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Bacteriophage T4, a model system for understanding the mechanism of type II topoisomerase inhibitors
Biochimica et Biophysica Acta 1400 (1998) 339^347
Review
Bacteriophage T4, a model system for understanding the mechanism of type II topoisomerase inhibitors Kenneth N. Kreuzer * Department of Microbiology, Duke University Medical Center, Durham, NC 27710, USA Accepted 22 April 1998
Abstract Bacteriophage T4 provides a simple model system for analyzing the mechanism of action of antitumor agents that inhibit DNA topoisomerases. The phage-encoded type II topoisomerase is sensitive to many of the same antitumor agents that inhibit mammalian type II topoisomerase, including m-AMSA, ellipticines, mitoxantrone and epipodophyllotoxins. Results from the T4 model system provided a convincing demonstration that topoisomerase is the physiological drug target and strong evidence that the drug-induced cleavage complex is important for cytotoxicity. The detailed molecular steps involved in cytotoxicity, and the mechanism of recombinational repair of inhibitor-induced DNA damage, are currently being analyzed using this model system. Studies with the T4 topoisomerase have also provided compelling evidence that topoisomerase inhibitors interact with DNA at the active site of the enzyme, with each class of inhibitor favoring a different subset of cleavage sites based on DNA sequence. Finally, analysis of drug-resistance mutations in the T4 topoisomerase have implicated certain regions of the protein in drug interaction and provided a strong link between the mechanism of action of the antibacterial quinolones, which inhibit DNA gyrase, and the various antitumor agents, which inhibit mammalian type II topoisomerase. 0167-4781 / 98 / $ ^ see front matter ß 1998 Elsevier Science B.V. All rights reserved. Keywords: Antitumor agent; Quinolone; Topoisomerase ; DNA repair; Bacteriophage; Aminoacridine
Contents 1.
Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
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2.
Topoisomerase is the physiological target for m-AMSA . . . . . . . . . . . . . . . . . . . . . . . . . .
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3.
Molecular mechanism of toxicity . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
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4.
Repair of the cleavage complex . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
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The inhibitor binding site . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
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6.
Topoisomerase mutations that alter drug sensitivity . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
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* Fax: +1 (919) 681-8911; E-mail: [email protected] 0167-4781 / 98 / $ ^ see front matter ß 1998 Elsevier Science B.V. All rights reserved. PII: S 0 1 6 7 - 4 7 8 1 ( 9 8 ) 0 0 1 4 5 - 6
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Acknowledgements . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
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References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
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1. Introduction The relatively simple bacteriophage T4 provides a very useful model system for determining the precise mechanism of action of antitumor agents that inhibit the mammalian type II DNA topoisomerases. The T4 and mammalian topoisomerases share several regions of conserved amino acid sequence [1] and display similar enzymatic properties, including catalysis of ATP-dependent relaxation, lack of intrinsic DNA supercoiling activity, and inhibition by singlestranded DNA [2]. The possibility that phage T4 could be used to study antitumor drug action ¢rst emerged when Liu and coworkers found that the aminoacridine m-AMSA (4P-(9-acridinylamino)methanesulfon-m-anisidide) inhibits both the mammalian and T4 topoisomerases [3,4]. In both cases, m-AMSA induces formation of the cleavage complex, suggesting a common mechanism of inhibition. The inhibition of the mammalian and phage enzymes by m-AMSA is not a coincidence ^ the phage enzyme is also sensitive to mitoxantrone (an anthracenedione), members of the ellipticine family, and two epipodophyllotoxins, all of which inhibit the mammalian enzyme [5]. As will be mentioned below, a variety of results from the T4 system closely parallel those from studies of mammalian systems, validating the use of this simple bacterial virus for analyzing antitumor drug action. The T4 model system has numerous advantages which facilitate rapid progress. The phage genome is a single duplex DNA molecule of just under 170 kb, which is now entirely sequenced. T4 has been an intensively studied prokaryotic system since the early days of molecular biology, so that powerful tools of classical and molecular genetics and biochemistry can be readily applied. With respect to the phage-encoded topoisomerase, drug-resistant mutants can be isolated by a simple 1-day procedure and analyzed genetically over the course of just a few weeks [6,7]. Furthermore, milligram amounts of wild-type or mutant topoisomerase can be puri¢ed to
homogeneity by a 3-column procedure that takes a few days [2,8]. From a broader perspective, T4 provides one of the most thoroughly studied systems of nucleic acid metabolism (replication, recombination, transcription, repair). The primary sequences of all phage proteins involved in these processes have been determined, and most of these proteins have been overproduced, puri¢ed and analyzed in simple in vitro systems. Given these numerous advantages, bacteriophage T4 provides an inexpensive, convenient and valuable model system for the analysis of antitumor drug action. 2. Topoisomerase is the physiological target for m-AMSA Genetic and biochemical analyses of m-AMSA-resistant T4 mutants provided convincing evidence that the phage-encoded topoisomerase is the physiological target for the antitumor drug in T4-infected bacterial cells. Growth of wild-type T4 (but not the Escherichia coli host) is prevented by m-AMSA, and drugresistant phage mutants were readily isolated simply by plating mutagenized phage on bacterial lawns grown in the presence of the inhibitor [6,7]. Topoisomerase puri¢ed from the drug-resistant phage displayed m-AMSA-resistance during in vitro reactions. The results of standard phage genetic crosses localized one drug-resistance mutation to gene 39 and another to gene 52; these two genes encode two of the three topoisomerase subunits. Furthermore, gene replacement techniques were used to prove that a single amino acid substitution in either gp39 or gp52 is su¤cient to confer m-AMSA-resistance [9]. 3. Molecular mechanism of toxicity Results from a variety of systems (e.g. E. coli, yeast and mammalian cells) indicate that the potent cytotoxicity of topoisomerase inhibitors results from
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stabilization of the cleavage complex rather than just simple enzyme inhibition (see [10^12]). The same is true in the phage T4 system. Thus, inhibition of the T4 topoisomerase by m-AMSA is more detrimental to phage growth than mutational elimination of the topoisomerase. Furthermore, the mutational inactivation of the topoisomerase actually protects T4 from the detrimental e¡ect of m-AMSA, clearly implicating the drug-induced cleavage complex in toxicity [13]. One of the strongest arguments that toxicity involves a form of DNA damage is the fact that a special form of DNA repair strongly reduces the effectiveness of topoisomerase inhibitors (see below). Also consistent with a key role of DNA damage, results from the T4 system uncovered two distinct pathways of mutagenesis induced by topoisomerase inhibitors. Ikeda and coworkers showed that oxolinic acid (a weak inhibitor of the T4 topoisomerase) induces illegitimate recombinants between non-homologous DNA molecules in vitro [14^16]. The sites of illegitimate recombination coincided with oxolinic acid-induced enzyme-mediated DNA cleavage sites, leading to a `subunit exchange' model in which two topoisomerase complexes at di¡erent sites trade `half enzymes' during the cleavage-resealing reaction cycle. The second pathway of mutagenesis was analyzed extensively by Ripley and coworkers, who found that m-AMSA and related topoisomerase inhibitors are potent frameshift mutagens in phage T4-infected cells [17,18]. Every frameshift mutation at a drug-induced hotspot had one endpoint at one of two phosphodiester bonds that were spaced four basepairs apart on opposite strands of the helix, and these two locations were precisely the sites of cleavage for a strong drug-induced enzyme cleavage site [18]. A variety of additional results provided convincing evidence that the topoisomerase is directly involved in drug-induced frameshift mutagenesis: (1) a mutation that inactivates topoisomerase eliminated frameshift mutagenesis [18]; (2) m-AMSA was more e¡ective both at inducing frameshift mutations and inducing enzyme-mediated DNA cleavage than its closely related isomer o-AMSA [18]; (3) DNA sequence alterations in the hotspot site that greatly reduced topoisomerase-mediated DNA cleavage also greatly reduced frameshift mutagenesis [19]; and (4) DNA sequence alterations in the hotspot site that shifted the site of
