(Received for publication, June 21, 1994; and in revised form, October 24, 1994)
From the
Previous studies have shown that formation of intermolecular DNA triplexes at sequences that overlap protein binding sites inhibits DNA binding by these proteins. We show that DNA cleavage by eukaryotic topoisomerase II is blocked by triplex formation at sites overlapping and adjacent to the triple binding site. To map precisely the boundaries of triplex interference, we constructed a vector containing enzyme binding sites of different lengths and flanked both 5` and 3` by DNA triplexes. We call this method Triplex Interference Mapping by Binding Element Replacement (TIMBER). Triplex regions within 3 bases 5` or 7 bases 3` of cleavage sites blocked DNA cleavage; triplex formation outside of this region had no effect upon cleavage activity. We conclude that topoisomerase II binding requires unhindered access to the major groove of a duplex DNA binding site in this 10-base region. In addition, the inclusion of topoisomerase II inhibitors yielded the same results for the triplex interference assays despite alterations in DNA cleavage site selection. The statistical analyses of over 500 topoisomerase II cleavage sites (in the presence or absence of inhibitors) suggest a model consistent with the region spanning -3 to +7 (relative to the cleavage site) containing most of the base-specific contacts for topoisomerase II. This triplex interference assay may prove valuable in the characterization of DNA binding sites for other proteins as well, particularly in conjunction with deletion analysis.
Eukaryotic topoisomerase II is a nuclear enzyme that modulates
the topological states of DNA via transient double strand breaks in DNA
coupled with strand passage(1) . The enzyme is essential for
the segregation of daughter chromosomes in
mitosis(2, 3, 4) ; it is also the
intracellular target for a number of classes of clinically valuable
anticancer agents(5, 6, 7) . Most of these
agents, including doxorubicin, amsacrine, ellipticine,
epipodophyllotoxins, and quinolones, act through the trapping of the
covalent enzyme-DNA complex, subsequently inducing chromosome
breaks(7, 9, 10) . Recently, another class of
antitumor compounds was shown to stabilize a topoisomerase
IIATP
DNA complex in a conformation incompatible with DNA
cleavage (11) .
Topoisomerase II is a sequence-specific
DNA-binding protein, but it makes double strand breaks in DNA sites
that are not palindromic and are diverse in sequence content;
characterizations of cleavage sites in the absence of inhibitors have
revealed quite degenerate consensus
sequences(12, 13, 14) . The presence of
inhibitors alters the base preferences around the cleavage site,
leading to the derivations of different druginduced consensus sequences
(for example, see (14, 15, 16) . These
consensus studies have yielded results that are inconsistent in terms
of defining the size of the sequence-specific DNA recognition element
for topoisomerase II. To approach this problem in a different way, we
devised a mapping strategy based on our observation that the formation
of a DNA triple helix, in which a homopyrimidine oligonucleotide bound
to a homopurine/homopyrimidine target duplex, prevented topoisomerase
II cleavage at sites present on the DNA duplex alone(17) . We
call this method Triplex Interference Mapping by Binding Element Replacement (TIMBER). ()
Intermolecular DNA
triplexes are formed under defined conditions when an appropriate
single strand oligonucleotide anneals to duplex DNA; these unusual DNA
structures offer new prospects for evaluating and modulating the
interaction between proteins and DNA(18, 19) . In this
DNA structure, pyrimidine oligonucleotides bind through the major
groove parallel to the Watson-Crick purine strand by Hoogsteen hydrogen
bonds. Thymine (T) in the third strand recognizes adenine-thymine (AT)
base pairs (TAT triplet) and protonated cytosine (CH+)
recognizes guanine-cytosine (GC) base pairs (CH+GC
triplet)(20, 21, 22, 23, 24, 25, 26, 27) .
Due to the requirement for protonation of cytosine, triplexes involving
C-rich sequences are more stable under acidic conditions (pH
5-6)(26, 28) . The pH stability of
intermolecular triplex formation thus depends on the sequence
composition of oligonucleotides employed; however, intermolecular
triplexes have been shown to exist at neutral
pH(21, 24, 25, 26, 29) .
