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INTRODUCTION |
Type IB topoisomerases modulate the topological state of DNA by
cleaving and rejoining one strand of the DNA duplex. Cleavage is a
transesterification reaction in which the scissile phosphodiester is
attacked by a tyrosine of the enzyme, resulting in the formation of a
DNA-(3'-phosphotyrosyl)-enzyme intermediate and the expulsion of a
5'-OH DNA strand. In the religation step, the DNA 5'-OH group attacks
the covalent intermediate resulting in expulsion of the active site
tyrosine and restoration of the DNA phosphodiester backbone. Vaccinia
topoisomerase is a prototype of the type IB topoisomerase family, which
includes eukaryotic nuclear and mitochondrial topoisomerase IB, the
poxvirus topoisomerases, and poxvirus-like topoisomerases encoded by
bacteria (1-4). The vaccinia enzyme is distinguished from the nuclear
topoisomerase I by its compact size (314 amino acids) and its site
specificity in DNA transesterification (5). Vaccinia topoisomerase
binds and cleaves duplex DNA at a pentapyrimidine target sequence
5'-(T/C)CCTTp
. The Tp
nucleotide (defined as the +1 nucleotide)
is linked to Tyr-274 of the enzyme.
The DNA features that contribute to target site recognition and
catalysis have been examined using synthetic substrates containing a
single CCCTT site. The DNase I footprint of vaccinia topoisomerase covers ~13 nucleotides upstream (5') and ~9-13 nucleotides
downstream (3') of the scissile bond (6). Exonuclease III footprinting suggests a two-part interaction of topoisomerase with the DNA 5' of the
site of covalent adduct formation (7). A transient margin of protection
from exonuclease III digestion, which extends to positions +13 to +14
on the scissile strand, gives way to a long-lived margin of protection
at positions +7 to +9. The tightly protected segment includes the
entire CCCTT
element that directs site-specific transesterification.
Modification interference, modification protection, analog
substitution, and UV cross-linking experiments indicate that vaccinia
topoisomerase makes contact with several nucleotide bases and the
sugar-phosphate backbone of DNA in the vicinity of the CCCTT
recognition site. For example, dimethyl sulfate protection and
interference experiments revealed interactions in the major groove with
the three guanine bases of the pentamer motif complementary strand
(3'-GGGAA) (8).
The contributions of individual phosphates to binding specificity were
initially inferred from the effects of phosphate ethylation on protein
binding (9). Ethylation of four phosphates on the scissile strand
(positions CpCpCpTpTp
within the pentamer motif) and three phosphates on the nonscissile strand (3'-GpGpGpApA) interfered with
topoisomerase-DNA complex formation. In a B-form structure of the
CCCTT-containing DNA substrate, the relevant topoisomerase-phosphate
contacts are arrayed across the minor groove of the double helix (9).
The major groove base-specific contacts that comprise the
topoisomerase-DNA interface are situated on the opposite face of the
DNA helix from the specific phosphate contacts and from the scissile
phosphate. These results suggested that topoisomerase binds
circumferentially to its target site in duplex DNA (9).
Subsequent structural analyses of the human and vaccinia topoisomerases
revealed that the type IB enzymes do indeed form a C-shaped protein
clamp around the DNA duplex (10, 11). The carboxyl catalytic domain
interacts with the minor groove face of the DNA at the cleavage site,
while the amino domain is positioned on the major groove side of the
target site. The crystal structures, together with functional studies
of the vaccinia enzyme (12, 13), imply that several of the catalytic
amino acids either coordinate the scissile phosphodiester from the
minor groove side or penetrate directly into the minor groove.
