Topoisomerase II can unlink replicating DNA by precatenane removal
2001; Springer Nature; Volume: 20; Issue: 22 Linguagem: Inglês
10.1093/emboj/20.22.6509
ISSN1460-2075
Autores Tópico(s)DNA and Nucleic Acid Chemistry
ResumoArticle15 November 2001free access Topoisomerase II can unlink replicating DNA by precatenane removal Isabelle Lucas Isabelle Lucas Present address: University of Washington, Department of Genetics, Seattle, WA, 98165 USA Search for more papers by this author Thomas Germe Thomas Germe Laboratoire de Génétique Moléculaire, Ecole Normale Supérieure, 46 rue d'Ulm, F-75230 Paris, Cedex 05, France Search for more papers by this author Marianne Chevrier-Miller Marianne Chevrier-Miller Laboratoire de Génétique Moléculaire, Ecole Normale Supérieure, 46 rue d'Ulm, F-75230 Paris, Cedex 05, France Search for more papers by this author Olivier Hyrien Corresponding Author Olivier Hyrien Laboratoire de Génétique Moléculaire, Ecole Normale Supérieure, 46 rue d'Ulm, F-75230 Paris, Cedex 05, France Search for more papers by this author Isabelle Lucas Isabelle Lucas Present address: University of Washington, Department of Genetics, Seattle, WA, 98165 USA Search for more papers by this author Thomas Germe Thomas Germe Laboratoire de Génétique Moléculaire, Ecole Normale Supérieure, 46 rue d'Ulm, F-75230 Paris, Cedex 05, France Search for more papers by this author Marianne Chevrier-Miller Marianne Chevrier-Miller Laboratoire de Génétique Moléculaire, Ecole Normale Supérieure, 46 rue d'Ulm, F-75230 Paris, Cedex 05, France Search for more papers by this author Olivier Hyrien Corresponding Author Olivier Hyrien Laboratoire de Génétique Moléculaire, Ecole Normale Supérieure, 46 rue d'Ulm, F-75230 Paris, Cedex 05, France Search for more papers by this author Author Information Isabelle Lucas2, Thomas Germe1, Marianne Chevrier-Miller1 and Olivier Hyrien 1 1Laboratoire de Génétique Moléculaire, Ecole Normale Supérieure, 46 rue d'Ulm, F-75230 Paris, Cedex 05, France 2Present address: University of Washington, Department of Genetics, Seattle, WA, 98165 USA *Corresponding author. E-mail: [email protected] The EMBO Journal (2001)20:6509-6519https://doi.org/10.1093/emboj/20.22.6509 PDFDownload PDF of article text and main figures. ToolsAdd to favoritesDownload CitationsTrack CitationsPermissions ShareFacebookTwitterLinked InMendeleyWechatReddit Figures & Info We have analysed the role of topoisomerase II (topo II) in plasmid DNA replication in Xenopus egg extracts, using specific inhibitors and two-dimensional gel electrophoresis of replication products. Topo II is dispensable for nuclear assembly and complete replication of plasmid DNA but is required for plasmid unlinking. Extensive unlinking can occur in the absence of mitosis. Replication intermediates generated in the absence of topo II activity have an increased positive superhelical stress (+ΔLk), suggesting a deficiency in precatenane removal. The geometry of replication intermediates cut by poisoning topo II with etoposide and purified by virtue of their covalent attachment to topo II subunits demonstrates that topo II acts behind the forks at all stages of elongation. These results provide direct evidence for unlinking replicating DNA by precatenane removal and reveal a division of labour between topo I and topo II in this eukaryotic system. We discuss the role of chromatin structure in driving DNA unlinking during S phase. Introduction Topoisomerases unlink DNA during and after replication (Ullsperger et al., 1995). Lk, the linking number of the parental DNA strands, must be reduced to zero for separation of daughter DNA molecules. ΔLk is the difference between Lk and Lk0, the value of Lk for the same DNA molecule in relaxed form. The unwinding of parental DNA by replicative helicases causes a compensatory (+)ΔLk that must be removed by topoisomerases. Champoux and Been (1980) suggested that the (+)ΔLk could take the form of (+) supercoils in the unreplicated region as well as windings of the two newly replicated regions around each other. Thus, topoisomerases could unlink replication intermediates (RIs) by acting either in front of or behind the fork. The form of (+)ΔLk in the replicated DNA was subsequently named precatenane, due to structural similarity to catenanes and to distinguish it from the (+)ΔLk in the unreplicated DNA (Ullsperger et al., 1995). Any (+)ΔLk not removed before termination would form catenanes, and these would be unlinked after replication. This model did not achieve immediate