Nucleic Acids Research, Vol. 20, No. 19 5027-5033
Incomplete reversion of double stranded DNA cleavage mediated by Drosophila topoisomerase II: formation of single stranded DNA cleavage complex in the presence of an anti-tumor drug VM26 Maxwell P.Lee and Tao-shih Hsieh* Department of Biochemistry, Duke University Medical Center, Durham, NC 27710, USA Received July 27, 1992; Revised and Accepted September 3, 1992
ABSTRACT Anti-tumor drug VM26 greatly stimulates topoisomerase 11 mediated DNA cleavage by stabilizing the cleavable complex. Addition of a strong detergent such as SDS to the cleavable complex induces the double stranded DNA cleavage. We demonstrate here that heat treatment can reverse the double stranded DNA cleavage; however, topoisomerase 11 remains bound to DNA even in the presence of SDS. This reversed complex has been shown to contain single strand DNA breaks with topoisomerase 11 covalently linked to the nicked DNA. Chelation of Mg+ + by EDTA and the addition of salt to a high concentration also reverse the double strand DNA cleavage, and like heat reversion, topoisomerase 11 remains bound to DNA through single strand DNA break. The reversion complex can be analyzed and isolated by CsCI density gradient centrifugation. We have detected multiple discrete bands from such a gradient, corresponding to protein/DNA complexes with 1, 2, 3,. .... topoisomerase 11 molecules bound per DNA molecule. Analysis of topoisomerase IVDNA complexes isolated from the CsCI gradient indicates that there are single stranded DNA breaks associated with the CsCI stable complexes. Therefore, topoisomerase Il/DNA complex formed in the presence of VM26 cannot be completely reversed to yield free DNA and enzyme. We discuss the possible significance of this finding to the mechanism of action of VM26 in the topoisomerase 11 reactions. INTRODUCTION DNA topoisomerases play an important role in modulating the structure of DNA in cells. Type 11 topoisomerases interconvert DNA topoisomers by breaking and rejoining both strands of duplex DNA in a more or less concerted manner, and passing a segment of DNA through the transient breaks (1-3). Several reactions are common to the eukaryotic topoisomerase II, such as relaxation of DNA supercoils, catenation/decatenation, and knotting/unknotting of circular DNA molecules. Knowledge of *
To whom correspondence should be addressed
the biological roles of eukaryotic topoisomerase H comes primarily from genetic studies in yeasts. Topoisomerase II functions in segregating the intertwined daughter chromosomes at the end of DNA replication (4-6), and it may play a role in the condensation of replicated chromosomes at the beginning of mitosis (7). Either topoisomerase I or topoisomerase H is required to relieve the superhelical tension generated during transcription or replication (4, 8-10). In addition, topoisomerase H is implicated as a major component in the nuclear matrix or scaffold structure (11-13). The suggested mechanism of DNA topoisomerase H, passage of DNA through a reversible double strand break, is strongly supported by the topoisomerase-mediated DNA cleavage reaction. The addition of a potent protein denaturant like sodium dodecyl sulfate (SDS) or strong alkali to a reaction mixture containing topoisomerase II and DNA results in double strand DNA breaks. The DNA cleavage is generated from a putative topoisomerase 11/DNA complex termed 'cleavable complex' which may be an intermediate in the strand passage reaction pathway. Detailed structural analysis of the cleavage product revealed that the breaks on the complementary DNA strands are staggered by 4 nucleotides and each subunit of topoisomerase 11 is covalently joined to the protruding 5' phosphoryl ends at the break (14-16). The double stranded DNA cleavage can be reversed by salt or EDTA before the addition of protein denaturants (15, 16). The salt reversibility of the cleavage reaction suggests that the cleavable complex is in equilibrium with other topoisomerase 11/DNA complexes, and topoisomerase H in the cleavable complex can dissociate from DNA after the reversion treatment. The reversibility of the topoisomerase H mediated DNA cleavage is one of the hallmarks of topoisomerase H reactions, which distinguish topoisomerase H mediated DNA cleavage from those catalyzed by nucleases. Topoisomerase H has been shown to be the primary target of some anti-tumor drugs (3, 17, 18). The cytotoxicity of these drugs is likely due to the chromosomal DNA breakage. Many in vivo and in vitro studies have demonstrated that these anti-tumor drugs act by stimulating topoisomerase II mediated DNA cleavage. It has been hypothesized that they stimulate topoisomerase II
5028 Nucleic Acids Research, Vol. 20, No. 19
mediated DNA cleavage by stabilizing the cleavable complex (19, 20). The double stranded DNA cleavage mediated by mammalian topoisomerase II in the presence of drugs has been shown to be reversible with respect to salt treatment (20) and heat treatment (21). However, the cytotoxic effects of some of the topoisomerase 11-targeting drugs like VM26 (teniposide or 4'-demethylepipodophyllotoxin ethylidene-,B-D-glucoside), are noticeably irreversible. For instance, the exposure of human lymphoid cells to VM26 inhibits traversal of the cell cycle and the treated cells that are reincubated in the drug-free medium cannot resume growth (22). During our studies of the interactions between Drosophila topoisomerase II and DNA, we investigated if VM26 stimulated double strand DNA cleavage by topoisomerase II could be reversed by salt or heating. We show in this report that the double strand DNA cleavage generated from the cleavable complex can be reversed, but a fraction of the cleavable complexes is converted into another form of cleavable complex in which only one of the topoisomerase subunits can cleave the phosphodiester DNA backbone bond. We discuss the possible significance of this finding to the mechanism of action of VM26 on topoisomerase reaction and its relevance to the cytotoxic action of VM26.
