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J. Biol. Chem., Vol. 275, Issue 47, 36506-36508, November 24, 2000
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From the Department of Chemistry and Chemical Biology, Harvard University, Cambridge, Massachusetts 02138
Received for publication, August 28, 2000
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ABSTRACT |
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 TOP
 ABSTRACT
 INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS AND DISCUSSION
REFERENCES
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Cytosine deamination and the misincorporation of
2'-dUrd into DNA during replication result in the presence of
uracil in DNA. Uracil-DNA glycosylases (UDGs) initiate the excision
repair of this aberrant base by catalyzing the hydrolysis of the
N-glycosidic bond. UDGs are expressed by nearly all
known organisms, including some viruses, in which the functional role
of the UDG protein remains unresolved. This issue could in principle be
addressed by the availability of designed synthetic inhibitors that
target the viral UDG without affecting the endogenous human UDG. Here, we report that double-stranded and single-stranded oligonucleotides incorporating either of two dUrd analogs tightly bind and inhibit the
activity of herpes simplex virus type-1 (HSV-1) UDG. Both inhibitors
are exquisitely specific for the HSV-1 UDG over the human UDG. These
inhibitors should prove useful in structural studies aimed at
understanding substrate recognition and catalysis by UDGs, as well as
in elucidating the biologic role of UDGs in the life cycle of herpesviruses.
Uracil-DNA glycosylases
(UDGs)1 are a highly
conserved class of DNA repair enzymes that initiates excision repair of
uracil in DNA by hydrolyzing the N-glycosidic bond (Fig.
1a). Uracil in DNA arises from the misincorporation of
deoxyuridine triphosphate (dUTP) during replication and from the
hydrolytic deamination of cytosine (1, 2). Cytosine deamination
generates highly mutagenic G:U mismatches that lead to G:C to A:T
transition mutations. Characterized by a high substrate specificity,
UDGs are able to distinguish between such structurally similar bases as
uracil and thymine. Unlike other DNA glycosylases, UDGs remove uracil from both single-stranded and double-stranded DNA, often with higher
efficiency for the single-stranded substrates (1, 3).
DNA glycosylases accomplish the formidable task of scanning a large
excess of normal DNA bases, recognizing and then catalyzing the
excision of damaged bases. A molecular-level description of how DNA
glycosylases achieve recognition and repair requires the formation of
stable protein-substrate complexes that can be studied by x-ray
crystallography or NMR. Such studies are not ordinarily possible
because of the transient nature of the enzyme-substrate interaction.
One way to circumvent this problem involves mutating the enzyme so as
to obtain a catalytically inactive form that retains the ability
to recognize its substrate (4, 5). An alternative approach is in effect
to "mutate" the substrate by preparing synthetic analogs that bind
the enzyme specifically but cannot be processed by it (6-8). Both
approaches have proven useful in structural and biochemical analysis of
base excision DNA repair (4-12). Powerful features of the substrate
modification approach are that it requires no prior knowledge of the
enzyme active site and that it can be structurally very
conservative, entailing changes of as little as a single atom.
UDGs have an unusually broad phylogenetic distribution, being present
in organisms from the simplest free-standing bacterium (13) to plants
(14) to man (15). Furthermore, UDGs have been identified in the genomes
of herpesvirus and poxvirus. HSV-1 UDG is dispensable for HSV-1
replication in cell culture, presumably because the cellular UDG can
supplant the activity of the viral enzyme (16, 17). However, an intact
HSV-1 UDG gene appears to be necessary for efficient viral replication
and reactivation in the murine nervous system (18). The difference in
the efficiency of viral replication and reactivation between the
in vitro and in vivo studies has been attributed
to the lack of detectable endogenous UDG expression in neurons, which
are terminally differentiated (19). Studies of HSV-1 in neural systems
are relevant because HSV-1 establishes latency in the neuronal ganglia
(20). Despite these results, the role of UDG in herpesvirus replication
and reactivation remains unclear. For example, variella-zoster virus (VZV), another member of the herpesvirus family, has been shown to
replicate in the absence of detectable endogenous or host UDG activity
(16). Human cytomegalovirus (CMV), another herpesvirus, is unlike HSV-1
or VZV in that the disruption of its UDG gene results in a longer
replication cycle with delayed DNA synthesis in tissue culture (21).
