From the Departments of Chemistry and Biology, University of Virginia, Charlottesville, Virginia 22901
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ABSTRACT |
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The DNA cleavage-ligation reaction of DNA
topoisomerase I was investigated employing synthetic DNA substrates
containing 3'-deoxyadenosine or 3'-deoxythymidine at specific sites and
acceptor oligonucleotides of different lengths. The modified
nucleotides were substituted systematically within the putative
enzyme-binding domain and also next to the high efficiency cleavage
site to determine the effect of single base changes on enzyme function.
Depending on the site of substitution, the facility of the cleavage and
ligation reactions were altered. The bases at positions 1 and
2 on
the noncleaved strand were found to be important for determining the
site of cleavage. Inclusion of 3'-deoxythymidine in the scissile strand at position
1 permitted the demonstration that topoisomerase I can
cleave and form a 2'
5'-phosphodiester linkage. Partial duplexes doubly modified at positions
4 or
6 in the noncleaved strand and at positions +1 or
1 within scissile strand were not good
substrates for topoisomerase I, showing that cleavage can depend
importantly on binding interactions based on structural alterations at
spatially separated sites. Substitution of a 3'-deoxynucleotide on the
scissile strand at position
6 enhanced formation of the ligation
product resulting from cleavage at site 1 and suppressed cleavage at
site 2.
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INTRODUCTION |
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The DNA topoisomerases alter DNA topology via the introduction of transient breaks in the phosphodiester backbone of this biopolymer (1, 2). These enzymes participate in the essential cellular processes of DNA replication, transcription, and recombination (3). DNA topoisomerases are classified into two groups based on their mode of DNA strand scission: the type I enzymes mediate the transient single-strand breakage of a DNA substrate, whereas the type II topoisomerases break both strands. Because of their essential role in the cell, the eukaryotic topoisomerases have become important targets for the development of antitumor agents (4).
The mechanism of DNA cleavage involves nucleophilic attack of a tyrosine OH group in the active site of topoisomerase I on the phosphate ester backbone, resulting in the attachment of tyrosine to the DNA through an oligonucleotide 3'-phosphate with concomitant release of an oligonucleotide having a free 5'-OH terminus (5). After strand passage of the free DNA strand around the unbroken strand, religation of the broken strand occurs by a process believed to involve reversal of the cleavage reaction. Under normal circumstances the cleavage and ligation reactions are tightly coupled with low steady-state concentrations of the covalent intermediate (6). However, the two partial reactions can be uncoupled in vitro, e.g. by site-specific cleavage of partially double-stranded substrates containing a high efficiency cleavage site (see Fig. 1) (7). Cleavage of the "suicide substrate" occurs without sequential religation due to the instability of the duplex involving the truncated strand downstream from the site of cleavage; loss of this oligonucleotide traps the topoisomerase I-DNA covalent intermediate. The covalently bound enzyme is catalytically competent, as may be judged by formation of the intact duplex upon admixture of an oligonucleotide complementary to the single-stranded region in the enzyme-DNA covalent binary complex (see Fig. 1B). The covalent binary complex also undergoes ligation with partially complementary acceptors containing a free 5'-OH group (8).
The topoisomerases I from all species characterized thus far recognize specific nucleotide sequences in their DNA substrates, which are cleaved with high efficiency (see Fig. 1) (9, 10). Such sites can be identified by footprinting analysis (11). Although the cleavage-ligation reactions have been studied in some detail, further investigation is needed to define the possible roles of single nucleotides within high efficiency sequences on enzyme binding, as well as cleavage and ligation (12). Different approaches have been utilized to determine the role of individual nucleotides, including phosphate ethylation (13) and deoxyguanosine N7-methylation and subsequent depurination to create abasic sites (11). Abasic sites within the scissile strand on the 5'-side of the high efficiency cleavage site rendered the DNA substrate refractory to cleavage by topoisomerase I (11). In addition, Pourquier et al. (14) have incorporated deoxyuridine and abasic sites into the noncleaved strand of a DNA substrate and investigated the effect on topoisomerase I-mediated cleavage. They found that the position of cleavage was altered and that the new site of cleavage was dependent on the location of the site modified.