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enzyme-mediated DNA cleavage also correspondingly shifted the positions of drug-induced frameshift mutations [19]. From these and other results, it is clear that mutations are generated from DNA discontinuities introduced by the topoisomerase and stabilized by the topoisomerase inhibitors. The detailed steps involved in drug-induced frameshift mutagenesis are currently under investigation (see [20]). One of the most pressing problems in the topoisomerase ¢eld is to understand the detailed mechanism of cytotoxicity. The generation of mutations presumably contributes, but is unlikely to account for the majority of drug-induced toxicity. Results from mammalian systems provide several clues. First, drug-induced toxicity is reversible, as is the drug-induced cleavage complex, if the drug is washed out relatively quickly. However, toxicity becomes irreversible with prolonged incubation, arguing that the reversible cleavage complex is converted into an irreversible form of DNA damage (see [21]). Second, DNA replication and transcription have been implicated in the cytotoxicity of topoisomerase (type I and II) inhibitors, suggesting that a crucial event in DNA damage involves nucleic acid synthesis on a template that contains a drug-induced cleavage complex [22^ 24]. As a ¢rst step towards understanding the consequence of a collision between a replication fork and a cleavage complex, we staged an in vitro showdown between a DNA helicase and a topoisomerase cleavage complex [25]. Every DNA replication fork contains a helicase, which opens the parental helix, and therefore the initial encounter between the replication fork and cleavage complex is likely to involve the helicase. The results were dramatic ^ the helicase was able to convert the normally reversible cleavage complex into an irreversible break, and at least one of the broken strands (the single strand that was not covalently attached) was released from the enzyme complex. These results are consistent with models in which a replication fork-associated helicase disrupts the cleavage complex, creating a frank DNA break. Production of a frank DNA break could be a critical step in cytotoxicity, frameshift mutagenesis (see above), and/or recombinational repair (see below). We are currently attempting more complete experiments, namely, analyzing the consequences of
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running a complete replication fork into a cleavage complex both in vivo and in vitro. 4. Repair of the cleavage complex As already mentioned, a special form of DNA repair is able to reduce the e¡ectiveness of topoisomerase inhibitors, presumably because this pathway can repair some fraction of the topoisomerase-mediated DNA damage. Thus, the sensitivity of Saccharomyces cerevisiae to m-AMSA is increased by mutations in the RAD52 gene, which encodes a protein involved in recombinational repair of radiation-induced damage and double-strand DNA breaks [26,27]. Likewise, mutations in the xrs genes of CHO cells increase sensitivity to both radiation and inhibitors of type II topoisomerase [28,29]. The T4 system is useful for investigating the repair of damage caused by topoisomerase inhibitors. Many pathways of DNA repair, including recombinational repair, have been extensively studied in the T4 system (see [30^32]). Indeed, the very ¢rst evidence for recombinational repair emerged from the T4 system a half century ago [33], and since that time, phage recombination proteins have been identi¢ed and characterized in biochemical detail (see [34,35]). Mutations that inactivate key T4 recombination proteins (UvsX, UvsY, UvsW, RNase H, gp59 and gp46/47) increase the sensitivity of the phage to mAMSA, consistent with a role of recombinational repair in drug sensitivity [13,36]. Most of these proteins have previously been shown to be involved in recombinational repair of radiation- and/or chemical-induced DNA damage. Two additional results are consistent with the proposed role of recombination in repairing m-AMSA-induced cleavage complexes. First, homologous recombination was found to be stimulated by the drug, presumably re£ecting productive recombinational repair reactions. Second, we detected m-AMSA-induced cleavage complexes within the infected cell, with cleavage occurring at the same DNA sites as in in vitro reactions with the puri¢ed topoisomerase [13]. Based on these results and the known activities of T4 recombination proteins, a plausible model for repair of topoisomerase-mediated DNA damage can now be formulated. The model is based on previous
models for the repair of double-strand breaks [37,38]. Perhaps the most interesting question regarding repair involves the very ¢rst step, namely conversion of the cleavage complex into a form of DNA damage that can be acted upon by recombination proteins. Whether this step is referred to as part of the repair process is, in a sense, a semantic point ^ this step could well be identical to the critical step in cytotoxicity described above. In that case, the ¢rst step may involve the collision of a replication fork helicase, or perhaps a transcribing RNA polymerase, with the cleavage complex. A very intriguing alternative is provided by the discovery of an enzyme from eukaryotic cells that can disjoin the covalent protein^ DNA linkage of the cleavage complex by directly breaking the phosphotyrosine linkage [39]. There is currently no direct evidence that this enzyme is involved in repair or processing of the cleavage complex in vivo, and such an activity has yet to be detected in any prokaryotic system. Once a protein-free DNA break is created, the remaining steps of the recombinational repair model can be readily correlated with known activities of T4 recombination proteins. Thus, an early step presumably involves erosion of the 5P ends at the break by means of a 5P to 3P exonuclease, perhaps either gp46/ 47 or RNase H. Next, the single-stranded 3P ends bordering the break would invade homologous duplex DNA in a well-studied strand invasion reaction catalyzed by UvsX (the T4 RecA analog) and UvsY proteins. The next proposed step is DNA replication primed by the invading 3P ends, using the homologous duplex as template. The assembly of the T4 replication machinery on recombination intermediates probably depends on gp59, a helicase-loading factor [40,41]. Finally, recent evidence implicates the UvsW protein in branch migration reactions [42], and therefore this protein could play a key role in resolution of the Holliday junctions that result from the repair reaction. This relatively simple adaptation of the Szostak model for double-strand break repair seems su¤cient to understand the repair of topoisomerase-mediated DNA damage. However, it should also be pointed out that recent experiments with nuclease-induced double-strand breaks argue that break repair may not follow the Szostak model in T4-infected cells [43,44]. In the George and Kreuzer [43] study, all
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detectable break repair was associated with very extensive DNA replication, much beyond the region of the break. This and other results led to the extensive chromosome replication (ECR) model, in which an invading 3P end from a broken DNA molecule establishes a new replication fork that duplicates the entire homologous template DNA [43]. 5. The inhibitor binding site It is now clear that the inhibitor binding site within the cleavage complex consists of both protein and DNA. Results from the T4 model system have signi¢cantly contributed to this realization, and those results will be summarized brie£y. The most compelling argument for an involvement of the topoisomerase in drug binding is the fact that simple amino acid substitutions can alter drug sensitivity. Perhaps the most revealing mutation was one that appeared to structurally rearrange the inhibitor binding site. This mutation (in gene 39, see below) caused resistance to some inhibitors, but increased sensitivity to others, and di¡erentially a¡ected sensitivity to compounds within the ellipticine family of antitumor agents [5]. The involvement of DNA in inhibitor binding was initially suggested by the fact that many (though not all) of the type II topoisomerase inhibitors are intercalating agents. During the 1980s and early 1990s, results from both the T4 and mammalian systems provided additional hints in this direction. Most notably, each chemical family of topoisomerase inhibitor was shown to induce a di¡erent set of DNA cleavage sites with a given enzyme, arguing that the inhibitor somehow in£uences the selection of cleavage sites [5,6,45^50]. Two relatively recent studies using the T4 model system have provided compelling evidence that the DNA substrate is indeed part of the inhibitor binding site [51,52]. In the ¢rst, a thorough mutational analysis was performed on a particular topoisomerase cleavage site, with almost every base substitution tested at each of 14 basepairs in and around the cleavage site (Fig. 1; [51]). Each of the mutant substrates was tested for the e¤ciency of topoisomerasemediated cleavage in the absence of drug and in the presence of m-AMSA, mitoxantrone, an ellipticine
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Fig. 1. Duplex oligonucleotide substrate for mutational analysis. The 30-bp substrate is schematically illustrated, with 8 bp on the left and 6 bp on the right indicated by solid lines. The positions of topoisomerase-mediated DNA cleavage are indicated by arrows. See [51].
