©1995 by The American Society for Biochemistry and Molecular Biology, Inc.
Determination of 5` and 3` DNA Triplex Interference Boundaries Reveals the Core DNA Binding Sequence for Topoisomerase II (*)

(Received for publication, June 21, 1994; and in revised form, October 24, 1994)

J. R. Spitzner (§) I. K. Chung (¶) Mark T. Muller (**)

From the Department of Molecular Genetics, Ohio State University, Columbus, Ohio 43210

ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES

ABSTRACT

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.


INTRODUCTION

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 IIbulletATPbulletDNA 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). (^1)

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 (TbulletAT 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 purbulletpyr 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.


MATERIALS AND METHODS

Enzyme and Materials

Topoisomerase II was purified from whole chicken blood according to procedures previously described(15, 31) . Restriction enzymes and T4 nucleotide kinase were purchased from Life Technologies, Inc. and U. S. Biochemical Corp., respectively. alpha- and -[P]ATP were from ICN. Oligonucleotides were synthesized on an Applied Biosystem DNA synthesizer (at the Ohio State University Biochemical Instrumentation Center). The topoisomerase II inhibitors 4`-(9-acridinylamino)methanesulfon-m-anisidide (m-AMSA)and VM-26 (teniposide) were provided by the National Cancer Institute divisions of synthetic and natural products, respectively. DNA manipulation, fragment end labeling, and DNA sequencing were performed as described elsewhere(32) .

Plasmid Constructions

The triplex plasmid pTX-0 was constructed as follows: pUC19 DNA was digested with HincII, and 14-bp homopurine-homopyrimidine fragments were inserted (see Fig. 1) such that a new SmaI site was generated in the middle of the 14-base elements (the original SmaI site of pUC19 was removed by previous manipulation). Constructs pTX-GT17, pTX-AC12, and pTX-N10 were prepared by inserting appropriate fragments into SmaI site of pTX-0.


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.



Band Shift Analysis

Formation of triplex structures was assayed using a modification of the band shift technique described previously(28) . End-labeled oligonucleotides (10 nM, 10,000 cpm) were mixed with 10 nM supercoiled or linearized DNA (20 µl, total volume) in triplex buffer (25 mM Tris acetate, pH 5.5, 70 mM NaCl, 10 mM MgCl(2), 10 mM 2-mercaptoethanol, 0.4 mM spermidine, 3 mM ATP, and 100 µg/ml bovine serum albumin). Reactions were incubated for 30 min at 25 °C, and, after addition of 2 µl of tracking dye (0.25% bromphenol blue, 40% sucrose), they were loaded onto a 1% agarose gel (running buffer, 40 mM Tris acetate, pH 5.5, 100 mM sodium acetate with circulation). After electrophoresis, the gel was stained with ethidium bromide, destained, photographed, dried, and exposed to film at -70 °C.

DNase I Footprintng and Topoisomerase II Cleavage Reactions

Reactions (20 µl) were carried out in triplex buffer (see above) and contained 5` end-labeled fragment (2 nM, 20,000 cpm) and the specified oligonucleotide at 3 µM. Reactions were preincubated 30 min at 25 °C before the addition of enzymes. For DNase I footprinting experiments, digestions were initiated by the addition of 1 µl of 40 mM CaCl(2) and 1 µl of 50 µg of DNaseI/ml. Reactions were incubated 1 min at 25 °C and terminated by the addition of 2 µl of 0.5 M EDTA. Topoisomerase II cleavage reactions were initiated by the addition of 8 units (5.6 nM) of purified chicken topoisomerase II in the presence or absence of drugs (m-AMSA at 50 µg/ml and VM26 at 500 µg/ml) as indicated, incubated 30 min at 30 °C, and then terminated by the addition of SDS to 1% followed by proteinase K digestion (50 µg/ml, 56 °C for 30 min). Samples were ethanol-precipitated and analyzed on an 8% sequencing gel.

