Excision of 5-Carboxylcytosine by Thymine DNA Glycosylase

Article abstract of DOI:10.1021/jacs.9b10376

5-Methylcytosine (mC) is an epigenetic mark that is written by methyltransferases, erased through passive and active mechanisms, and impacts transcription, development, diseases including cancer, and aging. Active DNA demethylation involves TET-mediated stepwise oxidation of mC to 5-hydroxymethylcytosine, 5-formylcytosine (fC), or 5-carboxylcytosine (caC), excision of fC or caC by thymine DNA glycosylase (TDG), and subsequent base excision repair. Many elements of this essential process are poorly defined, including TDG excision of caC. To address this problem, we solved high-resolution structures of human TDG bound to DNA with cadC (5-carboxyl-2′-deoxycytidine) flipped into its active site. The structures unveil detailed enzyme-substrate interactions that mediate recognition and removal of caC, many involving water molecules. Importantly, two water molecules contact a carboxylate oxygen of caC and are poised to facilitate acid-catalyzed caC excision. Moreover, a substrate-dependent conformational change in TDG modulates the hydrogen bond interactions for one of these waters, enabling productive interaction with caC. An Asn residue (N191) that is critical for caC excision is found to contact N3 and N4 of caC, suggesting a mechanism for acid-catalyzed base excision that features an N3-protonated form of caC but would be ineffective for C, mC, or hmC. We also investigated another Asn residue (N140) that is catalytically essential and strictly conserved in the TDG-MUG enzyme family. A structure of N140A-TDG bound to cadC DNA provides the first high-resolution insight into how enzyme-substrate interactions, including water molecules, are impacted by depleting the conserved Asn, informing its role in binding and addition of the nucleophilic water molecule.

Full text of DOI:10.1021/jacs.9b10376

Subscriber access provided by - Access paid by the | UCSF Library
Article
Excision of 5-carboxylcytosine by Thymine DNA Glycosylase
Lakshmi S. Pidugu, Qing Dai, Shuja Shafi Malik, Edwin Pozharski, and Alexander C Drohat
J. Am. Chem. Soc., Just Accepted Manuscript • DOI: 10.1021/jacs.9b10376 • Publication Date (Web): 06 Nov 2019
Downloaded from pubs.acs.org on November 7, 2019
Just Accepted
“Just Accepted” manuscripts have been peer-reviewed and accepted for publication. They are posted
online prior to technical editing, formatting for publication and author proofing. The American Chemical
Society provides “Just Accepted” as a service to the research community to expedite the dissemination
of scientific material as soon as possible after acceptance. “Just Accepted” manuscripts appear in
full in PDF format accompanied by an HTML abstract. “Just Accepted” manuscripts have been fully
peer reviewed, but should not be considered the official version of record. They are citable by the
Digital Object Identifier (DOI®). “Just Accepted” is an optional service offered to authors. Therefore,
the “Just Accepted” Web site may not include all articles that will be published in the journal. After
a manuscript is technically edited and formatted, it will be removed from the “Just Accepted” Web
site and published as an ASAP article. Note that technical editing may introduce minor changes
to the manuscript text and/or graphics which could affect content, and all legal disclaimers and
ethical guidelines that apply to the journal pertain. ACS cannot be held responsible for errors or
consequences arising from the use of information contained in these “Just Accepted” manuscripts.
is published by the American Chemical Society. 1155 Sixteenth Street N.W.,
Washington, DC 20036
Published by American Chemical Society. Copyright © American Chemical Society.
However, no copyright claim is made to original U.S. Government works, or works
produced by employees of any Commonwealth realm Crown government in the course
of their duties.

Page 1 of 11
Journal of the American Chemical Society
1
2
3
4
5
6
Excision of 5-carboxylcytosine by Thymine DNA Glycosylase
7
8
9
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
†
‡
†
†,§,*
and Alexander C. Drohat†,*
Lakshmi S. Pidugu, Qing Dai, Shuja S. Malik, Edwin Pozharski,
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
†
Department of Biochemistry and Molecular Biology, University of Maryland School of Medicine, Baltimore, MD 21201,
United States Department of Chemistry, The University of Chicago, Chicago, IL 60637, United States Center for
Biomolecular Therapeutics, Institute for Bioscience and Biotechnology Research, Rockville, MD 20850, United States
‡
§
ABSTRACT: 5-methylcytosine (mC) is an epigenetic mark that is written by methyltransferases, erased through passive and active
mechanisms, and impacts transcription, development, diseases including cancer, and aging. Active DNA demethylation involves
TET-mediated stepwise oxidation of mC to 5-hydroxymethylcytosine, 5-formylcytosine (fC), or 5-carboxylcytosine (caC), excision
of fC or caC by thymine DNA glycosylase (TDG), and subsequent base excision repair. Many elements of this essential process are
poorly defined, including TDG excision of caC. To address this problem, we solved high-resolution structures of human TDG bound
to DNA with cadC (5-carboxyl-2΄-deoxycytidine) flipped into its active site. The structures unveil detailed enzyme-substrate
interactions that mediate recognition and removal of caC, many involving water molecules. Importantly, two water molecules contact
a carboxylate oxygen of caC and are poised to facilitate acid-catalyzed caC excision. Moreover, a substrate-dependent conformational
change in TDG modulates the hydrogen bond interactions for one of these waters, enabling productive interaction with caC. An Asn
residue (N191) that is critical for caC excision is found to contact N3 and N4 of caC, suggesting a mechanism for acid-catalyzed base
excision that features an N3-protonated form of caC but would be ineffective for C, mC, or hmC. We also investigated another Asn
residue (N140) that is catalytically essential and strictly conserved in the TDG-MUG enzyme family. A structure of N140A-TDG
bound to cadC DNA provides the first high-resolution insight into how enzyme-substrate interactions, including water molecules, are
impacted by depleting the conserved Asn, informing its role in binding and addition of the nucleophilic water molecule.
INTRODUCTION
In particular, our understanding of how TDG recognizes and
excises fC and caC, but not their precursors (C, mC, hmC),
remains poor. TDG was discovered as an enzyme that excises
thymine from G·T mispairs, which can arise via mC
deamination and cause C→T mutations.
in mice causes embryonic lethality,
reflects the inability of other mammalian glycosylases to excise
fC or caC, an essential step of active DNA demethylation.
Chromatin structure appears to have a role in regulating TDG
activity, at least for G·T and G·fC pairs.
monofunctional glycosylases, TDG excises nucleobases from
Methylation of cytosine by DNA methyltransferases (DNMT)
produces 5-methylcytosine (mC), a prevalent mark for
epigenetic regulation that is erased through passive and active
mechanisms. An essential pathway for active DNA
demethylation in vertebrates includes mC oxidation by a TET
ten-eleven translocation) enzyme, excision of oxidized mC by
thymine DNA glycosylase (TDG) and base excision repair
(BER) (Figure 1). TET enzymes oxidize mC to give 5-
1
2-13
1
Depletion of TDG
1
4-15
a phenotype that likely
(
2
1
6-17
Like other
hydroxymethyl-C (hmC) and catalyze additional oxidation
reactions to yield 5-formyl-C (fC) or 5-carboxyl-C (caC).3-6
DNA by hydrolyzing the N-glycosyl bond of a deoxynucleotide
flipped into its active site.
activate the nucleobase leaving group (LG) for excision of caC
but not for fC or other target bases (U, T).
While mC and hmC are not excised by a DNA glycosylase,
TDG removes fC and caC and subsequent BER steps yield
DNA demethylation is required for
regulating cell differentiation during vertebrate development,
for transcriptional regulation in post-mitotic cells including
18-19
TDG employs acid catalysis to
7
-10
unmodified cytosine.
20-21
Acid catalysis is
effective for caC because it exists as a monoanion at
2
2
11
physiological pH, and N-glycosyl bond cleavage is hindered
neurons, and for suppressing tumor development, but many
aspects of this key epigenetic process are poorly defined.
2
1
by the poor LG quality of a departing caC dianion (Figure 2).
Protonation yields a neutral form of caC (amino, zwitterion, or
imino) and provides catalysis because a neutral base will depart
2
1
as a monoanion. However, the mechanism of acid-catalyzed
caC excision remains obscure, due in part to insufficient
structural information.
The mechanism for addition of the nucleophilic water
molecule in the hydrolytic reaction also remains unclear, for
TDG and the related bacterial MUG (mismatch uracil
glycosylase) enzymes. Previous studies indicate that
nucleophile addition requires an Asn residue that is strictly
conserved in the large TDG-MUG enzyme family (Asn140 in
2
3-26
human TDG).
reaction for these enzymes could involve
mechanism, where LG departure gives a discrete (short-lived)
Stepping back, we note that the overall
Figure 1. Methylation of cytosine and active demethylation by the
TET-TDG-BER pathway in vertebrates.
a
stepwise
ACS Paragon Plus Environment

