Published online 1 April 2009Nucleic Acids Research, 2009, Vol. 37, No. 113493–3500
Nontarget DNA binding shapes the dynamic
landscape for enzymatic recognition
of DNA damage
Joshua I. Friedman1, Ananya Majumdar2and James T. Stivers1,*
1Department of Pharmacology and Molecular Sciences, Johns Hopkins University School of Medicine,
725 North Wolfe Street Baltimore, MD 21205 and2Johns Hopkins Biomolecular NMR Center, 3400 North Charles
Street, Baltimore, MD 21218, USA
Received January 20, 2009; Revised February 20, 2009; Accepted February 22, 2009
The DNA repair enzyme human uracil DNA glycosy-
lase (UNG) scans short stretches of genomic DNA
and captures rare uracil bases as they transiently
emerge from the DNA duplex via spontaneous
base pair breathing motions. The process of DNA
scanning requires that the enzyme transiently
loosen its grip on DNA to allow stochastic move-
ment along the DNA contour, while engaging extra-
helical bases requires motions on a more rapid
timescale. Here, we use NMR dynamic measure-
ments to show that free UNG has no intrinsic
dynamic properties in the millisecond to microse-
cond and subnanosecond time regimes, and that
the act of binding to nontarget DNA reshapes the
dynamic landscape to allow productive millisecond
motions for scanning and damage recognition.
These results suggest that DNA structure and the
spontaneous dynamics of base pairs may drive the
evolution of a protein sequence that is tuned to
respond to this dynamic regime.
Biomolecules can have highly dynamic structures that
undergo motions over many different time scales and are
often essential to their biological functions. We are inter-
ested in the role of DNA and enzyme dynamics in the
recognition of damaged bases in DNA. One important
example is the recognition and removal of uracil in
DNA by the enzyme human uracil DNA glycosylase
(UNG), which uses an extrahelical base recognition mech-
anism (1). UNG is essential for the prevention of C!T
transition mutations arising from cytosine deamination
(2), cytotoxic U:A pairs arising from incorporation of
dUTP in DNA and for increasing Ig gene diversity
during the acquired immune response (3). A central
event in all of these UNG-mediated processes is the
singling out of target U:A or U:G base pairs in a back-
ground of approximately 109T:A or C:G base pairs in the
human genome. We have previously used NMR dynamic
measurements of free DNA and its complex with UNG to
establish that enzymatic discrimination of thymine from
uracil is initiated by dynamic opening of T:A and U:A
base pairs (4,5), leading to an extrahelical state of T or
U that places these bases in a transient exosite binding
pocket on the enzyme (Figure 1) (6). This capture mech-
anism requires UNG to scan along nontarget DNA, tran-
siently pausing to sample base pair opening events that
occur with an estimated rate of ?8ms?1under physiolog-
ical conditions (7). These intrinsic dynamics of DNA may
in turn guide the evolution of complementary dynamic
fluctuations in UNG that allow the enzyme to scan the
DNA chain and recognize transient states of damaged
bases in DNA.
Flexibility in structure is thought to be an important
aspect of enzyme regulation and function (8,9). For exam-
ple, if the free enzyme has a rigid structure, the binding
energy of the substrate may be used to drive formation of
the active state of the enzyme from an inactive form that
exists in the absence of substrate. Alternatively, the free
enzyme may be flexible and exist in a dynamic equilibrium
between active and inactive states. In such an enzyme,
substrate encounter may select for the active conformer
from the preexisting population. NMR dynamic studies
on several enzymes have found evidence for conformer
selection as the free proteins were observed in a skewed
equilibrium between active and inactive states in the
absence of substrate (10–14). Of course, any real process
would likely incorporate elements from both of these rigid
formalisms. Here, we report an intriguing example of
dynamics in the process of damage recognition by UNG
that is distinct from either of the formal models described
above. We find that the free enzyme is rigid in the milli-
second to microsecond and subnanosecond time regimes,
but that binding to nontarget DNA flattens its conforma-
tional free energy landscape allowing UNG to sample
*To whom correspondence should be addressed. Tel: +1 410 502 2758; Fax: +1 410 955 3023; Email: firstname.lastname@example.org
? 2009 The Author(s)
This is an Open Access article distributed under the terms of the Creative Commons Attribution Non-Commercial License (http://creativecommons.org/licenses/
by-nc/2.0/uk/) which permits unrestricted non-commercial use, distribution, and reproduction in any medium, provided the original work is properly cited.
states that are energetically inaccessible in the absence of
DNA. The energetic effects of nontarget DNA binding are
likely important factors that enable UNG to scan DNA
and capture rapidly emerging extrahelical bases.