acceptance because early electron microscopy (EM) observations of circular RIs showed supercoils but no precatenanes, suggesting that topoisomerases only act ahead of the fork. However, studies of plasmid replication with purified Escherichia coli enzymes suggested that precatenanes are important in unlinking during replication (Peng and Marians, 1993; Hiasa and Marians, 1994, 1996). Moreover, both (−) precatenanes and (−) supercoils were observed on purified RIs accumulated by replication of plasmids containing two termination sites in E.coli (Peter et al., 1998). An EM artefact apparently caused earlier studies to miss precatenanes. Finally, a topological analysis of knots trapped within arrested RIs suggested that (−) precatenanes exist in E.coli cells (Sogo et al., 1999). However, removing (−) precatenanes would just increase Lk. Arrested RIs from E.coli cells have a (−)ΔLk, presumably due to gyrase activity after replication arrest. Direct evidence for precatenanes on (+)ΔLk RIs, as predicted by Champoux and Been, has been lacking. In fact, (+)ΔLk RIs prepared by adding intercalating agents to purified (−)ΔLk RIs contain neither supercoils nor precatenanes. Instead, the (+) topological stress is relieved by re-annealing of the parental strands and formation of a Holliday junction, a process called fork reversal (Postow et al., 2001; J.B.Schvartzman, personal communication). It remains unclear, at least in bacteria, whether transient (not arrested) RIs carry a (+) or a (−)ΔLk and whether protein binding inside the cell prevents fork reversal and/or spinning and (+) precatenane formation. In bacteria, two type 2 topoisomerases can unlink replicating DNA. Gyrase introduces (−) supercoils in front of the forks and may suffice to overcome the (+)ΔLk generated by replication until late RI stages, while topoisomerase IV (topo IV) is responsible for decatenating complete replication products (reviewed in Levine et al., 1998). Studies with purified enzymes support a role for precatenane unlinking by topo IV (Peng and Marians, 1993; Hiasa and Marians, 1996). However, in topo IV mutants, newly synthesized plasmid DNA accumulates as catenanes with the same node number distribution as transient catenanes in wild-type cells (Zechiedrich and Cozzarelli, 1995). Thus, in vivo evidence for precatenane removal by topo IV is lacking. In fact, the recent discovery that topo IV relaxes (+) supercoils 20-fold faster than (−) supercoils suggests that it may unlink DNA in front of the fork as efficiently as gyrase (Crisona et al., 2000). Eukaryotes lack unconstrained (−) supercoils and the (−) supercoiling activity of gyrase. Thus, free (+) supercoils and possibly (+) precatenanes are expected to form during elongation. Both topo I and topo II can remove (+) supercoils in vitro. Studies with yeast mutants showed that replication can initiate in the absence of both enzymes, but elongation stops after a couple of thousand base pairs (Kim and Wang, 1989). Either topo I or topo II (but not topo III) can support further elongation and completion of S phase (Uemura and Yanagida, 1986; Brill et al., 1987). Topo II is strictly required at mitosis for separation of sister chromatids (Holm et al., 1985; Uemura and Yanagida, 1986). Similar observations were made for the replication of naked SV40 DNA in cell-free extracts (Yang et al., 1987) or with purified proteins (Ishimi et al., 1992b). The usual interpretation is that either topo I or topo II can remove (+) supercoils to drive elongation, while topo II is required to remove catenane crossings persisting after replication. However, it is unclear whether topo II relaxes (+) supercoils in front of the forks, like topo I, or removes (+) precatenanes behind the forks. Studies of SV40 minichromosome replication in cellular extracts or in infected cells suggest that topo II inhibition in some cases blocks elongation at the late RI stage (Richter et al., 1987; Ishimi et al., 1992a,b), but in other cases allows synthesis of complete catenated dimers, even though late RIs accumulate (Sundin and Varshavsky, 1981; Ishimi et al., 1995 and references therein; reviewed in Snapka, 1996). Thus, the implication of topo II in the late stages of DNA synthesis in higher eukaryotes is unclear. Another unresolved issue is what drives decatenation of daughter DNA molecules. In vitro, topo II catalyses both catenation and decatenation of DNA rings, and favours catenation at high DNA concentration (Krasnow and Cozzarelli, 1982). Given the high concentration