Statistical Analysis of Topoisomerase II Cleavage Site Data Sets

Previous analyses of topoisomerase II cleavage sites suggested that despite the double-stranded nature of these events, the sequence information common to all cleavage sites is conserved primarily on only one of the two strands(15) . The determination of cleavage site locations from sequencing gels does not reveal which of the two strands contains the information to specify the cleavage. To select the conserved strand from each cleavage pair, we used a computer algorithm that maximizes the information content of the data set (i.e. to yield the most nonrandom base proportions). Sites were taken in random order and tested to see which strand contributed to a greater information content from the set (33, 34) using .

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.




RESULTS

The Vector pTX-0 Forms DNA Triplexes at Two Adjacent Sequences

We previously observed that DNA triplex formation over a duplex DNA sequence containing topoisomerase II cleavage sites inhibited enzymatic cleavage at those sites; furthermore, this triplex interference was propagated over proximal cleavage sites flanking the triplex region(17) . Having shown that DNA triplex formation blocks topoisomerase II activity at sites both within and external to a triplex region(17) , the next experiments were designed to map, at base pair resolution, the exact 5` and 3` boundaries at which triplex formation interfered with topoisomerase II cleavage of DNA. Our previous experiments (17) were conducted in a neutral pH buffer (6.8). The following experiments were conducted at pH 5.5 (as described under ``Materials and Methods'') since triplexes with cytosine containing oligos are stabilized at lower pH(28) ; moreover, topoisomerase II displays very good DNA cleavage activity at this pH (36) . (^2)In terms of DNA relaxation activity, topoisomerase II has high catalytic activity pH 5.5 (90% of the activity seen at pH 6.8, data not shown). The plasmid pTX-0, shown in Fig. 1, contains two 14-base pair polypurine-polypyrimidine sequences (identical and in opposite orientation) with a central SmaI to allow insertion of additional DNA sequences between these two triplex-forming sites.

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.



Inhibition of Topoisomerase II Cleavage of pTX-0 Sequences by DNA Triplexes

For several reasons, topoisomerase II is particularly amenable to site mapping by triplex interference. The enzyme cleaves DNA at specific sites, making it possible to determine directly the binding site locations without resorting to band shifts or footprinting. In addition, while sequence-specific in its binding, topoisomerase II sites are found rather frequently (on average, one site every 25.9 bases in random DNA sequences, (15) ). Furthermore, a large number of binding sites have been characterized, and high affinity sites have been defined(13, 15, 37) .

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.



Triplex Interference: Analysis of Flanking Sequences Required by Topoisomerase II

To evaluate the minimal duplex DNA sequence required for topoisomerase II activity and to determine the exact boundaries of triplex interference, pTX-0 (triplex vector) was modified by inserting topoisomerase cleavage sites between the two 14-bp triplex blocks, such that sites would be at varying distances (both 5` and 3`) from the triplex boundaries. We took advantage of our previous results showing that topoisomerase II cleaves DNA at every second base in sequences of alternating purine-pyrimidine (RY), and that these sites are conserved when topoisomerase inhibitors are included(37) . The construct pTX-GT17 has a 17 bp (dGdT)-(dAdC) repeat cloned between the two triplex blocks in the SmaI site (see Fig. 1). DNase I footprinting revealed that both triplex regions were protected from digestion in the presence of the pyrimidine oligonucleotide (Fig. 5A, lane2); in addition, a novel hypersensitive site appeared at the 3` triplex boundary. Topoisomerase II cleavage reactions were also carried out on pTX-GT17 with or without oligonucleotides (lanes7-12). Topoisomerase II cleavages at triplex and flanking regions were inhibited by the pyrimidine oligonucleotides (compare lane7 with 8, 9 with 10, and 11 with 12). As reported previously(37) , topoisomerase II cleaves within the AC repeat at every second base (Fig. 5A, lanes7-10; in reactions lacking the cleavage-stimulating inhibitors, much longer exposures of the autoradiogram were required to visualize the cleavages, lanes11 and 12). The cleavage sites located centrally within the insert (through the AC repeat) were not significantly affected by triplex formation. Cleavage data on the pTX-GT17 target DNA are summarized in Fig. 5B. The sites marked x-1 and x-2 on the top strand were clearly inhibited by triplex formation; however, the enzyme could access the internal sites (e.g.y-1 to y-4). The paired sites on the bottom strand (4 bases 5`) reflect the results observed on the top strand, as marked in Fig. 5B (sequencing data not shown). From these data, it appears that 3 bases of duplex DNA 5` of a cleavage site are necessary for enzyme activity. For example, considering the top strand of a given topoisomerase II site, this corresponds to nucleotide positions -3 to -1 (cleavage at -1/+1).


