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Nucleic Acids Research Pages 984-992  

Human DNA topoisomerase II[beta] binds and cleaves four-way junction DNA in vitro
Introduction
Materials And Methods
   Preparation of DNA substrates
   Gel retardation analysis
   Cleavage reactions
   Gel filtration
Results
   Topoisomerase II[beta] binds short linear duplex DNA in a gel retardation assay
   Topo II[beta] binds a synthetic four-way junction in vitro
   Topo II[beta] has a higher affinity for four-way junction DNA than linear DNA
   The selectivity of DNA binding by topo II[beta]
   The effects of K+, Mg2+ and Ca2+ ions on DNA binding
   Topo II[beta] cleaves the centre of the four-way junction
Discussion
Acknowledgements
References

Human DNA topoisomerase II[beta] binds and cleaves four-way junction DNA in vitro

Human DNA topoisomerase II[beta] binds and cleaves four-way junction DNA in vitro

Katherine L. West+ and Caroline A. Austin*

School of Biochemistry and Genetics, The Medical School, The University of Newcastle Upon Tyne, Newcastle Upon Tyne NE2 4HH, UK

Received October 30, 1998; Revised and Accepted January 5, 1999

ABSTRACT

We have used gel retardation analysis to show that human DNA topoisomerase II[beta] can bind a 40 bp linear duplex containing a single DNA topoisomerase II[beta] cleavage site. Furthermore, we demonstrate for the first time that human DNA topoisomerase II[beta] binds to four-way junction DNA. This supports previous suggestions that topoisomerase II may be targeted to supercoiled DNA through the recognition of DNA cruciforms, helix-helix crossovers and hairpins. DNA topoisomerase II[beta] had a 4-fold higher affinity for the four-way junction than for the linear duplex, as demonstrated by protein titration and competition analysis. Furthermore, the DNA topoisomerase II[beta]:four-way junction complex was significantly more salt stable than the complex with linear DNA. The four-way junction contained potential topoisomerase II[beta] cleavage sites straddling the points of strand exchange, and indeed, topoisomerase II[beta] was able to cleave three of these four predicted sites. This indicates that topoiso-merase II[beta] can bind to the centre of the junction. Topoisomerase II has to bind both the transported and the gated DNA helices prior to strand passage, and it is possible that both helices are provided by the four-way junction in this case. The stable complex of DNA topoisomerase II[beta] with four-way junction DNA may provide an ideal substrate for further studies into the mechanism of substrate recognition and binding by DNA topoisomerase II.

INTRODUCTION

DNA topoisomerase II (E.C. 5.99.1.3; topo II) is essential for DNA replication and is also considered to play important roles in transcription and recombination (1-4). It is a dimeric enzyme (5-10) that catalyses the ATP-dependent relaxation of supercoiled DNA and the decatenation of replication products. This DNA strand passage activity requires topo II to bind two duplexes of DNA, termed the gate duplex and the transported duplex (9). Topo II transiently breaks the gate duplex, forming an intermediate in which each monomer is covalently linked to the 5[prime] end of one DNA strand by a phosphotyrosine linkage (11-13). The sites of cleavage on each DNA strand are staggered by 4 bp. ATP binding, and possibly hydrolysis of one of the two ATP molecules (14,15), stimulates a conformational change which is concomitant with the passage of the transported helix through the enzyme-bridged break in the gate helix (16,17). Hydrolysis of the second ATP is required to complete the catalytic cycle, and thus allow topo II to carry out the next round of strand passage (14,15,18,19).

The structures of a 92 kDa fragment of yeast topo II (9), the breakage-reunion domain of DNA gyrase A (20) and the ATPase domain of DNA gyrase B (21) have been determined by X-ray crystallography. Computer modelling (9,20), protein-DNA crosslinking (22) and protein footprinting (23) have all indicated that the gate duplex lies in a semi-circular groove leading to the active site tyrosine residues in the centre of the dimer. The DNA gyrase structure corresponds to the conformation of the dimer prior to DNA cleavage, while the yeast topo II structure is thought to represent the molecule after it has cleaved the gated helix and pulled the two DNA segments apart (9,20). It has been proposed that the transported DNA helix may be bound between the N-terminal ATPase domains prior to strand passage (9,21,24). However, the exact positions of all the domains within the native protein are not fully established, nor is it known how these domains move relative to each other during the catalytic cycle (9,25).