of DNA in vivo, one expects the equilibrium to be toward catenation. Mechanical separation of sister chromatids during mitosis has been proposed to drive decatenation (Holm, 1994; Duplantier et al., 1995). Although experiments in yeast and Xenopus show that topo II is required for mitotic chromosome condensation and segregation (reviewed in Holm, 1994), it is not known whether decatenation is postponed entirely until mitosis or already starts in S or G2 phases. To investigate these questions, we have studied the effect of topo II inhibition on DNA replication in Xenopus egg extracts. Because studying replication and topology of a long linear chromosome would be difficult, we focused on circular plasmid DNA. Any plasmid DNA incubated in Xenopus egg extracts is replicated under cell cycle control, but only after it has been assembled by the egg extract into chromatin and then into synthetic nuclei, in which replication occurs at discrete foci as in normal nuclei (Blow and Laskey, 1986; Blow and Sleeman, 1990; Cox and Laskey, 1991). Small plasmids ( 98%) inhibits decatenation of kinetoplast DNA by crude Xenopus egg extracts (Shamu and Murray, 1992; Takasuga et al., 1995; data not shown). We then analysed the effect of these drugs on plasmid replication (Figure 1A, upper row). pBR322 DNA was incubated in egg extracts in the presence of [α-32P]dATP for 90 min, cut with EcoRI and analysed using the two-dimensional gel technique of Brewer and Fangman (1987). Since pBR322 DNA contains a single EcoRI site and supports a single, randomly located initiation event, RIs consisted of bubbles and double Ys. Replicated plasmids (1× spot) and complete RI arcs were detected in the control or when 100 μM ICRF-193 or 200 μM VP-16 was added at the start of the incubation (Figure 1A, upper row). Similar results were obtained with 50 μM VM-26, 50–200 μM VP-16 or 50–200 μM ICRF-193 (not shown). Figure 1.Effect of topo II inhibition on nuclear assembly and replication of plasmid DNA in Xenopus egg extracts. (A) pBR322 DNA was incubated for 90 min in an egg extract in the presence of [α-32P]dATP, and in the presence or absence of 100 μM ICRF-193, 200 μM VP-16 and 1 mg/ml WGA as indicated. The DNA was purified, cut with EcoRI and analysed by two-dimensional gel electrophoresis. (B) Schematic diagram of two-dimensional gel patterns. The linear 1× spot corresponds to fully replicated plasmids. The three classes of RIs (O, bubble arc; Y, simple Y arc; H, double Y smear) are shown. The arc above the 1× spot consists of heterogeneously sized open circles formed by end joining of a minor population of broken molecules in the plasmid preparation. The faint labelling of the 1× spot in the WGA samples is presumably due to repair synthesis. (C) Light microscopic appearance of pseudo-nuclei assembled from pMM-36, a 36 kb plasmid (Lucas et al., 2000). pMM-36 DNA was incubated in egg extract for 3 h with 20 μM biotin-16-dUTP, and with or without 100 μM ICRF-193 or 200 μM VP-16 as indicated. Samples were supplemented with1 mg/ml Hoechst 33258 to show DNA, 0.1 vol. of fluorescein-tagged streptavidin (Amersham) to show biotin, and 12 μM rhodamine-tagged dextran 70S (Sigma) to show exclusion by the nuclear membrane, and were then viewed unfixed under fluorescence (Dextran, DNA, Biotin) or Nomarski optics (Nomar.). Some nuclei in the ICRF-193 sample showed a faint dextran signal, probably due to increased fragility and artefactual damage. An unambiguous example of strong dextran exclusion is shown by a smaller nucleus. Bar, 5 μm. Download figure Download PowerPoint This observation was surprising, since it was reported that replication of plasmid DNA depends on prior nuclear assembly (Blow and Sleeman, 1990) and that nuclear assembly is blocked by 40 μM VM-26 (Newport, 1987). We first verified that even with topo II inhibitors, plasmid DNA was assembled into pseudo-nuclei surrounded by a closed membrane that excludes fluorescent dextran and that replication occurred within these pseudo-nuclei (Figure 1C). Nevertheless, most of the DNA remained condensed at the centre of the pseudo-nuclei and detached from the nuclear envelope, and in some nuclei the DNA associated with the envelope replicated less efficiently. Secondly, we demonstrated that plasmid DNA replication in the presence or absence of topo II inhibitors was completely blocked by wheat germ agglutinin (WGA), an inhibitor of nuclear transport (Finlay et al., 1987) (Figure 1A, bottom row). WGA blocks sperm chromatin replication in