The DNA topoisomerases are the sirens of DNA biochemistry. They are attractive enzymes with elusive roles. Type I and type I1 topoisomerases are both capable of altering DNA topology; type I1 by making a transient double strand break which allows another DNA molecule (or another part of the same molecule) to pass through, type I performing a similar trick via a single strand break. They reduce the level of supercoiling of closed circular DNA molecules in model systems; we can assume they do the same with linear eukaryotic DNA in vivo, since this is constrained as loops, anchored to the nuclear matrix, and is effectively circular. The temptations that have led the unwary astray are classic ones: the facile extrapolation from prokaryotic to eukaryotic systems and the over-interpretation of experiments with inhibitors. A bacterial type I1 topoisomerase, gyrase, is able to introduce negative supercoils into closed double-stranded circles of DNA, a process that requires energy; eukaryotic type I1 enzymes isolated so far are able only to relax supercoils, though they still re uire ATP and, like gyrase, possess an ATPase activity(' . There is, of course, the possibility that a gyrase-like activity has been lost during preparation of the eukaryotic type I1 enzyme, but there is no problem in accounting for the introduction of supercoils in eukaryotic DNA without any involvement of gyrase. The bulk of eukaryotic supercoiling is due to the winding of DNA about the nucleosomes, and is apparently achieved 'passively', since while the nucleosomes are assembled during replication the nascent DNA is free to rotate; on final ligation, the supercoils are sealed in. But topoisomerase I1 is essential, at least in yeast. DiNardo et al.(2)used a Saccharomyces cerevisiae strain with a temperature-sensitive mutation in topoisomerase 11. The cells were synchronised at the permissive temperature; it was found that, on raising them to the non-permissive temperature, they underwent one and only one round of DNA replication. Arrest occurred in nuclear division. The reason for the arrest became clear when the structure of a 2pm plasmid that replicates naturally in these cells was analysed; it was found to be in the form of multiply intertwined catenated dimers, that would normally be separated after replication. In an in vitro SV40 replication system, too, topoisomerase

9

I1 was found to be essential for segregation of newly synthesised daughter molecules(3). The reason that daughter DNA molecules are intertwined is probably that some turns of the double helix (normally unwound in advance of the replication fork by topoisomerase I) remain in place as two adjacent replication forks approach each other, and are translated into turns of one daughter molecule about the other(4). The prokaryotic gyrase has a well-known inhibitor, novobiocin. Many people (this author included) have investigated the effects of novobiocin in eukaryotes, and have jumped to the conclusion that they are studying the inhibition of topoisomerase 11. Novobiocin does inhibit eukaryotic topoisomerase I1 in vitro but what should alert our suspicions is the fact that the prokaryotic gyrase is inhibited at a 1 0 0 0 ~lower concentration. In cultured animal cells, novobiocin strongly inhibits DNA replication(536),cell cycle progression from Go to S phase as well as tran~cription(~), and the repair of damage induced in DNA by UV and alkylating agents(69s). There are credible roles for topoisomerase I1 in all these processes but the evidence has been accumulating that the target for novobiocin in these experiments is not necessarily topoisomerase 11. Downes et u Z . ( ~ ) found that, at the concentrations employed to inhibit replication and repair, novobiocin profoundly disturbs the structure of mitochondria in human cells. It also reduces the ratio of intracellular ATP:ADP. Since both replication and repair require ATP, this, rather than an inhibition of topoisomerase 11, could explain the cellular effects of the drug. This interpretation has been strengthened with recent reports from the kicester University group of Elisha Orr and colleagues, who have looked for the target of novobiocin in yeast. They find that yeast topoisomerase I1 does not bind to novobiocin in vitro (unlike gyrase) and that novobiocin-resistant mutants are not defective in topoisomerase II(''). They set out to identify possible targets for the antibiotic by analysing the proteins from yeast that bind the drug strongly. Yeast cell extract was passed through a column of Sepharose linked to novobiocin. From this column, two protein species were eluted with 2 M KCl; three other proteins required 5111 urea for removal. One of this latter group was studied in detail("). It is a protein of molecular mass 50K, which is specifically eluted from the column by 1 0 m ATP. ~ The N-terminal region of 20 amino acids was sequenced, revealing a perfect homology with the yeast F1 ATP synthetase /%subunit, a mitochondria1 protein highly conserved throughout evolution. The protein (p50) was used to raise antibodies, which were applied to Western blots of total yeast proteins and of the proteins eluted from the novobiocin-Sepharose, a single band was recognised. The localisation of p50 in yeast mitochondria was confirmed by indirect immunofluorescence staining with the antibodies. The same protein was found (by Western blot analysis) in extracts of chicken liver and the cytoplasm of a human cell line,

