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Eukaryotic topoisomerase II cleavage is independent of duplex DNA conformation
BB
Biochim~ic~a etBiophysicaA~ta
iz;,! ELSEVIER
Biochimica et Biophysica Acta 1264 (1995) 209-214
Eukaryotic topoisomerase II cleavage is independent of duplex DNA conformation In Young Choi a In Kwon Chung a, *, Mark T. Muller
b
a Department of Biology, College of Science, Yonsei University, Seoul, 120-749, South Korea b Department of Molecular Genetics, The Ohio State Universi~., Columbus, OH 43210, USA Received 24 April 1995; accepted 27 June 1995
Abstract
Alternating purine-pyrimidine (RY) repeats have been identified in naturally occurring DNA and have many intriguing properties. Eukaryotic topoisomerase II displays significant cleavage activity at RY repeats (Spitzner et al. (1990) Nucleic Acids Res. 18, 1-11) due to the homology between RY repeat and the topoisomerase II consensus sequence. Cleavages are remarkably strong on duplex B form DNA. Certain RY elements are known to adopt altered DNA forms, such as Z-DNA, under the influence of superhelical stress. To investigate the dependence of topoisomerase II activity on DNA conformation, a plasmid containing a 40 bp of deoxyguanine-thymine repeat was constructed and the dependence of topoisomerase II cleavage patterns were compared. Although the degree of negative supercoiling strongly affected the overall efficiency of topoisomerase II cleavage, the sequence specificity was identical over a wide range of superhelical densities. These results suggest that topoisomerase II site specific action on duplex DNA is largely independent of DNA conformation. Moreover, since the GT target sequence is known to adopt a Z-DNA structure under conditions of superhelical density used in these experiments, the results reveal that topoisomerase II is a DNA binding protein capable of recognizing Z-DNA structure in eukaryotic cell. Keywords: Topoisornerase II; Non-B DNA structure; Z-DNA; Alternating purine-pyrimidine repeat; Etoposide
1. Introduction
It is now well established that DNA has a structurally polymorphic nature [1]. Alternating purine-pyrimidine repeats (which we refer to as RY repeats) are known to adopt unusual DNA structures such as cruciform and Z-DNA forms under physiological conditions [2-4]. ZDNA is of particular interest in considering the relationship between the negative superhelicity of DNA and the formation of non-B DNA structures. Because of the higher free energy of supercoiled DNA, alternating deoxyguanine-thymine (GT) repeats undergo local transitions from the right-handed B-DNA form into the left-handed Z-DNA form resulting in a favorable reduction in superhelicity. Since Z-DNA has been identified in crystalline systems [2] and biological systems [5], much progress has been made into the structural and chemical aspects of Z-DNA confor-
* Corresponding author. Fax: +822 312 5657. 0167-4781/95/$09.50 © 1995 Elsevier Science B.V. All rights reserved SSDI 0 1 6 7 - 4 7 8 1 ( 9 5 ) 0 0 1 4 4 - 1
mation. However, the biological significance of this structure in vivo is not known. Recently, 329 potential Z-DNA forming sequences were mapped in over one megabase pairs of human DNA encompassing 137 complete genes [6]. The distribution of these sequences relative to the location of the genes is distinctly nonrandom, being strongly clustered in the Y-end of the genes and in the promoter regions. Based upon an extensive data base of sequenced topoisomerase II cleavage sites, alternating RY repeats display considerable homology to the consensus sequence for eukaryotic topoisomerase II [7]. In fact, the repeat nature of GT (and other RY repeats) explains why topoisomerase II cleavages are clustered at RY elements with cleavages detected at every other base in repeats over 10 bp in length [8]. DNA topoisomerases transiently break and reseal the backbone bond of DNA, altering the linking number and allowing adjustment of DNA topology [9,10]. Biologically, topoisomerase II has been implicated in a variety of cellular processes such as transcription [11,12], DNA replica-
210
LY. Choi et al. / Biochimica et Biophysica Acta 1264 (1995) 209-214
tion and segregation of DNA molecules prior to cell division [13-16]. In terms of the enzymatic activity, an important feature of these enzymes is their ability to discriminate between various DNA sequences and DNA structures at which breakage/rejoining of DNA strands proceeds. In particular, topoisomerase II does not act on single strand DNA (that is not annealed); however, topoisomerase II recognized and cleaved single stranded substrates when annealed to form a novel structure characterized as parallel tetraplex DNA [17]. Glikin et al. reported that Drosophila topoisomerase II showed a significantly higher affinity for the mini-CG minicircle containing Z-DNA than the control minicircle lacking the (CG) 7 insert [18]. Although the enzyme formed more stable covalent complexes with mini-CG plasmid in which Z-conformation was predominant, specific breakage points were not detected. In this report, we have evaluated sequence specificity of topoisomerase II at a structurally polymorphic DNA sequence (40 bp of alternating GT) at different superhelical densities.
2.3. Topoisomerase H cleavage reaction
2. Materials and methods
3. Results
2.1. Plasmids and reagents
The plasmid pGT-40 was constructed as follows: two double-stranded fragments consisting of 18 bp-homoguanine and homocytosine oligonucleotides were ligated and replaced by a KpnI-BamHI fragment of pBS such that a new Smal site was generated in the middle of the 18 bp fragments (called pGT-0). To construct plasmid pGT-40, two oligonucleotides containing 40 bp-alternating deoxycytosine-adenine and deoxyguanine-thymine repeat, respectively, were annealed and cloned into the new Smal site of pGT-0 (Fig. 1A). Calf thymus topoisomerase I, human topoisomerase II and VP16 (etoposide) were obtained from TopoGEN (Columbus, OH). Radioactive nucleotides were purchased from Amersham International and restriction enzymes, T4 DNA ligase, and Klenow fragment of DNA polymerase I were from Promega. 2.2. Preparation of topoisomers and two-dimensional gel
electrophoresis
The topoisomers were prepared by incubating 10 /zg of plasmid DNA with increasing amount of ethidium bromide (0-15 /zM) in the presence of calf thymus topoisomerase I as previously described [19]. A mixture of topoisomers was loaded on a 1.5% agarose gel. In the first dimension, the gel was run for 18 h at 3.6 V / c m in TBE buffer (100 mM Tris-boric acid and 1 mM EDTA, pH 8.3). In the second dimension, the gel was rotated 90 ° to the first and was run under the same conditions except in the presence of 5 /xM chloroquine. Buffer was recirculated during electrophoresis.