We have reported previously that topoisomerase I can catalyze the
ligation to the covalent topoisomerase I-DNA binary complex of
complementary acceptor oligonucleotides having modified nucleophiles at
the 5' terminus; the resulting duplexes have altered connectivity at
the site of ligation (15). Recently, we have extended these findings by
employing a modified acceptor oligonucleotide having the 5'-terminal
deoxyadenosine linked to the remainder of the oligomer through a 2 5'-phosphodiester bond (see Fig. 1C). When employed in the presence of
a partial DNA duplex that afforded a single-stranded region
complementary to the acceptor upon cleavage by topoisomerase I, the
modified oligonucleotide was incorporated into a newly formed duplex
(16). The extent of (modified) duplex formation was only ~10% of
that achieved with the respective unmodified acceptor oligonucleotide
after 1 h, but the yield of the modified duplex could be increased
to 20% after a 13-h incubation (16). In addition, modified partial
duplexes were utilized as substrates; each contained a single
3'-deoxyadenosine moiety within the DNA topoisomerase I-binding region
on the scissile strand. The facility of cleavage or ligation was
altered in a fashion dependent on the location of the 3'-deoxyadenosine
moiety.
To further characterize the role of single nucleotides within the
recognition domain on substrate recognition and cleavage by
topoisomerase I, additional partial duplex substrates containing modifications in the scissile and noncleaved strand have been prepared.
Presently we demonstrate that (i) substitutions on the scissile or
noncleaved strands can affect the topoisomerase I-mediated cleavage and
ligation reactions, (ii) certain combinations of modified nucleotides
within the substrate can substantially enhance overall cleavage and
ligation efficiency, (iii) topoisomerase I can effect the cleavage and
ligation of a 2' 5'-phosphate ester linkage within the DNA
substrate, and (iv) alterations in efficiency of enzyme action caused
by the introduction of a 3'-deoxynucleotide at a specific site can
sometimes be compensated by introduction of the complementary
3'-deoxynucleotide at the same site on the opposite strand.
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EXPERIMENTAL PROCEDURES |
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T4 polynucleotide kinase and proteinase K were purchased from
U. S. Biochem Corp.; exonuclease III was from Life Technologies, Inc.
-Cyanoethyl phosphoramidites, activator solution, and the solid
support for oligonucleotide synthesis were obtained from Cruachem Inc.
Nensorb prep nucleic acid purification cartridges were from NEN Life
Science Products, and [
-32P]ATP (7000 Ci/mmol) was
obtained from ICN Pharmaceuticals. Scintillation counting was performed
on a Beckman LS-100C instrument using Beckman Ready Safe scintillation
fluid.
Gel electrophoresis was carried out on 20% polyacrylamide gels (19%
(w/v) acrylamide, 1% (w/v)
N,N-methylenebisacrylamide, 8 M urea)
in 90 mM Tris borate buffer, pH 8.3, containing 1 mM EDTA. Polyacrylamide gel loading solution included 10 M
urea, 1.5 mM EDTA, 0.05% (w/v) xylene cyanol, 0.05%
(w/v) bromphenol blue. Gels were visualized by autoradiography at
80 °C with Kodak XAR-2 film and quantified utilizing a Molecular
Dynamics 400E PhosphorImager equipped with ImageQuant version 3.2 software. DNA sequence analysis was performed by modification of the
traditional Maxam-Gilbert method (17) for short, single-stranded
deoxyoligonucleotides. Distilled, deionized water from a Milli-Q system
was used for all aqueous manipulations.