egg extracts by preventing nuclear formation as well as by blocking protein import into pre-formed nuclei (Cox, 1992). In contrast, WGA has no effect on DNA synthesis on single-stranded templates (Cox, 1992), nor on sperm chromatin replication when the need for nuclear assembly is circumvented by sequential addition of cytosolic and nuclear extracts (Walter et al., 1998). Therefore, WGA has no effect on DNA replication other than by inhibition of nuclear assembly. We conclude that replication of plasmid DNA occurs exclusively in pseudo-nuclei and that topo II inhibition does not abolish nuclear assembly nor replication of plasmid DNA in egg extracts. Nevertheless, phosphoimager quantification showed that the amount of RIs was increased and the 1× spot was decreased in the presence of topo II inhibitors. Thus, both the turnover of RIs and the completion of plasmid synthesis were slower. Forks accumulated at all stages of elongation and not specifically near termination (quantitations not shown). As demonstrated in other experiments (see Supplementary data available at The EMBO Journal Online), these differences are due to an ∼2-fold slowing of fork progression. However, replication forks can proceed at normal speed if topo II inhibitors are only added after nuclear assembly (see Supplementary data). Therefore, topo II is required to optimize nuclear assembly for replication but not to stimulate elongation per se. Topo II rapidly decatenates daughter duplexes in S phase Next, we analysed the effects of topo II inhibition on the topology of DNA replication. Aliquots of pBR322 replication reactions were removed at 90 and 240 min and analysed on two-dimensional gels without restriction enzyme digestion (Figure 2). The migration behaviour of the various forms of plasmid monomers, RIs and catenated dimers is diagrammed in Figure 2. Evidence for these assignments is based on treatment with DNase I and topo I (Brewer and Fangman, 1987; Brewer et al., 1988; Martin-Parras et al., 1998). Further evidence based on topo II treatment (Figure 3) and partial digestion with EcoRI or S1 nuclease (Figure 5) is reported here. Figure 2.Topological analysis of plasmid replication. pBR322 DNA was incubated in an egg extract in the presence of [α-32P]dATP and in the presence or absence of 100 μM ICRF-193. Samples were removed at 90 and 240 min, and analysed by two-dimensional gel electrophoresis without restriction enzyme digestion. Interpretative diagrams are shown on the right. L, linear molecules; CCC, covalently closed circles; OC, open circles; the numbers refer to the multimeric state (1, monomers, 2, dimers). CCC1/OC1, molecules that migrated as CCC1 during the first dimension, were subsequently nicked, and migrated as OC1 during the second dimension. Cat A, Cat B, Cat C refer to OC1–OC1, OC1–CCC1 and CCC1–CCC1 catenated dimers, respectively. OC RIs and CCC RIs are open and covalently closed circular replication intermediates, respectively. Download figure Download PowerPoint Figure 3.Identification of CCC RIs and catenated dimers by resolution with topo II. pBR322 was replicated in an egg extract without ICRF-193 for 90 min or with 100 μM ICRF-193 for 240 min, in order to maximize the accumulation of RIs and catenated dimers, respectively. Purified replication products were analysed by two-dimensional gel electrophoresis either directly (left) or after treatment with D.melanogaster topo II (right). Arrows point to CCC RIs (top) and arrowheads to catenanes (bottom left) or to OC and relaxed CCC monomers (bottom right). Download figure Download PowerPoint In the controls (Figure 2, top row), most replication products were open circular (OC) and covalently closed circular (CCC) monomers. RIs were abundant at 90 min but scarce at 240 min, indicating that most nuclei had finished replicating at that time. The migration behaviour of RIs is discussed later. A small amount of catenated dimers was detected at both time points. A-type (OC–OC) and B-type (OC–CCC) catenanes were resolved as two series of spots of decreasing intensity, each corresponding to a certain node number. Only catenanes with 2 to <10 nodes (1 to 40 nodes. The most intertwined catenanes probably had 70–80 nodes. The migration of C-type catenanes also differed from the control. The trail emanating downwards from the spot of lowly intertwined C-type catenanes to merge with the bottom ends of A- and B-type catenanes arcs presumably consisted of highly intertwined C-type catenanes. A similar electrophoretic