For nucleotide level mapping of cleavage sites, the reactions (final volume of 20/xl) contained cleavage buffer (30 mM Tris-HC1, pH 7.6, 60 mM KC1, 8 mM MgC12, 15 mM 2-mercaptoethanol, 3 mM ATP, 3 0 / x g / m l BSA), and the end labeled fragment (usually ( 1 - 3 ) . l04 DPM). Topoisomerase II inhibitor, VP16 (etoposide), was added at a final concentration of 500 /xM and the reactions were initiated by addition of the 25 ng of purified human topoisomerase II (p170), incubated 30 min at 30°C and terminated by addition of SDS to 1% followed by incubation with 50 /xg/ml proteinase K at 56°C for 30 min. For indirect end-labeling mapping, whole plasmids (1 /xg) were incubated with topoisomerase II in the presence or absence of etoposide. The purified samples were digested with ScaI and electrophoresed on a native 1.5% agarose gel. The DNA was transferred to nitrocellulose and was probed with a 357 bp of ScaI-BsmAI fragment (see Fig. 1A for location of the probe).
3.1. Structural transition of pGT-40 to a left-handed Z-DNA
Topoisomers were prepared from pGT-40 and analyzed by two-dimensional gel electrophoresis in order to reconstruct the superhelical density-dependent transition of Bto Z-DNA [19]. As shown in Fig. 1B, a structural transition was observed from topoisomer number 12 ( - 0 . 0 4 2 of superhelical density) to topoisomer number 14 ( - 0.049 of superhelical density). Beyond topoisomer number 14, the plasmid exists in a Z-DNA form (Fig. 1C). 3.2. Sequencing of topoisomerase H sites on the GT repeat of pGT-40
Topoisomerase II is highly reactive at alternating RY repeat sequences because the consensus recognition site is reiterated at every other base [7]. We sequenced topoisomerase II cleavage sites using a DNA fragment containing the GT repeat plus flanking sequences from pGT-40 (Fig. 2). In the presence of topoisomerase II inhibitor, etoposide, the enzyme cleaved the GT repeats at every other base; however, cleavages did not extend over the entire length of the repeat because the flanking GT sequences come under the influence of adjacent non-RY sites and the consensus match breaks down. Consistent with this idea is our finding that longer repeats gave longer runs of cleavages [7] (data not shown). In the absence of inhibitor, the intensities of cleavage bands within GT repeat were markedly reduced; however, the specificity of cleavage pattern was not altered. These results show that cleavage sites within the GT repeat have essentially the same sequence speci-
1.E Choi et al. / Biochimica et Biophysica Acta 1264 (1995) 209-214
A (GT)20 GTGTGTGTGTGTGTGTGTGTGTGTGTGTGTGTGTGTGTGT
B
C
211
In the presence of etoposide, pBS and pGT-40 showed identical cleavage patterns except that pGT-40 had an additional strong cleavage site which corresponded to the site of the GT repeat (Fig. 3B, marked by arrows). Cleavages were extremely weak in the absence of etoposide (see lanes 1, 2, 5 and 6) as expected. When supercoiled plasmid was used as input DNA in the absence of ATP, the cleavage site was mapped within the GT repeat (lanes 7, marked by arrow). Although the superhelical conformation of input plasmids did not affect the overall cleavage patterns (compare lanes 7 and 8), the supercoiled substrates enhanced the cleavage efficiency of the enzyme without altering the specificity at the resolution of agarose gels. These results were consistent with previous findings that Drosophila topoisomerase II interacted preferentially with supercoiled DNA over relaxed or nicked circular molecules in the absence of a nucleoside triphosphate cofactor [20]. 3.4. Titration o f topoisomerase H cleavages as a function o f enzyme concentration Although the enzyme cleaved both B- and Z-DNA structures at GT repeats, we wished to know whether more G + : Etoposide G A - + + : Topoll 1 2345
Fig. 1. Two-dimensional gel electrophoresis analysis of pGT-40. (A) Schematic diagram of pGT-40; see text for details of construction. The circle indicates the location of (GT)20 repeat of pGT-40 and probe used in this experiment. (B) Two-dimensional agarose gel electrophoresis was used to determine the superhelical density at which Z-DNA formed in plasmid DNA. Topoisomer numbers are indicated. (Panel C) pGT-40 at physiological superhelicity was analyzed on the same gel. ficity in the presence and absence of the topoisomerase II poison, etoposide. 3.3. Effects o f DNA conformation on topoisomerase H cleavage activi~ Since topoisomerase II cleavage sites are strongly clustered at GT repeats, we next determined whether topoisomerase II cleavage patterns were the same in alternative conformations of plasmid. Relaxed or supercoiled pGT-40 DNA targets were compared. Fig. 3A shows that there is no relaxation of the supercoiled form by topoisomerase II in the absence of ATP (compare lanes 1 and 5,3 and 7), although topoisomerase II cleavages were readily detected on both relaxed and supercoiled templates (lanes 7 and 8). Topoisomerase II cleavage reactions on intact plasmid DNA were performed and the cleavage sites were mapped by indirect end-labeling to compare the reactivity of the enzyme at the GT repeat of B- and Z-DNA. Topoisomerase II cleavages either in the presence or absence of etoposide were catalogued on pBS and pGT-40 (Fig. 3B).
(GT)20
Fig. 2. Sequencing topoisomerase II cleavage sites on (GT)20 repeat. Cleavage reactions were performed on a KpnI-SacI fragment (labeled 5' end at KpnI site) of pGT-40 as described in Section 2. Samples were loaded onto 8% sequencing gel. Lanes 1 and 2 are chemical sequencing marker: lane 3 is fragment alone: lanes 4 and 5 contained topoisomerase II in the absence and presence of etoposide, respectively. The position of the (GT)20 repeat is indicated by a bracket.