derivative, the epipodophyllotoxin VP16, and the quinolone oxolinic acid. Several interesting conclusions emerged. First, the DNA sequences £anking the cleavage site (Fig. 1; positions 32 through 36 and 32P through 34P) were important for cleavage e¤ciency, but the same bases were preferred at any particular position regardless of which inhibitor was present (note that each cleavage site position will be referred to as a single base for convenience and clarity, but in this mutational study, the base on the opposite strand was also changed to the correct complement). We refer to these £anking positions as `enzyme speci¢c', because the enzyme (rather than the inhibitor) imposes the base preferences. The preferred bases in these £anking regions showed dyad symmetry with respect to the cleavage site, indicating that both protomers of the topoisomerase homodimer interact with DNA in an analogous manner. Second, the preferred bases immediately 5P to the cleaved phosphodiester bonds (Fig. 1; 31 and 31P) are highly speci¢c to the inhibitor used in the cleavage reaction (again with a fairly strong degree of dyad symmetry between 31 and 31P). Thus, T or A was preferred in the presence of m-AMSA, C for mitoxantrone, and T for the ellipticine derivative. These results strongly suggest that the inhibitors interact in a speci¢c manner with the basepair adjacent (exterior) to each cleaved phosphodiester bond. The 31 and 31P positions are therefore referred to as `inhibitor speci¢c'. Third, the interior position adjacent to each cleaved phosphodiester bond showed indications of inhibitor speci¢city, although in a more complex manner than the exterior positions. Fourth and somewhat surprisingly, the central two basepairs (+2 and +2P) of the cleavage site were relatively unimportant, with only small variations in
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Fig. 2. Model for a topoisomerase cleavage complex trapped by an inhibitor. A generic topoisomerase inhibitor is depicted by the 3-ring chromophore, the enzyme by two symmetrical blobs, and the active-site tyrosine residues by `Tyr'. See [51,52].
cleavage with any of the mutations that were introduced. The inhibitor-speci¢c preferences at the basepairs immediately £anking the cleaved phosphodiester bonds is highly signi¢cant in understanding the nature of drug binding to the cleavage complex. These results imply that each family of inhibitor engages in preferential interactions with only one or two of the four possible basepairs, providing indirect evidence that the inhibitors bind in the immediate vicinity of the cleaved phosphodiester bonds (see model in Fig. 2). Furthermore, the results provide a conceptual explanation for the di¡erent sets of cleavage sites induced by the di¡erent families of inhibitors. The simplest interpretation is that each family of inhibitor can `select out' only a subset of possible topoisomerase cleavage sites. Consistent with this view, a very poor cleavage site for mitoxantrone (with T at both the 31 and 31P positions) could be converted into a very strong mitoxantrone site by substituting C at each of the two positions [51]. Substitution of only a single C provided an intermediate level of mitoxantrone-induced cleavage, arguing that one optimal site for drug binding is su¤cient to stimulate cleavage, but that two sites are much better than one. With regard to the value of the T4 model system, the inhibitor-speci¢c preferences at the basepairs £anking the cleaved phosphodiester bonds seem very similar between the mammalian and T4 enzymes (see [51]). This similarity argues that each family of inhibitor may select its preferred basepair(s) based on fairly simple chemical rules that are not in£uenced by the neighboring amino acid residues of the protein. This view is not inconsistent with the fact that amino
acid substitutions in topoisomerase can alter the inhibitor binding site ^ those alterations may a¡ect regions of the drug molecule that are not directly interacting with the basepairs at the cleavage site. The second set of experiments by Freudenreich provided very direct evidence that the inhibitor binds in the immediate vicinity of the cleaved phosphodiester bonds. A photoactivatible derivative of mAMSA, 3-azido-AMSA, was synthesized by Shieh et al. [53] and found to have very similar DNA binding properties as the parent drug. Freudenreich and Kreuzer [52] found that 3-azido-AMSA was as e¡ective as m-AMSA in inducing DNA cleavage with the T4 topoisomerase, and then went on to map the sites of photoinduced crosslinking of the inhibitor to the DNA molecule (crosslinking to the protein was not tested). Using primer-extension and piperidine-induced cleavage methods, the drug was shown to crosslink to one of the two bases in each of the four basepairs that £ank the two cleaved phosphodiester bonds (i.e. the basepairs indicated as 31, +1, 31P and +1P in Fig. 1). Crosslinking to these positions required the addition of topoisomerase and light activation of the 3-azido-AMSA, and, as expected, no light-induced crosslinking was detected in reactions with m-AMSA. These results clearly show that the inhibitor molecule is located precisely at the DNA sites cleaved by the topoisomerase. The simplest possibility is that the inhibitors intercalate into the internucleotide space at the cleaved phosphodiester bonds (see Fig. 2). A closely related possibility is that the inhibitors stack upon the terminal base at the broken 3P end, perhaps even when the two halves of the DNA are pulled apart by the enzyme to allow strand passage. 6. Topoisomerase mutations that alter drug sensitivity As mentioned above, m-AMSA-resistance mutations were genetically mapped to the T4 topoisomerase genes 39 and 52 [6,7] and gene replacement methods have been used to demonstrate that single amino acid substitutions in the topoisomerase can lead to antitumor drug resistance [9]. With the recent solution of crystal structures for large fragments of the yeast type II topoisomerase and bacterial DNA gyrase subunit A [54,55], it is now possible to correlate