Yeast topo II has a higher affinity for supercoiled DNA than relaxed or linear B form DNA (17,18,26,27), which raises the important question of how topo II recognises its preferred substrate, supercoiled DNA. Both negatively and positively supercoiled DNA contain regions of helix-helix juxtaposition or crossovers, as does catenated DNA. Electron microscopy and functional activity assays have shown that topo II can associate with DNA crossovers (28,29), and it is possible that topo II is targeted to its substrates through the recognition of these structures. There is also evidence that topo II recognises certain secondary structures which are unique to negatively supercoiled DNA. Topo II can bind (30,31) and cleave (32) Z DNA and indeed, has a higher affinity for Z DNA than B DNA (30,31). Furthermore, topo II can cleave DNA hairpins in a manner that is dependent on both the DNA sequence and the secondary structure of the hairpin (33). It has also been suggested that topo II can be targeted to supercoiled DNA through the recognition of cruciforms (34).

We wished to develop a gel retardation assay in order to study DNA binding by human topo II[beta] (35), as it is a high resolution technique which is sensitive to small changes in binding affinity. Although gel retardation has previously been used to study topo II:DNA binding, these studies all used fairly long DNA substrates (>180 bp) (26,36,37) or very high protein concentrations (38). We have developed experimental conditions that allow the binding of topo II[beta] to short DNA fragments to be studied by gel retardation. Using this assay, we show that topo II[beta] can bind a 40 bp duplex containing a single topo II cleavage site. Furthermore, we demonstrate that topo II[beta] can bind and cleave a synthetic four-way junction in vitro. The four-way junction contained the sequence of the 40 bp linear duplex along two adjacent arms, with the cleavage site straddling the point of strand exchange. The remaining two arms were comprised of sequences which do not contain topo II cleavage sites, except that a potential m-AMSA-inducible cleavage site was introduced across the junction of these two arms. Topo II[beta] cleaved this substrate at the predicted sites, indicating that the enzyme can bind and cleave the centre of the four-way junction.The nature of topo II[beta] binding to both four-way junction and linear DNA substrates is discussed in relation to the catalytic cycle of topo II, and to its activity in vivo.

MATERIALS AND METHODS

Human topo II[beta] was overexpressed from plasmid YephTOP2[beta]KLM (39) in the Saccharomyces cerevisiae strain JEL1[Delta]top1([alpha] leu2 trp1 ura3-52 prb1-1122 pep4-3 [Delta]his3::PGAL 10-GAL4 [Delta]top1), kindly provided by R. Hanai (Rikkyo University, Tokyo). Topo II[beta] was purified from yeast extracts according to a modified version (35) of the protocol of Worland and Wang (13).

Preparation of DNA substrates

The linear DNA substrate is comprised of 40 bp from pBR322 surrounding cleavage site C102 (40). Within pBR322, the 40 bp surrounding C102 are cleaved only weakly at one additional site, A95, in the presence of m-AMSA. To minimise topo II binding and cleavage at this secondary site, A95 was changed to G in the 40 bp substrate used here (41).

Oligonucleotides used:

C: 5[prime]-CGCAATCTGACAATGCGCTCATCGTCATCCTCGGCAC-3[prime]

D: 5[prime]-CGCGTGCCGAGGATGACGATGAGCGCATTGTCAGATT-3[prime]

O1 5[prime]-CTGGACGCAATCTGACAATGCGCTCATCGTCATCCTCGGCACGC-3[prime]

O2 5[prime]-CGGCGCGTGCCGAGGATGACGATGAGATAGGCGTTAACGCGG-3[prime]

O3 5[prime]-TAGGCCGCGTTAACGCCTATTTGCCCGGGAGTACCGGCAT-3[prime]

O4 5[prime]-AGGAATGCCGGTACTCCCGGGCAACGCATTGTCAGATTGCGT-3[prime]

Oligo O1 is the same as oligo C except that it has five and two additional bases at its 5[prime] and 3[prime] ends, respectively. Oligo O2 contains the first 23 bases of oligo D (underlined bases), and oligo O4 contains the last 14 bases of oligo D (bases in bold italics).Oligonucleotides were end-labelled with T4 PNK in the presence of [[gamma]-32P]ATP. The PNK was inactivated by incubation at 70°C for 30 min, and unincorporated nucleotides were removed using a G50-M Sephadex spin column. Oligo D was annealed with oligo C or oligo O1 to make linear substrates of 40 or 46 bp, respectively (the same results were obtained when either linear substrate was used). Oligos O1, O2, O3 and O4 were annealed to make the four-way junction. Annealing reactions contained 20 pmol of each oligo in 50 mM NaCl and 10 mM MgCl2. Samples were placed in a 70°C water bath which was allowed to cool to room temperature. Overhangs were filled in with DNA polymerase I Klenow fragment and dNTPs. Labelled DNA substrates were purified on 7.5% non-denaturing polyacrylamide gels. Four-way junction DNA was eluted from gel slices in 300 µl elution buffer (10 mM Tris-HCl pH 7.5, 1 mM EDTA, 100 mM NaCl and 10 mM MgCl2) overnight at 4°C. The four-way junction was stored at 4°C for up to three weeks. Gel slices containing linear DNA were macerated, then the DNA was eluted in TE by gentle agitation overnight at 22°C. Acrylamide particles were removed by centrifugation through a glass wool column. The linear DNA was precipitated with ethanol, resuspended in 300 µl TE or elution buffer, and stored at -20°C.