1. Y. Choi et al. / Biochimica et Biophysica Acta 1264 (1995) 209-214
212
,6, DNAform: I Ir I Ir I Ir ATP: + + + + . . . Etoposide: + + 123456
I Ir IIl . + + 789
linear supercoiled
~
relaxed B
pGT-40 r
I
pBS F-3
D N A f o r m : I I r 1 I r 1 Ir I Ir I Ir ATP:+ + + + . . . . . . Etoposide: _ _ + + _ _ + + + + M 1 2 3 4
5
6 7
8
9 10 11
Fig. 3. Mapping topoisomerase II sites in pGT-40 by indirect end-labeling. Cleavage reactions were performed on whole plasmid in the presence or absence of etoposide. (A) Following termination of the reactions, the samples were purified and loaded onto a 1.2% agarose gel containing 0.5 ~ g / m l concentration of ethidium bromide. The reaction conditions are indicated above the gel. (B) For the indirect-end labeling, the purified samples were digested with ScaI and electrophoresed on a native 1.5% agarose gel. The DNA was transferred to nitrocellulose for Southern blotting. In odd numbered lanes the input plasmid was supercoiled (indicated as I) and in even numbered lanes the input plasmid was relaxed (indicated as Ir). The arrow points to the cleavage site which corresponds to the location of the (GT)z0 repeat. Lane M contained a SmaI-Scal fragment of pBS as size reference of GT repeat site. The bands shown in lanes 5 and 6 were due to partial restriction digestion and were not seen in a repeat experiment.
relative band intensities. Although there is a minor variation of the cleavage efficiency in lane 8 as compared to lane 3 of Fig. 4, cleavage at control site was 1.68-fold enhanced in a supercoiled target (compare lanes 2 - 5 and 7-10). The enzyme also cleaved GT repeat (indicated by Z-DNA) when it existed both as Z-conformation and as B-conformation. The GT repeat of supercoiled DNA was cleaved 2.1-fold better than the corresponding site in relaxed DNA. These results indicate that the cleavage bias in supercoiled DNA exists at GT and non-GT repeats to about the same extent.
3.5. Titration of topoisomerase H cleavages as a function of negatiue superhelical density Two-dimensional gel electrophoresis data show that the B to Z equilibrium of pGT-40 was shifted toward Z-DNA at physiological superhelical densities (Fig. 1C). Since the structural transition of B- to Z-DNA is energetically favored in a more negatively supercoiled DNA, we compared targets of varying levels of negative superhelical density. Seven topoisomer populations of pGT-40 (having different average negative superhelical density ranging from 0 to - 0 . I ) were prepared and the cleavages were carried out to compare how different conformational states affect recognition of the GT repeat (Fig. 5). As the superhelical density of input pGT-40 was increased, we found a proportional increase in topoisomerase II cleavage activity in whole plasmid. Moreover, enzyme cleavages were es-
I
Supercoiled pGT-40
Relaxed pGT-40
Topo[l
Topoll
2 3 4
5
6 7
8 9 10 11 12
Control
subtle distinctions existed between the reactivity of the enzyme and the conformation of the substrate. To address this, we carried out an indirect end-labeling experiment utilizing constant DNA substrate (supercoiled or relaxed) and variable amount of enzyme. The reactions contained excess DNA; therefore, sites (or structures) with the highest enzyme cleavage avidity would be favored. All reactions were performed in the absence of ATP and contained the non-intercalating inhibitor, etoposide. As shown in Fig. 4, cleavages were strongly enhanced on negatively supercoiled template. The cleavage efficiencies at different sites were compared. Two cleavage bands from GT repeat (indicated by Z-DNA) and non-GT site (indicated by control) were scanned by densitometry to measure the
Z-DNA
Fig. 4. Titration of topoisomerase II cleavages on the relaxed or supercoiled pGT-40 as a function of the enzyme concentration. Cleavage reactions were performed on whole plasmid in the absence of ATP. The supercoiled plasmid (1 /xg, lanes 1-5) or relaxed plasmid (1 /~g, lanes 6-10) was incubated with 0, 12, 25, 50, 100 ng of purified human topoisomerase II, respectively. The cleavage site at GT repeat was indicated by Z-DNA and the cleavage sites of non-GT repeat was indicated by control. Lane M contained a SmaI-ScaI fragment of pBS as size reference of GT repeat site.
1. Y. Choi et al. / Biochimica et Biophysica Acta 1264 (1995) 209-214
pGT-40 I
- ~ xl02: MI
0
I
re?
1.5 2.5 3.5 4.5 7.5 10 m. 2 3 4 5 6 78
Z-DNA
Fig. 5. Titration of topoisomerase II cleavages as a function of negative superhelical density. Cleavage reactions were performed on 1 /zg of pGT-40 topoisomers with the indicated average superhelical density. The cleavage site at GT repeat was indicated by Z-DNA. Lane M contained a Srnal-ScaI fragment of pBS as size reference of GT repeat site. Lanes 1-7 contained plasmids with the different levels of superhelical density values as designated on the top of each lane. Lane 8 contained pBS as input plasmid.
sentially identical in GT and non-GT flanks, based upon densitometry. These results confirm that topoisomerase II recognizes and cleaves a left-handed Z-DNA about as efficiently as B-DNA.
