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DNA TOPOISOMERASEII: A REVIEW OF ITS INVOLVEMENTIN CHROMOSOME STRUCTURE, DNA REPLICATION,TRANSCRIPTIONAND MITOSIS

Hilary J. Anderson and Michel Roberge Dept. Biochemistry, Fac. Medicine, University of British Columbia, Vancouver, BC, Canada, V6T 1Z3

INTRODUCTION Topological problems arise during replication and transcription of DNA and during mitosis because of the double-helical structure of DNA and its enormous length in chromosomes (Cook, 1991; Wang, 1991). For example, during replication and transcription large protein complexes track along the DNA, producing positive supercoils ahead and negative supercoils behind (Liu and Wang, 1987). The local torsional stress generated must be relieved for replication and transcription to proceed. There exist two types of enzymes capable of manipulating DNA topology: type I topoisomerases introduce transient single-stranded cuts and change the DNA linking number in steps of one while type II topoisomerases transiendy break both strands of a DNA segment and pass another double-stranded segment through the transient break, thus changing the DNA linking number in steps of two. Topoisomerase II can relax, catenate, decatenate, knot and unknot DNA (see Wang, 1985; Vosberg, 1985 for comprehensive reviews). The topoisomerases are expected to play an important role in many cellular processes since the topology of DNA is crucial to its function. This review summarizes recent studies on the involvement of topoisomerase II in the structural organization of DNA in the nucleus, and in the processes of replication, transcription, and mitosis. STRUCTURALORGANIZATIONOF DNA IN THE NUCLEUS Immunocytochemistry has shown that topoisomerase II is present throughout the core of the long axis of the chromatids of metaphase chromosomes (Gasser et al., 1986, 1989), and in meiotic chromosomes (Moens and Eamshaw, 1989; Klein et al., 1992). It is also found in matrix or scaffold preparations of interphase nuclei (Eamshaw et al., 1985; Berrios et al., 1985; Gasser and Laemrnli, 1986). Such preparations also contain particular A/T-rich DNA sequences called MARs or SARs (for matrix or scaffold association regions; Mirkovitch et al., 1984; CockeriU and Garrard, 1986). Topoisomerase H binds preferentially and cooperatively to these sequences (Adachi et al., 1989) and cleaves them (Sperry et al., 1989) in vitro. These observations have led to the proposal that topoisomerase H serves an inaportant structural role by anchoring MARs or SARs to the nuclear matrix or scaffold, the intervening sequences forming large loops of DNA (Gasser and Laemmli, 1987). However, Sperry et al. (1989) observed that topoisomerase 1] does not bind strongly to all MARs in vitro and that it binds strongly to some sequences which are not MARs. The authors also point out that the nuclei of resting ceils have very low 0309-1651/92/080717-8/$03.00/0

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levels of topoisomerase H while their isolated nuclear matrices retain the ability to bind MARs, suggesting that other proteins must participate in loop attachment. Thus, although topoisomerase H binding sites are generally associated with MARs, they are neither necessary nor sufficient to specify a MAR. Kaufmann and Shaper (1991 ) have shown that the enrichment of topoisomerase H in nuclear matrix or scaffold preparations may be due in large part to the formation of insoluble topoisomerase H oligomers linked by intermolecular disulfide bonds during sample preparation. They were unable to demonstrate the existence of these crosslinked complexes in intact cells. Determining the actual sites of topoisomerase II activity in vivo can provide direct information about the intranuclear localization of the enzyme. This can be done by treating cells or nuclei with drugs which stabilize an intermediate in the topoisomerase II reaction called the cleavable complex. Treatment of cleavable complexes with protein denaturants results in the formation of double strand DNA breaks which may be detected directly (Liu, 1989). Reitman and Felsenfeld (1990) used this technique to map topoisomerase II sites in the chicken B-globin locus. They found that topoisomerase II acts preferentially at DNase I-hypersensitive sites, suggesting that its access to DNA is generally restricted to nucleosome-free regions. Only the most upstream site was compatible with an involvement in DNA loop anchorage as it was close to the end of the locus and was constitutive. This is in agreement with previous reports that topoisomerase II sites occur at high frequency in DNase I hypersensitive regions and that only a subset of the sites are found at MARs (Rowe et al., 1986; Muller and Mehta, 1988; Pommier et al., 1990). These results are consistent with the observation that in SV40 DNA reconstituted with nucleosomes in vitro, the access of topoisomerase II to DNA is restricted to linker DNA between nucleosomes and to nucleosome-free regions (Capranico et al., 1990). It is possible that two classes of topoisomerase II molecules occur in the nucleus: mobile molecules able to bind to exposed DNA sequences in chromatin, and structural molecules serving to anchor DNA loops (Reitman and Felsenfeld, 1990). It is interesting that at least two topoisomerase II isozymes exist in mammalian ceils (Chung et al., 1989) and that they have different subnuclear distributions (M.T. Muller, personal communication). MITOSIS