Gel retardation analysis

Binding reactions contained 1.4-417 nM topo II[beta] and 1-3 µl of labelled DNA (~1 ng) in 20 µl binding buffer (50 mM Tris-HCl pH 7.5, 1 mM EDTA, 1 mM DTT, 1 mg/ml BSA, 1% Triton X-100, 5% glycerol and 50 mM KCl). Reactions were incubated for 30 min on ice, then 6 µl of 8% Ficoll was added immediately prior to loading. Samples were run on 5% 80:1 polyacrylamide gels made in 50 mM Tris-glycine, pH 9.2 (42). Electrophoresis was carried out at 130 V for 4-5 h at 4°C. To calculate the apparent dissociation constant, binding reactions were carried out using a range of protein concentrations. A sigmoidal dose response curve with variable slope was fitted to the data:

y = min + (max -min)/{1+10^[(LogEC50-x)*hillslope]}

y (percentage of DNA bound) goes from min to max with a sigmoidal shape, and the hillslope parameter describes the slope of the curve. x is LOG(protein concentration), and EC50 is amount of protein required to bind 50% of the DNA. The dissociation constant (KD) was calculated from the equation:

KD = [topo II]eq[DNA]eq/[topo II:DNA complex]eq

When 50% of the DNA is bound, this simplifies to KD =[topo II] eq, and when the protein is in vast excess over the DNA, [topo II] eq is equivalent to the initial topo II concentration.

When directly comparing competition efficiencies on linear and four-way junction DNA, both substrates were made using the same batch of labelled O1 oligo. The specific activity of each substrate was the same, therefore, and the relative DNA concentrations were determined by Cerenkov counting. Equal concentrations of DNA were used in each experiment.

Cleavage reactions

Labelled DNA was incubated with topo II[beta] in 20 µl binding buffer in the presence of 10 mM Mg2+ or Ca2+ at 37°C for 30 min. m-AMSA or the non-hydrolysable ATP analogue, AMPPNP, were included where indicated. SDS and proteinase K were then added to 0.1% and 1 mg/ml respectively, and reactions incubated at 50°C for 30 min. The DNA was precipitated with ethanol prior to electrophoresis on a 15% sequencing gel in 0.5× TBE. Equal c.p.m. were loaded in each lane, alongside Maxam and Gilbert G+A sequencing reactions (43). Differences between the mobilities of sequencing markers and topo II cleavage products were accounted for as described previously (40,44).

Gel filtration

Topo II[beta]-linear DNA complexes were generated by incubating 40 µl of the labelled 40 bp duplex with or without 278 nM topo II[beta] in 200 µl binding buffer containing 20 µM m-AMSA, 10 mM Ca2+ and 1 mM AMPPNP. Samples were incubated at 37°C for 30 min, then topo II[beta]-cleaved DNA complexes were trapped by the addition of EDTA to 30 mM. Topo II[beta]:four-way junction complexes were generated by incubating 40 µl of labelled four-way junction in the presence or absence of 278 nM topo II[beta] in 200 µl binding buffer on ice for 30 min. Samples were run on a Superose 6 column (Pharmacia) at 4°C in buffer containing 50 mM Tris-HCl pH 7.5, 50 mM KCl, 5% glycerol, 0.5 mM EDTA, 1% Triton X-100. Fractions were collected, and the amount of labelled DNA present was assayed by Cerenkov counting. Samples were then freeze dried and topo II[beta] was detected by western blotting with topo II[beta]-specific antiserum.

RESULTS

Topoisomerase II[beta] binds short linear duplex DNA in a gel retardation assay

A polyacrylamide gel retardation assay was utilised to study the DNA binding characteristics of topo II[beta]. The linear DNA substrate was a 40 bp duplex containing a single topo II[beta] cleavage site which corresponds to site C102 on pBR322 (Fig. 1A) (40). The ability of topo II[beta] to bind the 40 bp duplex was tested under standard gel retardation assay conditions using 1× TBE (90 mM Tris-borate, 2 mM EDTA; ~pH 8.3) as the gel running buffer. No protein-DNA complexes were observed under these conditions, however, even when the TBE concentration was lowered to 0.5 or 0.25×. In contrast, a distinct protein-DNA complex was observed (Fig. 1B, lane 2) when using running buffer with a higher pH (50 mM Tris-glycine, pH 9.2) (42). A variety of different conditions were investigated in order to maximise the stability of the protein-DNA complex. More efficient complex formation was observed when binding reactions were performed on ice rather than at 25 or 37°C, and also when gels were run at 4°C (data not shown). The inclusion of 1 mg/ml BSA significantly reduced complex dissociation during electrophoresis. Furthermore, the presence of the non-ionic detergent Triton X-100 improved complex formation by 2-7-fold, presumably by reducing aggregation of topo II[beta] molecules (45,46). The inclusion of ATP or its non-hydrolysable analogue AMPPNP did not have a significant effect on complex formation (data not shown).