MATERIALS AND METHODS Enzymes and substrates The preparation and sources of enzymes and DNA substrates were described previously (23, 24). Radiolabeled DNA fragments were prepared from either pBR322 or 6/122b plasmid DNAs. 6/122b is a pBR322 derivative containing the Drosophila heat shock intergenic region. DNA fragments were dephosphorylated with calf intestine alkaline phosphatase, and 5'-ends were labeled with T4 polynucleotide kinase and -y-32P-ATP. To prepare labeled negatively supercoiled circular DNA, it was cyclized by the action of T4 DNA ligase in the presence of 6,tg/ml ethidium bromide. DNAs were separated and isolated from an agarose gel and purified through an NACS -52 column (Gibco-BRL, Gaithersburg, MD) according to the manufacturer's recommended procedures. The calf thymus type H DNA topoisomerase was purified according to the published procedure (25) and was a generous gift from Dr. Leroy Liu. Double strand DNA cleavage reaction and its reversion The reaction was performed in 20 td of solution containing 10 mM Tris-HCl pH 7.9, 10 mM MgCl2 50 mM KCI, 1.25 mM ATP, 50,g/ml BSA, 100 ,tg/ml VM26, 0.21 nM 1.6 Kb pBR322 Hinf I digested linear DNA fragment, and 2.5 nM Drosophila topoisomerase H. The reaction mixture was incubated at 30°C for 10 minutes and terminated by adding SDS to 1 % and proteinase K to 100 Atg/ml. Incubation was continued at 45°C for 30 minutes. To reverse the double stranded DNA cleavage, the reaction mixture was heated at 65°C for 10 minutes after the addition of 50 mM dithiothreitol, then followed by SDS and proteinase K treatment. The reversion could also be brought about by adding EDTA and NaCl to the reaction mixture to give a final concentration of 12 mM and 0.5 M, respectively and the incubation was continued at 30°C for 10 minutes before the treatment with SDS and proteinase K. For analyzing the topoisomerase H/DNA complexes, samples that were not treated with proteinase were also directly loaded onto an agarose gel containing 0. I% SDS.
CsCl density gradient equilibrium centrifugation The binding reaction was similar to that used in the cleavage reaction and the reaction volume was scaled up to 1 ml. The molar ratio of enzyme to DNA was increased to 24 (12 nM enzyme/0.5 nM DNA molecules). At the end of the incubation, the binding reaction was terminated by the addition of TE buffer (10 mM Tris-HCl pH 7.9, 0.1 mM EDTA) to a final volume of 1.8 ml. 40 1l of 0.5 M EDTA, 0.1 ml of 10 mg/ml BSA, and 2.8 ml of a stock solution of saturated CsCl were then added to give a solution with a density of 1.54 g/ml. The CsCl containing solution was spun at 42 k rpm (164,000 g) at 20°C for 44 h. To minimize the adsorption of enzyme/DNA complexes to the centrifuge tubes, it was necessary to coat the centrifuge tube with 0.2 mg/ml BSA for several hours before use. Fractions of about 100 A1 were collected from the bottom of the tube. 10 Al of each fraction was used to measure the radioactivity by liquid scintillation counting. The compositional densities of the collected fractions were determined by first measuring 10 Mt1 of sample in a graduated micropipet (Accupette) and then determining its weight with an analytical balance (Mettler AE163) to a precision of 0.01 mg. For the analysis of DNA and complexes isolated from the CsCl gradient, gradient fractions were diluted with 10 volumes of TE buffer before proteinase K digestion or treatment with various protein denaturing conditions.
RESULTS A fraction of the topoisomerase II remains bound to DNA after the reversion of cleavage reaction by either heat or salt treatment Several clinically important anti-tumor drugs have been shown to target at topoisomerase II (3, 17, 18). These anti-tumor drugs can stimulate topoisomerase H mediated DNA strand cleavage. The drug induced enhancement of DNA cleavage observed in vitro has provided a biochemical basis for the mechanism of how the drugs cause cell death and chromosomal rearrangement and fragmentation. DNA cleavage reaction stimulated by the antitumor drugs can be reversed by either salt or heat treatment (20, 21). During our studies of topoisomerase H cleavage reaction and its reversion, we made an observation indicating that after the reversion of topoisomerase II cleavage by either salt or heat treatment, a significant amount of DNA is still linked with topoisomerase H. In one of these experiments (shown in Fig. 1), topoisomerase II/DNA complexes were formed in the presence of VM26 at 30'C, followed by the addition of a strong detergent SDS to induce double stranded DNA breaks within the cleavable complex. The DNA products were analyzed by electrophoresis in an agarose gel containing SDS. The DNA cleavable complex was clearly demonstrated in this gel system by the generation of smaller DNA fragments, and the protease treatment produced even smaller DNA framents spreading toward the high-mobility end of the gel (Fig. 1, lanes 2 and 5). The reversion of the cleavage reaction could be readily monitored by this gel electrophoresis system. Heating the reaction mixture at 650C for 10 min. prior to the addition of SDS resulted in the disappearance of cleavage complex (Fig. 1, lane 3) and the religation of small DNA fragments into the full-length DNA (Fig. 1, lane 6). In the absence of protease treatment, heating step also converted the heterogeneous labeled DNA fragments of the cleavage complexes into a ladder of discrete bands (Fig. 1, lane 3). Proteolytic digestion converted the ladder of the bands into a
Nucleic Acids Research, Vol. 20, No. 19 5029 4PK 1 2 3
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I Figure 1. Heat reversion of the topoisomerase 11/linear DNA cleavable complex. A 1.6 Kb linear DNA fragment from pBR322 Hinf I digestion was 5' end labeled by y-32P-ATP with polynucleotide kinase. A standard 20 yd binding reaction mixture contained 10 mM Tris-HCI, pH 7.9, 50 mM KC1, 10 mM MgCl2, 0.1 mM EDTA, 50 itg/ml BSA, 1.25 mM ATP, 100 itg/ml VM26, 0.21 nM labeled DNA, and 2.5 nM purified Drosophila topoisomerase II homodimer. The reaction mixture was incubated at 30°C for 10 min, after which it was either kept at 30°C (lanes 1, 2, 4, and 5) or heated at 65°C (lanes 3 and 6) for 30 min in the presence of 50 mM dithiothreitol (DTT). The addition of DTT reduces the aggregation of topoisomerase II during the heating step, and it has no effect on the reversion of double strand breaks. 2 ul of 10% SDS stock solution was then added after the heat treatment. Half of the samples were treated with proteinase K (lanes 4 to 6, marked with +PK). All the samples were analyzed by electrophoresis in 1.5% agarose gel containing 0.1% SDS. The radioautographic intensity in lane 5 is lower because of the spreading of DNA fragments toward the lower end of the gel. Minus enzyme controls are in the lanes 1 and 4. The arrow points to the position of intact substrate DNA.