behaviour has been reported for C-type catenanes produced by exposure of replicating SV40 to ICRF-193, and has been attributed to an inverse relationship between catenation and superhelicity (Permana et al., 1994). Although this arc seemed continuous, we do not think it consisted of RIs because it accumulated with time (compare 90 and 240 min) and was converted to OC and CCC upon topo II treatment (Figure 3, bottom). In the 90 min sample (Figure 2, bottom left panel), only catenanes with >16 nodes were clearly detected. Thus, upon topo II inhibition, newly synthesized DNA essentially appeared in the form of highly intertwined catenated dimers. These results confirmed, as expected, that topo II is required for optimum unlinking of daughter duplexes in egg extracts. The topology of CCC RIs was analysed by treatment with topo II. Untreated CCC RIs migrated as a broad smear rising upwards from the CCC1 ladder and extending mainly to the right of OC RIs (Figures 2, top row and 3, top left). Topo II-treated, relaxed CCC RIs migrated to the left and very close to OC RIs (Figure 3, top right), as a thinner arc along which RIs were resolved essentially according to mass. Similar results were obtained with calf thymus topo I (Martin-Parras et al., 1998). Since (+)ΔLk RIs have no net writhe and migrate as relaxed RIs due to fork reversal (Postow et al., 2001; J.B.Schvartzman, personal communication), the faster migration of untreated RIs in the first dimension must be due to compaction by (−) precatenanes and (−) supercoils. In the second dimension, the high (+)ΔLk introduced by ethidium bromide caused all RIs to migrate as relaxed RIs. The ΔLk of untreated RIs had two potential sources: the (+)ΔLk generated by replication and the (−)ΔLk constrained by nucleosomes in unreplicated DNA. The electrophoretic behaviour of untreated RIs shows that when both topo I and topo II are active, the resulting ΔLk remains negative until the very last helix turns are replicated. The migration of CCC RIs from ICRF-193-treated extracts was different from that of control RIs. The signal distribution across the smear was shifted to the left, especially in the upper part of the gel, suggesting a globally less negative ΔLk (Figure 2). Since catenanes with up to 80 nodes were seen, the ΔLk of the latest RIs (>90% replicated) should range from 0 to +40. According to Postow et al. (2001) and J.B.Schvartzman (personal communication), these (+)ΔLk RIs will migrate as relaxed RIs of the same mass. Indeed, most of the signal from late RIs concentrated at the upper left corner of the smear, at the position of relaxed late RIs (compare with Figure 3, top right). The changes in the electrophoretic behaviour of CCC RIs upon topo II inhibition reveal that topo II actively unlinks DNA during elongation in a way that cannot be taken over fully by topo I. This less complete unlinking of RIs provides a simple explanation for the increase in catenane complexity observed upon topo II inhibition. The increase in ΔLk appears stronger for late RIs, suggesting that the unlinking action of topo II that cannot be taken over by topo I increases as replication progresses. This is suggestive of precatenane rather than supercoil removal because the larger the replicated region, the more the ΔLk should be expressed as precatenanes (Peter et al., 1998; reviewed in Snapka, 1996), and because topo I, which is abundant in these extracts, can remove supercoils but not precatenanes. Topo II acts behind the forks during replication elongation In order to map where topo II acts during replication, the location of VP-16-induced breaks on RIs was assessed by two-dimensional-gel electrophoresis. VP-16 induces both double- and single-stranded breaks (DSBs and SSBs) in plasmid DNA in egg extracts (see Supplementary data). If topo II acts in the replicated region, an SSB will not change the topology of RIs, and a DSB will convert CCC and OC RIs into two-tailed circular RIs (TTC RIs; Figure 4A). On the other hand, if topo II acts in the unreplicated region, an SSB will convert CCC RIs into OC RIs and a DSB will convert CCC and OC RIs into bubble-shaped fragments (Figure 4B). Finally, a collision between a replication fork and a cleavable complex in unreplicated DNA will result in sigma-shaped RIs (σRIs; in the case of an SSB) and simple Y RIs (in the case of a DSB) (Figure 4C). The resulting two-dimensional gel patterns should allow discrimination between these three scenarios. Figure 4.Mapping the sites of topo II action on