Journal of the American Chemical Society
oxacarbenium ion intermediate followed by nucleophile
Page 2 of 11
1
8-19, 27
1
2
3
4
5
6
7
8
9
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
addition.
A stepwise mechanism, with rate-limiting LG
2
8-29
departure, is indicated for UNG (uracil DNA glycosylase),
RESULTS
which is of the same superfamily as TDG-MUG enzymes.
Alternatively, the TDG-MUG reaction could follow a concerted
mechanism with a single oxacarbenium ion-like transition state
High-resolution structures of TDG bound to cadC DNA.
Determining the structure of an enzyme-substrate complex
requires an approach to halt catalytic activity while minimally
perturbing substrate interactions, which typically involves a
non-reactive substrate analog or a catalytically inactive enzyme
variant. We used both approaches to solve high-resolution
structures of TDG bound to DNA with cadC flipped into its
(TS) in which LG departure is greatly advanced over
nucleophile addition (a “loose” or “highly dissociative” TS).
Such a mechanism was indicated by a QM/MM study of the
3
0
TDG reaction (for fC excision). Previous structures of TDG
reveal that the side chain (carboxamide) oxygen of the
active site (Table S1). One structure, solved at 1.55 Å
resolution, includes wild-type TDG
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
31-32
conserved Asn contacts the nucleophile,
suggesting a key
82-308
and DNA containing
role in positioning the nucleophile. Our understanding of how
the conserved Asn promotes nucleophile addition for TDG-
MUG enzymes could be advanced by structures for wild-type
enzyme and a variant that lacks the conserved Asn, bound to a
common substrate and solved at sufficient resolution to observe
water molecules in the active site, but such structures have not
been reported to date.
F
2
΄-fluoroarabino-cadC (cadC ) (Figure 3a; PDB ID: 6U17). The
subtle 2΄-F substitution fully precludes TDG cleavage of the N-
glycosyl bond for cadC and other substrates (dT, dU, fdC),
likely by destabilizing the putative oxacarbenium ion-like
18,
transition state in the reaction for N-glycosyl bond cleavage.
24, 30, 32-35
We also determined a high-quality structure of N140A-
TDG82-308 bound to DNA with cadC in its active site (1.60 Å
resolution; PDB ID: 6U16). The N140A substitution halts
catalytic activity of TDG while not diminishing its binding
2
4-25, 36
affinity for many substrates.
The mutation prevents cadC
cleavage in our crystallization conditions, consistent with single
8
2-308
turnover kinetics experiments showing that N140A-TDG
lacks significant caC excision activity in solution (reaction half-
36
life 70 days; Figure S1A).
Figure 2. The caC base is a monoanion at physiological pH and N-
glycosyl bond hydrolysis is likely precluded by the poor leaving-
group (LG) quality of a departing caC dianion. For clarity, the
focus is on LG departure in a stepwise mechanism (oxacarbenium
ion intermediate), without showing details for nucleophile
addition (see text). Leaving group quality is improved by
protonation of the caC anion to give a neutral species (amine,
zwitterion, or imino), as shown by previously calculated N1
–
1
acidies, where the free energy of deprotonation (kcal mol ) in
water is 27.7 for the caC anion and 20.5, 13.3, and 16.0 for the
amino, zwitterion, and imino forms of neutral caC, respectively.
Figure 3. High-resolution structure of TDG bound to G·caC DNA.
8
2-308
F
(
a) Crystal structure of TDG
bound to DNA with cadC in its
2
1
82-308
active site (1.55 Å; PDB ID 6U17). TDG
molecules are red spheres, the target DNA strand (with cadC ) is
yellow and the complementary strand green (with N, blue; O, red;
is cyan, water
F
To address these problems, we solved high resolution
structures of TDG and N140A-TDG bound to DNA with cadC
(5-carboxyl-2΄-deoxycytidine) flipped into the active site. The
structures redefine the molecular basis for recognition and
hydrolytic excision of caC by TDG, including key roles for
P, orange). The 2F
shown for DNA. (b) Alignment of our new structure and a
previous structure of TDG
o c
–F electron density map, contoured at 1.0, is
1
11-308
F
bound to cadC DNA (3.01 Å;
3
3
PDB ID 3UOB). Coloring for the new structure is the same except
that residues 107-122 are dark blue. The previous structure
exhibited 2:1 binding, one TDG subunit (pink) bound at a G·caC
site and the adjacent subunit (dirty violet) at a nonspecific site.
water molecules that were not observed in previous structures.
Our new structures also reveal substrate-dependent
a
conformational switch that alters the hydrogen bonding
properties for one of these water molecules, enabling it to
interact productively with the carboxylate group of caC. The
results suggest a new mechanism for acid-catalyzed base
excision that is consistent with previous experimental findings
and would be specific for caC and ineffective for C, mC, or
hmC. Our results also reveal, for the first time, the structural
effects of depleting the carboxamide group of the conserved
Asn residue for TDG-MUG enzymes, including the effect on
active-site water molecules, informing the role of the Asn in
mediating nucleophile addition during N-glycosyl bond
hydrolysis.
Our results provide a new structural paradigm for how TDG
recognizes and excises caC from DNA. Previous structures of
F
TDG bound to cadC DNA, and of N140A-TDG bound to cadC
DNA, were solved at moderate resolution (3.0 Å), lack water
molecules, and were obtained from crystals produced with
DNA that yields 2:1 binding (TDG:DNA), with one TDG
subunit bound at a specific site (G·caC) and the other at a
33, 35, 37
nonspecific site (Figure 3b).
The 2:1 complex has an
interface between TDG subunits that disrupts or alters their
DNA interactions when compared to structures of TDG-DNA
3
1-32, 38
complexes that feature 1:1 binding,
the stoichiometry that
ACS Paragon Plus Environment

Page 3 of 11
Journal of the American Chemical Society
its binding affinity for G·caC substrate and its fC excision
is likely to be physiologically relevant.37, 39-40 Our new
2
1
1
2
3
4
5
6
7
8
9
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
structures were generated using DNA that gives 1:1 binding and
under conditions that yield high resolution and feature hundreds
of ordered water molecules, including some that mediate key
enzyme-substrate interactions, as detailed below. In addition,
activity were equivalent to that of wild-type TDG. To advance
these findings we quantified the caC excision activity of
8
2-308
N191A-TDG
using single-turnover kinetics, finding a rate

5
1
constant of kmax = (3.7  0.3)  10 min (Figure S1b). This
large 3750-fold reduction in activity, relative to wild-type TDG,
indicates a critical catalytic role for the N191 carboxamide, one
that is unique to caC excision.
8
2-308
the structures were produced using TDG
, a construct that
fully recapitulates the substrate binding affinity and glycosylase
3
1-32
activity of full length TDG (410 residues),
smaller construct (TDG
unlike the
1
11-308
) used for the previous structures.
Owing to the use of improved constructs for both TDG and
the DNA, our new structures feature 16 N-terminal residues
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
(107-122) that were absent in the original structures, due likely
to disorder associated with 2:1 binding (Figure 3b). These N-
terminal residues, which include an alpha helix (0), interact
with DNA and with other regions of TDG, including a loop (2-