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the location of drug-resistance mutations with their positions in the 3-dimensional structure of the enzyme. Although the structure of the T4 and mammalian enzymes are sure to be divergent from the yeast and bacterial enzymes in important aspects, it seems likely that the highly conserved regions (e.g. near the active site tyrosine) will turn out to have similar overall structures. The only mutation so far analyzed within gene 39 (encodes the ATPase portion of the enzyme) is a replacement of Glu457 with Lys (corresponds to yeast residue Glu495 ) [9]. This region of the protein has several conserved motifs but is quite far from the region of the active site tyrosines in the crystal structures. It is not obvious how to interpret the drug resistance of the Glu457 CLys mutant with respect to the structures, other than to speculate about massive conformational changes during the reaction cycle. Drug-resistance mutations have also been found in this general region of the E. coli and eukaryotic type II topoisomerases (see [56]). The mutations in gene 52 (cleavage-resealing subunit) are much more interpretable. Amino acids Asn78 and Ser79 in gp52 correspond to two notorious locations with regard to drug resistance in other topoisomerases (Ser83 and Ala84 of E. coli DNA gyrase, Gln740 and Ser741 of the S. cerevisiae enzyme, and Met762 and Ser763 of the human enzyme; see [57]). Ser83 is the most important residue of the bacterial enzyme with respect to quinolone resistance mutations, with Leu or Trp substitutions conferring the highest levels of resistance [58]. In addition, Ala84 to Pro mutations in the E. coli enzyme result in quinolone resistance [58]. Antitumor drug resistance mutations in the eukaryotic topoisomerase have also been found in this general region, although somewhat more spread out than in the case of the quinolone-resistance mutations in bacterial DNA gyrase [59]. Recently, a mutation of Ser741 CTrp in yeast topoisomerase was shown to alter sensitivity to etoposide [60]. Consistent with the importance of this region in inhibitor sensitivity, we found that three substitutions of T4 gp52 Asn78 caused an increased sensitivity to m-AMSA, while two substitutions at Ser79 caused resistance to the antitumor drug (these ¢ve were the only substitutions tested at these two positions) [9]. The drug sensitivities of these mutants
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were tested in vivo; no experiments have yet been done with enzymes puri¢ed from these mutants. A beautiful correlation emerged when the three-dimensional structure of the yeast topoisomerase was determined [54]. These two residues are placed almost precisely at the broken 3P end of the DNA in a model which was generated for the complex of enzyme and broken DNA (the structure of a DNA^enzyme co-crystal has not been solved, but rather the broken DNA was modeled into the structure of the enzyme based on both the location of the active site tyrosine and a structural homology to the CAP and histone H5 DNA binding region). The correlation relates to the inhibitor crosslinking results described above ^ those studies placed the inhibitor in the immediate vicinity of the broken 3P end of the DNA. Additional insights emerged from the substitutions at Asn78 and Ser79 of T4 gp52 [9]. First, the mutations that a¡ected sensitivity to m-AMSA also generally a¡ected sensitivity to the quinolone oxolinic acid, although sometimes in opposite ways. Second, the mutations of Asn78 displayed a provocative parallel to bacterial DNA gyrase with respect to quinolone sensitivity. The T4 topoisomerase is intrinsically much less sensitive to quinolones than is DNA gyrase. However, mutation of gp52 Asn78 to Ser, which is the corresponding residue in gyrase (Ser83 ), conferred a strong quinolone sensitivity to the T4 topoisomerase. Furthermore, substitution of Asn78 with Trp caused quinolone resistance, just as the Ser83 CTrp substitution does in bacterial DNA gyrase. This single residue is therefore critical in determining the level of sensitivity to the antibacterial quinolones. It is a somewhat sobering thought that a single amino acid substitution in the mammalian enzyme might also lead to strong sensitivity to commonly used antibacterials. In a more global sense, the simultaneous alteration of sensitivity to quinolones and antitumor drugs provides additional evidence for common, or at least overlapping, inhibitor binding sites for the antitumor and antibacterial agents (also see [5]). In summary, utilization of the phage T4 model system has provided important insights into the molecular mechanism of topoisomerase inhibition, as well as the physiological events involved in toxicity, repair and mutationally derived drug resistance. The insight gained from this and other simple model sys-
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tems should help guide experiments in the less tractable, but more clinically relevant, mammalian tumor cell systems. Acknowledgements I thank my colleagues, whose work is cited herein, for many stimulating discussions. Research in my laboratory is supported by Grant CA60836 from the National Cancer Institute.
References [1] E. Wycko¡, D. Natalie, J.M. Nolan, M. Lee, T. Hsieh, J. Mol. Biol. 205 (1989) 1^13. [2] K.N. Kreuzer, C.V. Jongeneel, Methods Enzymol. 100 (1983) 144^160. [3] T.C. Rowe, K.M. Tewey, L.F. Liu, J. Biol. Chem. 259 (1984) 9177^9181. [4] E.M. Nelson, K.M. Tewey, L.F. Liu, Proc. Natl. Acad. Sci. USA 81 (1984) 1361^1365. [5] A.C. Hu¡, K.N. Kreuzer, J. Biol. Chem. 265 (1990) 20496^ 20505. [6] A.C. Hu¡, J.K. Leatherwood, K.N. Kreuzer, Proc. Natl. Acad. Sci. USA 86 (1989) 1307^1311. [7] A.C. Hu¡, R.E. Ward IV, K.N. Kreuzer, Mol. Gen. Genet. 221 (1990) 27^32. [8] K.N. Kreuzer, S.H. Neece, in: M.-A. Bjornsti, N. Oshero¡ (Eds.), Methods in Molecular Biology: Protocols in DNA Topology and Topoisomerases, Humana Press, Totowa, NJ, 1998, pp. 171^177. [9] C.H. Freudenreich, C. Chang, K.N. Kreuzer, Cancer Res. 58 (1998) 1260^1267. [10] L.F. Liu, Annu. Rev. Biochem. 58 (1989) 351^375. [11] A.Y. Chen, L.F. Liu, Annu. Rev. Pharmacol. Toxicol. 34 (1994) 191^218. [12] K. Drlica, R.J. Franco, Biochemistry 27 (1988) 2253^2259. [13] S.H. Neece, K. Carles-Kinch, D.J. Tomso, K.N. Kreuzer, Mol. Microbiol. 20 (1996) 1145^1154. [14] H. Ikeda, Mol. Gen. Genet. 202 (1986) 518^520. [15] H. Ikeda, Proc. Natl. Acad. Sci. USA 83 (1986) 922^926. [16] M. Chiba, H. Shimizu, A. Fujimoto, H. Nashimoto, H. Ikeda, J. Biol. Chem. 264 (1989) 12785^12790. [17] L.S. Ripley, A. Clark, Proc. Natl. Acad. Sci. USA 83 (1986) 6954^6958. [18] L.S. Ripley, J.S. Dubins, J.G. deBoer, D.M. DeMarini, A.M. Bogerd, K.N. Kreuzer, J. Mol. Biol. 200 (1988) 665^ 680. [19] M. Masurekar, K.N. Kreuzer, L.S. Ripley, Genetics 127 (1991) 453^462.