Oligonucleotide Substrates--
Synthetic oligonucleotides were
purchased from Cruachem Inc. or synthesized on a Biosearch 8600 series
DNA synthesizer using standard phosphoramidite chemistry (18). The
oligonucleotides synthesized on the Biosearch DNA synthesizer were
deblocked and cleaved from the solid support by treatment with
concentrated NH4OH at 55 °C for 12 h. The oligomers
were purified by Nensorb chromatography. The Nensorb cartridge was
activated with 10 ml of methanol and then pre-equilibrated with 5 ml of
0.1 M TEAA1
buffer, pH 7.0. Deblocked oligonucleotides dissolved in 4 ml of 0.1 M TEAA, pH 7.0, were pipetted onto the resin. The resin was
then washed with 10 ml of 1:9 acetronitrile, 0.1 M TEAA, pH 7.0. The oligonucleotides were detritylated with 25 ml of 0.5% trifluoroacetic acid and washed with 10 ml of 0.1 M TEAA,
pH 7.0. Elution of the deblocked oligonucleotides was accomplished with 5 ml of water containing 35% methanol (v/v). All oligonucleotides were
purified on preparative 20% denaturing polyacrylamide gels; the DNA
was recovered by crush and soak and then by precipitation. The DNA was
5'-32P end-labeled with T4 polynucleotide kinase + [-32P]ATP (19).
Hybridization of Substrates-- Oligonucleotides were hybridized in 100 µl (total volume) of 10 mM Tris-HCl, pH 7.5, containing 40 mM NaCl, 5 mM MgCl2 and 5 mM CaCl2. The solution was heated to 80 °C for 5 min and then cooled slowly to room temperature under ambient conditions over a period of about 3 h. Due to the low DNA oligonucleotide concentrations, hybridization mixtures contained 0.13 pmol of the labeled strand and a 100-fold excess of the unlabeled strands to ensure complete hybridization of the labeled DNA.
Topoisomerase I-mediated Reaction-- The 5'-32P end-labeled partial duplex (6.5 fmol, 100,000 dpm) was incubated with calf thymus topoisomerase I (20) (8.8 ng) in the presence of a 1000-fold excess of (17- or 19-mer) acceptor oligonucleotide in a reaction mixture (total volume, 20 µl) containing 20 mM Tris-HCl, pH 7.5, 40 mM NaCl, 0.5 mM EDTA, 5 mM MgCl2, 5 mM CaCl2, and 1 mM dithiothreitol. The reaction mixtures were incubated at 37 °C for 60 min followed by proteolysis with 1 mg/ml proteinase K (37 °C for 1 h). The reaction mixtures were dissolved in 12 µl of loading solution (10 M urea, 1.5 mM EDTA, 0.05% bromphenol blue, and 0.05% xylene cyanol). The reaction mixtures were heat-denatured at 90 °C for 5 min and quick chilled on ice; 6.5-µl aliquots were applied to a 20% denaturing polyacrylamide gel (19% (w/v) acrylamide, 1% (w/v) N-methylenebisacrylamide, 8 M urea) and run at 50 W for 2-3 h in 90 mM Tris borate, pH 8.3, containing 1 mM EDTA.
Exonuclease III Digestion-- Reaction mixtures consisted of 10 µl (total volume) of 50 mM Tris-HCl, pH 8.0, containing 5 mM MgCl2, 1 mM dithiothreitol, 40,000 dpm of 5'-32P end-labeled duplex DNA and 2 µg of calf thymus DNA. The reactions were initiated by the addition of 66 units of exonuclease III. After 30 min, the reactions were terminated by the addition of 2 µl of 200 mM EDTA and 8 µl of loading solution. The reactions were heat-denatured at 90 °C for 5 min and quick chilled on ice; 5 µl was applied to a 20% denaturing polyacrylamide gel. The mobilities of the degradation products were compared with the products resulting from Maxam-Gilbert G and A+G base-specific reactions (17).