plasmid RIs by trapping topo II–DNA cleavable complexes with VP-16. Products expected after a single or a double-strand break behind (A) or in front of (B) the fork or after collision of the fork with the cleavable complex (C) are shown. The expected changes in two-dimensional gel patterns are shown at the bottom. Download figure Download PowerPoint Figure 5.Identification of σRIs and TTC RIs. pBR322 was replicated in an egg extract for 90 min. Purified replication products were partially digested with S1 nuclease (A) or with EcoRI (B) and analysed by two-dimensional gel electrophoresis. Predicted products (left), experiments (middle) and interpretative diagrams (right) are shown. For clarity, CCC RIs and catenanes are not shown. Download figure Download PowerPoint The migration behaviour of bubbles, simple Ys and OC RIs is well documented (Brewer and Fangman, 1987). To identify σRIs, purified RIs were partially digested with S1 nuclease and the products were analysed on two-dimensional gels (Figure 5A). S1 cut parental DNA at the single-stranded regions of the forks, converting OC RIs and CCC RIs into σRIs (one cut), simple Y RIs (two cuts at one fork) and linear plus OC fragments (cuts at both forks). The resulting gel allowed the unambiguous identification of σRIs as the eyebrow-shaped arc starting and extending leftwards from the OC spot, as previously suggested (Belanger et al., 1996; Martin-Parras et al., 1998). To identify TTC RIs, purified RIs were partially digested with EcoRI (Figure 5B). As pBR322 replication initiates randomly in egg extracts, EcoRI cut at random in the unreplicated region (bubbles) or in one (TTC RIs) or both (double Ys) replicated arms. The arc of TTC RIs started from the OC spot much like σRIs, but broadened in a triangular smear above the position where σRIs would migrate. The migration of TTC RIs with respect to σRIs was reminiscent of the migration of double Ys with respect to simple Ys. Presumably, TTC RIs of identical masses but different lengths of the two tails migrate similarly in the first dimension but differently in the second dimension, with asymmetric forms migrating faster (closer to σRIs). Figure 6A shows the two-dimensional gel patterns generated when 50 μM VP-16 was present in the extract. Control (no drug) gels are shown in Figures 2 and 3, top left panels. Topo II inhibition by VP-16 resulted in the accumulation of highly intertwined catenated dimers, as with ICRF-193. However, the inhibition of topo II by 50 μM VP-16 was less complete than with 100 μM ICRF-193, as judged by the distribution of catenane node number, the amount of OC and CCC monomers and the migration of CCC RIs. Importantly, we observed in the VP-16-treated samples a new, composite arc (arrow) consisting of material either absent or less abundant in control or ICRF-193-treated samples. The rightmost part of the signal consisted of a strong eyebrow of σRIs surmounted by a faint triangular smear of TTC RIs. This signal was consistent with a mixture of DSBs behind the forks and SSBs at the fork (pathways A/DSB + C/SSB in Figure 4). Alternatively, this signal could result from DSBs exclusively behind but predominantly close to the forks (pathway A/DSB only). The leftmost part of the signal prolonged the eyebrow, but not the triangular smear, upwards and leftwards beyond the 2× position. This arc of >2× σRIs probably arose by ligation of a nascent strand to the broken parental strand of a 1–2× σRI to create a rolling circle intermediate, as seen when an SSB is produced by DNase activity (Gourlie and Pigiet, 1983) or by topo I poisoning (Snapka, 1986). Alternatively, rolling circles could result from repair of a TTC RI by the action of a flap endonuclease, gap filling and ligation. Neither a bubble arc, a simple Y arc nor a convincing increase of OC RIs was detected, failing to support pathways B/SSB, B/DSB and C/DSB. Overall, these results suggest that topo II poisoning by VP-16 results in DSBs behind the forks and possibly SSBs only a short distance ahead of the forks. Figure 6.Topo II acts behind replication forks. pBR322 DNA was replicated for 90 min in the presence of 50 μM VP-16. The total replication products (A) or the phenol-extracted covalent topo II–DNA complexes (B) were analysed by two-dimensional gel electrophoresis. The composite arc of σRIs, TTC RIs and rolling circle RIs induced by VP-16 is shown by an arrow on the top left panel. Interpretative diagrams are shown on the right. Download figure Download PowerPoint
Referência(s)