Preparation of enzyme
BL21 (DE3) Codon Plus cells were transformed with
a PET21a plasmid containing the UNG gene under the
control of an Isopropyl b-D-1-thiogalactopyranoside
(IPTG)-inducible T7 promoter. For the backbone assign-
obtained by growing cells in morphaline propanol sulfonic
acid (MOPS) minimal media containing
labeled glucose and 99%2H2O resulting in ?70% deute-
rium incorporation at non-exchangeable positions. For
enzyme used in the dynamic experiments, cells were
grown in MOPS media containing 99%
99% deuterated glucose as the sole carbon source.
Heavy water and all other isotopically enriched chemicals
were obtained from Cambridge Isotope Laboratories
(CIL), Inc. The protocol for over expression and purifica-
tion of UNG has been described previously (15).
2H2O using a
Fluorescence anisotropy measurements made use of a
DNA duplex containing a 50-FAM labeled strand
(50-FAM-T1C2G3A4T5C6G7A8T9G10-30) hybridized to its
unlabeled complement strand. Both single strands were
synthesized in-house using standard solid phase phosphor-
amidite chemistry. The single-stranded oligonucleotides
were purified using a preparative Phenomenex Jupiter
column and their concentrations were measured with
UV/VIS absorbance using calculated molar extinction
coefficients. The FAM-labeled DNA strand was combined
with a 1.2-fold excess of its unlabeled complement and
hybridization was accomplished by heating to 508C and
slowly cooling to room temperature. A native 15% poly-
acrylamide gel was used to assess whether hybridization
was successful. Unlabeled DNA of the same sequence
50-C1T2G3G4A5T6C7C8A9G10-30was used in the NMR
Technologies (IDT), Inc. and purified as described above.
and wasorderedfrom IntegratedDNA
Enzyme–DNA dissociation constant measurements
The binding affinity of UNG for nontarget DNA was
determined by following the increase in fluorescence
anisotropy of the 50-FAM-labeled DNA as UNG was
added to the solution. Binding experiments were per-
formed at 208C using several NaCl concentrations in the
range 10–100mM in a buffer containing 10mM NaH2PO4
(pH 7.0), 1mM ethylenediaminetetraacetic acid (EDTA)
and 1mM Dithiothreitol (DTT). The anisotropy increase
as a function of UNG concentration was fitted to
Equation (1) where C=([Pt]+[Lt]+Kd), and the
nonlinear regression software program Prism was used to
obtain the dissociation constant (Kd). A standard buffer
10mM NaH2PO4(pH 7.0), 75mM NaCl, 1mM EDTA
and 1mM DTT were chosen for all NMR experiments
as it was found to be optimal for protein solubility and
stoichiometric DNA binding.
FUDG¼ ðFmax? FminÞC ?
NMR resonance assignments
The backbone NH, Ca, Cband C0resonance assignments
HN(CO)CACB, HNCO, HN(CA)CO (16) and HNN
(17) 3D correlation experiments. Experiments were pre-
formed using an 800MHz Varian INOVA system
equipped with a cryoprobe. Resultant data were processed
using the NMRPipe software package (18) and analyzed
using CARA (19). Assignments for UNG bound to non-
target DNA were obtained by tracking the chemical shift
perturbations of the backbone amide resonances as DNA
was added to a solution of enzyme.
2H-labeled UNG were made using
of the HNCACB,
Quantification of dynamics in UNG
Protein dynamic measurements were performed using
UNG (0.92mM) that was doubly labeled with15N and
2H. For measurements on the nontarget DNA complex,
approximately 4-fold stoichiometric excess of DNA
(4.0mM) was present to ensure that the enzyme was
?99% bound under experimental conditions. Millisecond
timescale dynamics were measured using a modified spin-
state selective TROSY detected pulse sequence with a
60ms constant time Carr–Purcell–Meibloom–Gill period
(TCPMG) and a 3.8kHz B1CPMG pulse strength (20).
Observed transverse relaxation rates were calculated by
comparison of measured peak intensities to those obtained
from a reference experiment without a CPMG delay period
using the Equation (2).