4. D i s c u s s i o n
We have investigated the effects of the superhelical-dependent structural transition of alternating purine-pyrimidine repeats on topoisomerase II cleavages. The binding and cleavage of topoisomerase II with minicircles containing Z - D N A inserts were recently reported by Glikin et al. [18]. Although topoisomerase II showed significantly higher affinity for the mini-CG minicircle containing ZDNA than the control mini construct lacking the (CG) 7 insert, a specific cleavage in the Z - D N A sequence was not detected in this system [18]. To further demonstrate the reactivity of topoisomerase II with Z-DNA structure, we performed the cleavage reactions on plasmid pGT-40 containing 40 bp of alternating GT. Two-dimensional gel electrophoresis data (Fig. 1C) show that this plasmid undergoes a conformational transition to Z-DNA form at physiological levels of negative superhelicity. Recently, Roca and Wang have proposed that eukaryotic topoisomerase II acts as an ATP-dependent molecular clamp; without ATP binding and hydrolysis, strand passage can not be completed by the enzyme [21]. When the supercoiled plasmids were used for enzyme substrates in the absence of ATP, all cleavage products must have originated from the supercoiled D N A since that is the only target available when the reactions were initiated. Under
213
these conditions, we were able to detect whether the topoisomerase II cleavages occurred at the GT repeat when it existed in a non-B D N A state. As shown in Fig. 3, the specific cleavages were mapped at the GT repeats when input plasmids were negative supercoiled. At physiological levels of negative superhelicity, it is likely that 'bubbles' of Z - D N A exist in equilibrium with B-DNA throughout. To push the equilibrium toward the Z-DNA form, more negatively supercoiled plasmids ( - 0.1 of superhelical density) were prepared. As superhelical density increases, the Z-helix propagates throughout the GT repeat by successive B - Z transitions. The results shown in Fig. 5 demonstrate that cleavage activity at GT and non-GT sites is equally dependent on the superhelical conformation. In other words, topoisomerase II cleavages are conformationally independent in the context of the superhelical dependent transitions in the GT insert. In negatively supercoiled DNA targets, both Z-DNA and B-DNA appear to be equally attractive as topoisomerase II cleavage sites. An important feature of the enzymatic activity of topoisomerase II is its ability to recognize a wide variety of unusual DNA conformations. This enzyme recognizes DNA regions of topoisomerase I1 consensus sequence which exist in non-B DNA forms, such as cruciform, Z-DNA, curved DNA [23] and parallel-stranded tetraplex DNA [17], forming stable covalent complexes. In contrast, topoisomerase II strongly impeded at regions of triplex DNA structures [8]. Recently, the binding activities of the 170 kDa and 180 kDa human topoisomerase II to curved and linear Z-DNA fragments were characterized, indicating that both enzymes showed higher affinity for these unusual DNA structures [23]. DNA structures that eukaryotic topoisomerase II acts upon are summarized in Table 1. In conclusion, our data show that eukaryotic topoisomerase II binds and cleaves DNA regions with left-handed Z-DNA conformation. These results suggest that eukaryotic topoisomerase II is an example of a eukaryotic protein which recognizes the Z-DNA. The structural features of a left-handed helix are therefore likely to be compatible with the catalytic cycle of DNA breakage, strand passage and resealing (although we have only tested the cleavage step in our experiments). Finally, while topoisomerase II cleav-
Table 1 DNA structures that eukaryotic topoisomerase I1 acts upon DNA structures Topoisomerase II References activity Duplex DNA in B form Duplex DNA in Z form Curved DNA Single strand DNA in hairpin Parallel strand DNA tetraplex Single strand DNA, not annealed Triplex DNA
yes yes yes yes yes no no
[18,23,24] [23] [25] [ 17,22] [17] [8]
214
LY. Choi et al. / Biochimica et Biophysica Acta 1264 (1995) 209-214
age activity is clearly stimulated by negative supercoiling, we did not detect alterations in sequence specificity as the DNA conformation was modulated. For this reason and based upon our previous studies [17], we conclude that topoisomerase II cleavage activity is largely independent of duplex DNA conformation.
Acknowledgements We thank Dr. J.R. Spitzner and Mr. J.M. Suh for helpful discussion and advice. This work was supported by grant from KOSEF, Korea, 1994, project 941-0500-026-1.
References [1] Wells, R.D. (1988) J. Biol. Chem. 263, 1095-1098. [2] Wang, A.H.J., Quigley, G.J., Crawford, J.L., Van Boom, J.H., Van der Marel, G. and Rich, A. (1979) Nature 282, 680-686. [3] Nordheim, A. and Rich, A. (1983) Nature 303, 674-678. [4] Rich, A., Nordheim, A. and Wang, A.H.J. (1984) Annu. Rev. Biochem. 53, 791-846. [5] Nordheim, A., Pardue, M.L., Lafer, E.M., Moiler, A., Stollar, B.D. and Rich, A. (1981) Nature 294, 417-422. [6] Schroth, G.P., Chou, P.J. and Shing Ho, P. (1992) J. Biol. Chem. 267, 11846-11855.
[7] Spitzner, J.R., Chung, I.K. and Muller, M.T. (1990) Nucleic Acids Res. 18, 1-11. [8] Spitzner, J.R., Chung, I.K. and Muller, M.T. (1995) J. Biol. Chem. 270, 5932-5943. [9] Vosberg, H.P. (1985) Curt. Top. Microbiol. Immunol. 114, 19-102. [10] Wang, J.C. (1985) Annu. Rev. Biochem. 54, 665-697. [11] Muller, M.T. and Mehta, V.B. (1988) Mol. Cell. Biol. 8, 3661-3669. [12] Liu, L.F. and Wang, J.C. (1984) Proc. Natl. Acad. Sci. USA 84, 7024-7027. [13] Uemura, T. and Yanagida, M. (1984) EMBO J. 3, 1737-1744. [14] DiNardo, S, Voelkel, K. and Sternglanz, R. (1984) Proc. Natl. Acad. Sci. USA 81, 2616-2620. [15] Holm, C., Goto, T., Wang, J.C. and Botstein, D. (1985) Cell 41, 553-563. [16] Snapka, R.M., Powelson, M.A. and Strayer, J.M. (1988) Mol. Cell. Biol. 8, 515-521. [17] Chung, I.K., Mehta, V.B., Spitzner, J.R. and Muller, M.T. (1992) Nucleic Acids Res. 20, 1973-1977. [18] Glikin, G.C., Jovin, T.M. and Arndt-Jovin, D.J. (1991) Nucleic Acids Res. 19, 7139-7144. [19] Albert, A.C., Roman, A.M., Bouche, G., Leng, M. and Rahmouni, A.R. (1994) J. Biol. Chem. 269, 19238-19244. [20] Osheroff, N. (1986) J. Biol. Chem. 261, 9944-9950. [21] Roca, J. and Wang, J.C. (1994) Cell 77, 609-616. [22] Chung, I.K. and Muller, M.T. (1991) J. Biol. Chem. 266, 9508-9514. [23] Bechert, T., Diekmann, S. and Arndt-Jovin, D.J. (1994) J. Biomol. Str. Dynamics 12, 605-619. [24] Arndt, D.J., Udvardy, A., Garner, M., Ritter, S. and Jovin, T.M. (1993) Biochemistry 32, 4862-4872. [25] Gale, K. and Osheroff, N. (1990) Biochemistry 29, 9538-9545.