[20] V.L. Kaiser, L.S. Ripley, Proc. Natl. Acad. Sci. USA 92 (1995) 2234^2238. [21] P. D'Arpa, L.F. Liu, Biochim. Biophys. Acta 989 (1989) 163^177. [22] C. Holm, J.M. Covey, D. Kerrigan, Y. Pommier, Cancer Res. 49 (1989) 6365^6368. [23] P. D'Arpa, C. Beardmore, L.F. Liu, Cancer Res. 50 (1990) 6919^6924. [24] S.N. Ebert, S.S. Shtrom, M.T. Muller, J. Virol. 64 (1990) 4059^4066. [25] M.T. Howard, S.H. Neece, S.W. Matson, K.N. Kreuzer, Proc. Natl. Acad. Sci. USA 91 (1994) 12031^12035. [26] J. Nitiss, J.C. Wang, Proc. Natl. Acad. Sci. USA 85 (1988) 7501^7505. [27] W.-K. Eng, L. Faucette, R.K. Johnson, R. Sternglanz, Mol. Pharmacol. 34 (1989) 755^760. [28] P.A. Jeggo, K. Caldecott, S. Pidsley, G.R. Banks, Cancer Res. 49 (1989) 7057^7063. [29] K. Caldecott, G. Banks, P. Jeggo, Cancer Res. 50 (1990) 5778^5783. [30] C. Bernstein, Microbiol. Rev. 45 (1981) 72^98. [31] C. Bernstein, S.S. Wallace, in: C.K. Mathews, E.M. Kutter, G. Mosig, P.B. Berget (Eds.), Bacteriophage T4, American Society for Microbiology, Washington, DC, 1983, pp. 138^ 151. [32] K.N. Kreuzer, J.W. Drake, in: J.D. Karam (Ed.), Molecular Biology of Bacteriophage T4, ASM Press, Washington, DC, 1994, pp. 89^97. [33] S. Luria, Proc. Natl. Acad. Sci. USA 33 (1947) 253^264. [34] G. Mosig, in: J.D. Karam (Ed.), Molecular Biology of Bacteriophage T4, ASM Press, Washington, DC, 1994, pp. 54^ 82. [35] K.N. Kreuzer, S.W. Morrical, in: J.D. Karam (Ed.), Molecular Biology of Bacteriophage T4, ASM Press, Washington, DC, 1994, pp. 28^42. [36] D.L. Woodworth, K.N. Kreuzer, Genetics 143 (1996) 1081^ 1090. [37] M.A. Resnick, J. Theor. Biol. 59 (1976) 97^106. [38] J.W. Szostak, T.L. Orr-Weaver, R.J. Rothstein, F.W. Stahl, Cell 33 (1983) 25^35. [39] S.W. Yang, A.B. Burgin Jr., B.N. Huizenga, C.A. Robertson, K.C. Yao, H.A. Nash, Proc. Natl. Acad. Sci. USA 93 (1996) 11534^11539. [40] J. Barry, B. Alberts, J. Biol. Chem. 269 (1994) 33049^33062. [41] S.W. Morrical, K. Hempstead, M.D. Morrical, J. Biol. Chem. 269 (1994) 33069^33081. [42] K. Carles-Kinch, J.W. George, K.N. Kreuzer, EMBO J. 16 (1997) 4142^4151. [43] J.W. George, K.N. Kreuzer, Genetics 143 (1996) 1507^1520. [44] J.E. Mueller, T. Clyman, Y.J. Huang, M.M. Parker, M. Belfort, Genes Dev. 10 (1996) 351^364. [45] W.E. Ross, T.C. Rowe, B.S. Glisson, J. Yalowich, L.F. Liu, Cancer Res. 44 (1984) 5857^5860. [46] K.M. Tewey, G.L. Chen, E.M. Nelson, L.F. Liu, J. Biol. Chem. 259 (1984) 9182^9187.
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K.N. Kreuzer / Biochimica et Biophysica Acta 1400 (1998) 339^347 [47] K.M. Tewey, T.C. Rowe, L. Yang, B.D. Halligan, L.F. Liu, Science 226 (1984) 466^468. [48] T.C. Rowe, G.L. Chen, Y.-H. Hsiang, L.F. Liu, Cancer Res. 46 (1986) 2021^2026. [49] G. Capranico, F. Zunino, K.W. Kohn, Y. Pommier, Biochemistry 29 (1990) 562^569. [50] G. Capranico, K.W. Kohn, Y. Pommier, Nucleic Acids Res. 18 (1990) 6611^6619. [51] C.H. Freudenreich, K.N. Kreuzer, EMBO J. 12 (1993) 2085^ 2097. [52] C.H. Freudenreich, K.N. Kreuzer, Proc. Natl. Acad. Sci. USA 91 (1994) 11007^11011. [53] T.-L. Shieh, P. Hoyos, E. Kolodziej, J.G. Stowell, W.M. Baird, S.R. Byrn, J. Med. Chem. 33 (1990) 1225^1230. [54] J.M. Berger, S.J. Gamblin, S.C. Harrison, J.C. Wang, Nature 379 (1996) 225^232.
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[55] J.H. Morais Cabal, A.P. Jackson, C.V. Smith, N. Shikotra, A. Maxwell, R.C. Liddington, Nature 388 (1997) 903^905. [56] P.R. Caron, J.C. Wang, in: T. Andoh, H. Ikeda, M. Oguro (Eds.), Molecular Biology of DNA Topoisomerases and Its Application to Chemotherapy, CRC Press, Boca Raton, FL, 1993, pp. 1^18. [57] P.R. Caron, J.C. Wang, in: L.F. Liu (Ed.), DNA Topoisomerases: Topoisomerase-Targeting Drugs, Academic Press, San Diego, CA, 1994, pp. 271^297. [58] A. Maxwell, J. Antimicrob. Chemother. 30 (1992) 409^416. [59] W.T. Beck, M.K. Danks, J.S. Woverton, M. Chen, B. Granzen, R. Kim, D.P. Suttle, in: DNA Topoisomerases: Topoisomerase-Targeting Drugs, L.F. Liu (Ed.), Academic Press, San Diego, CA, 1994, pp. 145^169. [60] J.L. Nitiss, Y.-X. Liu, Y. Hsiung, Cancer Res. 53 (1993) 89^ 93.
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Review
Bacteriophage T4, a model system for understanding the mechanism of type II topoisomerase inhibitors Kenneth N. Kreuzer * Department of Microbiology, Duke University Medical Center, Durham, NC 27710, USA Accepted 22 April 1998
Abstract Bacteriophage T4 provides a simple model system for analyzing the mechanism of action of antitumor agents that inhibit DNA topoisomerases. The phage-encoded type II topoisomerase is sensitive to many of the same antitumor agents that inhibit mammalian type II topoisomerase, including m-AMSA, ellipticines, mitoxantrone and epipodophyllotoxins. Results from the T4 model system provided a convincing demonstration that topoisomerase is the physiological drug target and strong evidence that the drug-induced cleavage complex is important for cytotoxicity. The detailed molecular steps involved in cytotoxicity, and the mechanism of recombinational repair of inhibitor-induced DNA damage, are currently being analyzed using this model system. Studies with the T4 topoisomerase have also provided compelling evidence that topoisomerase inhibitors interact with DNA at the active site of the enzyme, with each class of inhibitor favoring a different subset of cleavage sites based on DNA sequence. Finally, analysis of drug-resistance mutations in the T4 topoisomerase have implicated certain regions of the protein in drug interaction and provided a strong link between the mechanism of action of the antibacterial quinolones, which inhibit DNA gyrase, and the various antitumor agents, which inhibit mammalian type II topoisomerase. 0167-4781 / 98 / $ ^ see front matter ß 1998 Elsevier Science B.V. All rights reserved. Keywords: Antitumor agent; Quinolone; Topoisomerase ; DNA repair; Bacteriophage; Aminoacridine
Contents 1.
Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
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2.
Topoisomerase is the physiological target for m-AMSA . . . . . . . . . . . . . . . . . . . . . . . . . .
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3.
Molecular mechanism of toxicity . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
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Repair of the cleavage complex . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
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The inhibitor binding site . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
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6.
Topoisomerase mutations that alter drug sensitivity . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
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* Fax: +1 (919) 681-8911; E-mail: [email protected] 0167-4781 / 98 / $ ^ see front matter ß 1998 Elsevier Science B.V. All rights reserved. PII: S 0 1 6 7 - 4 7 8 1 ( 9 8 ) 0 0 1 4 5 - 6
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Acknowledgements . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
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References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
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1. Introduction The relatively simple bacteriophage T4 provides a very useful model system for determining the precise mechanism of action of antitumor agents that inhibit the mammalian type II DNA topoisomerases. The T4 and mammalian topoisomerases share several regions of conserved amino acid sequence [1] and display similar enzymatic properties, including catalysis of ATP-dependent relaxation, lack of intrinsic DNA supercoiling activity, and inhibition by singlestranded DNA [2]. The possibility that phage T4 could be used to study antitumor drug action ¢rst emerged when Liu and coworkers found that the aminoacridine m-AMSA (4P-(9-acridinylamino)methanesulfon-m-anisidide) inhibits both the mammalian and T4 topoisomerases [3,4]. In both cases, m-AMSA induces formation of the cleavage complex, suggesting a common mechanism of inhibition. The inhibition of the mammalian and phage enzymes by m-AMSA is not a coincidence ^ the phage enzyme is also sensitive to mitoxantrone (an anthracenedione), members of the ellipticine family, and two epipodophyllotoxins, all of which inhibit the mammalian enzyme [5]. As will be mentioned below, a variety of results from the T4 system closely parallel those from studies of mammalian systems, validating the use of this simple bacterial virus for analyzing antitumor drug action. The T4 model system has numerous advantages which facilitate rapid progress. The phage genome is a single duplex DNA molecule of just under 170 kb, which is now entirely sequenced. T4 has been an intensively studied prokaryotic system since the early days of molecular biology, so that powerful tools of classical and molecular genetics and biochemistry can be readily applied. With respect to the phage-encoded topoisomerase, drug-resistant mutants can be isolated by a simple 1-day procedure and analyzed genetically over the course of just a few weeks [6,7]. Furthermore, milligram amounts of wild-type or mutant topoisomerase can be puri¢ed to
homogeneity by a 3-column procedure that takes a few days [2,8]. From a broader perspective, T4 provides one of the most thoroughly studied systems of nucleic acid metabolism (replication, recombination, transcription, repair). The primary sequences of all phage proteins involved in these processes have been determined, and most of these proteins have been overproduced, puri¢ed and analyzed in simple in vitro systems. Given these numerous advantages, bacteriophage T4 provides an inexpensive, convenient and valuable model system for the analysis of antitumor drug action. 2. Topoisomerase is the physiological target for m-AMSA Genetic and biochemical analyses of m-AMSA-resistant T4 mutants provided convincing evidence that the phage-encoded topoisomerase is the physiological target for the antitumor drug in T4-infected bacterial cells. Growth of wild-type T4 (but not the Escherichia coli host) is prevented by m-AMSA, and drugresistant phage mutants were readily isolated simply by plating mutagenized phage on bacterial lawns grown in the presence of the inhibitor [6,7]. Topoisomerase puri¢ed from the drug-resistant phage displayed m-AMSA-resistance during in vitro reactions. The results of standard phage genetic crosses localized one drug-resistance mutation to gene 39 and another to gene 52; these two genes encode two of the three topoisomerase subunits. Furthermore, gene replacement techniques were used to prove that a single amino acid substitution in either gp39 or gp52 is su¤cient to confer m-AMSA-resistance [9]. 3. Molecular mechanism of toxicity Results from a variety of systems (e.g. E. coli, yeast and mammalian cells) indicate that the potent cytotoxicity of topoisomerase inhibitors results from
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stabilization of the cleavage complex rather than just simple enzyme inhibition (see [10^12]). The same is true in the phage T4 system. Thus, inhibition of the T4 topoisomerase by m-AMSA is more detrimental to phage growth than mutational elimination of the topoisomerase. Furthermore, the mutational inactivation of the topoisomerase actually protects T4 from the detrimental e¡ect of m-AMSA, clearly implicating the drug-induced cleavage complex in toxicity [13]. One of the strongest arguments that toxicity involves a form of DNA damage is the fact that a special form of DNA repair strongly reduces the effectiveness of topoisomerase inhibitors (see below). Also consistent with a key role of DNA damage, results from the T4 system uncovered two distinct pathways of mutagenesis induced by topoisomerase inhibitors. Ikeda and coworkers showed that oxolinic acid (a weak inhibitor of the T4 topoisomerase) induces illegitimate recombinants between non-homologous DNA molecules in vitro [14^16]. The sites of illegitimate recombination coincided with oxolinic acid-induced enzyme-mediated DNA cleavage sites, leading to a `subunit exchange' model in which two topoisomerase complexes at di¡erent sites trade `half enzymes' during the cleavage-resealing reaction cycle. The second pathway of mutagenesis was analyzed extensively by Ripley and coworkers, who found that m-AMSA and related topoisomerase inhibitors are potent frameshift mutagens in phage T4-infected cells [17,18]. Every frameshift mutation at a drug-induced hotspot had one endpoint at one of two phosphodiester bonds that were spaced four basepairs apart on opposite strands of the helix, and these two locations were precisely the sites of cleavage for a strong drug-induced enzyme cleavage site [18]. A variety of additional results provided convincing evidence that the topoisomerase is directly involved in drug-induced frameshift mutagenesis: (1) a mutation that inactivates topoisomerase eliminated frameshift mutagenesis [18]; (2) m-AMSA was more e¡ective both at inducing frameshift mutations and inducing enzyme-mediated DNA cleavage than its closely related isomer o-AMSA [18]; (3) DNA sequence alterations in the hotspot site that greatly reduced topoisomerase-mediated DNA cleavage also greatly reduced frameshift mutagenesis [19]; and (4) DNA sequence alterations in the hotspot site that shifted the site of