single band which comigrated with the substrate DNA (Fig. 1, lane 6), suggesting that the radioactive DNA in the ladder, migrating slower than the substrate DNA, were protein linked. While the heat treatment reverses some of the cleavable complexes into free DNA and protein, a significant fraction of the cleavable complex is converted into a protein-linked complex. Protease digestion of the reversion complex releases the full length substrate DNA. Ailaline gel electrophoretic analysis of this DNA indicated that it contained single stranded nicks (data not shown). The reversion complex generated from the cleavable complex therefore contains a nick in the DNA, to which topoisomerase 11 is covalently linked. Similar results were obtained when the reversion was effected by EDTA and salt treatment (data not shown, also see Fig. 2), suggesting an identical reversion complex can be formed with different reversion procedures. To further support the idea that the reversion product of the cleavable complex is a protein/DNA complex containing single stranded DNA breaks with a covalently linked topoisomerase subunit rather than free, intact DNA, we carried out similar reactions with radioactively labeled circular DNA substrates which should allow a more sensitive detection of the nicked DNA product in the reversion reaction. Topoisomerase 11/DNA cleavage complexes were induced by adding SDS to a reaction mixture containing VM26, and the reaction products were treated either with or without proteinase K. The protease treated samples were analyzed by agarose gel electrophoresis in the presence of ethidium bromide to distinguish between nicked and covalently closed circles (Fig. 2B), while the samples without protease treatment were analyzed by SDS agarose gel electrophoresis to reveal tightly linked protein/DNA complexes (Fig. 2A). Formation of cleavable complexes at multiple sites generated DNA fragments of various sizes, appearing as smears in the gel electrophoresis (Fig. 2A&2B, lanes 1 and 3). With an increasing
pl-]KN(.
Markers I 2 3 4 L -
(N.:
wi._SC
4
Figure 2. EDTA/salt reversion of the topoisomerase II/circular DNA cleavable complex. A 1.6 Kb DNA fragment from the heat shock intergenic region (24) was 5' end labeled and circularized in the presence of ethidium bromide by T4 ligase to generate negatively supercoiled DNA. Labeled circular DNA was isolated from the agarose gel and purified through an NACS-52 column. The cleavage condition was the same as described in Fig. 1 except that it contained 0.046 nM labeled DNA and 0.63 nM topoisomerase II (lanes 1 and 2), or 1.26 nM topoisomerase II (lanes 3 and 4). Reversion was effected by the addition of EDTA to 12 mM and NaCl to 0.5 M and the incubation continued for 10 minutes (lanes 2 and 4). Marker lanes indicate the positions of NC (nicked circle), SC (supercoiled circle), RC (relaxed circle), KNC (knotted, nicked circle), and L (linear DNA). A: Samples were analyzed by electrophoresis in a 1.5% agarose gel containing 0.1% SDS. B: Samples were treated with proteinase K and then analyzed on a 3% agarose gel containing 0.5 ,ug/ml ethidium.
topoisomerase II/DNA ratio, the sizes of the cleavage products decreased due to the multiple cleavage events per DNA molecule (compare lanes 1 and 3 of Fig. 2A&2B). Another aliquot of the same reaction mixture was treated with EDTA and salt to reverse the DNA cleavage reaction (Fig. 2A&2B, lanes 2 and 4). Similar to the results from the reversion products of linear DNA substrate (see Fig. 1), the reversion complex from circular DNA also showed multiple bands migrating slower than the nicked circles in the SDS/agarose gel (Fig. 2A, lanes 2 and 4). These multiple bands likely correspond to the circular DNA molecules with different amounts of covalently linked topoisomerase moiety. After the treatment of the reaction mixtures containing the reversion complexes with the proteinase K, we detected the formation of various DNA species including linear, nicked circular, and covalently closed circular molecules in the agarose gel containing ethidium bromide (Fig. 2B, lanes 2 and 4). The nicked circles include both unknotted and knotted DNA molecules. The detection of nicked, knotted DNA using this type of gel electrophoresis system was described earlier (26). All of the nicked DNA species account for more than 50% of the reversion products based on the densitometric analysis of the autoradiographs similar to the one shown in Fig. 2B. These results therefore suggest that most of the topoisomerase 11/DNA complexes reversed from the cleavable complex contain DNA nicks with covalently attached topoisomerase 11. We also detected some linear DNA after proteinase digestion (Fig. 2B, lanes 2 and 4). This is apparently due to some of the residual cleavable complex that was not reversed in these reactions. It is possible
5030 Nucleic Acids Research, Vol. 20, No. 19 _
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1Figure 3. Effects of ATP and ATPyS on cleavage reaction and its reversion. The DNA substrate and its cleavage reaction was the same as described in Fig. 1. There was no ATP co-factor in reactions shown in lanes 1 and 2, while those in lanes 3 and 4 contained 1.25 mM ATP-yS and lanes 5 and 6 contained 1.25 mM ATP. Lane 7 shows the minus enzyme control. Heating reversion was carried out at 65°C (lanes 2, 4, and 6). Samples were analyzed on a 1.5% agarose gel in the presence of 0.1 % of SDS. The arrow indicates the position of the substrate DNA.