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RESULTS |
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Oligonucleotide Synthesis-- The 3'-deoxynucleotide monomers were prepared according to published procedures (16, 21) and incorporated into the DNA substrates within the topoisomerase I-binding region on the scissile and noncleaved strands using standard protocols for solid phase phosphoramidite chemistry (18). Cleavage from the solid support and removal of the base and phosphate protecting groups was affected as described under "Experimental Procedures." All oligonucleotides were purified first by Nensorb column chromatography and then by preparative 20% denaturing polyacrylamide gel electrophoresis. The formation of modified duplexes was verified by exonuclease III digestion, which accurately reflected the position of the modified nucleotide in each of the partial duplexes (15) (data not shown).
Effect of Modifications within the Noncleaved Strand--
The
topoisomerase I-mediated cleavage and ligation reactions were
investigated using partial duplexes containing high efficiency topoisomerase I cleavage sequences (22) and a single 3'-deoxnucleotide within the noncleaved strand at various positions. The 17- and 19-nucleotide acceptor oligonucleotides were fully complementary to
those regions of the duplex that became single-stranded upon cleavage
at sites 1 and 2, respectively (Fig. 1).
We first investigated the topoisomerase I-mediated cleavage-ligation
reaction using partial duplexes singly modified within the noncleaved
strand at positions 1,
2,
4, or
6. The 5'-32P
end-labeled partial duplexes containing these modifications were
treated with calf thymus DNA topoisomerase I in the presence of the
17-mer acceptor oligonucleotide at 37 °C for 60 min. The reaction
mixtures were quenched by treatment with 1% SDS and proteolyzed with
proteinase K to digest the covalently bound enzyme. The reactions were
analyzed on a 20% denaturing polyacrylamide gel. As shown in Fig.
2A, the topoisomerase
I-mediated cleavage and ligation in the presence the 17-mer acceptor
oligomer afforded ligated products when the noncleaved strand was
modified at positions
4 or
6. Modifications at position
1 or
2
in the noncleaved strand diminished ligation with the 17-mer acceptor,
although the presence of some cleavage products was apparent. To
determine whether the modifications at positions
1 and
2 in the
noncleaved strand created a duplex that was a poor substrate for the
enzyme or simply created new preferred cleavage sites, the same set of reactions under the same conditions was carried out in the presence of
the 19-mer acceptor. As shown in Fig. 2B, the topoisomerase I-mediated cleavage-ligation reaction with the unmodified partial duplex using a 19-mer acceptor afforded only a very limited amount of
ligation product (cf. lanes 3 and 6), in good agreement with a previous study (23). In contrast, incubation of topoisomerase I with
partial duplexes containing a modified nucleoside at position
1 or
2 gave good yields of ligation product (Fig. 2B,
lanes 9 and 12) in the presence of the 19-mer
acceptors. The use of 19-mer acceptors with partial duplexes containing
a modification at position
4 or
6 in the noncleaved strand afforded
little ligation product (Fig. 2B, lanes 15 and
18). The results indicate clearly that the location of the
modified bases within the noncleaved strand can be important
determinants of the site of cleavage. As shown in Table
I, which indicates the relative
efficiency and site selectivity of cleavage in the absence of any
acceptor oligonucleotide, the site selectivity of cleavage paralleled
the position at which ligation ultimately obtained preferentially (cf.
Table I and Fig. 2). Interestingly, the overall efficiency of cleavage
was lower for substitutions of deoxynucleotides at positions
4 and
6 than for substitutions at positions
1 and
2.
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Effect of Modification of the Scissile Strand at Position
+1--
Following the experiments involving partial duplexes singly
modified at sites on the noncleaved strand, we investigated the topoisomerase I-mediated cleavage reaction using doubly modified partial duplexes. The same set of reactions that had been used for the
partial duplexes containing an unmodified scissile strand was carried
out with partial duplexes containing 3'-deoxyadenosine at position +1.
As shown in Fig. 3A,
modification at position +1 completely eliminated ligation of the
17-mer acceptor. Inspection of Fig. 3A suggests that this
modification diminished cleavage at site 1 and resulted in an increase
in cleavage at site 2 (Fig. 3A, lanes 5 and
6). The same set of experiments was then carried out with
the 19-mer acceptor. The partial duplex having a single modification at
the position +1 in the scissile strand and an unmodified noncleaved
strand afforded enhanced ligation involving site 2 cleavage (Fig.