Errors in the measured relaxation rates were estimated by
measuring variances in peak intensities obtained from
acquiring duplicate data points and propagating that
Figure 1. The uracil target search mechanism of human UNG. The
search process begins by diffusion to a nontarget site in DNA followed
by short-range scanning of the DNA helix. During a short scanning
event lasting about 5ms, UNG can trap thymine or uracil bases (X) in
a specific exosite binding pocket as they spontaneously emerge from the
DNA base stack with estimated rates of 8ms?1at 378C (7). A molec-
ular sieving mechanism is used to reject the methyl group of thymine
and allow uracil (U) to selectively proceed to the enzyme active site.
The scanning and target discrimination step is probed by the NMR
experiments in this work.
Nucleic Acids Research, 2009, Vol. 37,No. 11
error into the measured values for Robs
relaxation rates at each ?cpmgwere fitted to Equation (3),
which describes dynamic exchange between two states a
and b with an exchange constant kex¼ kforwardþ kreverse,
an amplitude factor (A ¼ papb?!2=kex, where papbis the
product of the fractional populations of the two exchan-
ging states), and ?!, the difference in resonance frequen-
cies between states a and b.
þ A 1 ?2?cpmg
2. The observed
??¼ R2vcpmg! 1
In order to determine the field dependent ‘A’ term from
Equation (3), experiments were conducted at 600 and
800MHz static magnetic field strengths. Peak intensities
were measured using SPARKY (21) and the data for each
residue at the two magnetic fields were fitted using Prism
software. The amplitude factors (A) and kexvalues for
each residue were determined by simultaneous nonlinear
regression fitting of the dispersion data at both fields to
Equation (3) (Supplementary Table 1). The kexterms from
the individually fitted residues were within error of one
another which justified a global fitting procedure where
a common kexwas shared by all dynamic residues.
Normal mode analysis
The structural coordinates for UNG bound to nontarget
DNA (pdb code 2OXM) were used as input for the
elNemo webserver (http://www.igs.cnrs-mrs.fr/elnemo/)
with the coordinates for the bound DNA molecule
removed. The five lowest frequency normal modes of
this structure were computed using the default parameters
by representing the protein backbone as rigid blocks con-
sisting of two amino acids each with an 8A˚ interaction
cutoff distance. The lowest frequency mode is shown in the
NMR assignments and dynamic behavior of free UNG
In order to probe the conformational dynamics of the
backbone amides of human UNG, it was first necessary
to make residue-specific NMR assignments for the 210
nonproline residues of the free enzyme. A standard
series of heteronuclear triple resonance NMR experiments
were performed using
(Supplementary Figure 1). Ultimately, 204 of the 210
backbone15N, Ca, C0and Cbresonances of nonproline
amino acids of UNG were assigned. Of the six unassigned
resonances (none of which were observed in these experi-
ments), three are located in the flexible N-terminus, two
are located in the dynamic DNA binding site (Ser247 and
Tyr248, see below) and the final residue (Leu207) is in
the protein core.
NMR is uniquely capable of detecting chemical
dynamics with rate constants spanning many orders of
magnitude (22). However, DNA scanning and base pair
opening occur on the millisecond timescale leading us to
focus on similar timescale motions that might be present
in the free or DNA-bound enzyme (1,4,7). This time
regime is best probed using relaxation dispersion NMR
experiments (23,24), which can quantify rates of exchange
of magnetic nuclei between different chemical environ-
ments [Equation (3)]. The exchange contribution to the
observed transverse relaxation rate [Robs
is measured as a function of an applied external field
(?cpmg), and may be obtained using Equation (3) provided
conditions of fast exchange hold. If ?cpmgis much smaller
than the rate of exchange between states, this field
will have little effect on the measured relaxation rate.
Conversely, if ?cpmgis of a much greater frequency than
the exchange rate between states, the exchange component
to transverse relaxation [Rex, the second term in Equation
(3)] will be attenuated, and Robs
rate [R2vcpmg! 1
The relaxation dispersion profiles for the vast majority
of the backbone amides of free
showed a flat ?cpmgfield dependence indicating that very
few residues possessed millisecond timescale dynamics
[selected residues of free UNG (blue) are shown in
Figure 2a]. The exceptions were Ser247, Tyr248 and
Ser169, which hydrogen bond to the DNA backbone in
the complex, and the resonance of Asn204 which is a key
catalytic residue that hydrogen bonds to H3 and O4 of the
fully extrahelical uracil base, but does not interact with
DNA in the nontarget complex (6). We also investigated
whether free UNG possessed rapid dynamics in the
nanosecond time regime by performing a heteronuclear
1H–15N Nuclear Overhauser Effect (NOE) experiment
(22). The measured heteronuclear
large and relatively constant over the protein structure
providing no evidence for significant dynamic motions in
this rapid time regime (Supplementary Figure 2).