that either the reversion is less efficient for the negatively supercoiled DNA substrate, or perhaps detection of double strand DNA breaks in the circular DNA is more sensitive than in the linear DNA. There is also sone covalently closed, circular DNA released after proteinase digestion (Fig. 2B, lanes 2 and 4), which was produced from the complete reversion of the cleavable complex to yield the free, intact DNA. Similar reversion products were also obtained from these circular DNA substrates using the heat treatment instead (data not shown).
Characterization of the cleavage reversion reaction We have demonstrated that VM26 stimulated double strand DNA cleavage by topoisomerase II can be reversed to single strand DNA cleavage with either heat or EDTA/salt treatment. To further analyze the cleavage reversion, the effects of temperature on the heat reversion efficiency were investigated. As the temperature increased from 45°C to 65°C, reversion of the cleavable complex gradually increased and reached a maximum by 65°C (data not shown). The midpoint of this reversion curve is around 55°C. The temperature dependence of the reversion of Drosophila topoisomerase cleavable complex is very similar to that of the mammalian enzyme (21). Chelation of Mg+ + in the reaction mixture by the addition of EDTA and an increase in the ionic strength by adding NaCl to 0.5 M can also reverse some of the cleavable complex into the protein-linked complex. Either salt or EDTA alone cannot induce a reversion quite as efficiently as the combination of them. The efficiency of reversion is not affected by VM26 over a wide range of concentrations. For the DNA cleavage mediated by Drosophila topoisomerase significant stimulation can be observed in a reaction containing 0.3 lAg/ml VM26 and the amount of DNA cleavage increases with higher VM26 concentrations up to the maximal cleavage at a VM26 concentration of 100 gig/ml. Both the heat and EDTA/salt reversion steps can efficiently reverse the cleavage reactions carried out in the range of 0.3 to 100 jig/ml VM26. In the absence of VM26, the presence of the cofactor ATP in the reaction has only a modest effect on the topoisomerase II mediated DNA cleavage and results in about a 2-fold enhancement in the double strand DNA cleavage (15). However, in the presence of VM26, ATP can markedly increase the cleavage efficiency, while its analog ATP-yS exerts a lesser effect (compare lanes 3 and 5 with lane 1 in Fig. 3). The extent of these 11,
o
Figure 4. Kinetics of reversion of the cleavable complex by EDTA/CsCl. The same DNA substrate as described in Fig. 1 was used to form topoisomerase II/DNA complexes under similar reaction conditions, except that DNA and enzyme concentrations were 10-fold higher (2.1 nM DNA and 25 nM topoisomerase II). An aliquot was taken out and salt was diluted with 10 volumes of TE, followed by the addition of SDS to 1% to induce DNA cleavage (lane 2). Reversion was initiated by the addition of EDTA to 12 mM and followed by CsCl to 5 M. Aliquots were withdrawn at various timepoints after the addition of EDTA/CsCl: 10 sec. (lane 3), 10 min. (lane 4), 3 h. (lane 5), and 20 h. (lane 6). CsCl salt was diluted out with 10 volumes of TE and followed by the addition of SDS to 1%. Lane 1 is the substrate DNA and the arrow marks the position of the substrate DNA.
stimulatory effects
quantified by titrating the amount of
was
enzyme necessary to give rise to the same level of DNA cleavage. It appears that ATP and its analog ATP-yS can stimulate the
cleavage about 10- and 2-fold, respectively (data not shown). Heat reversal of the cleavable complexes is equally effective either in the absence or presence of ATP cofactors (Fig. 3, lanes 2, 4, and 6), and these protein-linked reversion products can be converted into fill-length DNA after protease digestion (data not shown). The kinetics of reversion is rapid. A significant amount of reversion takes place within 1 min. after shifting the incubation temperature to 65°C or upon the addition of EDTA/NaCl (data not shown). Efficient reversion was also observed when the reversion was induced by adding EDTA to 12 mM and CsCl to 5 M (Fig. 4). A slower reversion rate was observed here because of the much higher concentration of enzyme and DNA used in this experiment (see Fig. 4 legend). It is interesting to note that even after a prolonged incubation of 22 hours in EDTA/CsCl, a substantial amount of protein-linked reversion complex still persists (Fig. 4, lane 6).