In the present study, we use a minor groove-specific interference
approach to delineate the dimensions of the minor groove interface
between vaccinia topoisomerase and its cleavage site. We introduce
7,8-diol 9,10-epoxide adducts of benzo[a]pyrene
(BP)1 at the exocyclic
N2-amino group of single deoxyguanosine (dG)
positions within the nonscissile strand of a suicide cleavage substrate
for vaccinia topoisomerase. These adducts are derived from trans
opening with inversion at C-10 of the
(+)-(7R,8S,9S,10R)- and
(
)-(7S,8R,9R,10S)- enantiomers of the diol epoxides in which the benzylic 7-hydroxyl group
and the epoxide oxygen are trans (see Fig. 1). NMR structures have
established that these BPdG adducts fit into the minor groove with no
significant perturbations of Watson-Crick base pairing or B-form helix
conformation (14, 33, 34). Moreover, the S and R
adducts have opposite orientations in the minor groove, such that the
S adduct points toward the 5' end of the modified strand,
whereas the R adduct points toward the 3' end of the
modified strand (see Fig. 1). Thus, trans opened BPdG adducts provide
an elegant means to gauge the effects of space occupancy within the minor groove.
This technique has been applied previously to human topoisomerase IB,
whereby a BPdG adduct was placed on the scissile strand at the
nucleoside immediately 3' of the phosphodiester that is preferentially
cleaved by the human enzyme in unmodified DNA (15, 16). The BPdG adduct
suppressed cleavage at the normal site but promoted alternative
cleavages elsewhere on both strands of the DNA substrate. This response
to interfering lesions is typical for human topoisomerase I, which
lacks stringent sequence specificity for DNA transesterification and
simply finds another site when confronted with an impediment (17).
Vaccinia topoisomerase is more amenable to quantitatively informative
interference studies because the pre-steady-state kinetic parameters
for transesterification are known (18-20) and the enzyme does not
relinquish its site specificity in response to a DNA lesion (21).
Here we introduce BPdG lesions into the G-rich nonscissile strand at
the three guanines within the 3'-GGGAA complement of the 5'-CCCTT
cleavage site (these are defined as the +5, +4, and +3 G residues) and
also at the two nucleosides immediately downstream of the cleavage site
(the
1 and
2 nucleosides). We observe a sharp margin of
interference effects, whereby +5 and
2 BPdG modifications are well
tolerated but +4, +3, and
1 BPdG adducts are severely deleterious.
The stereoselective effects at the
1 nucleoside (the R
diastereomer interferes, whereas the S diastereomer does not) delineate
at high resolution the downstream border of the minor groove interface.
We also apply the BPdG interference method to probe the interactions of
Escherichia coli exonuclease III with the DNA minor groove.
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EXPERIMENTAL PROCEDURES |
DNA Substrates--
Modified oligonucleotides containing single
chiral BPdG adducts (Fig. 1) were
synthesized and purified on a 1.5-µmol scale as described previously
(35). The modified dG was introduced by manual coupling using the
diastereomeric mixture of suitably protected R and
S C-10 adducted phosphoramidates (36). After completion of
the synthesis and removal of protecting groups, the resultant
diasteromeric oligonucleotides were purified and separated from each
other by high performance liquid chromatography (Table I).
Absolute configurations of the separated diastereomers were determined from their circular
dichroism spectra. The pyrene chromophore produces a positive band in
the 320-360-nm range for the R diastereomer and a negative
band for the S diastereomer (37). Unmodified
oligonucleotides were purchased from BIOSOURCE International. DNA concentrations were determined by UV absorbance at
260 nM. Unmodified 34-mer scissile strands were 5'
32P-labeled by enzymatic phosphorylation in the presence of
[
-32P]ATP and T4 polynucleotide kinase. The labeled
oligonucleotides were gel-purified and hybridized to standard or
BPdG-modified 18-mer oligonucleotides at a 1:4 molar ratio of 34-mer to
18-mer. Annealing reaction mixtures containing 0.2 M NaCl
and oligonucleotides as specified were heated to 80 °C and then
slow-cooled to 22 °C. The hybridized DNAs were stored at 4 °C.
The structures of the annealed duplexes are depicted in Figs.
2 and 5.

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Fig. 1.
Opposite orientation of trans
S and R BPdG adducts in the DNA minor
groove. Structures of the C-10 trans S and R
BPdG adducts and their diol epoxide precursors are shown in the
bottom panel beneath space-filling views of the NMR
structures of the chiral BP adducts (colored light blue) in
the minor groove of an 11-bp B-form duplex DNA (14). BP is
covalently attached to the DNA strand to the right side of the BP
moiety. The S diastereomer is oriented toward the 5' end of
the strand to which it is attached, and the R diastereomer
points toward the 3' end.