4) that contains an important catalytic residue (Thr197) and a
helix (1) that contains residues which contact the flipped base.
A comparison of protein backbone conformation in the new and
the previous structure of TDG bound to cadC DNA reveals
F
substantial variation, as indicated by a percentile-based spread
4
1
(
pbs) of 0.51 Å. The variance is most pronounced in two
helices (3, 6), one strand (4), and two loops (1-2, 2-4)
and is likely attributable to differences in structural resolution
3
1
as wells as binding stoichiometry. Indeed, smaller variances
are observed when comparing our new structure to structures of
8
2-308
TDG
bound to other substrates or to product DNA,
including G·U (pbs 0.14 Å; PDB ID 5HF7), G·fC (pbs 0.23 Å;
PDB ID 5T2W), or G·AP (pbs 0.14 Å; PDB ID 5FF8), which
31-32
all feature 1:1 binding and resolution of 1.54 Å to 2.2 Å.
TDG interactions with the caC base. Our structures reveal
detailed interactions with the caC base, including contacts
involving water molecules that have not been previously
observed (Figure 4). As such, our results provide a robust
structural accounting for findings that TDG binds tightly to
Figure 4. Structure of TDG82-308 bound to DNA with cadCF
flipped into the active site. The 2F –F electron density map,
o
c
F
G·caC DNA, with a dissociation constant (K
d
= 2.3 nM) that is
contoured at 1.0σ, is shown for cadC DNA and water molecules.
Dashed lines represent hydrogen bonds, with interatomic
3
9
about 100-fold greater than that for non-specific DNA. It is
important to note that while the discussion below focuses
distances (Å) shown. Water molecules contacting the carboxylate
of cadC are labeled (a, b). The 2΄-F substituent, colored cyan, is
partially obscured by C2΄, though its electron density is visible.
8
2-308
F
largely on wild-type TDG
bound to cadC DNA, the same
suite of interactions with the caC base, including water-
8
2-308
mediated contacts, are observed for N140A-TDG
bound to
A striking observation in the new structures is that two water
molecules contact a carboxylate oxygen of caC (Figures 4, 5,
S2). The waters are each coordinated by contacts with the 5΄
phosphate of the flipped cadC nucleotide and a backbone amide
nitrogen of TDG, such that each water can donate a hydrogen
bond to the carboxylate of caC. As such, these waters likely
contribute to substrate binding by stabilizing the flipped
conformation of cadC. Moreover, they are poised to facilitate
acid-catalysis by helping to transfer a proton to the carboxylate
of the anionic caC base. Importantly, the structures also reveal
a channel from the TDG surface to its active site that contains
many ordered water molecules and a hydrogen bonding
network that could potentially shuttle a proton from solvent to
the carboxylate of caC. Notably, the water molecules revealed
here to interact with the caC carboxylate have been observed in
cadC DNA (Figure S2a). Notably, these structural observations
account for findings that the N140A mutation has little effect
on the binding affinity of TDG for G·caC and other
24, 36
substrates.
Our structures reveal that the caC O2 oxygen is contacted by
two backbone amides (139, 140) and a water molecule that is
tightly bound by the hydroxyl of S271 and a backbone oxygen.
Together, these contacts could promote catalysis by stabilizing
negative charge developing on the O2 carbonyl oxygen, helping
to activate the substrate in the ground state or stabilize the
departing caC base in the transition-state of the reaction.
The structures also show that the N191 carboxamide oxygen
is within hydrogen-bonding distance to the N3 and exocyclic
N4 of caC. Notably, the N191 carboxamide nitrogen interacts
with the carboxylate of D126 through two ordered water
molecules, a hydrogen bond network that could contribute to
activating the caC leaving group, as discussed below. Both of
these residues are conserved in TDG from vertebrates. Previous
studies indicate that N191 could be essential for caC excision,
possibly by facilitating the formation of a neutral form of the
caC base that is protonated at N3, such as the zwitterion or the
imino species (Figure 2).21 Thus, N191A-TDG was shown to
lack substantial caC excision activity (in a 3 h reaction) while
8
2-308
previous structures of TDG
bound to other DNA substrates
3
1-32, 38
(dU, fdC),
but they do not contact U or fC and are thus
unlikely to mediate excision of these bases, which are removed
without acid catalysis.
ACS Paragon Plus Environment

Journal of the American Chemical Society
Page 4 of 11
the water molecule (“a”) are identical for dU and fdC DNA
1
2
3
4
5
6
7
8
9
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
(Figures 6, S3). It is also notable that the conformational switch
enlarges the solvent-filled channel from the TDG surface to its
active site, for cadC DNA (Figure 5b) relative to fdC DNA (not
shown). Several high-resolution structures TDG bound to
abasic DNA product reveal an 1-2 loop conformation
identical to that for G·caC DNA (not shown), which could
potentially facilitate departure of the excised base, a possibility
that warrants additional studies.
Figure 5. Binding pocket for the caC carboxylate and water
molecules that contact this group and populate a channel to the
surface. (a) Binding pocket for carboxyl of caC as seen in the
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
82-308
F
structure of TDG
bound to cadC DNA. Select residues of
TDG are shown in surface and stick format, with formatting
otherwise similar to that in Figure 4. The 2΄-F substituent is not
shown (for clarity). (b) Water-filled channel from the TDG active
site to its surface. The hydrogen bonding network (dashed lines)
could facilitate transfer of a solvent proton to caC. Water
molecules that contact the carboxylate of cadC are labeled (a, b).
Our structures also provide new definition regarding the role
of other TDG residues that interact with the caC base. We
confirm the previous finding that a backbone nitrogen (Y152)
forms a relatively short contact with a caC carboxylate
3
3
oxygen, which could contribute substantially to substrate
binding and caC excision. By contrast, our structures do not
support previous proposals that the caC carboxylate is contacted
by the side chain of either N157 or N230.33, 42 Notably, the
Figure 6. Substrate dependent conformational switch. (a) The new
8
2-308
F
structure of TDG
(cyan) bound to cadC DNA (yellow)
pocket surrounding the caC carboxylate includes hydrophobic
moieties of several residues (L124, A145, H151, Y152, P153)
(Figure 5a). Such an environment could favor a neutral relative
to an ionized group and could thereby elevate the pK of the caC
a
carboxylate, which could facilitate acid-catalyzed caC excision.
8
2-308
superimposed with the previous structure of TDG
bound to
F
fdC DNA (pink) (PDB ID 5T2W). Red (or pink) spheres
represent water molecules. For cadC DNA, the water molecule of
interest receives a hydrogen bond from a backbone nitrogen
(G154), while for fdC DNA, the corresponding water (pink)
A substrate-dependent conformational switch. The new
structures also reveal a remarkable substrate-dependent
conformational switch involving a TDG loop (1-2), which
modulates the hydrogen bond interactions of a water molecule
and thus its capacity to promote caC binding and excision
donates a hydrogen bond to a backbone oxygen (P153). Cis-trans
isomerization is shown for P155 (b) Effect of the conformational
switch on hydrogen bond interactions of the ordered water
molecule and its capacity to provide a hydrogen bond to the
carboxylate of caC.
(Figure 6a, water “a”; shown also in Figures 4, 5). The
Nucleophile binding and addition. A critical element of N-
glycosyl bond hydrolysis is coordination and addition of the
nucleophilic water molecule. Previous studies indicate that this
step requires an Asn that is strictly conserved in TDG-MUG
conformational change involves residues 153-156, as revealed
8
2-308
by comparing structures of TDG
bound to DNA containing
F
F
cadC or fdC , and its magnitude is remarkably large given that
cadC and fdC differ by a single atom. For cadC DNA, the water
molecule donates a hydrogen bond to the 5΄ phosphate and
receives a hydrogen bond from a backbone nitrogen (G154),
such that it can donate a hydrogen bond to the caC carboxylate
2
4-26
enzymes (Asn140 in human TDG),
but its role in TDG
hydrolysis of cadC remains unclear, due in part to insufficient
structural information. We addressed this problem using
structural and biochemical approaches. Single turnover kinetics
(Figure 6). By contrast, for fdC DNA, the water molecule
82-308
experiments show that N140A-TDG
has exceedingly low
donates a hydrogen bond to the 5΄ phosphate and to a backbone
oxygen. If the loop conformation observed for fdC DNA were
adopted for cadC DNA, the water molecule would likely
present a lone pair to the carboxylate of caC, which would be
unfavorable for binding or excision of caC. Importantly, the
conformation of this loop is identical in the high-resolution
caC excision activity; kmax = (6.8  0.7)  106 min (Figure
S1A). Thus, depleting the carboxamide of N140 causes a
1
36
20500-fold loss in caC activity, similar to the massive effects
2
4, 32
observed for other substrates (G·fC, G·T, G·U).
structure of TDG
The
8
2-308
F
bound to caC DNA reveals that the
putative nucleophilic water molecule is coordinated by the
carboxamide oxygen of N140 and the backbone oxygen of T197
82-308
structures reported here for wild-type- and N140A-TDG
bound to cadC DNA (not shown).
(Figure 7). Assignment of this water as the nucleophile is
The switch involves cis-trans isomerization for P155 and
large conformational changes for the flanking Gly residues
supported by the effect of removing the N140 carboxamide on
caC activity and findings of an identical nucleophile binding
(G154, G156). The trans isomer of P155 is seen for TDG bound
82-308
F
mechanism in structures of TDG
or fdC DNA.
bound to DNA with dU
In addition, the position of the nucleophile
and its distance from the electrophile (C1΄) are similar in all
to cadC DNA, and the cis isomer is adopted for fdC DNA.
Notably, both P155 isomers are populated in a 1.54 Å structure
F
31-32
8
2-308
of TDG
1-2 loop conformation is otherwise quite similar to fdC DNA
Figure S3). As such, the hydrogen bond interactions involving
bound to DNA with dU in its active site, but the
F
F
F
three structures (with caC , fdC , dU ). Notably, the N140
carboxamide group is positioned by close contacts from its
nitrogen to the hydroxyl of T197 and a backbone oxygen (195).