Figure 1. (A) The linear DNA substrate, created by annealing two oligonucleotides C and D. Bases added by Klenow fragment are shown as lower case. The topo II[beta] cleavage site is indicated by arrows a and b. (B) Gel retardation analysis of topo II[beta] binding to linear DNA. Topo II[beta] (250 nM) was incubated on ice in the presence or absence of anti-topo II[beta] antiserum or pre-immune serum for 2 h. Labelled linear DNA was added, and incubation was continued for 30 min prior to native polyacrylamide gel electrophoresis. The topo II[beta]-DNA complex is indicated by the open arrow, whereas the super-shifted complexes are indicated by the filled arrows. Additional bands (marked by asterisks) are due to contaminating DNA binding proteins in the topo II[beta] preparation.

The presence of topo II[beta] in the protein-DNA complex was tested using topo II[beta]-specific antiserum (Fig. 1B). Pre-incubation of topo II[beta] with antiserum prior to the addition of DNA reduced the mobility of the protein-DNA complex in the polyacrylamide gel (lane 4). In contrast, pre-incubation with pre-immune serum did not decrease the mobility of the complex (lane 3). These results strongly suggest that topo II[beta] forms specific complexes with the 40 bp linear duplex.

Topo II[beta] binds a synthetic four-way junction in vitro

Four-way junctions are the branchpoint created when four helices are held together by the covalent continuity of the component strands. Holliday junctions created during recombination are a naturally occurring four-way junction (47). In the absence of divalent cations the four-way junction has an extended planar structure (48) (Fig. 2A). However, in the presence of divalent cations the junction folds by pairwise coaxial stacking to generate an anti-parallel stacked X-structure, which is analogous to two juxtaposed helices (48). There are two possible conformations of this X-structure which differ as to which arms stack upon each other (conformers I and II in Fig. 2A).


Figure 2. (A) The four-way junction, created by annealing oligonucleotides O1-4. Arms A and B have the same sequence as the linear substrate (Fig. 1A), and the arrows correspond to the positions of cleavage sites a and b in the 40 bp duplex. The equivalent positions on arms C and D are also indicated by arrows, and the presence of adenine bases immediately 3[prime] to these sites should promote m-AMSA-stimulated cleavage by topo II[beta] (40,49). In the absence of divalent cations, the four-way junction has an extended, planar structure as shown in the top diagram. When magnesium ions are added, the junction folds into a stacked X-structure which can exist as either conformer I or II (48). (B) Gel retardation analysis of topo II[beta] interactions with four-way junction DNA. Topo II[beta] (56 nM) was pre-incubated with anti-topo II[beta] antiserum or pre-immune serum on ice for 2 h, prior to the addition of four-way junction DNA. After incubation for a further 30 min, samples were analysed by native polyacrylamide gel electrophoresis.

A synthetic four-way junction was created to investigate whether topo II can bind to this type of structure (Fig. 2A). The junction was designed to promote topo II[beta] binding to its centre, but to minimise binding to the arms. In order to minimise sequence differences between the linear and four-way junction substrates, arms A and B contained the sequence of the linear duplex, with the single cleavage site straddling the central junction. The sequences of arms C and D were chosen from regions of pBR322 which do not contain strong cleavage sites (40), except that a potential m-AMSA-inducible cleavage site was created across the junction of these two arms by the introduction of two adenine bases (Fig. 2A) (40,49). The potential cleavage sites straddling the centre of the junction were designed to enable detection of topo II[beta], should it happen to bind there.

Strikingly, topo II[beta] was able to bind this synthetic four-way junction in the gel retardation assay (Fig. 2B, lane 2). The protein:four-way junction complex could be supershifted by anti-topo II[beta] antiserum (lane 4), but not by pre-immune serum (lane 3), confirming the presence of topo II[beta] in the complex. The interaction of topo II[beta] with four-way junction DNA was not strongly dependent on low temperature binding conditions or on the presence of Triton X-100, in contrast to the interaction with linear DNA (data not shown).