3B, lanes 5 and 6). The enzymatic reactions involving partial duplexes containing modifications both at
position +1 in the scissile strand and positions 1 or
2 in the
noncleaved strand gave increased site 2 cleavage as compared with the
+1 substitution on the scissile strand alone (cf. lanes 5,
8, and 11). In these cases the enzyme can
efficiently catalyze the ligation reactions to afford full-length
products (Fig. 3B, lanes 9 and 12). However, the
substitutions at positions
4 or
6 in the noncleaved strand in
addition to the +1 substitution on the scissile strand resulted in
minimal ligation at best in the presence of the 17- or 19-mer acceptors
(Fig. 3, A and B, lanes 15 and
18).
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Effect of Modification of the Scissile Strand at Position
1--
Nucleotides in the scissile strand were then systematically
substituted to determine which were particularly important for recognition and cleavage by topoisomerase I. In addition to assessing the structural effects of a modification at position
1, we were particularly interested to see whether topoisomerase I would cleave the
DNA backbone at the unnatural 2'
5' linkage present in these substrates. As shown in Fig.
4A (lane 6),
topoisomerase I can cleave and ligate the unnatural 2'
5'
phosphodiester linkage at the topoisomerase I high efficiency cleavage
site in the modified partial duplex. The extent of ligation obtained
with this singly modified partial duplex was 10% of that obtained for
the unmodified partial duplex, as determined by phosphorimager
analysis. The partial duplexes containing a modification at position
1 in the scissile strand in addition to a modification at position
4 or
6 in the noncleaved strand were also substrates for the
cleavage-ligation reaction, although the enzyme catalyzed the
cleavage-ligation reaction to a lesser extent (~5%) relative to
unmodified partial duplex (Fig. 4A, lanes 15 and
18, and Fig. 5). The partial
duplex having a single modification at position
1 in the scissile
strand was cleaved and ligated with greater facility at site 2, as
verified by enzymatic reactions in the presence of the 19-mer acceptor (Fig. 4B, lane 6). Additional modification at
position
1 or
2 on the noncleaved strand along with the
modification at position
1 on the scissile strand had little further
effect on the extent of cleavage and ligation at site 2 (Fig.
4B, cf. lanes 6, 9, and 12). As shown previously for the modification at position +1
(Fig. 3B), regardless of the acceptor employed, there was
minimal ligation product formed when the substrate was modified at
position
4 or
6 on the noncleaved strand in addition to the
substitution at position
1 on the scissile strand (Fig. 4,
A and B, lanes 15 and
18).
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Effect of Modification of the Scissile Strand at Position
2--
Modified oligonucleotides were used to investigate the effect
on topoisomerase I-mediated cleavage and ligation of incorporating 3'-deoxynucleotides at position
2 of the scissile strand.
Interestingly, modification at position
2 did not significantly
affect substrate cleavage and ligation by topoisomerase I (Fig.
6A). Only an additional modification at position
1 on the noncleaved strand altered the cleavage site of the enzyme from site 1 to 2 (Fig. 6, A and
B, cf. lanes 9). This double modification
resulted in exclusive cleavage at site 2 as indicated by a ligated
product in presence of the 19-mer acceptor (Fig. 6B,
lane 9), whereas all other modifications on the noncleaved
strand afforded no ligated product with the 19-mer acceptor.
Interestingly, in this case modification at position
4 or
6 within
the noncleaved strand actually enhanced the extent of cleavage and
ligation at site 1 (Fig. 6A, lanes 15 and
18).
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Effect of Modification of the Scissile Strand at Position
4--
As described previously, modification at position
4 of the
scissile strand resulted in decreased ligation of the 17-mer acceptor oligonucleotide by shifting the substrate cleavage site (Fig. 7).