(
ACS Paragon Plus Environment

Page 5 of 11
Journal of the American Chemical Society
The catalytic importance of T197 is indicated by previous
findings that the T197A mutation causes a 32-fold loss in
glycosylase activity for a G·T substrate, and its strict
conservation in TDG-MUG enzymes. Importantly, the new
structure reveals a second water molecule that contacts the
N140 carboxamide oxygen, in addition to contacts with the
nucleophile and the 3΄ oxygen of cadC (“W2”, Figure 7). This
second N140-interacting water molecule, not seen in previous
structures, could potentially contribute to nucleophile addition,
as discussed below.
1
2
3
4
5
6
7
8
9
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
3
5
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
Figure 8. Effects of removing the N140 carboxamide. Structure of
8
2-308
N140A-TDG
(wheat) bound to DNA (light green) with cadC
flipped into its active site (PDB ID: 6U16). Aligned to this is the
8
2-308
F
structure of TDG
6
(white) bound to cadC DNA (PDB ID:
82-308
U17). Water molecules are red spheres N140A-TDG
and
82-308
82-308
magenta for TDG
. The putative nucleophile for TDG
is
labeled “n”; a corresponing water displaced by 1 Å for N140A-
8
2-308
TDG
is labeled “*” and its distance from the electrophile
(
C1΄) is 3.6 Å (thin dotted line). The second water that contacts
8
2-308
N140 of TDG
is labeled “W2”.
Figure 7. Coordination of the nucleophilic water molecule for
Removing the N140 carboxamide also exerts effects beyond
coordination of the nucleophile. The effects on TDG backbone
conformation are relatively small at the substitution site but
more pronounced for a loop (2-4; 192-204) that includes
T197, due to loss of two contacts between the loop and the N140
carboxamide nitrogen. In turn, this alters a contact involving a
backbone oxygen (198) and R275, the “plug” residue that fills
8
2-308
F
TDG
o c
bound to cadC DNA (PDB ID: 6U17). The 2F –F
electron density map, contoured at 1.0σ, is shown for water
molecules only (some omitted for clarity). Dashed lines represent
hydrogen bonds with interatomic distances (in Å) shown
(
otherwise 3.3 Å). The nucleophile (magenta sphere) and
electrophile (C1΄) are separated by 3.8 Å (thin dotted line).
3
5, 37
the DNA void generated by nucleotide flipping (Figure 8).
Effect of removing the carboxamide of N140. The
R275 contacts the phosphate of nucleotides adjacent to the
flipped nucleotide (cadC) and likely stabilizes the flipped
conformation. N140A-TDG exhibits a second conformation for
R275, which does not contact the DNA backbone and would
likely not hinder retrograde flipping of cadC, raising the
possibility that the N140A mutation could impact nucleotide
flipping. However, findings that the N140A mutation has little
effect on substrate binding affinity suggest a relatively small
mechanism of nucleophile binding and addition for TDG-MUG
8
2-308
enzymes is also informed by our structure of N140A-TDG
bound to cadC DNA (Figure 8). Importantly, this is the first
high-resolution structure of any TDG-MUG enzyme that
reveals the effect of depleting the carboxamide group of the
essential Asn, including effects on water molecules.
Remarkably, N140A-TDG binds a water molecule (“*”, Figure
8) using two of the three contacts observed for the nucleophilic
water of wild-type TDG (197 backbone oxygen, water W2).
The third contact, to the N410 carboxamide oxygen for wild-
type TDG, is replaced by a contact to the A140 backbone
oxygen for N140A-TDG. While the water molecule for N140A-
TDG exhibits a similar displacement (3.6 Å) from the
electrophile (C1΄ of cadC), its relative position in the active site
is altered by about 1 Å (for aligned structures). The absence of
significant caC excision activity for N140A-TDG indicates that
this water molecule (*) cannot serve as the nucleophile, due
likely to improper positioning and possibly additional factors,
as discussed below. Our structure also reveals that the N140A
substitution does not alter the binding of the second water
molecule (W2) that contacts the N140 carboxamide oxygen in
wild-type TDG; its position and two of its interactions are
retained for N140A-TDG (Figure 8). This observation supports
the conclusion that, although this water molecule contacts the
N140 carboxamide, it is not the nucleophile.
2
4, 36
effect on flipping.
Minimal effects of 2΄-F on enzyme-substrate interactions.
2΄-F-substituted deoxynucleotides have long been used to
generate
a stable enzyme-substrate (E-S) complex for
structural, biophysical, and biochemical studies of many DNA
2
4, 26, 32, 34, 40, 43-45
glycosylases.
The 2΄-F substitution is subtle and
is thought to not dramatically alter the conformation of a
nucleotide flipped into a glycosylase active site or perturb
enzyme interactions. However, this has not been directly
investigated by structural methods (to our knowledge), due
perhaps to experimental impediments. Such studies require an
enzyme mutation that halts base excision activity but does not
perturb interactions with the flipped nucleotide, and structures
of that variant enzyme bound to two DNA duplexes, with and
without a 2΄-F in the flipped nucleotide. Our structures show
that the N140A mutation satisfies the first requirement, as it
does not substantially perturb interactions with the flipped
nucleotide (Figures 4, S2). To address the second requirement,
ACS Paragon Plus Environment

Journal of the American Chemical Society
Page 6 of 11
we solved a structure of N140A-TDG82-308 bound to cadC DNA
F
second approach, our results provide the first robust structural
1
2
3
4
5
6
7
8
9
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
at 2.40 Å resolution (PDB ID: 6U15). Comparing our structures
evidence that removing the carboxamide of the catalytic Asn in
TDG-MUG enzymes hinders productive binding of the
essential nucleophilic water molecule while preserving
interactions with the flipped nucleotide. Indeed, we show that
this approach can be highly informative for studying the E-S
complex for TDG and we suspect it could be similarly
productive for bacterial MUG enzymes. However, the approach
does have some drawbacks. Removing the N140 carboxamide
causes structural changes in TDG that are unrelated to
nucleophile binding, as detailed above. Also, N140A-TDG
exhibits some residual activity, depending on the substrate (e.g.,
G·U DNA) such that base excision might occur during crystal
8
2-308
F
of N140A-TDG
bound to DNA with either cadC or cadC
indicates that TDG interactions with the caC base, including
water-mediated contacts to O2 and the carboxyl group, are not
substantially altered by the 2΄-F substitution, aside from minor
differences that could be attributed to the large difference in
structural resolution (1.60 Å, 2.40 Å). In addition, the
conformation of the flipped nucleotides in the active site are
remarkably similar (Figure 9). One minor difference is that a
82-308
water molecule seen in the structure of N140A-TDG
bound
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
to cadC DNA is missing in the corresponding structure with
F
cadC , due likely to steric clash with the 2΄-F substituent, but
2
4
the water seems unlikely to contribute substantially to substrate
binding or catalysis. These structural observations are
consistent with findings that the affinity of N140A-TDG for
binding G·U or G·T DNA is not substantially altered by a 2΄-F
growth. As noted above, this is not a problem for G·caC DNA
under our crystallization conditions. By contrast, the other
approach for studying an E-S complex, using wild-type enzyme
and DNA containing a 2΄-F-substituted substrate analog, has no
major shortcomings. Indeed, our structures indicate that the 2΄-
F substitution has little effect on the structure of the flipped
nucleotide or its enzyme interactions. Thus, our results show
that for TDG, and perhaps most other DNA glycosylases, using
wild-type enzyme and DNA with a 2΄-F-substituted nucleotide
is the most robust approach for studying the E-S complex.
Previous structural studies of MutY led to a similar
2
4
substitution in the target nucleotide (dU or dT). More broadly,
our results provide the first direct structural evidence that a 2΄-
F-arabino substitution does not substantially alter the
conformation of a nucleotide flipped into a DNA glycosylase
active site or perturb enzyme-substrate interactions.
4
6
conclusion. Notably, the 2΄-F substitution fully precludes base
excision activity for most DNA glycosylases, which is
important for crystallography given the incubation time needed
for crystal growth. Availability of the phosphoramidite form of
the 2΄-F deoxynucleotide can be an issue, as they can be
challenging to produce, though some are commercially
available.
Mechanism of acid-catalyzed caC excision. Previous
studies show that TDG employs acid catalysis to activate the
nucleobase leaving group (LG) for excision of caC but not for
2
1
excision of fC or other substrate bases (U, T). The unique need
of caC for LG activation is explained by its anionic ionization
state at physiological pH, which likely hinders N-glycosyl bond
cleavage due to the poor LG quality of a departing caC dianion
(Figure 2). Protonation of the caC anion yields a neutral form
of caC, including the amino, zwitterion, or imino species, which
are all predicted to have dramatically enhanced LG quality
when compared to the caC anion, as judged by N1 acidity
Figure 9. Effect of 2΄-F substituent on the conformation of cadC
82-308
flipped into the active site of N140A-TDG
. (a) Alignment of
bound to DNA containing cadC
lime) or cadC (yellow) reveals little difference in cadC
conformation. Water molecules are red spheres for cadC and
8
2-308
structures of N140A-TDG
F
(
F
F
21
magenta for cadC ; one water is seen for cadC but not cadC , due
likely to steric hindrance with 2΄-F (red dotted line). Dashed lines
represent hydrogen bonds with interatomic distances (Å). (b, c)
(Figure 2). Previous findings that candidate general acid
residues (His151, Tyr152) do not contribute to caC excision
indicate that acid-catalyzed excision does not involve direct
F
Closeup view of cadC and cadC nucleotides, flipped into the
21
protonation of caC by a general acid. Thus, the mechanism of
8
2-308
N140A-TDG
active site, indicate that 2΄-F has a minor effect
acid-catalyzed caC excision by TDG has remained unknown.
on sugar pucker (C1΄-exo, slight O4΄-exo). View is along a C4΄-
Our discovery here that two ordered water molecules contact
a caC carboxylate oxygen suggests a mechanism for transfer of
a proton to the carboxylate group of the caC anion to yield the
neutral amino form of caC, a much better leaving group relative
to the caC anion (Figure 10a). Notably, our structures also
reveal a channel from the protein surface to the active site which
contains ordered water molecules and a network of hydrogen
bonds that could potentially shuttle a proton from solvent to one
of the two waters that contacts the caC carboxylate (Figure 5b).
Together, these findings suggest one potential mechanism for
acid-catalyzed activation of the caC anion (Figure 10a).
C2΄ axis with C4΄ in foreground. 2F
are contoured at 1.0σ.
o C
–F electron density maps
DISCUSSION
Optimal approach for studying an E-S complex. Our
results provide new information that could help guide decisions
regarding the optimal approach for studying the enzyme-
substrate complex of a DNA glycosylase, where the goal is to
halt catalysis in a manner that minimally perturbs substrate
interactions. We took two common approaches, using wild-type
enzyme with a non-reactive substrate analog (2΄-F-cadC) and
mutant enzyme with natural substrate (cadC). Regarding the
ACS Paragon Plus Environment