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Table I
High performance liquid chromatography retention times and absolute
configurations of 18-mer oligonucleotides containing trans opened
N2-BPdG adducts at C-10
The modified base is underlined. Configurational assignments are based
on the long-wavelength (320-360 nm) CD bands of the oligonucleotides,
which are positive for R and negative for S
adducts.
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Fig. 2.
Effects of BPdG adducts at positions 1 and
2 of the nonscissile strand on the rate of DNA transesterification by
vaccinia topoisomerase. The 34-mer/18-mer CCCTT-containing
substrate is shown at the bottom of the figure. The site of
cleavage is indicated by a vertical arrow, and the 5'
32P-label on the scissile strand is denoted by an
asterisk. The unmodified control substrates containing a
1C:G base pair (panel A) or a 2C:G base pair
(panel B) are depicted in greater detail with the
phosphodiester backbones drawn as horizontal lines, and the
base pairs as vertical lines. The numerical coordinates of
the nucleotides are indicated above the control sequence in panel
A. The S and R diastereomers of the BPdG
adducts are depicted as horizontal bars in their respective
orientations from the site of covalent attachment to guanine on the
nonscissile strand. Cleavage rate constants and reaction endpoints for
transesterification by vaccinia topoisomerase are indicated to the
right of each structure.
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Vaccinia Topoisomerase--
Recombinant vaccinia topoisomerase
was produced in E. coli (BL21) by infection with
bacteriophage
CE6 (22) and then purified to apparent homogeneity
from the soluble bacterial lysate by phosphocellulose and Source S-15
chromatography steps. Protein concentration was determined by using the
dye-binding method (Bio-Rad) with bovine serum albumin as the standard.
DNA Transesterification--
Reaction mixtures containing (per
20 µl) 50 mM Tris-HCl (pH 7.5), 0.3 pmol 34-mer/18-mer
DNA, and 75 or 150 ng (2 or 4 pmol) of vaccinia topoisomerase were
incubated at 37 °C. Aliquots (20 µl) were withdrawn at the times
specified and quenched immediately with SDS (1% final concentration).
The products were analyzed by electrophoresis through a 10%
polyacrylamide gel containing 0.1% SDS. Free DNA migrated near the dye
front. Covalent complex formation was revealed by transfer of
radiolabeled DNA to the topoisomerase polypeptide. The extent of
covalent complex formation was quantified by scanning the dried gel
using a Fujifilm BAS-2500 PhosphorImager. A plot of the percentage of
input DNA cleaved versus time established the end point
values for cleavage. The data were then normalized to the end point
values (defined as 100%), and the cleavage rate constants
(kcl) were calculated by fitting the normalized
data to the equation 100
% cleavage(norm) = 100 e
kt. The values of kobs at the
saturating level of input topoisomerase (75 ng) and the actual end
point cleavage values are listed in Figs. 2 and 5.
Cleavage site-Specificity--
Reaction mixtures (20 µl)
containing 50 mM Tris-HCl (pH 7.5), 0.3 pmol of standard or
BPdG-modified 34-mer/18-mer DNA, and 75 ng of vaccinia topoisomerase
were incubated at 37 °C for either 30 s (standard,
2R,
2S, and +5R BPdG), 3 min (+5S and
1S BPdG), 4 h (
1R BPdG),
or 24 h (+4R and +4S BPdG). The reactions were quenched with 1%
SDS. Half of the sample was digested for 2 h at 37 °C with 10 µg of proteinase K, and the other half was not digested. The mixtures
were adjusted to 47% formamide, heat denatured, and analyzed by
electrophoreses through a 17% denaturing polyacrylamide gel containing
7 M urea in TBE (90 mM Tris borate, 2.5 mM EDTA). The reaction products were visualized by
autoradiographic exposure of the gel.
Exonuclease III Digestion--
5' 32P-labeled
BPdG-modified 18-mer oligonucleotides were hybridized to complementary
34-mer DNAs at a 1:4 molar ratio of labeled strand to 34-mer.