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).



Topoisomerase II Cleavages on pTX-N10 Containing One Cleavage Site between Two Triplex Blocks

In order to confirm that a 10-base pair duplex binding site is sufficient for topoisomerase II-mediated cleavage, we constructed the triplex plasmid pTX-N10, in which a single 10-bp topoisomerase II cleavage site was inserted into the SmaI site of pTX-0 (see Fig. 7B). Triplex formation is shown by the DNase I protection pattern in lane2 of Fig. 7A. Topoisomerase II cleavages on this fragment are shown in lanes6-9 of Fig. 7A. Again, the topoisomerase II cleavages were blocked directly over triplex regions and at flanking sites when triplexes were formed by the pyrimidine oligonucleotides. However, the cleavage site within the 10-base pair insert (Fig. 7A, the site indicated by arrow) was not inhibited by triplex formation (compare lanes6 and 7, 8 and 9). Furthermore, it is clear that while the addition of a topoisomerase II inhibitor (in this case, m-AMSA) may alter the sequence specificity of the enzyme, it does so within this 10-base minimal binding site, because the triplex interference results appear independent of drug inclusion.


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.



Statistical Analyses of Topoisomerase II Cleavage Sites

Our results suggest that most of the base-specific contacts of topoisomerase II with DNA lie in the 10-bp region spanning -3 to +7 relative to the cleavage site. We wanted to compare our triplex interference mapping results with results obtained from statistical analyses of large numbers of topoisomerase II cleavage sites to test whether the bulk of conserved base-specific contacts identified also lay within this 10-bp region. We used the measure of information content to reveal which positions (relative to the cleavage sites) contained conserved base occurrences within sets of topoisomerase II cleavage sites. This analysis identifies positions with nonrandom base frequencies, and, in contrast with -square analysis, the information content at a base position is independent of sample size; the maximum value is two bits of information for a position at which one particular base occurs in all sites examined, and the minimum value is zero bits for random base frequencies(33, 38) .

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, (^3)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) .^3 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.


DISCUSSION

DNA Triplex Inhibition of Topoisomerase II Cleavages

Repetitive topoisomerase II cleavage sites were cloned into the SmaI site of the pTX-0 vector (located between the two triplex-forming regions) in order to reveal the 5` and 3` boundaries of triplex interference. On all of the DNA targets tested, topoisomerase II cleavage sites were not affected if located at least 3 bases 3` and 7 bases 5` of triplex regions; sites located more proximally to the triplexes were inhibited. Therefore, we conclude that topoisomerase II has a minimal requirement of 10 base pairs of duplex DNA at a binding site. Topoisomerase II is a homodimeric enzyme; in its double-stranded cleavages, both subunits cleave the DNA, with a 4-base 5` overhang between the two paired cuts on opposite strands. Considering the double-stranded nature of topoisomerase II cleavages, the triplex interference data suggest a symmetrical 10-base pair recognition element for the enzyme (see Fig. 7B). These data support a model in which the cleavage step is carried out by each subunit of the homodimer acting on single strands, and both sites must be available in order for the enzyme to initiate cleavage activity. The data suggest that it needs access to the major groove over a span of 10 bp. It is likely that the enzyme makes contacts further 3` and 5` of the 10-bp site based upon nuclease footprinting experiments(40) , minimal site binding analyses(41) , and consensus sequence derivations (12, 15) . Our results suggest, however, that these external interactions are either not major groove contacts or are not essential for topoisomerase II activity.

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 4^9 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.