Page 7 of 11
Journal of the American Chemical Society
1
2
3
4
5
6
7
8
9
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
Figure 10. Potential mechanisms for acid-catalyzed excision of caC by TDG, with a focus on steps leading to N-glycosyl bond cleavage.
We note that subsequent steps, including potential mechanisms for nucleophile addition and product release, are not shown. (a) Activation
of the caC anion through protonation at its carboxylate to give the neutral caC amino; the mechanism involves proton shuttling through one
of the two water molecules shown here to contact the caC carboxylate. (b) Activaton of the caC anion through protonation at N3 to give the
neutral zwitterion; the “?” indicates that the source of the requisite proton is unclear. (c) A new mechanism for activtion of the caC anion
involves water-mediated protonation of the carboxylate to give the neutral caC amino and its conversion to the caC imino, mediated by
N191 and its iteraction (through water molecules) with a general base, D126. For all three mechanisms, some interactions with water
molecules or TDG groups are shown only in the most relevant steps or not at all (for clarity).
However, such a mechanism does not account for
biochemical and structural findings that point to an essential
role for N191 in acid catalysis of caC excision. Depleting the
carboxamide of N191 (via N191A mutation) reduces the caC
excision activity of TDG by 3750-fold (Figure S1b) but does
answer is that the corresponding nucleotides (dC, dmC,
dhmC) do not flip productively into the active site, which is
consistent with findings that TDG binds with reduced affinity
to DNA containing these nucleotides relative to DNA
3
3, 36, 39
containing fdC and cadC.
Still, this seems unlikely to
2
1
not alter its binding affinity for a G·caC substrate. The
N191A mutation does not alter TDG activity for G·fC
substrates and only modestly reduces activity for G·U and
G·T substrates (by 5- and 13-fold), and these three substrates
fully account for the absence of TDG activity for these
nucleotides.
We propose a new mechanism for acid-catalyzed caC
excision that would be specific for caC and is consistent with
previous findings and the results reported here (Figure 10c).
It begins with protonation of the caC anion at a carboxylate
oxygen to give the neutral amino form of caC, through a
proton transfer mechanism as described above. Accounting
for its essential role, the next step involves N191-mediated
conversion of the caC amino to its imino form, facilitated by
ordered water molecules and the carboxylate of D126, a
residue that is conserved in vertebrate TDG. As noted above,
leaving group quality is expected to be substantially improved
2
1, 24, 35
are processed without acid-catalysis.
These
observations, together with structural findings that the N191
carboxamide oxygen contacts N3 and N4 of caC, suggest that
N191 could mediate the formation of an N3-protonated form
of caC in the active site, thereby activating the caC anion for
excision.
Notably, direct protonation of the caC anion at N3 yields a
zwitterion, which is predicted to be a good leaving group and
could be another potential mechanism for acid-catalyzed
2
1
21
activation of caC excision (Figure 10b). However, our high-
resolution structures, which include hundreds of ordered
water molecules, do not reveal a water molecule that contacts
N3 or N4 of caC or the carboxamide oxygen of N191. As
such, the source of the proton that would be needed for direct
for the imino relative to the amino of caC (Figure 2). The
amino to imino conversion would begin with abstraction of a
proton from N4 of caC by the N191 carboxamide, facilitated
by proton shuttling to a general base, D126, through hydrogen
bonds involving two ordered water molecules (Figure 4). The
resulting negative charge on the caC base could be
accommodated through delocalization to the O2 oxygen,
stabilized by hydrogen bonds from two backbone amides and
an ordered water. Subsequent transfer of a proton back
through the D126-water-N191 network to N3 of caC would
yield the imino species. Notably, abstraction of the caC N4
proton would likely be enabled by the initial protonation step,
which converts the carboxylate, an electron-donating group,
protonation at N3 is unclear. Given the low pK
converting the anionic caC base to a neutral species,
a
of 4.2 for
2
1, 47
it
seems unlikely that the N3-protonated form of caC is
substantially populated in the TDG-DNA complex prior to
nucleotide flipping, but this possibility cannot be excluded.
Another concern regarding a mechanism involving activation
of the caC anion by direct protonation at N3 has to do with
specificity. TDG does not excise C, mC, or hmC from DNA,
yet the N3 acidity for these bases is similar to that for the caC
anion (assuming a mechanism that protonates exclusively at
to carboxylic acid,
a
strongly electron-withdrawing
–
substituent ( is –0.10 and 0.37 for -COO and -COOH,
m
21
48
N3 to give a zwitterion). Thus, if TDG were to catalyze caC
respectively).
Because this mechanism begins with
excision through direct protonation at N3, how would it avoid
acid-catalyzed excision of the other three bases? One potential
protonation at the carboxylate group to give an electron-
withdrawing substituent, it would not likely be effective for
ACS Paragon Plus Environment