Exonuclease III reaction mixtures (60 µl) containing 66 mM Tris-HCl (pH 8.0). 0.66 mM
MgCl2, 1.8 pmol of 18-mer/34-mer, and 1.0 unit of
E. coli exonuclease III (New England Biolabs) were incubated at 22 °C. Aliquots (10 µl) were withdrawn at the times specified and quenched by adding EDTA to 30 mM final
concentration. The samples were adjusted to 47% formamide, heat
denatured, and analyzed by electrophoresis through a 17% denaturing
polyacrylamide gel containing 7 M urea in TBE. The reaction
products were visualized by autoradiographic exposure of the gel.
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RESULTS |
Stereospecific Minor Groove Interference at the Nucleoside Flanking
the Cleavage Site--
A pair of 18-mer strands containing a single
S or R BPdG adduct at position
1 of the
nonscissile strand were synthesized and then annealed to 5'
32P-labeled 34-mer scissile strands to form "suicide"
substrates for vaccinia topoisomerase (Fig. 2A).
Transesterification results in covalent attachment of a 5'
32P-labeled 12-mer (5'-pCGTGTCGCCCTTp) to the enzyme via
Tyr-274. The unlabeled 22-mer 5'-OH leaving strand dissociates
spontaneously from the protein-DNA complex. Loss of the leaving strand
drives the reaction toward the covalent state so that the reaction can be treated kinetically as a first-order unidirectional process (18-20). The reaction of excess topoisomerase with the unmodified control substrate attained an end point at which 87% of the DNA was
converted to covalent topoisomerase-DNA complex, and the reaction was
complete within 20 s. The extent of transesterification after 5 s was 85% of the end point value. From this datum, we
calculated a single-turnover cleavage rate constant
(kcl) of 0.4 s
1 (Fig. 2).
Introduction of a chiral S BPdG at position
1 had only a
modest (4-fold) effect on the cleavage rate constant
(kcl = 0.1 s
1) and no effect on
the reaction end point (Figs. 2A and
3.). In contrast, the R
diastereomer at position
1 reduced the rate of transesterification by
a factor of 200 (kcl = 0.002 s
1)
without influencing the end point (Figs. 2A and 3). The
kobs for cleavage of either of the
1
BPdG-modified substrates did not increase when the concentration of
topoisomerase in the reaction mixture was increased 2-fold (not shown).
This indicated that the slowed cleavage rate was not caused by a defect
in the initial binding of topoisomerase to the substrate. The results
define a stereospecific interference effect at the
1 nucleoside,
whereby the R diastereomer elicits 50-fold greater
inhibition of transesterification than the S diastereomer.
Note that the interfering
1R BPdG adduct is oriented toward the
scissile phosphodiester in the minor groove, whereas the
non-interfering
1S adduct is pointing away from the cleavage site
(Fig. 2A).

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Fig. 3.
Kinetic analysis of cleavage of 1
BPdG-modified substrates. Transesterification reaction mixtures
contained 0.3 pmol of the 1S or 1R BPdG substrates and 75 ng (2 pmol) of topoisomerase. The extents of covalent adduct formation are
plotted as a function of reaction time.
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To address whether the
1 BPdG substitutions altered the site of
cleavage within the 34-mer scissile strand, the reaction products were
digested with proteinase K in the presence of SDS to remove the
covalently linked topoisomerase. The radiolabeled DNA reaction products
were then analyzed by denaturing polyacrylamide gel electrophoresis
(Fig. 4). Reaction of topoisomerase with
the unmodified control substrate resulted in the appearance of a
cluster of radiolabeled species migrating faster than the input
32P-labeled 34-mer strand (Fig. 4, lane 2). The
cluster consists of the 12-mer 5'-pCGTGTCGCCCTTp linked to one or more
amino acids of the topoisomerase. Detection of the covalent
oligonucleotide-peptide complex was completely dependent on prior
digestion of the sample with proteinase K (not shown). This is because
the labeled DNA does not migrate into the polyacrylamide gel when it is
bound covalently to the topoisomerase polypeptide. The instructive
finding was that the same cluster was produced by proteinase K
digestion of the covalent complex formed by reaction of topoisomerase
with the substrates containing
1R or
1S BPdG adducts on the
nonscissile strand (Fig. 4, lanes 3 and 4). Thus,
the site of covalent complex formation was unchanged by the BPdG
modifications. Any shift in the cleavage site, and hence the size of
the covalently bound oligonucleotide, would have been readily detected
by an altered mobility of the array of labeled oligonucleotide-peptide
complexes.