The availability of topoisomerase mutants in yeast has made it a popular subject for studies of topoisomerase function. Studies with mutants have clearly established that topoisomerase II is required for chromatid disjunction and chromosome segregation at anaphase (DiNardo et al., 1984; Holm et al., 1985, 1989; Ucmura et al., 1987). The role of topoisomerase II is most likely to unlink the intertwined duplex molecules generated by DNA replication (Wang, 1991 and see next section). More recent studies have focussed on the involvement of topoisomerase II in chromosome condensation. In current models of chromosome structure, the chromatin fibre is folded into loops and further coiled during the early stages of mitosis to attain maximum levels of compaction at metaphase (Boy de la Tour and Laemmli, 1988). Topoisomerase II might be expected to play a catalytic role as topological

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problems arise during chromosome condensation, as well as a possible structural role (Wood and Earnshaw, 1990; Adachi et al., 1991; Chung and Muller, 1991). It is not clear to what extent chromosomes condense during a normal mitosis in the yeast S. pombe because condensed chromosomes can be seen only in cells arrested in mitosis either by mutations or by drug treatments affecting tubulin function. However, Uemura et al. (1987) have observed that in cold-sensitive [~tubulin and temperature-sensitive topoisomerase H double mutants of S. pombe, chromosomes are not as condensed when topoisomerase II is inactivated by temperature shift. This suggests either that topoisomerase II plays a direct role in the final stages of condensation or that chromosome condensation cannot go to completion if the intertwined daughter duplexes generated during replication remain intertwined. Chromosome condensation is more readily observed and better defined in higher eukaryotic cells. Although topoisomerase H mutants axe not available, other experimental approaches have been devised. In one approach, topoisomerase II inhibitors were used to block the activity of the enzyme in cultured mammalian cells. Three different groups reported that treatment of G2-phase cells with the topoisomerase II inhibitor VM-26 prevents chromosome condensation and entry into mitosis (Lock and Ross, 1990; Roberge et al., 1990; Charron and Hancock, 1990). VM-26 also halts phosphorylation of histones HI and H3, which is strictly correlated with chromosome condensation (Roberge et al., 1990) and inhibits cdc2 kinase, a major regulator of entry into mitosis (Lock and Ross, 1990). VM-26 has no inhibitory effect on the activity of the kinase in vitro, indicating that its inhibitory effect in vivo is not direct (Lock and Ross, 1990; Roberge et al., 1990). One interpretation of these results is that chromosomes do not condense in the presence of VM-26 because topoisomerase II activity is required for the process of chromosome condensation. Another possibility is that the cleavable complex stabilized by VM-26 is recognized by the cell as DNA--damage; it is well known that cells with damaged DNA do not enter into mitosis. In early surf clam embryos, mitotic chromosome condensation was also blocked by VM-26 (Wright and Schatten, 1990). In another approach, the involvement of topoisomerase II in chromosome condensation was investigated in vitro. Interphase nuclei undergo chromosome condensation when added to extracts from mitotic cultured cells (Wood and Eamshaw, 1990) or Xenopus eggs (Adachi et al., 1991). Wood and Earnshaw (1990) found that the extent of chromosome condensation parallels the levels of topoisomerase 11 present in the interphase nuclei when the mitotic extracts are previously immtmodepleted of their topoisomerase II. Mature erythrocyte nuclei, which contain less than 100 molecules of topoisomerase II, do not undergo efficient condensation whereas nuclei from dividing cells, which contain at least 100 000 topoisomerase II molecules, undergo condensation. In a more extensive study, Adachi et al. (1991) also demonstrated that adding purified topoisomerase II back to hrmaunodepleted extracts reestablishes their ability to condense chromosomes completely. This is clear and strong evidence for a direct involvement of topoisomerase H in chromosome condensation. However, some questions remain. In both studies, some condensation occurred in nuclei containing very low levels of topoisomerase II. Does