Journal of the American Chemical Society
Page 8 of 11
acid-catalyzed excision of C, mC, or hmC. Additional studies
will be needed to test this and other potential mechanisms for
acid-catalyzed caC excision by TDG.
hydrogen bonds.30 A key catalytic role for T197 is indicated
by findings that the T197A mutation causes a 32-fold loss in
1
2
3
4
5
6
7
8
9
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
3
5
G·T activity, though this could also reflect impaired
nucleophile positioning. Our structures suggest the positive
charge arising upon nucleophile addition could also be
stabilized by the second water molecule. Moreover, this water
is poised to be the initial recipient of the proton that is released
by the nucleophile. It is assumed the proton would then be
transferred to solvent or possibly to the excised anionic base,
depending on conformational changes and interactions that
arise in the product complex. Additional studies will be
needed to test this and other possible mechanisms for
nucleophile addition during N-glycosyl bond hydrolysis by
enzymes in the TDG-MUG family.
Nucleophile binding and addition. Together, our
biochemical and structural results inform the mechanism of
nucleophile binding and addition in the hydrolytic reaction
catalyzed by TDG enzymes, and by extension, the related
bacterial MUG enzymes. This central catalytic function
requires an Asn that is strictly conserved in the TDG-MUG
2
4-26
82-
family (N140 for human TDG).
Our structure of TDG
3
08
F
bound to cadC DNA reveals an ordered water molecule
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
that contacts the N140 carboxamide oxygen and a backbone
oxygen (197) and likely represents the nucleophile for cadC
hydrolysis. Importantly, the same interactions and relative
position are observed for the putative nucleophile in high-
8
2-308
F
resolution structures of TDG
bound to DNA with fdC or
F
31-32
dU flipped into the active site.
Our structures also provide
the first robust structural information regarding the effects of
depleting the carboxamide of the conserved Asn for any
enzyme in the TDG-MUG family. As noted above, the
structures reveal that the nucleophile for wild-type TDG, and
a corresponding water molecule for N140A-TDG, are each
coordinated by three hydrogen bonds, two of which are
conserved. However, the water molecules occupy different
relative positions in the active site and are separated by 1 Å
Figure 11. Potential role for the conserved Asn and Thr residues
of TDG-MUG enzymes in positioning the nucleophilic water
molecule, stabilizing positive charge upon nucleophile addition,
and facilitating proton transfer to another water molecule. The
structural interactions are shown in Figure 7. Labels denote
residues in human TDG.
(in aligned structures). These observations provide a structural
context for findings that the N140A mutation reduces caC
activity by 20500-fold (Figure S1a) and causes similar activity
2
4, 32
reductions for other substrates.
The massive effect of the N140A mutation could reflect a
critical role for the conserved Asn in nucleophile positioning,
as suggested by the perturbed nucleophile location revealed
here for N140A-TDG. It seems unlikely that the activity loss
caused by the N140A mutation reflects a major role for the
conserved Asn in nucleophile activation, given that the
N140D mutation, which introduces a potential general base,
MATERIALS AND METHODS
Materials. We expressed and purified TDG82-308
comprising residues 82-308 of human TDG (410 total
,
3
1
residues), as described. Expression vectors for N140A- and
N191A variants were generated by site-directed
24
also reduces TDG activity dramatically, albeit to a far smaller
mutagenesis and the vector for the C276A variant was
25, 49
obtained from DNA 2.0 (Newark, CA). The variants were
extent than the N140A mutation.
While N140D-TDG
82-308
could potentially bind the nucleophile using a carboxylate
oxygen in a manner analogous to that for the carboxamide
oxygen of TDG, the carboxylate would likely disrupt contacts
with T197 and the 195 backbone oxygen that are formed by
the carboxamide nitrogen of wild-type TDG, which could
perturb nucleophile positioning. Moreover, it seems unlikely
that strong nucleophile activation would be needed for a
reaction that is likely to be either stepwise, with a highly
electrophilic oxacarbenium ion intermediate, or concerted
with a loose transition state in which LG departure is greatly
purified using the same methods as for wild-type TDG
.
Enzyme preparations were >99% pure, as judged by SDS-
PAGE (Coomassie-stained gel) and concentration was
3
9, 50
280
determined by absorbance at 280 nm,
using ε = 17.4
−
1
−1
82-308
mM cm
for TDG
(and the variants). Standard
oligodeoxynucleotides (ODNs) were obtained from IDT and
those containing 5-carboxyl-dC (cadC) were synthesized by
the Keck Foundation Biotechnology Research Laboratory at
Yale University. Also produced at Yale were ODNs
F
containing 2΄-fluoroarabino-substituted cadC (cadC ), using a
1
8-19, 27, 30
phosphoramidite that was synthesized as described.33, 51
advanced over nucleophile approach.
38, 45
ODNs were purified by reverse phase HPLC,
exchanged
Nevertheless, along with a key role in nucleophile
positioning, the conserved Asn could potentially stabilize
positive charge associated with nucleophile addition and
facilitate transfer of the released proton to a water molecule
into 0.02 M Tris-HCl pH 7.5, 0.04 M NaCl, and quantified by
5
2
absorbance. The DNA included a 28mer target strand, 5΄-
AGC TGT CCA TCG CTC AxG TAC AGA GCT G, where
F
x is cadC or cadC , and the complementary strand, 5΄-CAG
(Figure 11). As shown by our structures, the other active-site
CTC TGT ACG TGA GCG ATG GAC AGC T, such that
water (W2) contacts the N140 carboxamide, the nucleophilic
water, and the 3΄-oxygen of the flipped cadC nucleotide
(Figure 7). Stabilization of positive charge by N140 could
F
cadC is paired with dG and located in a CpG dinucleotide
5
2-53
context.
involve
a
hydrogen bond that is strengthened by
X-ray Crystallography. Samples used for crystallization
8
2-308
82-308
delocalization of negative charge to its carboxamide oxygen,
stabilized by hydrogen bonds from its nitrogen to T197 and a
backbone oxygen (105). A recent QM/MM study of the TDG
reaction suggests the positive charge could be delocalized to
the side chains of N140 and T197 through relatively strong
contained 0.35 mM TDG
(or N140A-TDG
) and 0.42
mM DNA in a buffer of 5 mM Tris-HCl pH 7.5, 0.13 M NaCl,
0.2 mM DTT, 0.2 mM EDTA. Crystals were grown at room
temperature (~22 C) by sitting drop vapor diffusion, using 1
μl of the TDG-DNA sample and 1 or 2 ul of mother liquor,
ACS Paragon Plus Environment