Previous findings have additionally revealed that pyrimidine
oligonucleotides can inhibit restriction endonuclease and methylase
activities at sites located in or near pur
pyr
tracts(18, 19) . Furthermore, triplex-forming
oligonucleotides blocked binding of the eukaryotic transcription factor
Sp1 to its consensus sequence(18) . These results clearly show
that DNA-binding proteins can be inhibited by oligonucleotide-directed
triplex formation. An interesting and useful extension of this idea
relates to recent reports using triplexes to limit the systemic
specificity of restriction enzyme in high complexity
genomes(30) .
In this report, we show that intramolecular DNA triplexes inhibit topoisomerase II induced breakage of DNA at positions flanking the triplex regions. We evaluated the ability of the DNA triplexes to interfere with topoisomerase II cleavage of DNA sites located over a range of distances away from the two triplex blocks. We found that triplex regions within 3 bases 5` or 7 bases 3` of a cleavage site on either DNA strand define a minimum 10-bp core-binding element required for topoisomerase II activity. This novel application of DNA triplexes should prove useful for defining duplex DNA binding minima for other proteins.
Figure 1: Partial sequence of the triplex vector pTX-0. The vector was constructed as described under ``Materials and Methods,'' and a restriction map is shown in the lowerfigure. Topoisomerase II sites were cloned into the unique SmaI site defined at the interface between the two triplex blocks. The upperdiagram shows the triplex blocks with their corresponding pyrimidine oligos used to drive triplex formation (see Fig. 2).
Figure 2: Band shift experiment of pUC19 and pTX-0 with pyrimidine oligonucleotide. End-labeled oligonucleotides were incubated with either pUC19 and pTX-0 under conditions optimal for triplex formation as described under ``Materials and Methods.'' Samples were loaded onto a 1% agarose gel and following electrophoresis, the gel was stained with ethidium bromide and photographed (B), dried, and exposed to X-ray film (A). The odd numbered lanes contained supercoiled (sc) DNA, and even lanes contained EcoRI-digested linear (lin) DNA; open circular DNA (oc) is marked in panelB. Pyrimidine oligonucleotides were added in lanes1, 2, 5, and 6. Purine oligonucleotides were added in lanes3, 4, 7, and 8.
where H is the entropy (in bits), calculated as follows:
in which p is the probability of base occurrence at position x taken from the base proportions of the already aligned sites, plus one strand of the current site. For example, as the 10th selected site is analyzed, the base proportions of the previously aligned nine sites are used, with one orientation of the 10th site added in; this information content is compared with that with the opposite strand sequence added in. After a strand has been selected for the last site of a data set, the process is repeated selecting the sites in a different random order. After 10-20 such repetitions, the alignment that yielded the greatest total information content is used, and the base proportions of this set are determined. This method was used for the analyses in Fig. 8, A-C, termed the consensus single strand method; the information content, in bits, at each position was then determined as in Stormo(33, 35) . In Fig. 8, D and E, both the sequenced strand cleavage site and the complementary strand site (located 4 bases 5`) were pooled for all sites, with no strand selection; the information content at each position was calculated directly from the frequencies of all pooled sites. All computations were performed using the EDEN Genesys computer software system (TEAM Associates, Westerville, OH).
Figure 8: Information content analyses of topoisomerase II cleavage site data sets. The information content (in bits), is plotted against the sequence position relative to the cleavage site for sets of topoisomerase II sites analyzed by consensus single strands (A-C) or pooled double strands (D and E), as described in the text. The origins of the data are indicated. The bracket marks the 10-base region from -3 to +7, which was found by triplex interference to be the essential duplex binding region; the identities of the bases at the most highly conserved positions are indicated in the boxed region.