Analysis of the reversion complexes by CsCI density gradient Since the reversed topoisomerase 11/DNA complex was stable in EDTA and 5 M CsCl this prompted us to analyze and isolate these complexes by CsCl density gradient equilibrium ,
centrifugation. In such a gradient, radiolabeled linear pBR322 DNA forms a single band with a measured density of 1.72 g/cm3 (Fig. 5A). For pBR322 DNA with a base composition of 54% guanosine and cytosine, this measured buoyant density is close to the calculated value based upon the known relation between buoyant density of DNA and its base composition (27). Only one peak, at the density expected for free DNA, is apparent when topoisomerase II/DNA complexes are formed in the absence of VM26 (Fig. SB), indicating that the majority of topoisomerase II/DNA complex formed in the absence of VM26 is not stable in a high concentration of CsCl. Most of the noncovalent protein/DNA complexes are expected to dissociate in such a high salt solution. When topoisomerase H/DNA complexes
Nucleic Acids Research, Vol. 20, No. 19 5031
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Incomplete reversion of double stranded DNA cleavage mediated by Drosophila topoisomerase II: formation of single stranded DNA cleavage complex in the presence of an anti-tumor drug VM26 Maxwell P.Lee and Tao-shih Hsieh* Department of Biochemistry, Duke University Medical Center, Durham, NC 27710, USA Received July 27, 1992; Revised and Accepted September 3, 1992
ABSTRACT Anti-tumor drug VM26 greatly stimulates topoisomerase 11 mediated DNA cleavage by stabilizing the cleavable complex. Addition of a strong detergent such as SDS to the cleavable complex induces the double stranded DNA cleavage. We demonstrate here that heat treatment can reverse the double stranded DNA cleavage; however, topoisomerase 11 remains bound to DNA even in the presence of SDS. This reversed complex has been shown to contain single strand DNA breaks with topoisomerase 11 covalently linked to the nicked DNA. Chelation of Mg+ + by EDTA and the addition of salt to a high concentration also reverse the double strand DNA cleavage, and like heat reversion, topoisomerase 11 remains bound to DNA through single strand DNA break. The reversion complex can be analyzed and isolated by CsCI density gradient centrifugation. We have detected multiple discrete bands from such a gradient, corresponding to protein/DNA complexes with 1, 2, 3,. .... topoisomerase 11 molecules bound per DNA molecule. Analysis of topoisomerase IVDNA complexes isolated from the CsCI gradient indicates that there are single stranded DNA breaks associated with the CsCI stable complexes. Therefore, topoisomerase Il/DNA complex formed in the presence of VM26 cannot be completely reversed to yield free DNA and enzyme. We discuss the possible significance of this finding to the mechanism of action of VM26 in the topoisomerase 11 reactions. INTRODUCTION DNA topoisomerases play an important role in modulating the structure of DNA in cells. Type 11 topoisomerases interconvert DNA topoisomers by breaking and rejoining both strands of duplex DNA in a more or less concerted manner, and passing a segment of DNA through the transient breaks (1-3). Several reactions are common to the eukaryotic topoisomerase II, such as relaxation of DNA supercoils, catenation/decatenation, and knotting/unknotting of circular DNA molecules. Knowledge of *
To whom correspondence should be addressed
the biological roles of eukaryotic topoisomerase H comes primarily from genetic studies in yeasts. Topoisomerase II functions in segregating the intertwined daughter chromosomes at the end of DNA replication (4-6), and it may play a role in the condensation of replicated chromosomes at the beginning of mitosis (7). Either topoisomerase I or topoisomerase H is required to relieve the superhelical tension generated during transcription or replication (4, 8-10). In addition, topoisomerase H is implicated as a major component in the nuclear matrix or scaffold structure (11-13). The suggested mechanism of DNA topoisomerase H, passage of DNA through a reversible double strand break, is strongly supported by the topoisomerase-mediated DNA cleavage reaction. The addition of a potent protein denaturant like sodium dodecyl sulfate (SDS) or strong alkali to a reaction mixture containing topoisomerase II and DNA results in double strand DNA breaks. The DNA cleavage is generated from a putative topoisomerase 11/DNA complex termed 'cleavable complex' which may be an intermediate in the strand passage reaction pathway. Detailed structural analysis of the cleavage product revealed that the breaks on the complementary DNA strands are staggered by 4 nucleotides and each subunit of topoisomerase 11 is covalently joined to the protruding 5' phosphoryl ends at the break (14-16). The double stranded DNA cleavage can be reversed by salt or EDTA before the addition of protein denaturants (15, 16). The salt reversibility of the cleavage reaction suggests that the cleavable complex is in equilibrium with other topoisomerase 11/DNA complexes, and topoisomerase H in the cleavable complex can dissociate from DNA after the reversion treatment. The reversibility of the topoisomerase H mediated DNA cleavage is one of the hallmarks of topoisomerase H reactions, which distinguish topoisomerase H mediated DNA cleavage from those catalyzed by nucleases. Topoisomerase H has been shown to be the primary target of some anti-tumor drugs (3, 17, 18). The cytotoxicity of these drugs is likely due to the chromosomal DNA breakage. Many in vivo and in vitro studies have demonstrated that these anti-tumor drugs act by stimulating topoisomerase II mediated DNA cleavage. It has been hypothesized that they stimulate topoisomerase II
5028 Nucleic Acids Research, Vol. 20, No. 19
mediated DNA cleavage by stabilizing the cleavable complex (19, 20). The double stranded DNA cleavage mediated by mammalian topoisomerase II in the presence of drugs has been shown to be reversible with respect to salt treatment (20) and heat treatment (21). However, the cytotoxic effects of some of the topoisomerase 11-targeting drugs like VM26 (teniposide or 4'-demethylepipodophyllotoxin ethylidene-,B-D-glucoside), are noticeably irreversible. For instance, the exposure of human lymphoid cells to VM26 inhibits traversal of the cell cycle and the treated cells that are reincubated in the drug-free medium cannot resume growth (22). During our studies of the interactions between Drosophila topoisomerase II and DNA, we investigated if VM26 stimulated double strand DNA cleavage by topoisomerase II could be reversed by salt or heating. We show in this report that the double strand DNA cleavage generated from the cleavable complex can be reversed, but a fraction of the cleavable complexes is converted into another form of cleavable complex in which only one of the topoisomerase subunits can cleave the phosphodiester DNA backbone bond. We discuss the possible significance of this finding to the mechanism of action of VM26 on topoisomerase reaction and its relevance to the cytotoxic action of VM26.