Page 9 of 11
Journal of the American Chemical Society
which was 30% (w/v) PEG 4000, 0.2 M ammonium acetate,
.1 M sodium acetate, pH 6.0. Crystals were cryo-protected
Rao,
A.,
Conversion
of
5-methylcytosine
to
5-
1
2
3
4
5
6
7
8
9
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
0
hydroxymethylcytosine in mammalian DNA by MLL partner TET1.
Science 2009, 324 (5929), 930-5.
using mother liquor supplemented with 18% ethylene glycol
and flash cooled in liquid nitrogen. X-ray diffraction data
were collected at the Stanford Synchrotron Radiation
Lightsource (SSRL beamlines 12-2). Images were processed
using XDS and MOSFLM and scaled with Aimless from the
4
. He, Y. F.; Li, B. Z.; Li, Z.; Liu, P.; Wang, Y.; Tang, Q.; Ding,
J.; Jia, Y.; Chen, Z.; Li, L.; Sun, Y.; Li, X.; Dai, Q.; Song, C. X.;
Zhang, K.; He, C.; Xu, G. L., Tet-Mediated Formation of 5-
Carboxylcytosine and Its Excision by TDG in Mammalian DNA.
Science 2011, 333 (6047), 1303-1307.
5. Ito, S.; Shen, L.; Dai, Q.; Wu, S. C.; Collins, L. B.; Swenberg,
J. A.; He, C.; Zhang, Y., Tet Proteins Can Convert 5-Methylcytosine
to 5-Formylcytosine and 5-Carboxylcytosine. Science 2011, 333
(6047), 1300-1303.
6. Pfaffeneder, T.; Hackner, B.; Truss, M.; Munzel, M.; Muller,
M.; Deiml, C. A.; Hagemeier, C.; Carell, T., The Discovery of 5-
Formylcytosine in Embryonic Stem Cell DNA. Angew Chem Int Ed
Engl 2011, 50 (31), 7008-7012.
5
4-57
CCP4
http://smb.slac.stanford.edu/facilities/software/xds).
Resolution cutoff was determined based on CC1/2 values.
Structures were solved by molecular replacement using
program
suite,
assisted
by
autoxds
(
5
8
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
59
82-308
Phaser, and a prior structure of DNA-bound TDG
as the
search model (PDB ID: 5T2W). Refinement was performed
6
0
using BUSTER-TNT, and model building was performed
6
1
62-63
using Coot. TLS refinement utilized the TLSMD server,
7
. Maiti, A.; Drohat, A. C., Thymine DNA glycosylase can rapidly
3
8
as described. The data collection and refinement statistics
are provided in Supplementary Table S1. Structural figures
were made with PyMOL (http://www.pymol.org).
excise 5-formylcytosine and 5-carboxylcytosine: Potential
implications for active demethylation of CpG sites. J Biol Chem
2011, 286 (41), 35334-35338.
8
. Song, C. X.; Szulwach, K. E.; Dai, Q.; Fu, Y.; Mao, S. Q.; Lin,
Enzyme activity assays. Glycosylase activity was
L.; Street, C.; Li, Y.; Poidevin, M.; Wu, H.; Gao, J.; Liu, P.; Li, L.;
Xu, G. L.; Jin, P.; He, C., Genome-wide profiling of 5-formylcytosine
reveals its roles in epigenetic priming. Cell 2013, 153 (3), 678-91.
9. Shen, L.; Wu, H.; Diep, D.; Yamaguchi, S.; D'Alessio, A. C.;
Fung, H. L.; Zhang, K.; Zhang, Y., Genome-wide analysis reveals
TET- and TDG-dependent 5-methylcytosine oxidation dynamics.
Cell 2013, 153 (3), 692-706.
10. Weber, A. R.; Krawczyk, C.; Robertson, A. B.; Kusnierczyk,
A.; Vagbo, C. B.; Schuermann, D.; Klungland, A.; Schar, P.,
Biochemical reconstitution of TET1-TDG-BER-dependent active
DNA demethylation reveals a highly coordinated mechanism. Nat
Commun 2016, 7, 10806.
8
2-308
determined for TDG
variants acting on DNA containing
a G·caC pair using single turnover kinetics experiments
performed under saturating enzyme conditions.3
1, 64
Detailed
methods are given in the Supporting Information.
ASSOCIATED CONTENT
Supporting Information
Table S1, Figures S1-S3, Supplementary Methods.
Accession Codes
11. Schomacher, L.; Niehrs, C., DNA repair and erasure of 5-
methylcytosine in vertebrates. BioEssays 2017, 39 (3).
Coordinates and structure factor amplitudes have been deposited
with the Protein Data Bank (accession codes 6U15, 6U16, 6U17).
1
2. Neddermann, P.; Jiricny, J., The purification of a mismatch-
specific thymine-DNA glycosylase from HeLa cells. J Biol Chem
993, 268 (28), 21218-24.
AUTHOR INFORMATION
Corresponding Authors
1
13. Neddermann, P.; Gallinari, P.; Lettieri, T.; Schmid, D.; Truong,
O.; Hsuan, J. J.; Wiebauer, K.; Jiricny, J., Cloning and expression of
human G/T mismatch-specific thymine-DNA glycosylase. J Biol
Chem 1996, 271 (22), 12767-12774.
*
*
epozharskiy@som.umaryland.edu
adrohat@som.umaryland.edu
1
4. Cortazar, D.; Kunz, C.; Selfridge, J.; Lettieri, T.; Saito, Y.;
Macdougall, E.; Wirz, A.; Schuermann, D.; Jacobs, A. L.; Siegrist,
F.; Steinacher, R.; Jiricny, J.; Bird, A.; Schar, P., Embryonic lethal
phenotype reveals a function of TDG in maintaining epigenetic
stability. Nature 2011, 470 (7334), 419-423.
Notes
The authors declare no competing financial interest.
ACKNOWLEDGMENT
1
5. Cortellino, S.; Xu, J.; Sannai, M.; Moore, R.; Caretti, E.;
The studies were supported by a grant from the National Institutes
of Health, National Institute of General Medical Sciences (R01-
GM072711, to ACD) and the National Human Genome Research
Institute (K01-HG006699, to QD). X-ray diffraction data were
collected at the Stanford Synchrotron Radiation Lightsource,
SLAC National Accelerator Laboratory, supported by the U.S.
Department of Energy, Office of Science, Office of Basic Energy
Sciences under Contract No. DE-AC02-76SF00515. The SSRL
Structural Molecular Biology Program is supported by the DOE
Office of Biological and Environmental Research and the
National Institutes of Health, National Institute of General
Medical Sciences (P41GM103393). The contents of this
publication are solely the responsibility of the authors and do not
necessarily represent the official views of NIGMS or NIH.
Cigliano, A.; Le Coz, M.; Devarajan, K.; Wessels, A.; Soprano, D.;
Abramowitz, L. K.; Bartolomei, M. S.; Rambow, F.; Bassi, M. R.;
Bruno, T.; Fanciulli, M.; Renner, C.; Klein-Szanto, A. J.; Matsumoto,
Y.; Kobi, D.; Davidson, I.; Alberti, C.; Larue, L.; Bellacosa, A.,
Thymine DNA glycosylase is essential for active DNA
demethylation by linked deamination-base excision repair. Cell 2011,
146 (1), 67-79.
16. Tarantino, M. E.; Dow, B. J.; Drohat, A. C.; Delaney, S.,
Nucleosomes and the three glycosylases: High, medium, and low
levels of excision by the uracil DNA glycosylase superfamily. DNA
Repair (Amst) 2018, 72, 56-63.
1
7. Deckard, C. E., 3rd; Banerjee, D. R.; Sczepanski, J. T.,
Chromatin Structure and the Pioneering Transcription Factor FOXA1
Regulate TDG-Mediated Removal of 5-Formylcytosine from DNA.
J. Am. Chem. Soc. 2019, 141 (36), 14110-14114.
1
8. Drohat, A. C.; Maiti, A., Mechanisms for enzymatic cleavage
REFERENCES
of the N-glycosidic bond in DNA. Org. Biomol. Chem. 2014, 12 (42),
8367-8378.
19. Stivers, J. T.; Jiang, Y. L., A mechanistic perspective on the
chemistry of DNA repair glycosylases. Chem. Rev. 2003, 103 (7),
2729-59.
1
.
Kohli, R. M.; Zhang, Y., TET enzymes, TDG and the dynamics
of DNA demethylation. Nature 2013, 502 (7472), 472-9.
Drohat, A. C.; Coey, C. T., Role of Base Excision "Repair"
Enzymes in Erasing Epigenetic Marks from DNA. Chem Rev 2016,
16 (20), 12711-12729.
Tahiliani, M.; Koh, K. P.; Shen, Y.; Pastor, W. A.; Bandukwala,
H.; Brudno, Y.; Agarwal, S.; Iyer, L. M.; Liu, D. R.; Aravind, L.;
2
.
1
20. Maiti, A.; Drohat, A. C., Dependence of substrate binding and
catalysis on pH, ionic strength, and temperature for thymine DNA
3
.
ACS Paragon Plus Environment