Biochim~ic~a etBiophysicaA~ta
iz;,! ELSEVIER
Biochimica et Biophysica Acta 1264 (1995) 209-214
Eukaryotic topoisomerase II cleavage is independent of duplex DNA conformation In Young Choi a In Kwon Chung a, *, Mark T. Muller
b
a Department of Biology, College of Science, Yonsei University, Seoul, 120-749, South Korea b Department of Molecular Genetics, The Ohio State Universi~., Columbus, OH 43210, USA Received 24 April 1995; accepted 27 June 1995
Abstract
Alternating purine-pyrimidine (RY) repeats have been identified in naturally occurring DNA and have many intriguing properties. Eukaryotic topoisomerase II displays significant cleavage activity at RY repeats (Spitzner et al. (1990) Nucleic Acids Res. 18, 1-11) due to the homology between RY repeat and the topoisomerase II consensus sequence. Cleavages are remarkably strong on duplex B form DNA. Certain RY elements are known to adopt altered DNA forms, such as Z-DNA, under the influence of superhelical stress. To investigate the dependence of topoisomerase II activity on DNA conformation, a plasmid containing a 40 bp of deoxyguanine-thymine repeat was constructed and the dependence of topoisomerase II cleavage patterns were compared. Although the degree of negative supercoiling strongly affected the overall efficiency of topoisomerase II cleavage, the sequence specificity was identical over a wide range of superhelical densities. These results suggest that topoisomerase II site specific action on duplex DNA is largely independent of DNA conformation. Moreover, since the GT target sequence is known to adopt a Z-DNA structure under conditions of superhelical density used in these experiments, the results reveal that topoisomerase II is a DNA binding protein capable of recognizing Z-DNA structure in eukaryotic cell. Keywords: Topoisornerase II; Non-B DNA structure; Z-DNA; Alternating purine-pyrimidine repeat; Etoposide
1. Introduction
It is now well established that DNA has a structurally polymorphic nature [1]. Alternating purine-pyrimidine repeats (which we refer to as RY repeats) are known to adopt unusual DNA structures such as cruciform and Z-DNA forms under physiological conditions [2-4]. ZDNA is of particular interest in considering the relationship between the negative superhelicity of DNA and the formation of non-B DNA structures. Because of the higher free energy of supercoiled DNA, alternating deoxyguanine-thymine (GT) repeats undergo local transitions from the right-handed B-DNA form into the left-handed Z-DNA form resulting in a favorable reduction in superhelicity. Since Z-DNA has been identified in crystalline systems [2] and biological systems [5], much progress has been made into the structural and chemical aspects of Z-DNA confor-
* Corresponding author. Fax: +822 312 5657. 0167-4781/95/$09.50 © 1995 Elsevier Science B.V. All rights reserved SSDI 0 1 6 7 - 4 7 8 1 ( 9 5 ) 0 0 1 4 4 - 1
mation. However, the biological significance of this structure in vivo is not known. Recently, 329 potential Z-DNA forming sequences were mapped in over one megabase pairs of human DNA encompassing 137 complete genes [6]. The distribution of these sequences relative to the location of the genes is distinctly nonrandom, being strongly clustered in the Y-end of the genes and in the promoter regions. Based upon an extensive data base of sequenced topoisomerase II cleavage sites, alternating RY repeats display considerable homology to the consensus sequence for eukaryotic topoisomerase II [7]. In fact, the repeat nature of GT (and other RY repeats) explains why topoisomerase II cleavages are clustered at RY elements with cleavages detected at every other base in repeats over 10 bp in length [8]. DNA topoisomerases transiently break and reseal the backbone bond of DNA, altering the linking number and allowing adjustment of DNA topology [9,10]. Biologically, topoisomerase II has been implicated in a variety of cellular processes such as transcription [11,12], DNA replica-
210
LY. Choi et al. / Biochimica et Biophysica Acta 1264 (1995) 209-214
tion and segregation of DNA molecules prior to cell division [13-16]. In terms of the enzymatic activity, an important feature of these enzymes is their ability to discriminate between various DNA sequences and DNA structures at which breakage/rejoining of DNA strands proceeds. In particular, topoisomerase II does not act on single strand DNA (that is not annealed); however, topoisomerase II recognized and cleaved single stranded substrates when annealed to form a novel structure characterized as parallel tetraplex DNA [17]. Glikin et al. reported that Drosophila topoisomerase II showed a significantly higher affinity for the mini-CG minicircle containing Z-DNA than the control minicircle lacking the (CG) 7 insert [18]. Although the enzyme formed more stable covalent complexes with mini-CG plasmid in which Z-conformation was predominant, specific breakage points were not detected. In this report, we have evaluated sequence specificity of topoisomerase II at a structurally polymorphic DNA sequence (40 bp of alternating GT) at different superhelical densities.