2, Equation (3)]
will approach the intrinsic
1H–15N NOE’s were
Nontarget DNA binding induces millisecond-timescale
dynamics in UNG
To investigate the scanning and target search steps
(Figure 1), we then interrogated the dynamics of UNG
while it was bound to a 10-mer nontarget DNA duplex.
Using fluorescence anisotropy methods, the equilibrium
dissociation constant for a fluorescein end-labeled version
of the 10-mer duplex was first determined to ensure that
UNG would be fully saturated with DNA in the NMR
dynamic experiments (KD=29?0.4mM) (Figure 3a).
This KDvalue is low enough to ensure that 99% of the
UNG molecules are DNA-bound at the millimolar
enzyme and DNA concentrations used in the NMR
experiments (see below). To obtain assignments for the
nontarget complex, the 10-mer duplex was titrated into
an NMR sample of UNG and
were acquired at each titration point. The complex was
in fast exchange with the free enzyme and DNA on the
chemical shift timescale, allowing the resonance assign-
ments for the complex to be obtained by following the
chemical shift changes over the course of the titration.
Ambiguous assignments in the complex were resolved
ment. Backbone amides whose chemical shifts were
directly perturbed by DNA binding exclusively mapped
1H–15N HSQC spectra
15N-edited 3D HMQC-NOESY-HSQC experi-
Nucleic Acids Research, 2009,Vol.37, No. 113495
to the DNA binding surface as indicated by comparison
with the crystallographic structure of the nontarget com-
plex (Figure 3b, c) (6). Thus, the solution complex recapit-
ulates the crystallographic binding mode, facilitating
We then performed relaxation dispersion measurements
on the UNG–DNA complex at two static field strengths
(600 and 800MHz). In contrast to the free enzyme, a select
group of residues in the complex displayed field-dependent
dispersion profiles and elevated exchange contributions
to transverse relaxation (Figure 2a, green data points)
(Supplementary Figure 3). These interesting residues are
found in five elements of the primary sequence that
we have designated as catalytic strand, phosphate pincers
I and II, minor groove finger and second sphere
(Figure 2a–c). The catalytic group comprises residues
144–148 and contains Gln144, Asp145 and His148 all of
which have been implicated in base flipping or catalysis
(25). Phosphate pincer regions I and II comprise residues
246–249 and residue Ser169, respectively. The pincer
regions contact the phosphate backbone of the DNA
strand containing the extrahelical base in both the early
exosite complex and the final catalytic complex with
uracil. These interactions are important in providing the
Figure 2. Dynamics of free UNG and its complex with nontarget DNA. (a) Global fits of the relaxation dispersion profiles of the amide nitrogens of
the UNG–DNA complex (green). Selected residues of the UNG–DNA complex were used to globally optimize a single exchange rate constant
[kex, Equation (3)]. All residues are located in close proximity to the DNA binding pocket of UNG. With the exception of Ser169, horizontal lines
are drawn through the data for free UNG (blue) to emphasize the ?cpmgfield independent behavior for resonances of the free enzyme. (b) Dynamic
regions that interact with the DNA strand containing the extrahelical thymine in the exosite (PDB 2OXM). The locations of the backbone amides
used in the global fitting in (a) are colored red in this view. The extrahelical thymine is shown in ball and stick representation and the other DNA
strand is omitted for clarity. (c) Global view of the dynamics and chemical shift perturbations induced by nontarget DNA binding. All backbone
amides in UNG that show significant exchange in the DNA-bound state are colored red. The width of the backbone of UNG is drawn proportional
to the amide chemical shift perturbation brought about by nontarget DNA binding (Figure 3c). The magnitudes of the chemical shift changes
between free and bound UNG are not well correlated with the exchange contribution to the line widths in the bound state indicating that exchange
involves two bound states. The bound DNA is shown in cyan looking down the helical axis.
Nucleic Acids Research, 2009, Vol. 37,No. 11
binding energy that drives the uracil base from the exosite
to the active site (Figure 1). The finger region is comprised
of residues 270–274 and projects into the DNA minor
groove, filling the hole left behind by the flipped base.
Although residues Leu272 and Val274 in this region can
be fitted to relaxation dispersion curves (Figure 2a), resi-
dues 270, 271 and 273 cannot, due to extremely weak
signals arising from unfavorable exchange parameters.