MATERIALS AND METHODS Enzymes and substrates The preparation and sources of enzymes and DNA substrates were described previously (23, 24). Radiolabeled DNA fragments were prepared from either pBR322 or 6/122b plasmid DNAs. 6/122b is a pBR322 derivative containing the Drosophila heat shock intergenic region. DNA fragments were dephosphorylated with calf intestine alkaline phosphatase, and 5'-ends were labeled with T4 polynucleotide kinase and -y-32P-ATP. To prepare labeled negatively supercoiled circular DNA, it was cyclized by the action of T4 DNA ligase in the presence of 6,tg/ml ethidium bromide. DNAs were separated and isolated from an agarose gel and purified through an NACS -52 column (Gibco-BRL, Gaithersburg, MD) according to the manufacturer's recommended procedures. The calf thymus type H DNA topoisomerase was purified according to the published procedure (25) and was a generous gift from Dr. Leroy Liu. Double strand DNA cleavage reaction and its reversion The reaction was performed in 20 td of solution containing 10 mM Tris-HCl pH 7.9, 10 mM MgCl2 50 mM KCI, 1.25 mM ATP, 50,g/ml BSA, 100 ,tg/ml VM26, 0.21 nM 1.6 Kb pBR322 Hinf I digested linear DNA fragment, and 2.5 nM Drosophila topoisomerase H. The reaction mixture was incubated at 30°C for 10 minutes and terminated by adding SDS to 1 % and proteinase K to 100 Atg/ml. Incubation was continued at 45°C for 30 minutes. To reverse the double stranded DNA cleavage, the reaction mixture was heated at 65°C for 10 minutes after the addition of 50 mM dithiothreitol, then followed by SDS and proteinase K treatment. The reversion could also be brought about by adding EDTA and NaCl to the reaction mixture to give a final concentration of 12 mM and 0.5 M, respectively and the incubation was continued at 30°C for 10 minutes before the treatment with SDS and proteinase K. For analyzing the topoisomerase H/DNA complexes, samples that were not treated with proteinase were also directly loaded onto an agarose gel containing 0. I% SDS.
CsCl density gradient equilibrium centrifugation The binding reaction was similar to that used in the cleavage reaction and the reaction volume was scaled up to 1 ml. The molar ratio of enzyme to DNA was increased to 24 (12 nM enzyme/0.5 nM DNA molecules). At the end of the incubation, the binding reaction was terminated by the addition of TE buffer (10 mM Tris-HCl pH 7.9, 0.1 mM EDTA) to a final volume of 1.8 ml. 40 1l of 0.5 M EDTA, 0.1 ml of 10 mg/ml BSA, and 2.8 ml of a stock solution of saturated CsCl were then added to give a solution with a density of 1.54 g/ml. The CsCl containing solution was spun at 42 k rpm (164,000 g) at 20°C for 44 h. To minimize the adsorption of enzyme/DNA complexes to the centrifuge tubes, it was necessary to coat the centrifuge tube with 0.2 mg/ml BSA for several hours before use. Fractions of about 100 A1 were collected from the bottom of the tube. 10 Al of each fraction was used to measure the radioactivity by liquid scintillation counting. The compositional densities of the collected fractions were determined by first measuring 10 Mt1 of sample in a graduated micropipet (Accupette) and then determining its weight with an analytical balance (Mettler AE163) to a precision of 0.01 mg. For the analysis of DNA and complexes isolated from the CsCl gradient, gradient fractions were diluted with 10 volumes of TE buffer before proteinase K digestion or treatment with various protein denaturing conditions.
RESULTS A fraction of the topoisomerase II remains bound to DNA after the reversion of cleavage reaction by either heat or salt treatment Several clinically important anti-tumor drugs have been shown to target at topoisomerase II (3, 17, 18). These anti-tumor drugs can stimulate topoisomerase H mediated DNA strand cleavage. The drug induced enhancement of DNA cleavage observed in vitro has provided a biochemical basis for the mechanism of how the drugs cause cell death and chromosomal rearrangement and fragmentation. DNA cleavage reaction stimulated by the antitumor drugs can be reversed by either salt or heat treatment (20, 21). During our studies of topoisomerase H cleavage reaction and its reversion, we made an observation indicating that after the reversion of topoisomerase II cleavage by either salt or heat treatment, a significant amount of DNA is still linked with topoisomerase H. In one of these experiments (shown in Fig. 1), topoisomerase II/DNA complexes were formed in the presence of VM26 at 30'C, followed by the addition of a strong detergent SDS to induce double stranded DNA breaks within the cleavable complex. The DNA products were analyzed by electrophoresis in an agarose gel containing SDS. The DNA cleavable complex was clearly demonstrated in this gel system by the generation of smaller DNA fragments, and the protease treatment produced even smaller DNA framents spreading toward the high-mobility end of the gel (Fig. 1, lanes 2 and 5). The reversion of the cleavage reaction could be readily monitored by this gel electrophoresis system. Heating the reaction mixture at 650C for 10 min. prior to the addition of SDS resulted in the disappearance of cleavage complex (Fig. 1, lane 3) and the religation of small DNA fragments into the full-length DNA (Fig. 1, lane 6). In the absence of protease treatment, heating step also converted the heterogeneous labeled DNA fragments of the cleavage complexes into a ladder of discrete bands (Fig. 1, lane 3). Proteolytic digestion converted the ladder of the bands into a
Nucleic Acids Research, Vol. 20, No. 19 5029 4PK 1 2 3
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I Figure 1. Heat reversion of the topoisomerase 11/linear DNA cleavable complex. A 1.6 Kb linear DNA fragment from pBR322 Hinf I digestion was 5' end labeled by y-32P-ATP with polynucleotide kinase. A standard 20 yd binding reaction mixture contained 10 mM Tris-HCI, pH 7.9, 50 mM KC1, 10 mM MgCl2, 0.1 mM EDTA, 50 itg/ml BSA, 1.25 mM ATP, 100 itg/ml VM26, 0.21 nM labeled DNA, and 2.5 nM purified Drosophila topoisomerase II homodimer. The reaction mixture was incubated at 30°C for 10 min, after which it was either kept at 30°C (lanes 1, 2, 4, and 5) or heated at 65°C (lanes 3 and 6) for 30 min in the presence of 50 mM dithiothreitol (DTT). The addition of DTT reduces the aggregation of topoisomerase II during the heating step, and it has no effect on the reversion of double strand breaks. 2 ul of 10% SDS stock solution was then added after the heat treatment. Half of the samples were treated with proteinase K (lanes 4 to 6, marked with +PK). All the samples were analyzed by electrophoresis in 1.5% agarose gel containing 0.1% SDS. The radioautographic intensity in lane 5 is lower because of the spreading of DNA fragments toward the lower end of the gel. Minus enzyme controls are in the lanes 1 and 4. The arrow points to the position of intact substrate DNA.