Topo II[beta] has a higher affinity for four-way junction DNA than linear DNA

To determine the affinity of topo II[beta] for the two DNA substrates, the efficiency of DNA binding was assayed over a range of protein concentrations. Incubation of increasing amounts of topo II[beta] with the linear 40 bp duplex resulted in a sigmoidal increase in complex formation (Fig. 3A). In the gel shown in Figure 3A, >50% of the DNA was bound when 167 nM topo II[beta] was added (lane 6). The KD was calculated as 130 nM (s.d.n-1 60 nM, n = 9). Topo II[beta] bound four-way junction DNA more strongly than the linear 40 bp duplex, as shown in Figure 3B. More than 50% of the DNA was bound in a single complex (complex a) when 21 nM of topo II[beta] was added (Fig. 3B, lane 6). Incubation with higher protein concentrations resulted in the sequential formation of two further complexes of lower mobility (complexes b and c), and a reduction in the level of complex a (lanes 8-12). The dissociation constant for topo II[beta] on four-way junction DNA was 29 nM (s.d.n-1 12 nM, n = 8). These results indicate that topo II[beta] has, on average, a 4-fold higher affinity for the four-way junction than for the 40 bp duplex under these conditions.


Figure 3. Topo II[beta] binds four-way junction DNA more strongly than linear DNA in the gel retardation assay. (A) Linear DNA was incubated with 0, 56, 83, 111, 139, 167, 194, 222, 250, 278, 333 or 389 nM topo II[beta] in lanes 1-12, respectively. (B) Four-way junction DNA was incubated with 0, 3, 7, 10, 14, 21, 28, 42, 56, 69, 83 or 111 nM topo II[beta] in lanes 1-12, respectively. Topo II[beta]:DNA complexes a, b and c are marked. Equimolar amounts of DNA were used in (A) and (B). Representative gels are shown. (C) The percentage of input DNA bound by topo II[beta] quantified from (A) and (B) [all three complexes in (B) were included] is plotted against the amount of protein added.

Topo II[beta] exists in solution predominantly as a stable dimer (5-8,10), with a small proportion of the protein forming tetramers and higher order multimers (45). Gel filtration was used to investigate the nature of the topo II[beta]-DNA complexes. Both the topo II[beta]-linear DNA complex and the smallest topo II[beta]-four-way junction complex, complex a, and eluted between protein size markers of 200 and 443 kDa (data not shown). This indicates that the topo II[beta] bound to each substrate is not comprised of tetramers or higher order multimers (720 kDa and above), but is likely to be dimeric (360 kDa). The higher order complexes of topo II[beta] with the four-way junction, complexes b and c, are probably generated by additional topo II[beta] molecules binding by protein-DNA interactions to exposed regions of the four-way junction in the initial complex.

The selectivity of DNA binding by topo II[beta]

The specificities of the topo II[beta]-DNA interactions were investigated by performing the binding reactions in the presence of non-specific competitor DNAs. Figure 4 (open circles) shows that increasing concentrations of poly(dI-dC)@poly(dI-dC) diminished the binding of topo II[beta] to the 40 bp duplex. Complex formation by 250 nM topo II[beta] was reduced by 50% in the presence of a 7-fold excess of competitor DNA. Poly(dA-dT)@poly(dA-dT), poly(dI-dC)@poly(dI-dC) and sonicated salmon sperm DNA were equally efficient at competing for topo II[beta] binding to linear DNA (data not shown). These data indicate that topo II[beta] does not have a strong selectivity towards the DNA sequence of the 40 bp duplex. In contrast to the results with the 40 bp duplex, a 50-fold excess of competitor DNA was required to reduce the binding of 250 nM topo II[beta] to the four-way junction by 50% (Fig. 4, triangles), suggesting that topo II[beta] has a higher specificity for the four-way junction than for the linear duplex. The efficiency of competition was dependent on the concentration of topo II[beta] in the reaction, as a 19-fold excess inhibited 50% of complex formation by 139 nM topo II[beta] (Fig. 4, squares), and only an 8-fold excess of competitor was required to inhibit 50% of complex formation by 56 nM topo II[beta] (Fig. 4, crosses). Due to the low specificity of topo II[beta] for the DNA substrates, and the correlation between competitor efficiency and protein concentration, subsequent experiments were carried out in the absence of competitor unless otherwise stated.


Figure 4. The effects of competitor DNA on DNA binding by topo II[beta]. Binding reactions were carried out in the presence of increasing excesses of poly(dI-dC)@poly(dI-dC) competitor, and contained: linear DNA and 250 nM topo II[beta] (open circles), four-way junction DNA and 250 nM topo II[beta] (triangles), four-way junction DNA and 139 nM topo II[beta] (squares), four-way junction DNA and 56 nM topo II[beta] (crosses). The concentration of labelled DNA was the same in each experiment. Topo II[beta]-DNA complexes were quantified as percentages of maximum complex formation, and are plotted against the fold excess of competitor DNA.

