or gene
expression analysis, motivated the conversion of a DNAtemplate polymerase into an RNA-reading polymerase,
which was achieved by screening thermostable KlenTaq
variants in which amino acids in immediate proximity
to the 2ʹ‑O of the RNA template base paired with the
incoming dNTP are mutated223–225. The KlenTaq mutant
transcribes RNA into DNA, and the latter can be ampli‑
fied using reverse transcription-PCR224. Similarly, a
KOD1 enzyme variant has been prepared that discrim‑
inates between 2ʹ‑O‑methylated and unmethylated
RNA226. Directed evolution221 also enabled engineering
of a thermostable Stoffel fragment in Taq polymerase
such that the resulting enzyme is an efficient RNA
polymerase227. Taq polymerase variants that can rec‑
ognize and amplify C2ʹ‑modified DNA228 also exist.
Evolution of the high-fidelity polymerase KOD results
in it being able to use RNA templates efficiently and
gives it proofreading activity for both DNA and RNA
templates222. Lastly, we note that one bacteriophage T4
polymerase variant, an exonuclease-deficient L412M
mutant, can replicate difficult DNA sequences. The
evolved enzyme can process sequences with high GC
content or tracks of mononucleotide repeats with an
enhanced ability to incorporate fluorophore-labelled
nucleotides229.
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Conclusion
The study of DNA polymerases — the enzymes most
essential for the existence and understanding of life —
brings chemists and biologists together. This Review
describes the molecular basis for the function of these
enzymes, emphasizing that high-fidelity polymerases
have well-aligned TSs for match incorporation but
distorted TSs for mismatches. Fidelity is lowered when
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Acknowledgements
The authors acknowledge financial support from the Ministry
of Science and Technology (Grant Nos MOST103‑2113‑M0 01 ‑ 01 6 ‑ M Y 3 , M O S T 10 5 ‑ 0 210 ‑ 01 ‑ 1 2 ‑ 01 a n d
MOST106‑0210‑01‑15‑04) to M.-D.T. and a US National
Institutes of Health intramural grant (DK036146‑08) to W.Y.
Competing interests statement
The authors declare no competing interests.
Publisher’s note
Springer Nature remains neutral with regard to jurisdictional
claims in published maps and institutional affiliations.
How to cite this article
Wu, W.-J., Yang, W. & Tsai, M.-D. How DNA polymerases catalyse replication and repair with contrasting fidelity. Nat. Rev.
Chem. 1, 0068 (2017).
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