Incubation of an end-labeled pyrimidine oligo probe (14-mer) with plasmid pTX-0 (see Fig. 1), resulted in a clear band-shift of the probe to a position coincident with the plasmid (Fig. 2A). As reported previously, these results can be explained by formation of intermolecular DNA triplexes(28) . Triplexes formed with closed circular supercoiled (sc) and linear (lin) pTX-0 DNA (Fig. 2A, lanes5 and 6; compare with the same gel stained with ethidium bromide and shown in Fig. 2B). Negative controls show that pUC19 DNA did not band shift the pyrimidine oligo (Fig. 2A, lanes1-4) and purine oligos did not bind pTX-0 (Fig. 2A, lanes7 and 8); thus, the pyrimidine oligo forms complexes specifically with pTX-0. The reactions and gel electrophoresis in Fig. 2were carried out with approximately the same molar concentrations of oligo and plasmid (10 nM) in the pH 5.5 buffer.
As triplexes block DNase I access at the homopurine-homopyrimidine sequences(17, 18, 22) , DNase I footprinting was performed to localize sites of pyrimidine oligo binding in pTX-0 (Fig. 3). The 14-mer pyrimidine oligo specifically protected the homopurine-homopyrimidine sequences (Fig. 3, lane6); however, footprints were not observed when a 14-mer purine oligo was used (Fig. 3, lane7), and the pUC 19 DNA fragments were not protected by any of the oligonucleotides tested (Fig. 3, lanes1-3). These results, combined with the gel shift data in Fig. 2A, show that pyrimidine oligonucleotides formed stable intermolecular triplexes with pTX-0 at the sequences of both target sites under our conditions. In Fig. 3, a DNase I hypersensitive site can be seen at the 3` boundary of the triplex region; a similar effect was observed at the 5` triplex boundary in Chung et al.(17) .
Figure 3: DNase I footprinting of triplexes. DNase I digestions were performed as described under ``Materials and Methods'' on an RsaI-PvuII fragment (labeled at the RsaI site). Lanes1-4 contained the pUC19 fragment as a control, and lanes5-8 contained the corresponding pTX-0 fragment, which differs in that it has two 14-base pair homopurine-homopyrimidine regions (bracketed). Purine and pyrimidine oligonucleotides were included as indicated above the gel lanes. Lanes4 and 8 show chemical sequencing markers. The filledbar marks the DNaseI protected region.
The sequencing gel in Fig. 4shows the results of topoisomerase II cleavage reactions (in the presence or absence of the inhibitors m-AMSA and VM-26) on either a pUC19 sequence (lanes1 and 2) or on the equivalent region of pTX-0 containing the two triplex target sites (lanes4-9). Incubation of the pUC19 fragment with the pyrimidine 14-mer used in Fig. 3did not alter the specificity of topoisomerase II cleavage. In contrast, in the reactions on pTX-0, all topoisomerase II sites (for no drug, m-AMSA or VM-26) within and immediately flanking the triplex forming sequences were blocked by binding of the 14-mer (compare lanes5, 7, and 9 with 4, 6, and 8, respectively, in Fig. 4); external to this region, triplex formation did not alter the cleavage patterns. This experiment revealed that the inhibition of topoisomerase II cleavage by DNA triplexes is independent of the presence of drugs, and suggested that, in all cases, the enzyme requires contact with a contiguous region of nontriplex (presumably B-form) DNA spanning a minimum of 2 bases 5` and 6 bases 3` of a cleavage site. The experiments described below were conducted in order to further define this core duplex sequence required for topoisomerase II activity.
Figure 4: Inhibition of topoisomerase II cleavages intermolecular triplexes. Topoisomerase II cleavage reactions were performed on the pUC-19 or pTX-0 fragments described in the legend to Fig. 3and in the presence (lanes2, 5, 7, and 9) or absence (lanes1, 4, 6, and 8) of pyrimidine oligonucleotides. Lanes containing the pUC19 or pTX-0 fragments are indicated as are the constituents of each reaction (inhibitor and oligo concentrations are specified under ``Materials and Methods''). Bracket shows the position of the two 14-base pair homopurine-homopyrimidine regions.