single band which comigrated with the substrate DNA (Fig. 1, lane 6), suggesting that the radioactive DNA in the ladder, migrating slower than the substrate DNA, were protein linked. While the heat treatment reverses some of the cleavable complexes into free DNA and protein, a significant fraction of the cleavable complex is converted into a protein-linked complex. Protease digestion of the reversion complex releases the full length substrate DNA. Ailaline gel electrophoretic analysis of this DNA indicated that it contained single stranded nicks (data not shown). The reversion complex generated from the cleavable complex therefore contains a nick in the DNA, to which topoisomerase 11 is covalently linked. Similar results were obtained when the reversion was effected by EDTA and salt treatment (data not shown, also see Fig. 2), suggesting an identical reversion complex can be formed with different reversion procedures. To further support the idea that the reversion product of the cleavable complex is a protein/DNA complex containing single stranded DNA breaks with a covalently linked topoisomerase subunit rather than free, intact DNA, we carried out similar reactions with radioactively labeled circular DNA substrates which should allow a more sensitive detection of the nicked DNA product in the reversion reaction. Topoisomerase 11/DNA cleavage complexes were induced by adding SDS to a reaction mixture containing VM26, and the reaction products were treated either with or without proteinase K. The protease treated samples were analyzed by agarose gel electrophoresis in the presence of ethidium bromide to distinguish between nicked and covalently closed circles (Fig. 2B), while the samples without protease treatment were analyzed by SDS agarose gel electrophoresis to reveal tightly linked protein/DNA complexes (Fig. 2A). Formation of cleavable complexes at multiple sites generated DNA fragments of various sizes, appearing as smears in the gel electrophoresis (Fig. 2A&2B, lanes 1 and 3). With an increasing
pl-]KN(.
Markers I 2 3 4 L -
(N.:
wi._SC
4
Figure 2. EDTA/salt reversion of the topoisomerase II/circular DNA cleavable complex. A 1.6 Kb DNA fragment from the heat shock intergenic region (24) was 5' end labeled and circularized in the presence of ethidium bromide by T4 ligase to generate negatively supercoiled DNA. Labeled circular DNA was isolated from the agarose gel and purified through an NACS-52 column. The cleavage condition was the same as described in Fig. 1 except that it contained 0.046 nM labeled DNA and 0.63 nM topoisomerase II (lanes 1 and 2), or 1.26 nM topoisomerase II (lanes 3 and 4). Reversion was effected by the addition of EDTA to 12 mM and NaCl to 0.5 M and the incubation continued for 10 minutes (lanes 2 and 4). Marker lanes indicate the positions of NC (nicked circle), SC (supercoiled circle), RC (relaxed circle), KNC (knotted, nicked circle), and L (linear DNA). A: Samples were analyzed by electrophoresis in a 1.5% agarose gel containing 0.1% SDS. B: Samples were treated with proteinase K and then analyzed on a 3% agarose gel containing 0.5 ,ug/ml ethidium.
topoisomerase II/DNA ratio, the sizes of the cleavage products decreased due to the multiple cleavage events per DNA molecule (compare lanes 1 and 3 of Fig. 2A&2B). Another aliquot of the same reaction mixture was treated with EDTA and salt to reverse the DNA cleavage reaction (Fig. 2A&2B, lanes 2 and 4). Similar to the results from the reversion products of linear DNA substrate (see Fig. 1), the reversion complex from circular DNA also showed multiple bands migrating slower than the nicked circles in the SDS/agarose gel (Fig. 2A, lanes 2 and 4). These multiple bands likely correspond to the circular DNA molecules with different amounts of covalently linked topoisomerase moiety. After the treatment of the reaction mixtures containing the reversion complexes with the proteinase K, we detected the formation of various DNA species including linear, nicked circular, and covalently closed circular molecules in the agarose gel containing ethidium bromide (Fig. 2B, lanes 2 and 4). The nicked circles include both unknotted and knotted DNA molecules. The detection of nicked, knotted DNA using this type of gel electrophoresis system was described earlier (26). All of the nicked DNA species account for more than 50% of the reversion products based on the densitometric analysis of the autoradiographs similar to the one shown in Fig. 2B. These results therefore suggest that most of the topoisomerase 11/DNA complexes reversed from the cleavable complex contain DNA nicks with covalently attached topoisomerase 11. We also detected some linear DNA after proteinase digestion (Fig. 2B, lanes 2 and 4). This is apparently due to some of the residual cleavable complex that was not reversed in these reactions. It is possible
5030 Nucleic Acids Research, Vol. 20, No. 19 _
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1Figure 3. Effects of ATP and ATPyS on cleavage reaction and its reversion. The DNA substrate and its cleavage reaction was the same as described in Fig. 1. There was no ATP co-factor in reactions shown in lanes 1 and 2, while those in lanes 3 and 4 contained 1.25 mM ATP-yS and lanes 5 and 6 contained 1.25 mM ATP. Lane 7 shows the minus enzyme control. Heating reversion was carried out at 65°C (lanes 2, 4, and 6). Samples were analyzed on a 1.5% agarose gel in the presence of 0.1 % of SDS. The arrow indicates the position of the substrate DNA.