2.3. Topoisomerase H cleavage reaction
2. Materials and methods
3. Results
2.1. Plasmids and reagents
The plasmid pGT-40 was constructed as follows: two double-stranded fragments consisting of 18 bp-homoguanine and homocytosine oligonucleotides were ligated and replaced by a KpnI-BamHI fragment of pBS such that a new Smal site was generated in the middle of the 18 bp fragments (called pGT-0). To construct plasmid pGT-40, two oligonucleotides containing 40 bp-alternating deoxycytosine-adenine and deoxyguanine-thymine repeat, respectively, were annealed and cloned into the new Smal site of pGT-0 (Fig. 1A). Calf thymus topoisomerase I, human topoisomerase II and VP16 (etoposide) were obtained from TopoGEN (Columbus, OH). Radioactive nucleotides were purchased from Amersham International and restriction enzymes, T4 DNA ligase, and Klenow fragment of DNA polymerase I were from Promega. 2.2. Preparation of topoisomers and two-dimensional gel
electrophoresis
The topoisomers were prepared by incubating 10 /zg of plasmid DNA with increasing amount of ethidium bromide (0-15 /zM) in the presence of calf thymus topoisomerase I as previously described [19]. A mixture of topoisomers was loaded on a 1.5% agarose gel. In the first dimension, the gel was run for 18 h at 3.6 V / c m in TBE buffer (100 mM Tris-boric acid and 1 mM EDTA, pH 8.3). In the second dimension, the gel was rotated 90 ° to the first and was run under the same conditions except in the presence of 5 /xM chloroquine. Buffer was recirculated during electrophoresis.
For nucleotide level mapping of cleavage sites, the reactions (final volume of 20/xl) contained cleavage buffer (30 mM Tris-HC1, pH 7.6, 60 mM KC1, 8 mM MgC12, 15 mM 2-mercaptoethanol, 3 mM ATP, 3 0 / x g / m l BSA), and the end labeled fragment (usually ( 1 - 3 ) . l04 DPM). Topoisomerase II inhibitor, VP16 (etoposide), was added at a final concentration of 500 /xM and the reactions were initiated by addition of the 25 ng of purified human topoisomerase II (p170), incubated 30 min at 30°C and terminated by addition of SDS to 1% followed by incubation with 50 /xg/ml proteinase K at 56°C for 30 min. For indirect end-labeling mapping, whole plasmids (1 /xg) were incubated with topoisomerase II in the presence or absence of etoposide. The purified samples were digested with ScaI and electrophoresed on a native 1.5% agarose gel. The DNA was transferred to nitrocellulose and was probed with a 357 bp of ScaI-BsmAI fragment (see Fig. 1A for location of the probe).
3.1. Structural transition of pGT-40 to a left-handed Z-DNA
Topoisomers were prepared from pGT-40 and analyzed by two-dimensional gel electrophoresis in order to reconstruct the superhelical density-dependent transition of Bto Z-DNA [19]. As shown in Fig. 1B, a structural transition was observed from topoisomer number 12 ( - 0 . 0 4 2 of superhelical density) to topoisomer number 14 ( - 0.049 of superhelical density). Beyond topoisomer number 14, the plasmid exists in a Z-DNA form (Fig. 1C). 3.2. Sequencing of topoisomerase H sites on the GT repeat of pGT-40
Topoisomerase II is highly reactive at alternating RY repeat sequences because the consensus recognition site is reiterated at every other base [7]. We sequenced topoisomerase II cleavage sites using a DNA fragment containing the GT repeat plus flanking sequences from pGT-40 (Fig. 2). In the presence of topoisomerase II inhibitor, etoposide, the enzyme cleaved the GT repeats at every other base; however, cleavages did not extend over the entire length of the repeat because the flanking GT sequences come under the influence of adjacent non-RY sites and the consensus match breaks down. Consistent with this idea is our finding that longer repeats gave longer runs of cleavages [7] (data not shown). In the absence of inhibitor, the intensities of cleavage bands within GT repeat were markedly reduced; however, the specificity of cleavage pattern was not altered. These results show that cleavage sites within the GT repeat have essentially the same sequence speci-
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A (GT)20 GTGTGTGTGTGTGTGTGTGTGTGTGTGTGTGTGTGTGTGT
B
C
211
In the presence of etoposide, pBS and pGT-40 showed identical cleavage patterns except that pGT-40 had an additional strong cleavage site which corresponded to the site of the GT repeat (Fig. 3B, marked by arrows). Cleavages were extremely weak in the absence of etoposide (see lanes 1, 2, 5 and 6) as expected. When supercoiled plasmid was used as input DNA in the absence of ATP, the cleavage site was mapped within the GT repeat (lanes 7, marked by arrow). Although the superhelical conformation of input plasmids did not affect the overall cleavage patterns (compare lanes 7 and 8), the supercoiled substrates enhanced the cleavage efficiency of the enzyme without altering the specificity at the resolution of agarose gels. These results were consistent with previous findings that Drosophila topoisomerase II interacted preferentially with supercoiled DNA over relaxed or nicked circular molecules in the absence of a nucleoside triphosphate cofactor [20]. 3.4. Titration o f topoisomerase H cleavages as a function o f enzyme concentration Although the enzyme cleaved both B- and Z-DNA structures at GT repeats, we wished to know whether more G + : Etoposide G A - + + : Topoll 1 2345
Fig. 1. Two-dimensional gel electrophoresis analysis of pGT-40. (A) Schematic diagram of pGT-40; see text for details of construction. The circle indicates the location of (GT)20 repeat of pGT-40 and probe used in this experiment. (B) Two-dimensional agarose gel electrophoresis was used to determine the superhelical density at which Z-DNA formed in plasmid DNA. Topoisomer numbers are indicated. (Panel C) pGT-40 at physiological superhelicity was analyzed on the same gel. ficity in the presence and absence of the topoisomerase II poison, etoposide. 3.3. Effects o f DNA conformation on topoisomerase H cleavage activi~ Since topoisomerase II cleavage sites are strongly clustered at GT repeats, we next determined whether topoisomerase II cleavage patterns were the same in alternative conformations of plasmid. Relaxed or supercoiled pGT-40 DNA targets were compared. Fig. 3A shows that there is no relaxation of the supercoiled form by topoisomerase II in the absence of ATP (compare lanes 1 and 5,3 and 7), although topoisomerase II cleavages were readily detected on both relaxed and supercoiled templates (lanes 7 and 8). Topoisomerase II cleavage reactions on intact plasmid DNA were performed and the cleavage sites were mapped by indirect end-labeling to compare the reactivity of the enzyme at the GT repeat of B- and Z-DNA. Topoisomerase II cleavages either in the presence or absence of etoposide were catalogued on pBS and pGT-40 (Fig. 3B).