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Fig. 4.
BPdG adducts do not affect the cleavage site
specificity of vaccinia topoisomerase. Cleavage reactions and
subsequent treatments of the reaction products were performed as
described under "Experimental Procedures." An autoradiogram of the
polyacrylamide gel is shown. The control substrates contained no
modifications ( ). Other substrates contained BPdG adducts in the
nonscissile strands at the positions specified. The positions of the
34-mer scissile strand and the cluster of covalent peptide-DNA
complexes are indicated on the right.
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BPdG Adducts at the
2 Nucleoside Do Not Interfere with
Transesterification--
We altered the sequence of the DNA strand 3'
of the CCCTT cleavage site so as to place a C:G base pair at the
2
position (Fig. 2B). Synthetic 18-mer strands containing a
single R or S BPdG adduct at the
2 nucleoside
of the nonscissile strand were annealed to the complementary 5'
32P-labeled 34-mer scissile strand to form a 34-mer/18-mer
suicide substrate. The reaction of topoisomerase with the unmodified
control substrate attained an end point of 89% covalent enzyme-DNA
formation with a cleavage rate constant of 0.25 s
1 (Fig.
2B). The
2S and
2R BPdG adducts had no interfering
effects on the rate or extent of transesterification. Indeed, the
cleavage of both
2 adducts was about twice as fast as the unmodified
DNA. (A rate constant of 0.5 s
1 is at the upper limit of
what we can measure by manual assay.) Analysis of the cleavage products
by PAGE after proteinase K digestion showed that the
2R and
2S BPdG
modifications did not alter the site of topoisomerase
transesterification to the scissile strand (Fig. 4, lanes 7 and 8). Taken together, the
1 and
2 BPdG interference experiments define the "downstream" margin of the minor groove interface between vaccinia topoisomerase and its target site, said
margin being between the +1 and
1 base pairs (Fig. 2).
Effects of BPdG Adducts within the 3'-GGGAA Sequence of the
Nonscissile Strand--
Previous studies suggested that N7 methylation
of the +5, +4, and +3 guanines in the major groove interfered with
noncovalent binding of vaccinia topoisomerase to its target site (8).
To explore the minor groove interface with the DNA on the covalently held side of the scissile phosphodiester, we introduced R
and S BPdG adducts at the +5, +4, and +3 nucleosides of the
3'-GGGAA sequence and measured the rate and extent of single turnover
transesterification by vaccinia topoisomerase on the BPdG-modified
substrates (Fig. 5). The unmodified
substrate was cleaved to an extent of 93% of the input-labeled DNA
with an apparent rate constant of 0.37 s
1. The +5R BPdG
modification had no significant effect on either the rate or extent of
transesterification (k+ 5R = 0.25 s
1; 87% end point cleavage). The +5S isomer had only a
modest (3-fold) slowing effect on the cleavage rate and little impact
on the end point (k+ 5S = 0.12 s
1; 80% yield) (Fig. 5). The electrophoretic mobility of
the cluster of proteinase K-digested reaction products formed with the
+5S and +5R BPdG substrates was unaltered compared with the cluster produced by cleavage of unmodified DNA (Fig. 4, lanes 13 and
14).

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Fig. 5.
Effects of BPdG adducts at positions +3, +4,
and +5 of the nonscissile strand on the rate and extent of DNA
transesterification by vaccinia topoisomerase. The full structure
of the 34-mer/18-mer CCCTT-containing substrate is shown at the bottom
of the figure. Detailed views of the CCCTT target site in the
unmodified control DNA and the BPdG-modified substrates are illustrated
with the numerical coordinates of the nucleotides indicated above the
unmodified scissile strand sequence. The S and R
diastereomers of the BPdG adducts are depicted as horizontal
bars in their respective orientations from the site of covalent
attachment to guanine on the nonscissile strand. The topoisomerase
cleavage rate constants and cleavage endpoints are indicated to the
right of each structure.