Figure 5: Topoisomerase II cleavages on pTX-GT17. PanelA, reactions were carried out on a HpaII-EcoRI fragment (labeled at the HpaII site) of pTX-GT17 (pTX-0 containing a 17-bp insert of GT). Lanes1-3 show DNase I footprinting protection, and lanes7-12 show topoisomerase II cleavage reactions. Oligonucleotides and topoisomerase II inhibitors (VM is VM-26, AM is m-AMSA, and ND is no drug) are indicated above the gel. Brackets to the left mark the two homopurine-homopyrimidine regions and 17 base-pair CA repeat. PanelB, summary of topoisomerase II cleavages on pTX-GT17 fragment. The 17-bp insert and partial flanking triplex regions are shown. The x and y designators correspond to sites marked in the sequencing gel in A. Sites marked by dottedlines correspond to cleavages that were inhibited by triplex formation.
A second plasmid was constructed to examine the cleavage site inhibition when a shorter RY repeat was inserted. The plasmid, pTX-AC12 contains a 12-bp (dAdC)-(dGdT) repeat between the two triplex blocks. DNase I footprinting shows the expected pattern of protection over the triplex regions (Fig. 6A, lane3). Topoisomerase II cleavages were also analyzed (Fig. 6A). The cleavage site located symmetrically in the middle of the AC repeat (marked z) was not affected by triplex formation; however, cleavage frequency of the flanking site y (2 bases 3` of triplex block) was significantly reduced by triplex formation, as were sites x-1, x-2, and x-3 (see summary, Fig. 6B). Cleavage protection by triplex formation on the bottom strand was identical to that of top strand (Fig. 6B, data not shown). These results, in conjunction with the protection pattern of pTX-GT17, indicate that the minimal sequence requirement for topoisomerase II cleavage is 10 base pairs of double strand DNA, spanning the sequence from -3 to +7 (cleavage at -1/+1). It also appears from these data that paired sites (defined as the top and bottom strand cleavages staggered by 4 bp; see (31) ) are both hindered by triplex formation, even when one of the paired sites is clearly in a freely accessible duplex region. For example, considering only the bottom strand in Fig. 6B, site y should be unhindered by triplex formation (it is between the two paired z sites, which were unaffected by the triplex); however, site y is blocked presumably because its paired site (on the top strand) is hindered by the 5` top strand triplex insulator.
Figure 6: Topoisomerase II cleavages on pTX-AC12. PanelA, reactions were carried out on a RsaI-PvuII fragment (labeled at the RsaI site) of pTX-AC12 (containing a 12-bp insert of AC in pTX0). Lanes2-4 are DNase I footprinting, and lanes6-11 are topoisomerase II cleavage reactions. Reaction conditions are shown above each gel lane. PanelB is a summary of topoisomerase II cleavages on the pTX-AC12 fragment (the top strand sites are shown in A; the bottom strand cleavages are from a different gel that is not shown). The cleavages are marked as x, y, and z and correspond to sites marked in A (see text).
Figure 7: Topoisomerase II cleavages on pTX-N10. PanelA, the reactions were performed on the RsaI-PvuII fragment (labeled at the RsaI site) of pTX-N10. The lanes1-3 show the DNase I footprinting data (sequencing ladders are in lanes4 and 5). Lanes6-9 show topoisomerase II cleavage data; individual reactions are as marked above each lane. The singlearrow indicates the topoisomerase II cleavage within the 10-bp insert. PanelB, summary of topoisomerase II cleavage on pTX- N10 fragment.
A total of 519 topoisomerase
II cleavage sites were analyzed, from four different sets of sites.
These data sets were 134 VM26-induced chicken enzyme sites, ()111 VM26-induced mouse topoisomerase II
sites(39) , 197 m-AMSA-induced mouse enzyme
sites(39) , and 77 Drosophila topoisomerase II sites
observed in the absence of inhibitors(12) .