that either the reversion is less efficient for the negatively supercoiled DNA substrate, or perhaps detection of double strand DNA breaks in the circular DNA is more sensitive than in the linear DNA. There is also sone covalently closed, circular DNA released after proteinase digestion (Fig. 2B, lanes 2 and 4), which was produced from the complete reversion of the cleavable complex to yield the free, intact DNA. Similar reversion products were also obtained from these circular DNA substrates using the heat treatment instead (data not shown).
Characterization of the cleavage reversion reaction We have demonstrated that VM26 stimulated double strand DNA cleavage by topoisomerase II can be reversed to single strand DNA cleavage with either heat or EDTA/salt treatment. To further analyze the cleavage reversion, the effects of temperature on the heat reversion efficiency were investigated. As the temperature increased from 45°C to 65°C, reversion of the cleavable complex gradually increased and reached a maximum by 65°C (data not shown). The midpoint of this reversion curve is around 55°C. The temperature dependence of the reversion of Drosophila topoisomerase cleavable complex is very similar to that of the mammalian enzyme (21). Chelation of Mg+ + in the reaction mixture by the addition of EDTA and an increase in the ionic strength by adding NaCl to 0.5 M can also reverse some of the cleavable complex into the protein-linked complex. Either salt or EDTA alone cannot induce a reversion quite as efficiently as the combination of them. The efficiency of reversion is not affected by VM26 over a wide range of concentrations. For the DNA cleavage mediated by Drosophila topoisomerase significant stimulation can be observed in a reaction containing 0.3 lAg/ml VM26 and the amount of DNA cleavage increases with higher VM26 concentrations up to the maximal cleavage at a VM26 concentration of 100 gig/ml. Both the heat and EDTA/salt reversion steps can efficiently reverse the cleavage reactions carried out in the range of 0.3 to 100 jig/ml VM26. In the absence of VM26, the presence of the cofactor ATP in the reaction has only a modest effect on the topoisomerase II mediated DNA cleavage and results in about a 2-fold enhancement in the double strand DNA cleavage (15). However, in the presence of VM26, ATP can markedly increase the cleavage efficiency, while its analog ATP-yS exerts a lesser effect (compare lanes 3 and 5 with lane 1 in Fig. 3). The extent of these 11,
o
Figure 4. Kinetics of reversion of the cleavable complex by EDTA/CsCl. The same DNA substrate as described in Fig. 1 was used to form topoisomerase II/DNA complexes under similar reaction conditions, except that DNA and enzyme concentrations were 10-fold higher (2.1 nM DNA and 25 nM topoisomerase II). An aliquot was taken out and salt was diluted with 10 volumes of TE, followed by the addition of SDS to 1% to induce DNA cleavage (lane 2). Reversion was initiated by the addition of EDTA to 12 mM and followed by CsCl to 5 M. Aliquots were withdrawn at various timepoints after the addition of EDTA/CsCl: 10 sec. (lane 3), 10 min. (lane 4), 3 h. (lane 5), and 20 h. (lane 6). CsCl salt was diluted out with 10 volumes of TE and followed by the addition of SDS to 1%. Lane 1 is the substrate DNA and the arrow marks the position of the substrate DNA.
stimulatory effects
quantified by titrating the amount of
was
enzyme necessary to give rise to the same level of DNA cleavage. It appears that ATP and its analog ATP-yS can stimulate the
cleavage about 10- and 2-fold, respectively (data not shown). Heat reversal of the cleavable complexes is equally effective either in the absence or presence of ATP cofactors (Fig. 3, lanes 2, 4, and 6), and these protein-linked reversion products can be converted into fill-length DNA after protease digestion (data not shown). The kinetics of reversion is rapid. A significant amount of reversion takes place within 1 min. after shifting the incubation temperature to 65°C or upon the addition of EDTA/NaCl (data not shown). Efficient reversion was also observed when the reversion was induced by adding EDTA to 12 mM and CsCl to 5 M (Fig. 4). A slower reversion rate was observed here because of the much higher concentration of enzyme and DNA used in this experiment (see Fig. 4 legend). It is interesting to note that even after a prolonged incubation of 22 hours in EDTA/CsCl, a substantial amount of protein-linked reversion complex still persists (Fig. 4, lane 6).
Analysis of the reversion complexes by CsCI density gradient Since the reversed topoisomerase 11/DNA complex was stable in EDTA and 5 M CsCl this prompted us to analyze and isolate these complexes by CsCl density gradient equilibrium ,
centrifugation. In such a gradient, radiolabeled linear pBR322 DNA forms a single band with a measured density of 1.72 g/cm3 (Fig. 5A). For pBR322 DNA with a base composition of 54% guanosine and cytosine, this measured buoyant density is close to the calculated value based upon the known relation between buoyant density of DNA and its base composition (27). Only one peak, at the density expected for free DNA, is apparent when topoisomerase II/DNA complexes are formed in the absence of VM26 (Fig. SB), indicating that the majority of topoisomerase II/DNA complex formed in the absence of VM26 is not stable in a high concentration of CsCl. Most of the noncovalent protein/DNA complexes are expected to dissociate in such a high salt solution. When topoisomerase H/DNA complexes
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