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Phasing the BPdG modifications 1 or 2 nucleotides closer to the
scissile phosphodiester dramatically reduced the rate and the extent of
the DNA cleavage reaction (Fig. 5). The reactions with the +4R, +4S,
+3R, and +3S BPdG-modified substrates occurred with similar rates of
approach to the reaction endpoints (kcl = 0.0002 s
1), which were attained when 18%, 6.3%, 7.4%, and
5.8% of the input DNA was transferred to the topoisomerase
polypeptide. Neither the rate nor the end point increased when the
concentration of topoisomerase was doubled, implying that the reaction
was not limited by the noncovalent binding step. Rather, we surmise
that the majority of the topoisomerase binding events are nonproductive with respect to transesterification and that there is not a free equilibrium between productive and nonproductive binding modes (at
least not within the 24-h time-frame in which the reactions were
monitored). Although the yields were low, the mobility of the
proteinase K-digested products formed on the +4R- and +4S-modified substrates was typical of the pattern seen with the unmodified DNA
(Fig. 4, lanes 11 and 12). We suspect that the
presence of one additional labeled species migrating above the main
cluster of DNA-peptide complexes reflects incomplete proteolysis. (Were there a shift in the cleavage site, we would expect to see phasing of
the entire cluster rather than only one additional species.)
The >1000-fold rate decrement elicited by the +4 and +3 BPdG adducts
illustrates the strong requirement for access to the DNA minor groove
within the segment spanning the +4C:G, +3C:G, and +2T:A base pairs.
Comparison to the benign effects of the +5 BPdG modifications
delineates an upstream margin for the minor groove interface between
the +5C:G and +4C:G base pairs (Fig. 5 and "Discussion" below).
Effects of BPdG Adducts on Exonuclease III--
E. coli
exonuclease III catalyzes unidirectional digestion of duplex DNA from
the 3' end to liberate 5' dNMP products. The phosphodiesterase activity
of exonuclease III is impeded by various chemical modifications of the
phosphate backbone (21, 23, 24). Here we explored the effects of BPdG
adducts in the minor groove on exonuclease III. 5'
32P-labeled 18-mers containing the unmodified or
BPdG-modified complement of the topoisomerase cleavage site were
annealed to an unlabeled complementary 34-mer strand, and the duplexes
were incubated with exonuclease III. The 5'-labeled digestion products
were resolved by denaturing gel electrophoresis (Fig.
6). All of the unmodified control 18-mer
was converted after 10-25 min to 5'-labeled species 6-7 nucleotides
in length. A ladder of partially digested strands was evident at 2 min.
Strands containing 3' dCMP termini were underrepresented in the ladder
in keeping with the intrinsically faster rate of hydrolysis of dCMP by
exonuclease III (25). Initial digestion of the +5, +4, and +3
BPdG-modified DNAs by exonuclease III was unaffected as gauged by the
rate of decay of the full-length 18-mer strand (Fig. 6). Note that the
electrophoretic mobility of the BPdG strands is retarded compared with
the unmodified control strand and that the R diastereomer
migrates more slowly than the S diastereomer in each case.
Electrophoretic mobility differences for DNAs containing R
and S BPdG diastereomers have been reported previously (38).
These differences notwithstanding, we were able to assign the 3' ends
of each of the digestion products using the partial digest ladder of
each substrate seen at the 2-min time point.

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Fig. 6.
Exonuclease III digestion of BPdG-modified
DNAs. 5' 32P-labeled control and BPdG-containing
18-mer oligonucleotides were annealed to a 34-mer to form the tailed
duplex molecule shown below the autoradiogram. The DNAs were digested
with exonuclease III for 0, 2, 10, or 25 min, and the products were
analyzed by PAGE. The nucleotide sequences of the 18-mers are displayed
next to the cleavage ladders with each letter specifying the 3'
nucleotide of the indicated radiolabeled species. Black
diamonds mark the sites of BPdG modification.