and mitochondria of mammalian and plant cells were stained with the antibodies. Bacterial novobiocin-binding proteins, separated by the same technique of novobiocin-Sepharose chromatography, include one of similar molecular mass to p50 that cross-reacts with the anti-p50 antibodies. However, there is very little homology in amino acid sequence between p50 and the B subunit of gyrase (apart from a motif that may represent the common novobiocinbinding domain). So the ATPase target for novobiocin in yeast, and other organisms, is apparently distinct from the gyrase of bacteria. The effect of novobiocin on yeast mitochondria is striking; they become condensed and stain intensely with the DNA-binding dye DAPI or with the anti-p50 antibodies. Mammalian cell mitochondria incubated with novobiocin for two hours become similarly distorted and condensed, as visualised with the anti-p50 antibodies. In the earlier report of novobiocin effects on mitochondria in human cells('), mitochondria were examined by electron microscopy. A progressive swelling of mitochondria was seen, followed by rupture of membranes and collapse of the organelles. So, clearly, one of the major effects of novobiocin is to bind to the p-subunit of the F1ATP synthetase and to interfere with ATP synthesis. However, this does not account for all of the activity of novobiocin. po strains of yeast lack mitochondrial DNA and have energetically inactive mitochondria (they grow by fermentation alone). Yet they, too, are susceptible to growth inhibition by novobiocin. It is likely that the other four novobiocin-binding proteins isolated by the Leicester group include further targets for novobiocin toxicity. One of these, of 200K molecular mass, is yeast myosin heavy chain(12). Another is a 52K protein, which also binds to DNA and is associated with the nuclear membrane. A mutant allele of the gene coding for this protein confers novobiocin resistance in yeast (ref. 13, and personal communication, E. Orr). Similarly, in mammalian cells, novobiocin has effects in addition to those mediated via disruption of ATP production. We recently looked(14), yet again, at the question of whether topoisomerase I1 is involved in DNA repair, using a novel inhibitor, fostriecin, which blocks topoisomerase I1 with apparently a higher specificity than novobiocin. We found that fostriecin caused a strong inhibition of replicative DNA synthesis, but only after a delay of over 30min - consistent with the proposed role for topoisomerase I1 at a late stage in replication rather than at the replication fork itself. However, there was no effect at all of fostriecin on repair of UV-damaged DNA; topoisomerase I1 can therefore be excluded from that process. Downes et and Snyder(16) reported a similar failure of another type of topoisomerase I1 inhibitor (VP-16 and

mAMSA) to inhibit DNA repair. We examined(14)the possibility that novobiocin blocks repair through ATP depletion, by employing a permeabilised cell system in which ATP - essential for DNA repair - is supplied exogenously. We found that repair was still strongly inhibited by novobiocin and that increasing the concentration of ATP did not decrease the effectiveness of the drug. So we await with interest the characterisation of other proteins that act as novobiocin targets.

References 1. HALLIGAN, B. D., EDWARDS, K. A.