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enzyme-mediated DNA cleavage also correspondingly shifted the positions of drug-induced frameshift mutations [19]. From these and other results, it is clear that mutations are generated from DNA discontinuities introduced by the topoisomerase and stabilized by the topoisomerase inhibitors. The detailed steps involved in drug-induced frameshift mutagenesis are currently under investigation (see [20]). One of the most pressing problems in the topoisomerase ¢eld is to understand the detailed mechanism of cytotoxicity. The generation of mutations presumably contributes, but is unlikely to account for the majority of drug-induced toxicity. Results from mammalian systems provide several clues. First, drug-induced toxicity is reversible, as is the drug-induced cleavage complex, if the drug is washed out relatively quickly. However, toxicity becomes irreversible with prolonged incubation, arguing that the reversible cleavage complex is converted into an irreversible form of DNA damage (see [21]). Second, DNA replication and transcription have been implicated in the cytotoxicity of topoisomerase (type I and II) inhibitors, suggesting that a crucial event in DNA damage involves nucleic acid synthesis on a template that contains a drug-induced cleavage complex [22^ 24]. As a ¢rst step towards understanding the consequence of a collision between a replication fork and a cleavage complex, we staged an in vitro showdown between a DNA helicase and a topoisomerase cleavage complex [25]. Every DNA replication fork contains a helicase, which opens the parental helix, and therefore the initial encounter between the replication fork and cleavage complex is likely to involve the helicase. The results were dramatic ^ the helicase was able to convert the normally reversible cleavage complex into an irreversible break, and at least one of the broken strands (the single strand that was not covalently attached) was released from the enzyme complex. These results are consistent with models in which a replication fork-associated helicase disrupts the cleavage complex, creating a frank DNA break. Production of a frank DNA break could be a critical step in cytotoxicity, frameshift mutagenesis (see above), and/or recombinational repair (see below). We are currently attempting more complete experiments, namely, analyzing the consequences of
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running a complete replication fork into a cleavage complex both in vivo and in vitro. 4. Repair of the cleavage complex As already mentioned, a special form of DNA repair is able to reduce the e¡ectiveness of topoisomerase inhibitors, presumably because this pathway can repair some fraction of the topoisomerase-mediated DNA damage. Thus, the sensitivity of Saccharomyces cerevisiae to m-AMSA is increased by mutations in the RAD52 gene, which encodes a protein involved in recombinational repair of radiation-induced damage and double-strand DNA breaks [26,27]. Likewise, mutations in the xrs genes of CHO cells increase sensitivity to both radiation and inhibitors of type II topoisomerase [28,29]. The T4 system is useful for investigating the repair of damage caused by topoisomerase inhibitors. Many pathways of DNA repair, including recombinational repair, have been extensively studied in the T4 system (see [30^32]). Indeed, the very ¢rst evidence for recombinational repair emerged from the T4 system a half century ago [33], and since that time, phage recombination proteins have been identi¢ed and characterized in biochemical detail (see [34,35]). Mutations that inactivate key T4 recombination proteins (UvsX, UvsY, UvsW, RNase H, gp59 and gp46/47) increase the sensitivity of the phage to mAMSA, consistent with a role of recombinational repair in drug sensitivity [13,36]. Most of these proteins have previously been shown to be involved in recombinational repair of radiation- and/or chemical-induced DNA damage. Two additional results are consistent with the proposed role of recombination in repairing m-AMSA-induced cleavage complexes. First, homologous recombination was found to be stimulated by the drug, presumably re£ecting productive recombinational repair reactions. Second, we detected m-AMSA-induced cleavage complexes within the infected cell, with cleavage occurring at the same DNA sites as in in vitro reactions with the puri¢ed topoisomerase [13]. Based on these results and the known activities of T4 recombination proteins, a plausible model for repair of topoisomerase-mediated DNA damage can now be formulated. The model is based on previous
models for the repair of double-strand breaks [37,38]. Perhaps the most interesting question regarding repair involves the very ¢rst step, namely conversion of the cleavage complex into a form of DNA damage that can be acted upon by recombination proteins. Whether this step is referred to as part of the repair process is, in a sense, a semantic point ^ this step could well be identical to the critical step in cytotoxicity described above. In that case, the ¢rst step may involve the collision of a replication fork helicase, or perhaps a transcribing RNA polymerase, with the cleavage complex. A very intriguing alternative is provided by the discovery of an enzyme from eukaryotic cells that can disjoin the covalent protein^ DNA linkage of the cleavage complex by directly breaking the phosphotyrosine linkage [39]. There is currently no direct evidence that this enzyme is involved in repair or processing of the cleavage complex in vivo, and such an activity has yet to be detected in any prokaryotic system. Once a protein-free DNA break is created, the remaining steps of the recombinational repair model can be readily correlated with known activities of T4 recombination proteins. Thus, an early step presumably involves erosion of the 5P ends at the break by means of a 5P to 3P exonuclease, perhaps either gp46/ 47 or RNase H. Next, the single-stranded 3P ends bordering the break would invade homologous duplex DNA in a well-studied strand invasion reaction catalyzed by UvsX (the T4 RecA analog) and UvsY proteins. The next proposed step is DNA replication primed by the invading 3P ends, using the homologous duplex as template. The assembly of the T4 replication machinery on recombination intermediates probably depends on gp59, a helicase-loading factor [40,41]. Finally, recent evidence implicates the UvsW protein in branch migration reactions [42], and therefore this protein could play a key role in resolution of the Holliday junctions that result from the repair reaction. This relatively simple adaptation of the Szostak model for double-strand break repair seems su¤cient to understand the repair of topoisomerase-mediated DNA damage. However, it should also be pointed out that recent experiments with nuclease-induced double-strand breaks argue that break repair may not follow the Szostak model in T4-infected cells [43,44]. In the George and Kreuzer [43] study, all
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detectable break repair was associated with very extensive DNA replication, much beyond the region of the break. This and other results led to the extensive chromosome replication (ECR) model, in which an invading 3P end from a broken DNA molecule establishes a new replication fork that duplicates the entire homologous template DNA [43]. 5. The inhibitor binding site It is now clear that the inhibitor binding site within the cleavage complex consists of both protein and DNA. Results from the T4 model system have signi¢cantly contributed to this realization, and those results will be summarized brie£y. The most compelling argument for an involvement of the topoisomerase in drug binding is the fact that simple amino acid substitutions can alter drug sensitivity. Perhaps the most revealing mutation was one that appeared to structurally rearrange the inhibitor binding site. This mutation (in gene 39, see below) caused resistance to some inhibitors, but increased sensitivity to others, and di¡erentially a¡ected sensitivity to compounds within the ellipticine family of antitumor agents [5]. The involvement of DNA in inhibitor binding was initially suggested by the fact that many (though not all) of the type II topoisomerase inhibitors are intercalating agents. During the 1980s and early 1990s, results from both the T4 and mammalian systems provided additional hints in this direction. Most notably, each chemical family of topoisomerase inhibitor was shown to induce a di¡erent set of DNA cleavage sites with a given enzyme, arguing that the inhibitor somehow in£uences the selection of cleavage sites [5,6,45^50]. Two relatively recent studies using the T4 model system have provided compelling evidence that the DNA substrate is indeed part of the inhibitor binding site [51,52]. In the ¢rst, a thorough mutational analysis was performed on a particular topoisomerase cleavage site, with almost every base substitution tested at each of 14 basepairs in and around the cleavage site (Fig. 1; [51]). Each of the mutant substrates was tested for the e¤ciency of topoisomerasemediated cleavage in the absence of drug and in the presence of m-AMSA, mitoxantrone, an ellipticine
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Fig. 1. Duplex oligonucleotide substrate for mutational analysis. The 30-bp substrate is schematically illustrated, with 8 bp on the left and 6 bp on the right indicated by solid lines. The positions of topoisomerase-mediated DNA cleavage are indicated by arrows. See [51].