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Placement of R and S BPdG adducts at position +3
resulted in a strong kinetic roadblock to exonuclease III digestion
located 2-4 nucleotides 3' of the site of the modification that was
apparent at 2 min (the adduct sites are indicated by
in Fig. 6).
The enzyme progressed to within 2-3 nucleotides of the adduct site by
25 min and no further. A similar kinetic pattern of arrest was seen
with the +4 and +5 BPdG adducts, except that the arrest sites were
phased by 1 nucleotide (for +4 BPdG) and 2 nucleotides (for +5
BPdG) in the 3' direction. In each case, exonuclease III was blocked
within 2 min at positions 2-4 nucleotides 3' of the modified base and
hydrolysis could not proceed beyond the position 2 nucleotides
3' of the adduct (Fig. 6). We discerned no significant stereoisomer
effects on the digestion ladder at positions +3 or +4. In the case of
the +5R BPdG-modified substrate, exonuclease III was sharply arrested 3 nucleotides prior to the lesion, whereas about half of the +5S isomer
was digested to within 2 nucleotides of the lesion (Fig. 6). These
results underscore the notion that exonuclease III surveys the minor
groove ahead of the active site of catalysis and that space-filling
lesions in the minor groove strongly impede its progress.
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DISCUSSION |
The Topoisomerase Minor Groove Interface--
By introducing
chiral BPdG adducts at five positions of the nonscissile strand, we
have defined the dimensions of the functional interface of vaccinia
topoisomerase with the DNA minor groove. We observe discrete margins
for interference effects on upstream (+) and downstream (
) of the
scissile phosphodiester, whereby +5 and
2 BPdG modifications have
little effect on the rate or extent of transesterification, while +4,
+3, and
1 BPdG adducts interfere strongly. The simple interpretation
of the data is that BPdG inhibition of transesterification is a
consequence of steric exclusion of constituents of the enzyme from the
DNA minor groove. This view is consistent with NMR data showing that
single R and S BPdG adducts do not perturb base
pairing or B-form helical conformation in a model synthetic
oligonucleotide (14, 33, 34). Alternatively, the BPdG adducts in the
minor groove may interfere with a topoisomerase-induced conformational
change of the DNA (entailing unpairing of the +1T base) that precedes
the transesterification reaction (26, 27).
The stereoselective effects at the
1 nucleoside (the R
diastereomer interferes, whereas the S diastereomer does
not) delineate at reasonably high resolution the downstream border of
the minor groove interface. Fig. 7 uses
the NMR structures (14, 33, 34) to model the
1S and
1R
BPdG-modified duplexes; the position of the scissile phosphodiester
(+1) on the CCCTT strand is indicated by the arrow. Note
that the non-interfering S diastereomer has the aromatic
pyrenyl moiety pointing away from the scissile phosphate, whereas the
strongly interfering R diastereomer has the pyrenyl component pointing toward the +1 phosphate. It is evident that the
50-fold greater interference by the R diastereomer
correlates with occlusion of the minor groove over the +1T:A base pair
and part of the +2T:A base pair, said surface being exposed in the model of the S diastereomer. The critical area is
highlighted by the blue ellipse in Fig. 7. One can similarly
model the
2BPdG substrates on the NMR structure by shifting the
scissile phosphate by one position (downward in the view shown in Fig.
7). This maneuver indicates that neither the
2R or
2S BPdG adducts
overlap the critical interfering surface, thereby explaining why the
2BPdG adducts do not inhibit the transesterification reaction.

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Fig. 7.
Downstream margin of the topoisomerase
minor groove interface. Models of B-DNA containing 1R and 1S
BPdG adducts on the nonscissile strand are shown with the site of
transesterification of topoisomerase to the scissile strand (+1)
indicated by the yellow arrows. The observed rate
constants for topoisomerase cleavage of the respective diastereomers
are shown below the structures.