A N D LIU.L. F. (1985). Purification and characterization of a type I1 DNA topoisomerase from bovine calf thymus. J. Biol. Chem. 260. 2475-2482. 2 DINARDO, S.. VOELKEL,K. AND STERNGLANZ, R. (1984). DNA topoisomerase 11 mutant of Saccharomyces cerevisiae: topoisomerase 11 is required for segregation of daughter molecules at the termination of DNA replication. Proc. Natl Acad. Sci. USA 81. 2616-2620. 3 YANG,L.. Worn, M. S., LI, J. J.. KELLY, T. J. AND LIV,L. F. (1987). R o l c ~ of DNA topoisomerases in simian virus 40 DNA replication in vitro. Proc. Narl Acad. Sci. USA 84. 950-954. 4 SUNDIN, 0. AND VARSHAVSKY, A. (1981). Arrest of segregation leads to accumulation of highly intertwined catenated dimers: dissection of the final stages of SV40 DNA replication. Cell 25, 659-669. 5 MATTERN, M. R. AND PAINTER, R. B. (1979). Dependence of mammalian DNA replication on DNA supercoiling. 11. Effects of novobiocin o n DNA synthesis in Chinese hamster ovary cells. Biochim. Biophys. Acra 563,306-312. 6 COLLINS, A. A N D JOHNSON, R. T. (1979). Novobiocin: an inhibitor of thc repair of UV-induced but not X-ray-induced damage in mammalian cells. Nacl. Acids Res. 7. 1311-1320. 7 ALLER,P. A N D BASERGA. R. (1986). Selective increases of c-myc mRNA levels by methylglyoxal-bis (guanylhydrazone) and novobiocin in serum stimulated fibroblasts. J. Cell. Physiol. 128, 362-366. 8 MATTERN. M. R., PAONE,R. F. A N D DAY.R. S. (1982). Eukaryotic DNA repair is blocked at different steps by inhibitors of DNA topoisomerases and of DNA polymerases &and /3. Biochim. BiophyA. Acta 697, 6-13. 9 DOWNES,C. S., ORD.M. J., MULLINGER, A . M., COLLINS, A. R. S. A N D JOHNSON, R. T. (1985). Novobiocin inhibition of DNA excision repair may occur through effects on mitochondrial structure and ATP metabolism, not on repair topoisomerases. Carcinogenesis 6 , 1343-1352. 10 POCKLINGTON, M. J., JENKINS, J . R. A N D ORR,E. (1990). The effect of novobiocin on yeast topoisomerase type 11. Molec. Gen. Genet. 220. 256-260. 11 JENKINS, J. R., POCKLINGTON, M. J. A N D ORR,E. (1990). The F, ATP synthetase &subunit: a major yeast novobiocin binding protein. J. Cell Scr. 96. 675-682. 12 WATTS,F. Z., MILLER, D. M. AND ORR,E. (1985). Identification of myosin heavy chain in Saccharomyces cerevisiae. Nature 316. 83-85. 13 POCKI.INGTON, M. J.. JOHNSTON, L.. JENKINS, J. R. AND ORR,E. (1990). The omnipotent suppressor SUP45 affects nucleic acid metabolism and mitochondrial structure. Yeast 6. in press. 14 GEDIK.C. M. A N D COLLINS,A. R. (1990). Comparison of effects of fostriecin, novobiocin, and camptothecin, inhibitors of DNA topoisomerases, on DNA replication and repair in human cells Nucl. Acids Res. 18. 1007-1013. 15 DOWNES, C. S., MULLINGER, A. M. A N D JOHNSON, R. T. (1987). Action of etoposide (VP-16-123) on human cells: no evidence for topoisomerase 11 involvement in excision repair of u.v.-induced DNA damage, nor for mitochondrial hypersensitivity in ataxia telangiectasia. Carcinogenesis 8. 1613-1618. 16 SNYDER, R. D. (1987). 1s DNA topoisomerase involved in the UV excision repair process? New evidence from studies with DNA intercalating and nonintercalating antitumor agents. Photochem. Photobiol. 45. 105-111.

Andrew Collins is at the Department of Molecular and Cell Biology, University of Aberdeen, Marischal College, Aberdeen AB9 1AS, Scotland.