(GT)20
Fig. 2. Sequencing topoisomerase II cleavage sites on (GT)20 repeat. Cleavage reactions were performed on a KpnI-SacI fragment (labeled 5' end at KpnI site) of pGT-40 as described in Section 2. Samples were loaded onto 8% sequencing gel. Lanes 1 and 2 are chemical sequencing marker: lane 3 is fragment alone: lanes 4 and 5 contained topoisomerase II in the absence and presence of etoposide, respectively. The position of the (GT)20 repeat is indicated by a bracket.
1. Y. Choi et al. / Biochimica et Biophysica Acta 1264 (1995) 209-214
212
,6, DNAform: I Ir I Ir I Ir ATP: + + + + . . . Etoposide: + + 123456
I Ir IIl . + + 789
linear supercoiled
~
relaxed B
pGT-40 r
I
pBS F-3
D N A f o r m : I I r 1 I r 1 Ir I Ir I Ir ATP:+ + + + . . . . . . Etoposide: _ _ + + _ _ + + + + M 1 2 3 4
5
6 7
8
9 10 11
Fig. 3. Mapping topoisomerase II sites in pGT-40 by indirect end-labeling. Cleavage reactions were performed on whole plasmid in the presence or absence of etoposide. (A) Following termination of the reactions, the samples were purified and loaded onto a 1.2% agarose gel containing 0.5 ~ g / m l concentration of ethidium bromide. The reaction conditions are indicated above the gel. (B) For the indirect-end labeling, the purified samples were digested with ScaI and electrophoresed on a native 1.5% agarose gel. The DNA was transferred to nitrocellulose for Southern blotting. In odd numbered lanes the input plasmid was supercoiled (indicated as I) and in even numbered lanes the input plasmid was relaxed (indicated as Ir). The arrow points to the cleavage site which corresponds to the location of the (GT)z0 repeat. Lane M contained a SmaI-Scal fragment of pBS as size reference of GT repeat site. The bands shown in lanes 5 and 6 were due to partial restriction digestion and were not seen in a repeat experiment.
relative band intensities. Although there is a minor variation of the cleavage efficiency in lane 8 as compared to lane 3 of Fig. 4, cleavage at control site was 1.68-fold enhanced in a supercoiled target (compare lanes 2 - 5 and 7-10). The enzyme also cleaved GT repeat (indicated by Z-DNA) when it existed both as Z-conformation and as B-conformation. The GT repeat of supercoiled DNA was cleaved 2.1-fold better than the corresponding site in relaxed DNA. These results indicate that the cleavage bias in supercoiled DNA exists at GT and non-GT repeats to about the same extent.
3.5. Titration of topoisomerase H cleavages as a function of negatiue superhelical density Two-dimensional gel electrophoresis data show that the B to Z equilibrium of pGT-40 was shifted toward Z-DNA at physiological superhelical densities (Fig. 1C). Since the structural transition of B- to Z-DNA is energetically favored in a more negatively supercoiled DNA, we compared targets of varying levels of negative superhelical density. Seven topoisomer populations of pGT-40 (having different average negative superhelical density ranging from 0 to - 0 . I ) were prepared and the cleavages were carried out to compare how different conformational states affect recognition of the GT repeat (Fig. 5). As the superhelical density of input pGT-40 was increased, we found a proportional increase in topoisomerase II cleavage activity in whole plasmid. Moreover, enzyme cleavages were es-
I
Supercoiled pGT-40
Relaxed pGT-40
Topo[l
Topoll
2 3 4
5
6 7
8 9 10 11 12
Control
subtle distinctions existed between the reactivity of the enzyme and the conformation of the substrate. To address this, we carried out an indirect end-labeling experiment utilizing constant DNA substrate (supercoiled or relaxed) and variable amount of enzyme. The reactions contained excess DNA; therefore, sites (or structures) with the highest enzyme cleavage avidity would be favored. All reactions were performed in the absence of ATP and contained the non-intercalating inhibitor, etoposide. As shown in Fig. 4, cleavages were strongly enhanced on negatively supercoiled template. The cleavage efficiencies at different sites were compared. Two cleavage bands from GT repeat (indicated by Z-DNA) and non-GT site (indicated by control) were scanned by densitometry to measure the
Z-DNA
Fig. 4. Titration of topoisomerase II cleavages on the relaxed or supercoiled pGT-40 as a function of the enzyme concentration. Cleavage reactions were performed on whole plasmid in the absence of ATP. The supercoiled plasmid (1 /xg, lanes 1-5) or relaxed plasmid (1 /~g, lanes 6-10) was incubated with 0, 12, 25, 50, 100 ng of purified human topoisomerase II, respectively. The cleavage site at GT repeat was indicated by Z-DNA and the cleavage sites of non-GT repeat was indicated by control. Lane M contained a SmaI-ScaI fragment of pBS as size reference of GT repeat site.
1. Y. Choi et al. / Biochimica et Biophysica Acta 1264 (1995) 209-214
pGT-40 I
- ~ xl02: MI
0
I
re?
1.5 2.5 3.5 4.5 7.5 10 m. 2 3 4 5 6 78
Z-DNA
Fig. 5. Titration of topoisomerase II cleavages as a function of negative superhelical density. Cleavage reactions were performed on 1 /zg of pGT-40 topoisomers with the indicated average superhelical density. The cleavage site at GT repeat was indicated by Z-DNA. Lane M contained a Srnal-ScaI fragment of pBS as size reference of GT repeat site. Lanes 1-7 contained plasmids with the different levels of superhelical density values as designated on the top of each lane. Lane 8 contained pBS as input plasmid.
sentially identical in GT and non-GT flanks, based upon densitometry. These results confirm that topoisomerase II recognizes and cleaves a left-handed Z-DNA about as efficiently as B-DNA.