The effects of K+, Mg2+ and Ca2+ ions on DNA binding

The binding of linear DNA by topo II[beta] was dependent on the concentration of KCl in the binding reactions (Fig. 5, filled diamonds). As the KCl concentration was raised from 50 mM to 1 M, complex formation decreased by 85%. In contrast, binding of the four-way junction by topo II[beta] was much less dependent on salt concentration, with 56% of the complexes remaining at 1 M KCl (Fig. 5, open squares). The increased salt resistance of the topo II[beta]:four-way junction complex became apparent atKCl concentrations of 400 mM and above (P < 0.01). The complex of topo II[beta] with four-way junction DNA is thus significantly more salt stable than the complex with linear DNA. The effect of divalent cations on topo II[beta] binding to DNA was also studied. Topo II requires divalent cations both for ATP binding and hydrolysis, and for DNA cleavage (50). Neither Mg2+ nor Ca2+ were required for topo II[beta] binding to either the 40 bp duplex or the four-way junction, as demonstrated in Figure 3. Furthermore, inclusion of 10 mM Mg2+ or Ca2+ did not have any effect on the affinity of topo II[beta] for either DNA template, as determined by titration curves (data not shown).


Figure 5. The effect of cation concentration on DNA binding by topo II[beta]. Binding reactions were carried out at varying KCl concentrations, and contained either linear DNA and 83 nM topo II[beta] (filled diamonds) or four-way junction DNA and 42 nM topo II (open squares). Topo II[beta]-DNA complexes were quantified as percentages of maximum complex formation and are plotted against salt concentration. Points represent the means from 11 experiments carried out with different DNA preparations and varying protein concentrations (28-208 nM) in the presence and absence of competitor DNA (up to 50-fold excess). Error bars represent one standard deviation from the mean.

Topo II[beta] cleaves the centre of the four-way junction

The cleavage site which is present in the linear duplex corresponds to site C102 in pBR322. Site C102 is cleaved strongly in the presence of the topo II poison m-AMSA and is also cleaved weakly in its absence (40). Figure 6 shows that topo II[beta] can still cleave site C102 in the context of the 40 bp duplex. Under the conditions used here, cleavage in the presence of Mg2+ alone is barely detectable (Fig. 6, lane 2). The addition of the topo II poison, m-AMSA, increases cleavage significantly (Fig. 6, lane 3). The use of Ca2+ rather than Mg2+ also stimulates DNA cleavage (Fig. 6, lane 4), and cleavage is strongest in the presence of Ca2+ and m-AMSA (Fig. 6, lane 5). Similar results have been previously obtained using a related short duplex from the same region of pBR322 (51).


Figure 6. Topo II[beta] cleavage of the linear 40 bp duplex labelled at both 5[prime] ends. Topo II[beta] and the linear duplex were incubated with 10 mM Mg2+ (lanes 1-3) or 10 mM Ca2+ (lanes 4 and 5) in the presence (lanes 3 and 5) or absence (lanes 2 and 4) of 10 µg/ml m-AMSA. SDS and proteinase K were then added to denature the protein-DNA complexes. Samples were processed for sequencing gel electrophoresis as described in Materials and Methods. Arrows a and b indicate products produced by cleavage of the two DNA strands at sites a and b (Fig. 1A).

To determine whether the complex of topo II[beta] with four-way junction DNA represented a functional interaction, the ability of topo II[beta] to cleave this substrate was examined. Figure 7A shows topo II[beta] cleavage reactions carried out on the four-way junction when it was radiolabelled at the 5[prime] end of either one of the four oligonucleotides. In the absence of m-AMSA, the major cleavage sites were at C21 on oligo 1, A25 on oligo 2 and A20 on oligo 3. These three sites occur near the centre of the junction on arms A, B and C respectively (Fig. 7B), and are the same as those predicted from cleavage analyses on linear DNA sequences (Fig. 2A) (40,49). Cleavage at these sites was observed at the lowest protein concentration tested (14 nM), at which 36% of the DNA was bound in the gel retardation assay (Fig. 3B, lane 5). These results demonstrate that topo II can efficiently bind and cleave the centre of the four-way junction.


Figure 7. Topo II[beta] cleaves near the centre of the four-way junction. (A) Four-way junction DNA was labelled on oligo 1 (lanes 1-7), 2 (lanes 8-14), 3 (lanes 15-21) or 4 (lanes 22-28). Cleavage reactions contained 10 mM Ca2+ and 2 mM AMPPNP, and were carried out as in the legend to Figure 6. Samples were electrophoresed on 15% sequencing gels alongside chemical sequencing reactions performed on the same DNA substrates. Cleavage sites are identified by the base immediately 3[prime] to cleavage, and m indicates stimulation in the presence of m-AMSA. Lanes 1, 8, 15 and 22, no protein added; lanes 2, 9, 16 and 23, 14 nM topo II[beta]; lanes 3, 10, 17 and 24, 28 nM topo II[beta]; lanes 4, 11, 18 and 25, 56 nM topo II[beta]; lanes 5, 12, 19 and 26, 14 nM topo II[beta] and 20 µM m-AMSA; lanes 6, 13, 20 and 27, 28 nM topo II[beta] and 20 µM m-AMSA; lanes 7, 14, 21 and 28, 56 nM topo II[beta] and 20 µM m-AMSA. (B) The cleavage sites mapped in (A) and other similar experiments are indicated on the four-way junction. The cleavage efficiency at each site is indicated by the size of the arrow: thick, very strong cleavage; thin, medium cleavage; dotted, weak cleavage.