Potent and highly specific inhibitors of viral UDGs could serve as
valuable tools to elucidate the in vivo role of the enzyme
in herpesvirus replication and virulence. The availability of such
molecules could provide the impetus to pursue HSV-1 UDG as a novel
target for anti-viral therapy (22).
Despite the potential therapeutic interest in HSV-1 UDGs, few synthetic
inhibitors of UDGs have been reported. The most potent non-protein UDG
inhibitors thus far reported have IC50 values in the
micromolar range (23, 24). Here, we report the design, synthesis, and
evaluation of two inhibitors of UDGs. Our results demonstrate that
these two oligonucleotide-based inhibitors bind and inhibit HSV-1 UDG
while having little effect on the human enzyme.
Enzymes--
HSV-1 and human UDG (UDG Oligonucleotide Substrates/Competitors--
All
oligonucleotides were synthesized by standard Electrophoretic Mobility Shift Assays (EMSA)--
The
standard binding reaction mixture (20 µl) contained varying amounts
of protein and 0.4 fmol of 32P-labeled oligonucleotides in
100 µM NaCl, 0.5 mM EDTA, 0.5 mM dithiothreitol, 5% glycerol, 50 mM Tris, pH 7.4. After 30 min of incubation at room temperature, the samples were resolved on a
10% native polyacrylamide gel in 0.5× Tris-borate-EDTA running buffer. The thermodynamic dissociation constants (Kd = [protein][DNA]/[protein-DNA complex]) were measured as the
concentration of the protein at which half of the target DNA is bound,
under conditions in which [DNA] Cleavage Inhibition Assays--
The standard reaction mixture
(20 µl) contained 20 fmol of 32P-labeled dU/G duplex
oligonucleotides, varying dilutions of enzyme and 1 pmol of competitor
oligonucleotide duplex. The reaction buffers were as follows: human UDG
(60 mM NaCl, 20 mM Tris-HCl, 1 mM
EDTA, 1 mM dithiothreitol, pH 8); HSV-1 UDG (20 mM Tris-HCl, 10 mM EDTA, pH 8). The reactions
were incubated for 30 min at 37 °C for HSV-1 UDG and 10 min at
30 °C for human UDG and were stopped by the addition of 10 µl of
formamide loading dye and 5 µl of 2 M piperidine. The
sample mixtures were then boiled for 30 min prior to resolving on a
20% polyacrylamide gel.
Design and Synthesis of UDG Inhibitors--
Like all DNA
glycosylases, UDG processes its substrate through a transition state
having partial dissociative (SN1) character, wherein
positive charge is accumulated on the sugar ring, especially at C-1' and O-1' (27-29). Inhibitors have been designed
either to mimic this transition state or to destabilize it
electronically. The latter principle was employed in this study.
Namely, analog 1 (2'-
The Binding Interactions between HSV-1 and Human UDGs and the
Inhibitors--
The binding affinities of the HSV-1 and human UDGs for
the designed inhibitors were evaluated by EMSA (Fig.
2). HSV-1 UDG binds the double-stranded
(ds)
UDGs are known to excise uracil from both single-stranded (ss) and
dsDNA. We therefore tested the binding of HSV-1 and human UDGs to
inhibitors 1 and 2 in ssDNA. Although HSV-1 UDG
does not bind detectably to the ssfhUrd inhibitor, HSV-1 UDG retained
nanomolar affinity for the ss Specific Inhibition of HSV-1 UDG Activity by Potential Uses for UDG Inhibitors--
Here, we have described the
design and evaluation of nanomolar inhibitors of HSV-1 UDGs. These UDG
inhibitors have several potential applications. Although previous
structural analyses of several UDGs, notably of human,
Escherichia coli, and HSV-1 UDGs (4, 30-34), have
elucidated certain key features of uracil excision repair, tight
complexes of a native UDG enzyme with an intact DNA substrate or
substrate analog have not previously been available for structural
studies. The precise mechanism by which these enzymes scan the genome
to locate and excise uracil residues remains unknown. The present
inhibitors allow the formation of such stable complexes and are thus
expected to shed further insight into the process of substrate
recognition and catalysis by UDGs.