4. D i s c u s s i o n
We have investigated the effects of the superhelical-dependent structural transition of alternating purine-pyrimidine repeats on topoisomerase II cleavages. The binding and cleavage of topoisomerase II with minicircles containing Z - D N A inserts were recently reported by Glikin et al. [18]. Although topoisomerase II showed significantly higher affinity for the mini-CG minicircle containing ZDNA than the control mini construct lacking the (CG) 7 insert, a specific cleavage in the Z - D N A sequence was not detected in this system [18]. To further demonstrate the reactivity of topoisomerase II with Z-DNA structure, we performed the cleavage reactions on plasmid pGT-40 containing 40 bp of alternating GT. Two-dimensional gel electrophoresis data (Fig. 1C) show that this plasmid undergoes a conformational transition to Z-DNA form at physiological levels of negative superhelicity. Recently, Roca and Wang have proposed that eukaryotic topoisomerase II acts as an ATP-dependent molecular clamp; without ATP binding and hydrolysis, strand passage can not be completed by the enzyme [21]. When the supercoiled plasmids were used for enzyme substrates in the absence of ATP, all cleavage products must have originated from the supercoiled D N A since that is the only target available when the reactions were initiated. Under
213
these conditions, we were able to detect whether the topoisomerase II cleavages occurred at the GT repeat when it existed in a non-B D N A state. As shown in Fig. 3, the specific cleavages were mapped at the GT repeats when input plasmids were negative supercoiled. At physiological levels of negative superhelicity, it is likely that 'bubbles' of Z - D N A exist in equilibrium with B-DNA throughout. To push the equilibrium toward the Z-DNA form, more negatively supercoiled plasmids ( - 0.1 of superhelical density) were prepared. As superhelical density increases, the Z-helix propagates throughout the GT repeat by successive B - Z transitions. The results shown in Fig. 5 demonstrate that cleavage activity at GT and non-GT sites is equally dependent on the superhelical conformation. In other words, topoisomerase II cleavages are conformationally independent in the context of the superhelical dependent transitions in the GT insert. In negatively supercoiled DNA targets, both Z-DNA and B-DNA appear to be equally attractive as topoisomerase II cleavage sites. An important feature of the enzymatic activity of topoisomerase II is its ability to recognize a wide variety of unusual DNA conformations. This enzyme recognizes DNA regions of topoisomerase I1 consensus sequence which exist in non-B DNA forms, such as cruciform, Z-DNA, curved DNA [23] and parallel-stranded tetraplex DNA [17], forming stable covalent complexes. In contrast, topoisomerase II strongly impeded at regions of triplex DNA structures [8]. Recently, the binding activities of the 170 kDa and 180 kDa human topoisomerase II to curved and linear Z-DNA fragments were characterized, indicating that both enzymes showed higher affinity for these unusual DNA structures [23]. DNA structures that eukaryotic topoisomerase II acts upon are summarized in Table 1. In conclusion, our data show that eukaryotic topoisomerase II binds and cleaves DNA regions with left-handed Z-DNA conformation. These results suggest that eukaryotic topoisomerase II is an example of a eukaryotic protein which recognizes the Z-DNA. The structural features of a left-handed helix are therefore likely to be compatible with the catalytic cycle of DNA breakage, strand passage and resealing (although we have only tested the cleavage step in our experiments). Finally, while topoisomerase II cleav-
Table 1 DNA structures that eukaryotic topoisomerase I1 acts upon DNA structures Topoisomerase II References activity Duplex DNA in B form Duplex DNA in Z form Curved DNA Single strand DNA in hairpin Parallel strand DNA tetraplex Single strand DNA, not annealed Triplex DNA
yes yes yes yes yes no no
[18,23,24] [23] [25] [ 17,22] [17] [8]
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age activity is clearly stimulated by negative supercoiling, we did not detect alterations in sequence specificity as the DNA conformation was modulated. For this reason and based upon our previous studies [17], we conclude that topoisomerase II cleavage activity is largely independent of duplex DNA conformation.
Acknowledgements We thank Dr. J.R. Spitzner and Mr. J.M. Suh for helpful discussion and advice. This work was supported by grant from KOSEF, Korea, 1994, project 941-0500-026-1.
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[7] Spitzner, J.R., Chung, I.K. and Muller, M.T. (1990) Nucleic Acids Res. 18, 1-11. [8] Spitzner, J.R., Chung, I.K. and Muller, M.T. (1995) J. Biol. Chem. 270, 5932-5943. [9] Vosberg, H.P. (1985) Curt. Top. Microbiol. Immunol. 114, 19-102. [10] Wang, J.C. (1985) Annu. Rev. Biochem. 54, 665-697. [11] Muller, M.T. and Mehta, V.B. (1988) Mol. Cell. Biol. 8, 3661-3669. [12] Liu, L.F. and Wang, J.C. (1984) Proc. Natl. Acad. Sci. USA 84, 7024-7027. [13] Uemura, T. and Yanagida, M. (1984) EMBO J. 3, 1737-1744. [14] DiNardo, S, Voelkel, K. and Sternglanz, R. (1984) Proc. Natl. Acad. Sci. USA 81, 2616-2620. [15] Holm, C., Goto, T., Wang, J.C. and Botstein, D. (1985) Cell 41, 553-563. [16] Snapka, R.M., Powelson, M.A. and Strayer, J.M. (1988) Mol. Cell. Biol. 8, 515-521. [17] Chung, I.K., Mehta, V.B., Spitzner, J.R. and Muller, M.T. (1992) Nucleic Acids Res. 20, 1973-1977. [18] Glikin, G.C., Jovin, T.M. and Arndt-Jovin, D.J. (1991) Nucleic Acids Res. 19, 7139-7144. [19] Albert, A.C., Roman, A.M., Bouche, G., Leng, M. and Rahmouni, A.R. (1994) J. Biol. Chem. 269, 19238-19244. [20] Osheroff, N. (1986) J. Biol. Chem. 261, 9944-9950. [21] Roca, J. and Wang, J.C. (1994) Cell 77, 609-616. [22] Chung, I.K. and Muller, M.T. (1991) J. Biol. Chem. 266, 9508-9514. [23] Bechert, T., Diekmann, S. and Arndt-Jovin, D.J. (1994) J. Biomol. Str. Dynamics 12, 605-619. [24] Arndt, D.J., Udvardy, A., Garner, M., Ritter, S. and Jovin, T.M. (1993) Biochemistry 32, 4862-4872. [25] Gale, K. and Osheroff, N. (1990) Biochemistry 29, 9538-9545.