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BPdG adducts at the +4 and +3 guanines of the 3'-GGGAA recognition
sites decreased the rate and the extent of transesterification by
vaccinia topoisomerase. The decreased yield of product was not
corrected by increasing enzyme concentration, implying that initial
binding was not rate-limiting but was predominantly
nonproductive. The sequence specificity of vaccinia topoisomerase is
dictated principally at the level of transesterification chemistry
rather than noncovalent binding. The enzyme cleaves exclusively at
(T/C)CCTT or related pentapyrimidine sites, but the noncovalent binding affinity for CCCTT-containing DNA is only 8-fold higher than its affinity for non-CCCTT DNA under the standard reaction conditions (28).
The transition from nonproductive to productive binding entails the
assembly of a catalytically competent active site (which is not
preformed in the free enzyme) in which the essential functional groups
(Arg-130, Lys-167, Arg-223, His-265, and Tyr-274) are oriented properly
at the scissile phosphodiester (10, 12, 13). The decreased cleavage
seen with the +4 and +3 BPdG adducts may reflect their inhibition of
this conformational transition, which could entail penetration of the
enzyme into the minor groove within the CCCTT element.
A notable finding was that the +5S BPdG adduct has only a 3-fold effect
on the cleavage rate constant and little impact on the end point,
whereas the +4R BPdG modification strongly suppressed the rate and
extent of the reaction (Fig. 5). This disparity is surprising at first
given that the +5S adduct (which points toward the scissile phosphate)
and the +4R adduct (which points away from the scissile phosphate) are
predicted to overlap substantially in the minor groove. We speculate
that the functional differential between +4R and +5S BPdG modifications
arises because the three nonplanar hydroxyl substituents of the
cyclohexene ring of the +4R BPdG adduct occlude the topoisomerase
interface, whereas the edge of the planar pyrenyl ring of +5S BPdG does
not penetrate into the enzyme's interaction zone in the minor groove.
Our results fix a sharp (and structurally subtle) upstream margin of
the minor groove interface of vaccinia topoisomerase near the +4C:G
base pair.
Minor Groove Interactions of Exonuclease III--
We also applied
the BPdG interference method to probe the interactions of exonuclease
III with the DNA minor groove. Exonuclease III employs a one-step
in-line mechanism in which an activated water attacks the scissile
phosphodiester (which is coordinated to an essential divalent cation)
and expels the 3'-O of the upstream nucleotide of the DNA
strand (29). Exonuclease III is clearly impeded from hydrolyzing the
two phosphodiester linkages immediately 3' of a BPdG adduct; indeed it
displays feeble activity toward the phosphodiesters located 3 or 4 nucleotides 3' of the BPdG nucleoside. We presume that the occupancy of
the minor groove precludes the approach of the active site to these
protected phosphodiesters because the enzyme itself penetrates the
minor groove when it binds to the 3' terminal dNMP of the substrate.
Exonuclease III is structurally related to DNase I and to the abasic
nuclease HAP1 (29, 30). DNase I binds to DNA in the minor groove (31),
and models of exonuclease III and HAP1 DNA interactions also invoke
minor groove contacts, including contacts of a conserved loop with
nucleotides 5' of the scissile phosphodiester (30). We showed
previously that a 2'-5' phosphodiester modification arrested
exonuclease III at positions 1 and 2 nucleotides prior to the encounter
of its active site with the 2'-5' linkage, which was itself poorly
hydrolyzed (21). Taken together, the 2'-5'-phosphate and BPdG
interference experiments indicate that exonuclease III surveys the
phosphate backbone and the minor groove advance of the active site.
Although the S and R diastereomers of trans BPdG
are oppositely oriented in the minor groove of duplex DNA, we did not
observe consistent differences in the sites or duration of the blocks to exonuclease III digestion by the S versus
R adducts. This contrasts with the stereoselective
impediments to exonucleolytic digestion of a BPdG-modified
single-stranded DNA oligonucleotide by snake venom and
spleen phosphodiesterases (32). Snake venom phosphodiesterase, which
has the same 3' to 5' directionality as exonuclease III, is arrested
either at the adducted guanine nucleotide (R diastereomer) or else 1 nucleotide 3' of the adduct (S diastereomer).
Thus, exonuclease III senses the BPdG adduct at a greater distance than does snake venom phosphodiesterase.