Viral infections represent a major human health concern with few
therapeutic options available. The fact that many members of the
poxvirus and herpesvirus families carry a functional UDG in their
limited genome and that other viruses, including the human
immunodeficiency virus type 1, actively package host UDG protein into
viral particles suggests a potentially important functional role for
this enzyme (35). However, the precise function of UDGs in viral
infection remains unclear. Highly selective and specific inhibitors
described in this communication provide new reagents to probe the
in vivo role of viral UDGs. Although oligonucleotides often
suffer from poor cell permeability, there is reason to be optimistic in
the case of these inhibitors, as oligonucleotides containing as few as
7 nucleotides inhibit HSV-1 UDG in vitro.2
INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS AND DISCUSSION
REFERENCES
EXPERIMENTAL PROCEDURES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS AND DISCUSSION
REFERENCES
84) were generous gifts
from Prof. Laurence H. Pearl (Department of Biochemistry and Molecular
Biology, University College, London) and Prof. Hans Krokan
(UNIGEN Center for Molecular Biology, The Norwegian University of
Science and Technology), respectively.
-cyanoethyl solid
phase chemistry on a 1 µmol scale using an ABI model 392 DNA
synthesizer and were purified by denaturing polyacrylamide gel
electrophoresis. End-radiolabeling reactions were performed with
T4 polynucleotide kinase (New England Biolabs) and
32P]ATP (PerkinElmer Life Sciences). Duplexes
were prepared by annealing with 10-fold excess of complementary strand.
The radiolabeled strand of each duplex is the top strand, as shown in
Sequence 1. The phosphoramidite of
2'--flouro-2'-deoxyuridine FdUrd) was synthesized according to
published procedures (25). The phosphoramidite of furan
homouridine (fhUrd) was synthesized by an adaptation of a published
procedure (26).2
View larger version (14K):
[in a new window]
Sequence 1.
Kd. Data
from at least three titration gels were averaged to obtain the reported
Kd values, which have standard errors of ± 50%. Example gels and binding data used to determine the experimental
equilibrium constants are available as supplemental information.
RESULTS AND DISCUSSION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS AND DISCUSSION
REFERENCES
-fluoro-2'-deoxyuridine, FdUrd)
(Fig. 1c) possesses a
2'-fluoro substituent, which destabilizes the transition state by
withdrawing electron density from the already electron-deficient C-1'
center. Analog 2 (fhUrd) (Fig. 1c)
interposes a CH2 unit between the base and its sugar,
thereby depriving the transition state of assistance from the lone pair
electrons on O-1'. The increased distance from C-1' to the
base in 2 may resemble the lengthening of the glycosidic
bond that occurs during displacement of the base (Fig. 1b).
Both analogs possess an attached uracil base, which UDG is expected to
recognize specifically. Indeed, analog 1 has previously been
shown to bind and inhibit the human G/T glycosylase at subnanomolar
concentrations (12).
View larger version (14K):
[in a new window]
Fig. 1.
The action of UDG and designed inhibitors of
UDG. a, UDG catalyzes the hydrolysis of the
N-glycosidic bond between uracil and the deoxyribose
backbone. b, the proposed transition state for glycosidic
bond cleavage by UDG, c, designed inhibitors of UDG.
FdUrd and fhUrd nucleoside analogs were synthesized and
incorporated into the middle of 25mer oligonucleotides using standard
solid phase phosphoramidite chemistry. For double-stranded inhibitors,
inhibitor oligonucleotides were annealed with complementary oligonucleotides containing a G opposite the analog.