Weak cleavage in the absence of m-AMSA was also observed at two additional sites on oligo O2 (e.g. G12m), indicating that some of the topo II[beta] is bound to arm B, rather than at the central junction. Interestingly, the addition of m-AMSA did not further increase cleavage at the centre of the junction, in contrast to its cleavage stimulation at the equivalent sites on the linear substrate (Fig. 6). However, it did induce cleavage at additional sites on arms B and D at the higher concentrations of topo II[beta], e.g. sites C11m and G12m on oligo O2 and site C9m on oligo O4. m-AMSA is thought to intercalate between the DNA bases and the active tyrosine residue of topo II[beta] (52,53), so it is possible that the DNA structure at the centre of the junction prevents m-AMSA from binding and stimulating DNA cleavage at these sites; instead, the drug may be targeting the topo II[beta] to additional sites on the junction arms where the sequence is favourable for cleavage stimulation.

It is not possible to deduce the proportion of topo II[beta] bound at the different sites from the cleavage efficiencies, as the relationship between cleavage efficiency and DNA binding is not known, i.e. topo II[beta] may bind efficiently to sites where it can only induce weak cleavage, and vice versa. Furthermore, when topo II[beta] is bound at the centre of the junction, the topology of the protein-DNA interaction may not be optimal for cleavage due to the paths of the interchanging DNA strands. Consequently, the cleavage efficiencies at these central sites may under-represent the proportion of topo II which is bound there. Indeed, cleavage of arms A and B at the centre of the junction was ~100-fold less efficient than cleavage of the 40 bp duplex, which has the same sequence (data not shown).

DISCUSSION

We have developed conditions for a polyacrylamide gel retardation assay which allows the study of DNA binding by eukaryotic topo II. Footprinting studies have shown that topo II binds at sites where it cleaves DNA, protecting 25-28 bp from enzymatic activity (54,55). However, topo II has a very low sequence specificity, and will cleave, on average, once every 25 bp in vitro (56). A short 40 bp duplex containing a single topo II[beta] cleavage site was used, therefore, to restrict topo II[beta] to a single position and to minimise the binding of more than one dimer to the DNA. The gel retardation analysis presented here shows that topo II[beta] does form a stable complex with the 40 bp duplex. DNA binding did not require divalent cations, and was maximal at low salt concentrations, in agreement with earlier work (22,26,50,54). The KD of topo II[beta] on the linear duplex was 130 nM, which is higher than previous estimates of 10 nM for Drosophila topo II (54), and 8 nM for S.cerevisiae topo II (22).

We have shown for the first time that topo II[beta] was able to bind a four-way junction DNA substrate which contained the sequence of the linear substrate across two of its arms. Topo II[beta] had a 4-fold higher affinity for four-way junction DNA than linear DNA, with a dissociation constant of 29 nM. Eukaryotic type II topoisomerases are highly conserved, and we expect that other members of this family will display a similar preference towards four-way junction DNA. This dissociation constant is comparable to those of other four-way junction-binding proteins, for example, yeast endonuclease CCE1 (48), linker histone H1 and HMG-1 (57) have KDs of 1, 16 and 80 nM, respectively.

In the absence of m-AMSA, topo II[beta] was able to cleave the four-way junction near its centre. Topo II cleaves DNA to generate a 4 bp staggered cut, and the junction was designed such that potential cleavage sites for topo II[beta] straddled points of strand exchange. In the absence of drugs, three of the four DNA strands were cleaved by topo II[beta] at the predicted sites. Cleavage occurred predominantly on arms A and B, with weaker cleavage on arm C and no cleavage on arm D. The four-way junction exists as a stacked X structure under these reaction conditions (Fig. 2A) (48,58,59). It is not known which of the two possible conformers is preferred by this junction, although it is likely that the two forms are in dynamic equilibrium (58,59) (Fig. 2A). It is interesting that most cleavage occurred on arms A and B, as this is consistent with the predominant conformer containing A-on-B and C-on-D stacking (Fig. 2A, conformer I). Stacking of arms A and B would generate a continuous helix containing the previously identified topo II[beta] binding and cleavage site (40). The cleavage of arm C could occur by topo II[beta] binding weakly to the C-D helix and cleaving only one DNA strand. Alternatively, cleavage of arm C could occur when the four-way junction adopts the alternative conformer with A-on-D and B-on-C stacking (Fig. 2A, conformer II). Topo II[beta] could then bind preferentially to the B-C helix and cleave both the B and C arms. Coaxial stacking of the helical arms of a four-way junction has previously been demonstrated using the restriction enzyme MboII, which cleaves DNA 7 bp away from its recognition site (58). The recognition site was placed on one helical arm, and cleavage was observed 7 bp away on a second arm, which is consistent with the formation of a co-linear helix between the two arms (58).