Analyses of Base Information Content in Sets of Topoisomerase II Cleavage Sites

We sought to test the validity of our triplex interference model by comparing its results with those of statistical analyses to determine which base positions were conserved among topoisomerase II sites. We determined the base conservation of topoisomerase II binding sites (by position relative to the cleavage site) using the measure of information content(15, 38) , which indicates the deviation from random base frequencies at each position. Four sets of topoisomerase II cleavage sites were selected to cover a range of origin of the enzyme and the presence or absence of different inhibitors. Both sets of VM26-induced topoisomerase II cleavage sites (134 chicken enzyme sites in Fig. 8A and 111 mouse enzyme sites in Fig. 8B) exhibited the majority of base conservation within the 10-bp region spanning -3 to +7, with each possessing only a single strongly conserved base, a cytosine at position -1 relative to the cleavage site. The information content plot of 197 m-AMSA-induced mouse topoisomerase II sites analyzed using the consensus single strands revealed conserved bases at five positions (-3, -2, +1, +4, +7), all of which are within the 10-bp TIMBER model core element. Fig. 8D shows a plot of the same data, but with the 197 pairs of sites (from each strand of the cleavage sites) pooled and treated as 394 individual sites, all aligned by the position of DNA cleavage. This method obviates the need for consensus single strand selections and reveals base information content that is conserved symmetrically in topoisomerase II sites; the bases common to these cleavage sites are located at positions -3, +1, +4, and +7 relative to the cleavage sites (i.e. within the -3 to +7 core). Finally, the information content of 77 Drosophila topoisomerase II cleavage sites (observed in the absence of inhibitors), was analyzed symmetrically as 154 sites (Fig. 8E); conserved base occurrences are found throughout the -3 to +7 region, but little information is found outside this 10-bp region. The statistical analyses of topoisomerase II sites reported by others (39, 45, 46) is in close agreement with the -3/+7 conserved region derived by the TIMBER model.

General Considerations for Triplex Interference Mapping of DNA Binding Sites

An important parameter in characterizing a DNA binding protein is determining the size of a DNA binding site and in particular, the region in which base-specific contacts occur. Generally this is addressed using a battery of biochemical and chemical nucleases to search for protected versus accessible sites. Triplex interference analysis complements these existing methods by allowing the investigator to combine the power of triplex selectivity(29, 30) with a systematic reduction in access to the major groove of the DNA site. Recent studies of DNA triplexes have shown that a variety of techniques permit their formation under physiological conditions so that our triplex interference assay may be easily extended for use in other systems. For example, Duval-Valentin et al.(1992) (48) showed that triplexes formed at pH 7.8 and inhibited in vitro transcription, and that chemical modifications to the polypyrimidine oligonucleotide further stabilized its binding to duplex DNA. In addition, Roy (49) demonstrated an inhibition of gene transcription by formation of triplexes using a purine-rich oligonucleotide. Dervan and co-workers (8) have published a number of reports characterizing conditions for stabilizing DNA triplexes. The triplex interference assay may prove quite useful in conjunction with the traditional deletion analysis of a protein's DNA binding sites.


FOOTNOTES

*
This work was supported in part by funds from TopoGEN, Inc. (P. O. Box 20607, Columbus, OH 43220) and by Public Health Service Grant AI128362. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore by hereby marked ``advertisement'' in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

§
Supported by Public Health Services Postdoctoral Grant GM-14335. Present address: Dept of Biology, Massachusetts Institute of Technology, 77 Massachusetts Ave., Cambridge, MA 02139.

Present address: Yonsei University, Dept. of Biology, College of Science, 134 Sinchon-don, Seotaemoonku, Seoul, Korea 120-749.

**
To whom all correspondence should be addressed: Dept. of Molecular Genetics, Ohio State University, 484 W. 12 Ave., Columbus, OH 43210. Tel.: 614-292-1914; Fax: 614-292-4702.

(^1)
The abbreviations used are: TIMBER, triplex interference mapping by binding element replacement; bp, base pair(s); m-AMSA, 4`-(9-acridinylamino)methanesulfon-m-anisidide.

(^2)
J. R. Spitzner, I. K. Chung, and M. T. Muller, unpublished observations.

(^3)
J. R. Spitzner, submitted for publication.


ACKNOWLEDGEMENTS

We thank Dr. Joseph Spitzner (TEAM Associates, Westerville, Ohio 43081, U.S.A.) for providing the EDEN-GENESYS computer software used to analyze the cleavage data sets. We thank D. Luo for characterizing pH effects on topoisomerase II activity. Critical reading of the paper by C. Sommer-Furbee and D. Subramanian is gratefully acknowledged.


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