Inclusion of 3'-deoxyadenosine at position
4 in the scissile strand
in addition to modification at positions
1 or
2 in the noncleaved
strand altered cleavage from site 1 to site 2 essentially completely
(Fig. 7). Interestingly, when there was a "compensatory" modification at
4 on the noncleaved strand, the product of cleavage and ligation at site 1 was restored, consistent with possible stabilization of the duplex, which could enable effective ligation by
topoisomerase I. Modification at
6 on the noncleaved strand disrupted
ligation by the enzyme with both the 17- and 19-mer. Although
topoisomerase I is clearly cleaving the substrate at site 2 in the
presence of the position
6 modification on the noncleaved strand,
this modification presumably alters binding of the enzyme, thereby
affecting ligation relative to the normal duplex.
Effect of Modification of the Scissile Strand at Position
6--
The substitution at position
6 resulted in cleavage largely
at site 1 and concomitant enhancement of ligated product formation (Fig. 8). Interestingly, modifications at positions
1 and
2 on the
noncleaved strand that previously altered cleavage by topoisomerase I
to site 2 when present as the sole modification (16) or in combination
with the presence of 3'-deoxyadenosine at position
4 on the scissile
strand (Fig. 7), afforded cleavage
predominantly at site 1 when combined with modification at position
6
on the scissile strand (Fig. 8).
Additional modifications on the noncleaved strand at positions
4 or
6 resulted in cleavage virtually exclusively at site 1 (Fig. 8). The
substrate having modifications on both strands at position
6 gave
enhanced ligation at site 1 relative to the unmodified substrate (Fig.
8). In the presence of a 19-mer acceptor, the modification at position
2, combined with modification at position
6 on the scissile strand,
exhibited the greatest amount of ligation due to partial cleavage at
site 2.
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DISCUSSION |
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Eukaryotic DNA topoisomerase I participates in the control of DNA topology. The enzyme can relax supercoiled DNA by transiently cleaving one strand of the DNA substrate and then passing and religating the cleaved strand via a 3'-linked DNA-enzyme covalent intermediate containing a phosphorotyrosine linkage (15, 24). Previously, we reported that DNA topoisomerase I can promote the rearrangement of DNA structure; nucleotide insertions and deletions were noted in addition to alteration of the DNA backbone (15, 23). The enzyme trapped using "suicide substrates" can undergo ligation with exogenous DNA acceptors, affording structural transformations of the DNA (25, 26).
The minimum DNA duplex region required for topoisomerase I-mediated
reaction in vitro was determined as an optimized sequence containing nine nucleotides on the scissile strand and five nucleotides on the noncleaved strand (9) (cf. Fig. 1A). This sequence is cleaved with the same specificity by eukaryotic topoisomerase I from
various organisms (26) and functions as a high efficiency site for DNA
relaxation (27). To determine which nucleotides contribute to the
sequence specificity and facility of catalysis by the eukaryotic
enzyme, previous studies have utilized DNA containing methylated
nucleotides, abasic sites, and uracil substitutions. Abasic sites at
positions 2 to
7 on the scissile strand rendered the substrate
refractory to cleavage by topoisomerase I. An abasic site at position
1 had little effect on enzyme function (11). Recently, it was
reported that an abasic site on the noncleaved strand at positions
1
to
4 suppressed DNA cleavage at the normal site and created new
cleavage sites. An abasic site at position
6 on the noncleaved strand
increased the extent of cleavage (14). Neither study involved a
substrate having two modifications within the topoisomerase I-binding
region, although double modifications could potentially facilitate a
better understanding of the role of DNA structure on topoisomerase
I-mediated cleavage, relaxation, and ligation.