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this mean that the first stages of condensation do not require topoisomerase II? Or did some residual topoisomerase 1I remain which was sufficient to initiate condensation? Second, these studies did not address directly the question of whether topoisomerase II is required for its catalytic activity or for a structural role. Chromosome decondensation as cells enter the G1 phase has been comparatively little studied. In the surf clam, sperm nuclear decondensation in fertilized oocytes and mitotic chromosome decondensation took place normally in the presence of VM-26, suggesting that topoisomerase II activity is not required for chromosome decondensation (Wright and Schatten, 1990). However, the possibility that the drug-induced DNA breaks themselves caused decondensation was not addressed. DNA REPLICATION Tracking of the large DNA replication machinery along template DNA is expected to require a swivel to prevent the accumulation of the positive and negative supercoils generated ahead of and behind the moving replication fork (Cook, 1991; Wang, 1991). Other topological problems would occur when converging replication forks meet, depending on the relative rate of unraveling of the intertwined parental strands and progeny strand synthesis. Two extreme cases illustrate the problems: when unraveling is slower, the replicated DNA molecules will be intertwined; when completion of the progeny strand synthesis is slower, the daughter DNA molecules will be unlinked but gapped (Wang, 1991). Genetic studies in yeast (Brill et al., 1987; Goto and Wang, 1984; Uemura and Yanagida, 1984) have indicated that either topoisomerase I or topoisomerase II can serve as a swivel for DNA replication, but that topoisomerase I is probably the major one (Kim and Wang, 1989). Studies on replicating SV40 DNA have shown that topoisomerase II inhibitors only slow down the replication of the last 5% of the genome, implying that for most of the replication, topoisomerase I is used as a swivel (Snapka et al., 1988). Other results in mammalian cells support this view; camptothecin, a topoisomerase I inhibitor, blocks replication immediately while the topoisomerase II inhibitor fostriecin blocks DNA replication, but after a delay, again suggesting a role only at a late stage of replication (Gedik and Collins, 1990). In mammalian cells VM-26 induces lesions preferentially in nascent DNA (Nelson et al., 1986) perhaps because topoisomerase II acts as a swivel near the replication fork. This observation has been confirmed using a variety of topoisomerase II inhibitors (Woynarowski et al., 1988). As outlined in the previous section, topoisomerase II is required for the separation of replicated chromosomes, which otherwise remain intertwined. Topoisomerase II inhibitors result in the production of catenated SV40 genome circles in vivo (Snapka et al., 1988), again indicating a role for topoisomerase H in the decatenation of daughter duplexes. Replication of the linear adenovirus genome is insensitive to topoisomerase H inhibitors and the inhibitors do not induce doublestrand cleavages in newly-replicated DNA, suggesting that topoisomerase II is not required for replication of short linear genomes (Wong and Hsu, 1990). Therefore, it seems that topoisomerase II activity is required to separate the products of DNA replication, at least in circular or large linear genomes and may act

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as a swivel. The question remains of whether replication and unlinking of the daughter duplexes normally occur simultanously, or whether replication is normally completed with extensive intertwining of daughter strands, followed by decatenation, perhaps driven by chromosome condensation, only at the time of mitosis. TRANSCRIPTION It is not clear whether topoisomerase 11 plays any significant role in transcription. For methodological reasons, most studies have been able to address the issue only indirectly and the results have often been inconclusive. Studies with plasmids in yeast mutants have shown that it is topoisomerase I and not topoisomerase II that relieves most of the torsional stress generated during transcription (Brill and Stemglanz, 1988; Giaver and Wang, 1988). In higher eukaryotes, topoisomerase I activity is tightly linked to transcription of the c-fos gene, both temporally mad physically (Stewart et al., 1990), whereas the intensity of cleavage at only a few topoisomerase II sites in the ~-globin locus depends on active transcription (Reitman and Felsenfeld, 1990). It has also been pointed out that topoisomerase II concentration decreases to very low levels in tenninaUy differentiated and quiescent cells (Schaak et al., 1990) yet these are transcriptionally active. Muller and Mehta (1988) have shown that in the ~-globin gene topoisomerase H activity is not necessary to maintain the "open" conformation characteristic of chromatin permissive to transcription. But could it be needed to establish this state? That this might be the case was proposed by Hart et al. (1985) who showed that the induction by heat shock of HSP70 transcription in Drosophila cells is blocked by the inhibition of topoisomerase II by novobiocin. However, this effect may result from histone precipitation by the novobiocin (Schaak et al., 1990) or from disruption of protein-protein or protein-DNA interactions essential for transcription initiation (Webb and Jacob, 1988). Inhibition of topoisomerase H by VM-26 does not reduce transcription--of HeLa heat shock genes or adenoviral genes (Schaak et al., 1990). But, to add to the confusion, inhibition with ellipticine does block transcription of adenoviral genes and HeLa genes (Wong and Hsu, 1990). ACKNOWLEDGEMENTS We thank Mark T. Muller for helpful discussions. This work was supported by grant MT-11375 and a Scholarship to MR from the Medical Research Cotmcil of Canada. REFERENCES Adachi Y., Kiis E. and Laemmli U.K. (1989) Preferential, cooperative binding of DNA topoisomerase H to scaffold-associated regions. EMBO J. 8: 3997-4006. Adachi Y., Luke M. and Laemmli U.K. (1991) Chromosome assembly in vitro: Topoisomerase H is required for condensation. Cell 64: 137-148. Berrios M., Osheroff N. and Fisher P.A. (1985) In situ localization of DNA topoisomerase II, a major polypeptide component of the Drosophila nuclear matrix. Proc. Natl. Acad. Sci. USA 82: 4142-4146. Boy de la Tour E. and Laemmli U.K. (1988) The metaphase scaffold is helically folded: sister chromatids have predominantly opposite helical handedness. Cell