FU inhibitor tightly with an apparent Kd of
4 nM. Under the same experimental conditions, the dsFU
inhibitor fails to form a specific gel shift complex with human UDG. A
25mer containing inhibitor 2 was tested in the same way
using EMSA and was found similarly to bind with high specificity to
only the viral UDG, albeit with lower affinity (Kd = 160 nM).
View larger version (35K):
[in a new window]
Fig. 2.
EMSA assay comparing the binding affinities
of HSV-1 and human UDG for ds FdUrd (a) and dsfhUrd
(b). Concentration of 32P-labeled
oligonucleotides = 0.4 nM.
FdUrd inhibitor (Kd = 47 nM). Neither uracil analog in ssDNA bound the human
UDG even at concentrations as high as 1 µM. Thus, the
FdUrd inhibitor is exquisitely specific for the viral over the human
UDG, irrespective of whether the DNA is single- or double-stranded.
FdUrd and
fhUrd Inhibitors--
To determine whether binding of the analogs to
UDG results in inhibition of catalytic activity, we analyzed the
ability of the analogs to interfere with cleavage of a natural
substrate by UDG. In this assay, a 5'-32P-labeled
uracil-containing substrate is incubated with UDG in the presence or
absence of unlabeled competitor analog. Processing of the natural
substrate creates an abasic site, which undergoes strand cleavage to
generate shortened oligonucleotide products upon treatment with aqueous
piperidine. As shown in Fig.
3a, a 50-fold excess of
dsFU gives nearly complete inhibition (~90%) of HSV-1 UDG
activity (lane 4). The ssFU oligonucleotide also inhibited HSV-1 UDG activity strongly (lane 7). Under the
conditions of these assays, the fhUrd analog inhibited cleavage by
~40% in dsDNA (lane 5) but not to any significant extent
in ssDNA (lane 8). By contrast, neither analog caused
pronounced inhibition of human UDG, although FU in both ssDNA and
dsDNA showed evidence of modest inhibition (<25% relative to
nonspecific DNA, Fig. 3b). Overall, the trends seen in
cleavage inhibition assays closely parallel those in binding assays,
consistent with the notion that these analogs target the enzyme active
site.
View larger version (33K):
[in a new window]
Fig. 3.
Cleavage inhibition of HSV-1 UDG
(a) and human UDG (b) using both
single-stranded and double-stranded FdUrd and fhUrd analogs.
Concentration of 32P labeled uracil-containing
substrate = 1 nM; concentration of competitor = 50 nM. ns, nonspecific competitor containing C
in the place of U.
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ACKNOWLEDGEMENTS |
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We thank Prof. Lawrence H. Pearl and
Prof. Hans E. Krokan for their generous gifts of HSV-1 and human
UDGs and Orlando Schärer for synthesis of the FU
oligonucleotides. We thank Xiaoyan Zhang, Karl Haushaulter, and Derek
Norman for critical readings of this manuscript, and members of the
Verdine laboratory for helpful discussions.
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FOOTNOTES |
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* The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
The on-line version of this article (available at
http://www.jbc.org) contains Figs. S1 and S2.
To whom correspondence should be addressed: Dept. of Chemistry and
Chemical Biology, Harvard University, 12 Oxford St., Cambridge, MA
02138. Tel.: 617-495-5323; Fax: 617-495-8755; E-mail:
verdine@chemistry.harvard.edu.
Published, JBC Papers in Press, August 31, 2000, DOI 10.1074/jbc.C000585200
2 Y. Sekino, S. D. Bruner, and G. L. Verdine, unpublished results.
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ABBREVIATIONS |
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The abbreviations used are:
UDG, uracil-DNA
glycosylase;
HSV-1, herpes simplex virus type-1;
VZV, variella-zoster
virus;
FdUrd, 2'--fluoro-2'-deoxyuridine;
fhUrd, furan
homouridine;
EMSA, electrophoretic mobility shift assay;
ds, double-stranded;
ss, single-stranded.
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REFERENCES |
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TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS AND DISCUSSION
REFERENCES
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