Two abasic sites would be expected to destabilize the DNA duplex dramatically (28, 29). In contrast with the effects of abasic sites on DNA stability, it has been reported that the insertion of two 3'-deoxyadenosines within a 16-nucleotide duplex reduced the Tm by only 11-12 °C, depending on the sites of substitution (30, 31). In an earlier study, we utilized as substrates a few modified partial duplexes, each of which contained a single 3'-deoxyadenosine within the topoisomerase I-binding region on the scissile strand. Depending on the location of the 3'-deoxyadenosine substitution, the facility of the topoisomerase I-mediated cleavage or ligation reaction was altered (16). Presently, we have modified the partial duplex systematically to assess the importance of specific nucleotides by incorporating their 3'-deoxynucleotide counterparts. Depending on the site of substitution, this particular modification can significantly modulate the effects of the enzyme by altering cleavage site or facility of cleavage or ligation, presumably reflecting alterations in substrate binding by the enzyme.
Initially, 3'-deoxynucleotides were incorporated into the noncleaved
strand to observe the effect on cleavage and ligation. These data (Fig.
2) indicate that modifications at positions 1 and
2 alter the
cleavage site, because ligation was observed in presence of the 19-mer
acceptor but not in presence the 17-mer acceptor. The same trend was
observed in the absence of any acceptor (Table I). It could be argued
that these modifications affect accessibility of the enzyme to cleavage
site 1 rather than the affinity of the enzyme for this substrate
per se, given the increase in cleavage at site 2. Introduction of 3'-deoxynucleotides into positions
4 and
6 had a
lesser effect on cleavage and ligation at site 1; little change in the
amount of ligation was noted for the substrate modified at positions
4 or
6. This was true despite the fact that the extent of cleavage
for these modified partial duplexes in the absence of acceptor
oligonucleotides was only a small fraction of that observed for the
unmodified partial duplex. This clearly indicates that the acceptors
can facilitate the overall process of cleavage and ligation. It is
interesting that the formation of ligation products from the substrates
modified at positions
4 and
6 resulted essentially exclusively from
cleavage at site 1. Suppression of cleavage at site 2 might be thought
to be logical because, for example, position
4 on the noncleaved
strand bears the same relationship to site 2 that position
2 does to
site 1 (cf. Figs. 2 and 6).
Alternatively, 3'-deoxynucleotides were incorporated within the
scissile strand. Beginning with a modification at position +1,
3'-deoxynucleotides were also introduced systematically at positions
1,
2,
4, and
6 within the scissile strand (Fig. 3). These
modified oligonucleotides were then utilized in coupled cleavage-ligation reactions. Subsequently, the modified scissile strands were combined with modified noncleaved strands to obtain more
information about topoisomerase I function.
3'-Deoxynucleotide incorporation at position +1 in the scissile strand
significantly altered substrate cleavage from site 1 to site 2, contrary to the enhancement of cleavage demonstrated when uracil and
abasic sites were incorporated at these positions (14). The results
were also contrary to experiments in which depurination of +1 caused
accumulation of cleaved intermediates (11). In the presence of the
19-mer acceptor, topoisomerase I cleaved the partial duplex
predominantly at site 2; the intermediate underwent ligation to form
the 30-mer product. Further modifications at positions 1 or
2 had
no effect on ligation when the 19-mer acceptor was present, whereas
further modification at position
4 or
6 on the noncleaved strand
decreased ligation even in the presence of the 19-mer acceptor (Fig.
3).
The substitution of a 3'-deoxynucleotide at position 1 was of special
interest because it permitted investigation of the ability of the
enzyme to cleave the DNA backbone at the site of an unnatural linkage.
Topoisomerase I did effect cleavage at site 1 in this substrate (Fig.
4), although most of the cleavage was redirected to site 2. The
intermediate cleavage products formed at sites 1 and 2 both underwent
ligation in the presence of the 17- and 19-mer acceptor
oligonucleotides, respectively. As noted for the substrates lacking any
scissile strand modification (Fig. 2), additional modification of the
partial duplexes at positions
1 and
2 on the noncleaved strand
suppressed cleavage at site 1; modifications at position
4 and
6
suppressed cleavage at site 2.