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55: 937-944. Brill S.J., DiNardo S., Voelkel-Meiman K. and Stemglanz R. (1987) Need for DNA topoisomerase activity as a swivel for DNA replication and for transcription of ribosomal RNA. Nature (Lond.) 326: 414-416. Brill S.J. and Stemglanz R. (1988) Transcription-dependent DNA supercoiling in yeast DNA topoisomerase mutants. Cell 54: 403-411. Capranico G., Jaxel C., Roberge M., Kohn K.W. and Pommier Y. (1990) Nucleosome positioning as a critical determinant for the DNA cleavage sites of mammalian DNA topoisomerase II in reconstituted simian virus 40 chromatin. Nucl. Acids Res. 18: 4553-4559. Charron M. and Hancock R. (1990) DNA topoisomerase II is required for formation of mitotic chromosomes in chinese hamster ovary cells: Studies using the inhibitor 4'-demethylepipodophyllotoxin 9-(4,6-O-thenylidene-b-D-glucopyranoside). Biochemistry 29:9531-9537. Chung I.K. and Muller M.T. (1991) Aggregates of oligo(dG) bind and inhibit topoisomerase II activity and induce formation of large networks. J. Biol. Chem. 266: 9508-9514. Chung T.D.Y., Drake F.H., Tan K.B., Per S.R., Crooke S.T. and Mirabelli C.K. (1989) Characterization and immunological identification of cDNA clones encoding two human DNA topoisomerase II isozymes. Proc. Natl. Acad. Sci. USA 86: 9431-9435. Cockerill P.N. and Garrard W.T. (1986) Chromosomal loop anchorage of the kappa hmnunoglobulin gene occurs next to the enhancer in a region containing topoisomerase II sites. Cell 44: 273-282. Cook P.R. (1991) The nucleoskeleton and the topology of replication. Cell 66: 627635. DiNardo S., Voelkel K. and Stemglanz R. (1984) DNA topoisomerase 11 mutant of Saccharomyces cerevisiae: topoisomerase II is required for segregation of daughter molecules at the termination of DNA replication. Proc. Natl. Acad. Sci. USA 81: 2616-2620. Eamshaw W.C., Halligan B., Cooke C.A., Heck M.M.S. and Liu L.F. (1985) Topoisomerase II is a structural component of mitotic chromosome scaffolds. J. Cell Biol. 100: 1706-1715. Gasser S.M., Amati B.B., Cardenas M.E. and Hofmann J.F.-X. (1989) Studies on scaffold attachment sites and their relation to genome function. Int. Rev. Cytol. 119: 57-95. Gasser S.M. and Laemmli U.K. (1986) Cohabitation of scaffold binding regions with upstream/erdaancer elements of three developmentally regulated genes of D. melanogaster. Cell 46: 521-530. Gasser S.M. and Laemmli U.K. (1987) A glimpse at chromosomal order. Trends Genet. 3: 16-22. Gasser S.M., Laroche T., Falquet J., Boy de la tour E. and Laemmli U.K. (1986) Metaphase chromosome structure: Involvement of topoisomerase II. J. Mol. Biol. 188: 613-629. Gedik C.M. and Collins A.R. (1990) Comparison of the effects of fostriecin,

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DNA topoisomerase II: a review of its involvement in chromosome structure, DNA replication, transcription and mitosis.

Cell Biology International Reports, VoL 16, No. 8, 1992 71 7 DNA TOPOISOMERASEII: A REVIEW OF ITS INVOLVEMENTIN CHROMOSOME STRUCTURE, DNA REPLICATIO...
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