The
data were treated for either a single strand (consensus sequence type)
analysis or a pooled double strand analysis method (see
``Materials and Methods''). The information content plots in Fig. 8, A and B, show that for both VM26 data
sets, the bulk of the total conserved base information does lie in the
10-bp region between -3 and +7; in fact, the only strongly
conserved position for each is at -1, which in each case
represents a high frequency of cytosines. The analysis of the m-AMSA sites in Fig. 8C also reveals that the
bases conserved among these sites are located within this 10-bp region;
the identities of these preferred bases are indicated. Fig. 8D shows the analysis of these same 197 m-AMSA sites analyzed by the pooled double strand method; the
plot appears quite similar to that of Fig. 8C. Finally,
the information content plot in Fig. 8E, representing
77 ``no drug'' Drosophila topoisomerase II sites,
also reveals that the bases conserved among these sites are found in
the 10-bp region spanning -3 to +7 relative to the cleavage
site. Thus, for these topoisomerase II cleavage data, the base-specific
information is found in the region identified as the core binding
element by our TIMBER results and is independent of the origin of the
enzyme or whether inhibitors were included in the reactions.
The TIMBER analysis strongly suggests a 10-bp binding core
for topoisomerase II. The question persists as to whether a duplex
element smaller than 10 bp (say 9 bp) might be sufficient for cleavage
activity. It would be difficult to prove that 9 bp is necessary and
sufficient for cleavage since failure to detect cleavage in the
presence of triplexes might result if the 9-bp test sequence simply is
not a topoisomerase II cleavage site. Parenthetically, the
topoisomerase II consensus sequences defined to date are larger than 9
bp; thus, only trial and error could be used to design an appropriate
insert less than 10 bp to test. Since there are 4 possible
sequence combinations in a 9-mer experiment, proving that cleavage never occurs is not practical; however, we consider that a
9-mer would not be a cleavage substrate in a TIMBER experiment for the
following reason. Placement of a cleavage site anywhere within the 9 bp
will violate the 5` or 3` positional requirements for cleavage as
defined in Fig. 5Fig. 6Fig. 7. For example,
consider the following 9-mer insert experiment with potential cleavages
at sites 1 to 4 (N is any base and top site 1 is paired with bottom
site 1).
Our data clearly show that cleavage inhibition is symmetrical (i.e. impeded at both top and bottom strand sites when either site is within 2 bases of a triplex). Thus, top site 1 is eliminated by analogy with top strand site x-1 in Fig. 5B. Since the bottom strand site x-1 (Fig. 5B) is also blocked by triplex, we can also eliminate bottom strand site 1 in the 9-mer. Site 2 cleavages in the 9-mer above are eliminated by the equivalent experiment summarized in Fig. 6B site y (topstrand) and its corresponding paired bottom strand site. By the same reasoning, one can see that neither bottom site 3 nor 4 would be cleavable by topoisomerase II (since both are <3 bases from the triplex). While we cannot categorically prove that a sequence exists less than 10 bp that might be cleavable in the triplex, the statistical analysis of cleavage sites indicates that a 10-bp window from -3 to +7 is conserved in topoisomerase II cleavage sites (see below). Moreover, as noted above, nuclease footprinting data (40) and minimal site binding experiments (41) suggest that the core is equal to or greater than 10 bp as opposed to smaller than 10 bp.
The conformation of triplex/duplex junctions may also be considered in interpreting our results. For example, Hartman et al.(42) identified distortions at these junctions, and we observed DNaseI hypersensitivity at the 3` triplex/duplex boundary. However, Huang et al.(43) reported no inhibition of protein binding by triplex formation immediately 5` of the minimal DNA binding element. Furthermore, the topoisomerase II cleavage sites characterized in Fig. 8show strong base preferences immediately at the triplex boundaries, suggesting that the junction does not impede enzyme activity. It is possible that topoisomerase II contacts a larger core binding site than indicated by these TIMBER experiments due to ``breathing'' of the triplex strand binding or to displacement of adjacent triplex base pairs by enzyme binding; however, rigorous tests of these dynamic interactions would be difficult. An important question concerning the mechanism for the stabilization of topoisomerase II-DNA covalent complexes by these anti-cancer agents is how they alter the interaction of the protein with its substrates. These agents are known to modify enzyme cleavage patterns(39, 44, 45, 46, 47) . The triplex interference results, however, revealed the same protection results regardless of whether drugs were included in the reactions. This suggests that the topoisomerase II inhibitors interact with the protein-DNA intermediate only at sequences proximal to the cleavage sites and do not alter the size of the recognition element.
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