Journal of the American Chemical Society
Page 10 of 11
glycosylase: Insights into recognition and processing of G.T
mispairs. DNA Repair 2011, 10 (5), 545-553.
1. Maiti, A.; Michelson, A. Z.; Armwood, C. J.; Lee, J. K.; Drohat,
A. C., Divergent mechanisms for enzymatic excision of 5-
formylcytosine and 5-carboxylcytosine from DNA. J. Am. Chem.
Soc. 2013, 135 (42), 15813-22.
22. Sumino, M.; Ohkubo, A.; Taguchi, H.; Seio, K.; Sekine, M.,
Synthesis and properties of oligodeoxynucleotides containing 5-
carboxy-2'-deoxycytidines. Bioorg Med Chem Lett 2008, 18 (1), 274-
39. Morgan, M. T.; Maiti, A.; Fitzgerald, M. E.; Drohat, A. C.,
1
2
3
4
5
6
7
8
9
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
Stoichiometry and affinity for thymine DNA glycosylase binding to
specific and nonspecific DNA. Nucleic Acids Res 2011, 39 (6), 2319-
2329.
40. Buechner, C. N.; Maiti, A.; Drohat, A. C.; Tessmer, I., Lesion
search and recognition by thymine DNA glycosylase revealed by
single molecule imaging. Nucleic Acids Res 2015, 43 (5), 2716-2729.
41. Pozharski, E., Percentile-based spread: a more accurate way to
2
compare crystallographic models. Acta Crystallogr
Crystallogr 2010, 66 (Pt 9), 970-978.
D Biol
7
.
2
3. Gallinari, P.; Jiricny, J.,
glycosylases related to human thymine-DNA glycosylase. Nature
996, 383 (6602), 735-8.
4. Maiti, A.; Morgan, M. T.; Drohat, A. C., Role of two strictly
A
new class of uracil-DNA
42. Hashimoto, H.; Zhang, X.; Cheng, X., Activity and crystal
structure of human thymine DNA glycosylase mutant N140A with 5-
carboxylcytosine DNA at low pH. DNA Repair (Amst) 2013, 12 (7),
535-540.
43. Stivers, J. T.; Pankiewicz, K. W.; Watanabe, K. A., Kinetic
mechanism of damage site recognition and uracil flipping by
Escherichia coli uracil DNA glycosylase. Biochemistry 1999, 38 (3),
952-63.
44. Chepanoske, C. L.; Porello, S. L.; Fujiwara, T.; Sugiyama, H.;
David, S. S., Substrate recognition by Escherichia coli MutY using
substrate analogs. Nucleic Acids Res 1999, 27 (15), 3197-204.
45. Dow, B. J.; Malik, S. S.; Drohat, A. C., Defining the Role of
Nucleotide Flipping in Enzyme Specificity Using (19)F NMR. J. Am.
Chem. Soc. 2019, 141 (12), 4952-4962.
46. Lee, S.; Verdine, G. L., Atomic substitution reveals the
structural basis for substrate adenine recognition and removal by
adenine DNA glycosylase. Proc Natl Acad Sci U S A 2009, 106 (44),
18497-502.
47. Dai, Q.; Sanstead, P. J.; Peng, C. S.; Han, D.; He, C.;
Tokmakoff, A., Weakened N3 Hydrogen Bonding by 5-
Formylcytosine and 5-Carboxylcytosine Reduces Their Base-Pairing
Stability. ACS Chem Biol 2016, 11 (2), 470-7.
48. Hansch, C.; Leo, A.; Taft, R. W., A Survey of Hammett
Substituent Constants and Resonance and Field Parameters. Chem.
Rev. 1991, 91 (2), 165-195.
49. Hashimoto, H.; Hong, S.; Bhagwat, A. S.; Zhang, X.; Cheng,
X., Excision of 5-hydroxymethyluracil and 5-carboxylcytosine by the
thymine DNA glycosylase domain: its structural basis and
implications for active DNA demethylation. Nucleic Acids Res 2012,
40 (20), 10203-14.
50. Gill, S. C.; von Hippel, P. H., Calculation of protein extinction
coefficients from amino acid sequence data. Anal. Biochem. 1989,
182 (2), 319-26.
1
2
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
conserved residues in nucleotide flipping and N-glycosylic bond
cleavage by human thymine DNA glycosylase. J Biol Chem 2009,
2
84 (52), 36680-36688.
25. Hardeland, U.; Bentele, M.; Jiricny, J.; Schar, P., Separating
substrate recognition from base hydrolysis in human thymine DNA
glycosylase by mutational analysis. J Biol Chem 2000, 275 (43),
3
2
3449-33456.
6. Barrett, T. E.; Scharer, O. D.; Savva, R.; Brown, T.; Jiricny, J.;
Verdine, G. L.; Pearl, L. H., Crystal structure of a thwarted mismatch
glycosylase DNA repair complex. EMBO J 1999, 18 (23), 6599-
6
2
609.
7. Berti, P. J.; McCann, J. A., Toward a detailed understanding of
base excision repair enzymes: transition state and mechanistic
analyses of N-glycoside hydrolysis and N-glycoside transfer. Chem.
Rev. 2006, 106 (2), 506-55.
2
8. Werner, R. M.; Stivers, J. T., Kinetic isotope effect studies of
the reaction catalyzed by uracil DNA glycosylase: evidence for an
oxocarbenium ion-uracil anion intermediate. Biochemistry 2000, 39
(46), 14054-14064.
29. Dinner, A. R.; Blackburn, G. M.; Karplus, M., Uracil-DNA
glycosylase acts by substrate autocatalysis. Nature 2001, 413 (6857),
52-755.
0. Naydenova, E.; Dietschreit, J. C. B.; Ochsenfeld, C., Reaction
7
3
Mechanism for the N-Glycosidic Bond Cleavage of 5-
Formylcytosine by Thymine DNA Glycosylase. J Phys Chem B
2
3
019, 123 (19), 4173-4179.
1. Coey, C. T.; Malik, S. S.; Pidugu, L. S.; Varney, K. M.;
Pozharski, E.; Drohat, A. C., Structural basis of damage recognition
by thymine DNA glycosylase: Key roles for N-terminal residues.
Nucleic Acids Res 2016, 44 (21), 10248-10258.
32. Pidugu, L. S.; Flowers, J. W.; Coey, C. T.; Pozharski, E.;
Greenberg, M. M.; Drohat, A. C., Structural Basis for Excision of 5-
Formylcytosine by Thymine DNA Glycosylase. Biochemistry 2016,
51. Dai, Q.; Lu, X.; Zhang, L.; He, C., Synthesis of DNA oligos
containing
2'-deoxy-2'-fluoro-D-arabinofuranosyl-5-
carboxylcytosine as hTDG inhibitor. Tetrahedron 2012, 68 (26),
5145-5151.
52. Morgan, M. T.; Bennett, M. T.; Drohat, A. C., Excision of 5-
halogenated uracils by human thymine DNA glycosylase: Robust
activity for DNA contexts other than CpG. J Biol Chem 2007, 282
(38), 27578-27586.
53. Waters, T. R.; Swann, P. F., Kinetics of the action of thymine
DNA glycosylase. J Biol Chem 1998, 273 (32), 20007-20014.
54. Kabsch, W., Xds. Acta Crystallogr D Biol Crystallogr 2010, 66
(Pt 2), 125-32.
55. Evans, P. R., An introduction to data reduction: space-group
determination, scaling and intensity statistics. Acta Crystallogr D
Biol Crystallogr 2011, 67 (Pt 4), 282-92.
56. Winn, M. D.; Ballard, C. C.; Cowtan, K. D.; Dodson, E. J.;
Emsley, P.; Evans, P. R.; Keegan, R. M.; Krissinel, E. B.; Leslie, A.
G.; McCoy, A.; McNicholas, S. J.; Murshudov, G. N.; Pannu, N. S.;
Potterton, E. A.; Powell, H. R.; Read, R. J.; Vagin, A.; Wilson, K. S.,
Overview of the CCP4 suite and current developments. Acta
Crystallogr D Biol Crystallogr 2011, 67 (Pt 4), 235-42.
57. Battye, T. G.; Kontogiannis, L.; Johnson, O.; Powell, H. R.;
Leslie, A. G., iMOSFLM: a new graphical interface for diffraction-
5
3
5 (45), 6205-6208.
3. Zhang, L.; Lu, X.; Lu, J.; Liang, H.; Dai, Q.; Xu, G. L.; Luo, C.;
Jiang, H.; He, C., Thymine DNA glycosylase specifically recognizes
5
-carboxylcytosine-modified DNA. Nat Chem Biol 2012, 8 (4), 328-
3
30.
3
4. Scharer, O. D.; Kawate, T.; Gallinari, P.; Jiricny, J.; Verdine, G.
L., Investigation of the mechanisms of DNA binding of the human
G/T glycosylase using designed inhibitors. Proc Natl Acad Sci U S A
1997, 94 (10), 4878-4883.
3
5. Maiti, A.; Noon, M. S.; Mackerell, A. D., Jr.; Pozharski, E.;
Drohat, A. C., Lesion processing by a repair enzyme is severely
curtailed by residues needed to prevent aberrant activity on
undamaged DNA. Proc Natl Acad Sci U S A 2012, 109 (21), 8091-
8096.
3
6. Coey, C. T.; Drohat, A. C., Defining the impact of sumoylation
on substrate binding and catalysis by thymine DNA glycosylase.
Nucleic Acids Res 2018, 46 (10), 5159-5170.
3
7. Maiti, A.; Morgan, M. T.; Pozharski, E.; Drohat, A. C., Crystal
Structure of Human Thymine DNA Glycosylase Bound to DNA
Elucidates Sequence-Specific Mismatch Recognition. Proc Natl
Acad Sci USA 2008, 105 (26), 8890-8895.
image processing with MOSFLM. Acta Crystallogr
Crystallogr 2011, 67 (Pt 4), 271-81.
D Biol
3
8. Malik, S. S.; Coey, C. T.; Varney, K. M.; Pozharski, E.; Drohat,
58. Karplus, P. A.; Diederichs, K., Linking crystallographic model
and data quality. Science 2012, 336 (6084), 1030-3.
59. McCoy, A. J.; Grosse-Kunstleve, R. W.; Storoni, L. C.; Read,
A. C., Thymine DNA glycosylase exhibits negligible affinity for
nucleobases that it removes from DNA. Nucleic Acids Res 2015, 43
(19), 9541-9552.
ACS Paragon Plus Environment

Page 11 of 11
Journal of the American Chemical Society
R. J., Likelihood-enhanced fast translation functions. Acta
Crystallogr D 2005, 61, 458-464.
0. Bricogne, G.; Blanc, E.; Brandl, M.; Flensburg, C.; Keller, P.;
Paciorek, W.; Rovers, i. P.; Sharff, A.; Smart, O. S.; Vonrhein, C.;
Womack, T. O. BUSTER version 2.10.2, Global Phasing Ltd.:
Cambridge, United Kingdom, 2011.
62. Painter, J.; Merritt, E. A., Optimal description of a protein
structure in terms of multiple groups undergoing TLS motion. Acta
Crystallogr D Biol Crystallogr 2006, 62 (Pt 4), 439-50.
63. Painter, J.; Merritt, E. A., TLSMD web server for the generation
of multi-group TLS models. J. Appl. Crystallogr. 2006, 39, 109-111.
64. Coey, C. T.; Drohat, A. C., Kinetic Methods for Studying DNA
Glycosylases Functioning in Base Excision Repair. Methods in
enzymology 2017, 592, 357-376.
1
2
3
4
5
6
7
8
9
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
6
61. Emsley, P.; Cowtan, K., Coot: model-building tools for
molecular graphics. Acta Crystallogr D 2004, 60 (Pt 12 Pt 1), 2126-
2
132.
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
Table of Contents artwork
ACS Paragon Plus Environment