The reaction cycle of topo II requires that the enzyme interacts with two DNA helices, the gate duplex and the transported helix. The interaction of topo II with two DNA helices is also thought to be important for both DNA cleavage (60) and ATP hydrolysis (24,61). The fact that topo II binds the four-way junction more strongly than linear DNA suggests that the four-way junction may provide both the gated and the transported helices for topo II[beta]. Topo II[beta] can cleave the four-way junction near its centre, which indicates that at least some of the enzyme is bound there.

The four-way junction in its stacked X conformation is analogous to two adjacent duplexes, and thus it is possible that the topo II[beta]-junction complex corresponds to the reaction cycle intermediate immediately prior to cleavage and strand passage by topo II, with the two juxtaposed helices corresponding to the gated and transported helices. Alternatively, the major site for topo II binding may be on one of the junction arms, where cleavage is not apparent in the absence of m-AMSA. A second arm could contribute the transported helix, thus stabilising the complex. The stability of this topo II[beta]:junction complex should make it an ideal substrate for further studies on the nature of topo II-DNA interactions, for example using protein footprinting or X-ray crystallographic techniques.

It is notable that topo II[beta] binds the four-way junction equally well in the presence or absence of magnesium ions, despite the fact that the structure of the junction is different under the two conditions (48). Junction-resolving endonucleases also bind four-way junctions independently of divalent cations, and they all appear to distort the junction to generate novel structures (reviewed in 48; 62). In each case, the junction is unstacked or unfolded by the protein, possibly to allow access to the scissile bonds by the enzyme active sites. If topo II[beta] binds predominantly at the centre of the junction, it could distort the junction in a cation-independent manner upon binding. Alternatively, the topo II dimer may be flexible enough to accommodate both the planar and the stacked X forms of the junction. Indeed, topo II has been shown to cleave a variety of DNA structures, including Z DNA, single stranded DNA containing a hairpin, and parallel-stranded tetraplex DNA (32,33,63).

The observation that topo II[beta] binds efficiently to the four-way junction supports previous suggestions that the enzyme may be targeted to supercoiled DNA through the recognition of DNA crossovers (17,18,26-28). Whether the two helices of DNA bound at a crossover represent the gate and transported segments poised for strand passage is not clear, however. It has been proposed that topo II may in fact have three DNA binding sites, two for binding helix-helix crossovers, and one for binding the transported helix (64). Topo II was shown to simplify DNA topology beyond that found at thermodynamic equilibrium, and the three DNA binding site model was suggested as a mechanism whereby topo II may assay DNA topology (64).

The ability of topo II to bind two helices simultaneously has important implications for its roles in segregating chromosomes at mitosis, and in anchoring chromatin loops to the chromosome scaffold (reviewed in 65). Interestingly, topo II has been shown to be a component of the Drosophila chromatin accessibility complex, CHRAC, along with ISWI (66). The strand passage activity of topo II did not appear to be required for chromatin remodelling by CHRAC, so it is possible that topo II is important for CHRAC DNA binding, or for interactions with other proteins (66). It has been suggested that the region where DNA enters and exits the nucleosome is structurally similar to a four-way junction, and indeed the linker histones H1 and H5 bind four-way junctions and DNA crossovers more strongly than linear DNA (67-69). Interestingly, the yeast chromatin remodelling complex, SWI/SNF, binds to nucleosomes and can also bind to DNA cruciforms (70).

ACKNOWLEDGEMENTS

We are grateful to A. G. West and J. Quinn for invaluable advice and assistance, and for critical reading of the manuscript. We would also like to thank L. M. Fisher for assistance with the preparation of this paper. We acknowledge financial support from the BBSRC, North of England Childrens Cancer Research Fund, and Wellcome Grant 044848.

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*To whom correspondence should be addressed. Tel: +44 191 222 8864; Fax: +44 191 222 7424; Email: caroline.austin@ncl.ac.uk
+Present address: Laboratory of Molecular Carcinogenesis, National Cancer Institute, National Institutes of Health, Bethesda, MD 20892, USA

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