Finally, the second sphere residues 153–159 form part of
an extended loop that runs anti-parallel to and contacts
the strand containing the catalytic residues Gln144,
Asp145 and His148.
Of the dozen amides that could be cleanly fitted to
Equation (3), similar values of kexwere extracted even
though these groups were dispersed in the primary
sequence and in regions of different secondary structure
(Supplementary Table 1). This result suggested that a
single global motion around the DNA binding site, with
a rate constant kex, was sufficient to model the dispersion
data. Indeed, global fitting of the entire set of curves
yielded a single exchange rate kex=900?200s?1(green
curves, Figure 2a). The observed effects cannot arise from
exchange of the enzyme between a free and bound
state because (i) the association rate constant of human
UNG for DNA (k=7?107M?1s?1, see Supplementary
Figure 4) (7,26,27) makes the observed pseudo-first-
order exchange rate between free and bound enzyme
(kex=kon[DNA]+koff) >280000s?1under the condi-
tions of the NMR dynamic studies ([DNA]=4mM).
This large exchange rate is over 300-fold greater than
the measured exchange rates, and in general, is too rapid
for measurements by NMR relaxation dispersion experi-
ments; (ii) the measured line widths of the exchanged
broadened resonances were independent of DNA concen-
tration; and (iii) the magnitudes of the chemical shift
changes between free and bound UNG are not well corre-
lated with the exchange contribution to the line widths in
the bound state (Figure 2c).
It should be noted that the dispersion profiles for
the majority of amino acids shown in Figure 2a have ele-
vated R2obsvalues even at the highest ?cpmgcompared to
the nondynamic residues of free and bound UNG
(Supplementary Figure 2). This indicates the presence of
additional contributions to the NMR line widths for these
amide resonances that could not be refocused by the exter-
nal applied field [vcpmg, Equation (3)]. We were unable to
refocus these exchange contributions to the line widths
using an off-resonance
capable of sampling frequencies as fast as 5000s?1(data
not shown) (28). The origin of this additional contribution
is unknown, but must involve motions that are slower
than the rotational correlation time of UNG. As with
the free enzyme, no fast nanosecond-timescale dynamics
were observed in the complex, as evidenced by the large
and invariant heteronuclear amide NOE’s (Supplementary
15N R1rrelaxation experiment
This study provides the first characterization of a DNA
repair enzyme dynamically inspecting undamaged DNA.
The data clearly reveal millisecond time scale motions of
UNG that are induced upon binding to nontarget DNA.
Since the induced dynamic behavior is localized to resi-
dues known to be involved in nontarget DNA binding and
target searching (6), the motions are likely to be function-
ally relevant to the scanning and target search steps of
DNA repair as depicted in Figure 1. Although NMR is
a powerful method for detecting dynamic motions of bio-
molecules, the measurements by themselves do not reveal
the structural changes that are being sampled. Thus, other
experimental observations must be utilized to link the
observed dynamics to structure and the process of search-
ing and recognizing DNA damage.
The regions of UNG that show induced dynamics upon
binding to nontarget DNA reflect a subset of regions that
undergo a clamping movement upon binding to both non-
target and target DNA (6,29). The relative displacements
in the backbone amide positions of UNG at early and
late points along the reaction coordinate are shown in
Figure 4a. The early displacements, which are relatively
large, involve transitioning from the free enzyme to the
Figure 4a). The late displacements, which are very small,
reflect structural changes that occur upon transitioning
from the exosite complex to the specific uracil complex
(green line, Figure 4a). Thus, the enzyme has largely
Figure 3. Nontarget DNA binding to human UNG. (a) Fluorescence
anisotropy measurements were used to determine the dissociation con-
stant of UNG for a 50-fluorescein-labeled 10-mer duplex DNA of the
sequence 50TCGATCGATG 30. (b) The crystal structure of human
UNG bound to nontarget DNA (yellow) showing the central thymidine
in the transient exosite specific for T and U (Figure 1) (6). (c) The same
view of UNG as panel (b) but the heteronuclear (1H–15N) weighted
chemical shift perturbations of its backbone amides upon addition of
nontarget DNA are color coded onto the surface. The DNA molecule
is removed for clarity.