In contrast, the substrate having 3'-deoxythyimidine at position 2
(scissile strand) exhibited unaltered cleavage and ligation at site 1 (Fig. 6). However, cleavage at site 2 was completely suppressed in
analogy with the effects of substitution at position +1 on cleavage at
site 1 (cf. Figs. 3 and 6). However, additional modification on the
noncleaved strand did alter the cleavage-ligation patterns at sites 1 and 2. The substrate additionally modified at position
1 on the
noncleaved strand underwent cleavage and ligation poorly, and only at
site 2, whereas additional modification at position
2 on the
noncleaved strand also resulted in limited cleavage and ligation but
only at site 1. Additional substitutions at positions
4 or
6 on the
noncleaved strand afforded substrates whose cleavage and ligation at
site 1 was enhanced (Fig. 6, cf. lanes 3, 15, and
18).
Introduction of 3'-deoxyadenosine at position 4 of the noncleaved
strand resulted in cleavage and ligation almost exclusively at site 2 (Fig. 7). This is consistent with the interpretation that the presence
of the 3'-deoxynucleotide actually has a facilitating effect on
cleavage at the closer cleavage site, fully consistent with the effects
noted upon modification at position
2 on the scissile strand (cf.
Figs. 6 and 7). Additional modification at site
1 or
2 on the
noncleaved strand had minimal further effects on the ratio of
cleavage-ligation at site 1 versus site 2.
The most dramatic effects occurred in the presence of a modification at
position 6 on the scissile strand. Cleavage occurred exclusively at
site 1 in the presence of the substrate lacking any modification on the
noncleaved strand. The same was also true for partial duplexes having
3'-deoxynucleotides at position
4 or
6. Even in the presence of
modifications at positions
1 or
2 on the noncleaved strand, which
frequently alter the cleavage from site 1 to site 2, topoisomerase I
cleaved the substrate predominantly at site 1. Further, most of the
substrates afforded a greater extent of cleavage and ligation than the
unmodified partial duplex substrate (Fig. 8). This constitutes strong
evidence that alteration of DNA structure at a site distant from the
actual cleavage site can have a strong facilitating effect on DNA
cleavage and ligation.
Of particular interest was an analysis of the effects of introducing
complementary 3'-deoxynucleotides at the same site on both strands.
Substitutions at positions 1 and
2 on both strands (Figs. 4 and 6,
respectively) had the effect of shifting topoisomerase I-mediated
cleavage to those sites (2 and 1, respectively) farther from the
position of modification. Interestingly, when the modifications were
present at sites distant from both cleavage sites, the compensatory modifications enhanced both the facility and specificity of cleavage. This was apparent for modification at positions
4, which restored virtually full cleavage-ligation to site 1 (Fig. 2) and especially for
modification at positions
6, which resulted in strong enhancement of
cleavage-ligation specifically at site 1 (Fig. 8).
Overall, substitution at position 2 on the noncleaved strand
generally directs cleavage to site 2 and thereby greater ligation at
this site. The exceptions occur where the modification occurs opposite
a complementary modification and in the presence of a substrate
modified at position
6 on the scissile strand. Interestingly, modification at position
1 on the noncleaved strand alters cleavage to site 2 in almost every case. The single exception involves substrates modified at position
6 on the scissile strand. Because the
modification of DNA at a specific site must alter overall DNA
structure, the present results do not permit firm conclusions to be
drawn about the specific sites of topoisomerase I-DNA interaction. However, the patterns of response to DNA structural alterations argue
strongly that specific common enzyme-DNA contacts do control the
specificity and efficiency of DNA substrate cleavage and ligation by
topoisomerase I.
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ACKNOWLEDGEMENT |
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We thank Xiangyang Wang for the sample of calf thymus DNA topoisomerase I used in these experiments.
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FOOTNOTES |
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* This work was supported by Research Grant CA-53913 awarded by the National Cancer Institute.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.
To whom correspondence should be addressed: Dept. of Chemistry,
University of Virginia, Charlottesville, VA 22901.
1 The abbreviation used is: TEAA, triethylammonium acetate.
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