Nucleic Acids Research, 2009,Vol.37, No. 113497
assumed its catalytically active closed conformation at the
exosite complex even though the flipped base is only one-
sixth of the way along the 1608 rotation leading to the
active site. The inference is that the observed dynamic
fluctuations of the DNA bound enzyme reflects exchange
observed in the exosite crystal structure and another
bound state that is intermediate between the free state
and the exosite complex. Such fluctuations on the milli-
second time scale are consistent with the estimated milli-
second life time of UNG at individual base pairs as it
Oscillations between a looser binding state and a closed
state that allows sampling of base pair opening events
provide a plausible mechanism for scanning and pausing
along the DNA contour to inspect the duplex for uracil
If an open to closed conformational transition is
responsible for the observed dynamic behavior of the
DNA-bound enzyme, then it would be expected that the
the DNA contour(7).
equilibrium scaffold of UNG would possess low-energy
motions that allow this type of reversible clamping
motion. To investigate this possibility, we performed a
normal mode analysis (NMA) of UNG using the crystal-
lographic coordinates from the exosite complex (pdb code
2OXM) (30). The lowest frequency mode obtained from
this analysis resulted in atom displacements that recapit-
ulate those observed in moving from the free enzyme to
the exosite complex (dashed line, Figure 4a). The two
extrema from this lowest frequency mode are shown in
Figure 4b with the exosite DNA duplex included for refer-
ence. A video of the complete trajectory is available as
Supplementary Video 1 online. The mechanism of short-
range DNA scanning that is suggested by this analysis
involves oscillation of the enzyme between an open state
that allows stochastic, thermally driven translocation
along the DNA strand and a closed state where the
pincer and finger regions interact more intimately
with the DNA major and minor grooves (Figure 4b).
The closed state seen in the exosite complex is especially
Figure 4. Backbone atom displacements of UNG upon DNA binding and lowest frequency NMA of the enzyme. (a) Comparison between amide
nitrogen displacement in the lowest frequency normal mode of the exosite DNA complex (black dashed line) and the observed amide displacements
between free UNG (pdb code 2OXM) and the exosite complex (pdb code 1AKZ) (blue line). (b) The two extrema of the lowest frequency normal
mode of UNG indicate an open to closed conformational transition. A video of the atom displacements in this normal mode trajectory is available as
Supplementary Video 1.
Nucleic Acids Research, 2009, Vol. 37,No. 11
well poised for the rapid capture of thymine and uracil
bases that spontaneously emerge from the DNA base
A surprising observation is that UNG is not dynamic
until it binds to nontarget DNA. This property distin-
guishes UNG from several previously studied enzyme sys-
tems where pre-existing and catalytically competent
dynamic motions were detected in the free enzyme
(10–14). The induced flexibility of the UNG backbone
upon DNA binding is distinct from several transcription
factors that are highly dynamic in the free state and
assume a rigid conformation only upon binding to their
cognate sequences (31,32). Given the high concentration
of nonspecific DNA binding sites in the cell nucleus
and UNG’s micromolar affinity for nontarget DNA
(Figure 3a), the enzyme would be expected to be bound
to DNA at all times in vivo. This environment would select
for dynamic properties of the enzyme–DNA complex that
optimize efficient repair rather than properties of the free
enzyme which may be under different selection pressures.
A functional model for the role of nontarget DNA
binding in the function of UNG is summarized in
Figure 5. The absence of significant dynamics in the free
enzyme indicates that free UNG inhabits a narrow con-
formational energy well and is unable to sample confor-
mations that resemble the bound state. Thus, the free
energy of DNA binding is used to populate unstable
states that are inaccessible in the absence of DNA, and
it is only upon binding to nontarget DNA that dynamic
modes relevant to the search process become activated.
The bound states of UNG are most reasonably assigned
to a weakly interacting open conformation that is compe-
tent for sliding along the DNA contour and resembles the
free enzyme. The second closed state resembles the early
exosite structure and is poised to detect extrahelical bases.
The closed state may also possess additional dynamic
motions in the sub-millisecond regime that allow efficient
capture of transiently emerging bases. These findings
reveal how the free energy of DNA binding can be used
to modulate the conformational and dynamic landscape of
an enzyme allowing it to scan the DNA contour and
respond to intrinsic base pair dynamics.
Supplementary Data is available at NAR Online.
The content of the publication does not necessarily reflect
the views or policies of the Department of Health and
Human Services, nor does the mention of trade names,
commercial products, or organizations imply endorsement
by the US Government.
National Institutes of Health (grant GM56834-13 to
J.T.S.). Funding to open access charge: GM56834-13.
Conflict of interest statement. None declared.
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Figure 5. The free energy of nontarget DNA binding is used to alter
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