derivative, the epipodophyllotoxin VP16, and the quinolone oxolinic acid. Several interesting conclusions emerged. First, the DNA sequences £anking the cleavage site (Fig. 1; positions 32 through 36 and 32P through 34P) were important for cleavage e¤ciency, but the same bases were preferred at any particular position regardless of which inhibitor was present (note that each cleavage site position will be referred to as a single base for convenience and clarity, but in this mutational study, the base on the opposite strand was also changed to the correct complement). We refer to these £anking positions as `enzyme speci¢c', because the enzyme (rather than the inhibitor) imposes the base preferences. The preferred bases in these £anking regions showed dyad symmetry with respect to the cleavage site, indicating that both protomers of the topoisomerase homodimer interact with DNA in an analogous manner. Second, the preferred bases immediately 5P to the cleaved phosphodiester bonds (Fig. 1; 31 and 31P) are highly speci¢c to the inhibitor used in the cleavage reaction (again with a fairly strong degree of dyad symmetry between 31 and 31P). Thus, T or A was preferred in the presence of m-AMSA, C for mitoxantrone, and T for the ellipticine derivative. These results strongly suggest that the inhibitors interact in a speci¢c manner with the basepair adjacent (exterior) to each cleaved phosphodiester bond. The 31 and 31P positions are therefore referred to as `inhibitor speci¢c'. Third, the interior position adjacent to each cleaved phosphodiester bond showed indications of inhibitor speci¢city, although in a more complex manner than the exterior positions. Fourth and somewhat surprisingly, the central two basepairs (+2 and +2P) of the cleavage site were relatively unimportant, with only small variations in
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Fig. 2. Model for a topoisomerase cleavage complex trapped by an inhibitor. A generic topoisomerase inhibitor is depicted by the 3-ring chromophore, the enzyme by two symmetrical blobs, and the active-site tyrosine residues by `Tyr'. See [51,52].
cleavage with any of the mutations that were introduced. The inhibitor-speci¢c preferences at the basepairs immediately £anking the cleaved phosphodiester bonds is highly signi¢cant in understanding the nature of drug binding to the cleavage complex. These results imply that each family of inhibitor engages in preferential interactions with only one or two of the four possible basepairs, providing indirect evidence that the inhibitors bind in the immediate vicinity of the cleaved phosphodiester bonds (see model in Fig. 2). Furthermore, the results provide a conceptual explanation for the di¡erent sets of cleavage sites induced by the di¡erent families of inhibitors. The simplest interpretation is that each family of inhibitor can `select out' only a subset of possible topoisomerase cleavage sites. Consistent with this view, a very poor cleavage site for mitoxantrone (with T at both the 31 and 31P positions) could be converted into a very strong mitoxantrone site by substituting C at each of the two positions [51]. Substitution of only a single C provided an intermediate level of mitoxantrone-induced cleavage, arguing that one optimal site for drug binding is su¤cient to stimulate cleavage, but that two sites are much better than one. With regard to the value of the T4 model system, the inhibitor-speci¢c preferences at the basepairs £anking the cleaved phosphodiester bonds seem very similar between the mammalian and T4 enzymes (see [51]). This similarity argues that each family of inhibitor may select its preferred basepair(s) based on fairly simple chemical rules that are not in£uenced by the neighboring amino acid residues of the protein. This view is not inconsistent with the fact that amino
acid substitutions in topoisomerase can alter the inhibitor binding site ^ those alterations may a¡ect regions of the drug molecule that are not directly interacting with the basepairs at the cleavage site. The second set of experiments by Freudenreich provided very direct evidence that the inhibitor binds in the immediate vicinity of the cleaved phosphodiester bonds. A photoactivatible derivative of mAMSA, 3-azido-AMSA, was synthesized by Shieh et al. [53] and found to have very similar DNA binding properties as the parent drug. Freudenreich and Kreuzer [52] found that 3-azido-AMSA was as e¡ective as m-AMSA in inducing DNA cleavage with the T4 topoisomerase, and then went on to map the sites of photoinduced crosslinking of the inhibitor to the DNA molecule (crosslinking to the protein was not tested). Using primer-extension and piperidine-induced cleavage methods, the drug was shown to crosslink to one of the two bases in each of the four basepairs that £ank the two cleaved phosphodiester bonds (i.e. the basepairs indicated as 31, +1, 31P and +1P in Fig. 1). Crosslinking to these positions required the addition of topoisomerase and light activation of the 3-azido-AMSA, and, as expected, no light-induced crosslinking was detected in reactions with m-AMSA. These results clearly show that the inhibitor molecule is located precisely at the DNA sites cleaved by the topoisomerase. The simplest possibility is that the inhibitors intercalate into the internucleotide space at the cleaved phosphodiester bonds (see Fig. 2). A closely related possibility is that the inhibitors stack upon the terminal base at the broken 3P end, perhaps even when the two halves of the DNA are pulled apart by the enzyme to allow strand passage. 6. Topoisomerase mutations that alter drug sensitivity As mentioned above, m-AMSA-resistance mutations were genetically mapped to the T4 topoisomerase genes 39 and 52 [6,7] and gene replacement methods have been used to demonstrate that single amino acid substitutions in the topoisomerase can lead to antitumor drug resistance [9]. With the recent solution of crystal structures for large fragments of the yeast type II topoisomerase and bacterial DNA gyrase subunit A [54,55], it is now possible to correlate
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the location of drug-resistance mutations with their positions in the 3-dimensional structure of the enzyme. Although the structure of the T4 and mammalian enzymes are sure to be divergent from the yeast and bacterial enzymes in important aspects, it seems likely that the highly conserved regions (e.g. near the active site tyrosine) will turn out to have similar overall structures. The only mutation so far analyzed within gene 39 (encodes the ATPase portion of the enzyme) is a replacement of Glu457 with Lys (corresponds to yeast residue Glu495 ) [9]. This region of the protein has several conserved motifs but is quite far from the region of the active site tyrosines in the crystal structures. It is not obvious how to interpret the drug resistance of the Glu457 CLys mutant with respect to the structures, other than to speculate about massive conformational changes during the reaction cycle. Drug-resistance mutations have also been found in this general region of the E. coli and eukaryotic type II topoisomerases (see [56]). The mutations in gene 52 (cleavage-resealing subunit) are much more interpretable. Amino acids Asn78 and Ser79 in gp52 correspond to two notorious locations with regard to drug resistance in other topoisomerases (Ser83 and Ala84 of E. coli DNA gyrase, Gln740 and Ser741 of the S. cerevisiae enzyme, and Met762 and Ser763 of the human enzyme; see [57]). Ser83 is the most important residue of the bacterial enzyme with respect to quinolone resistance mutations, with Leu or Trp substitutions conferring the highest levels of resistance [58]. In addition, Ala84 to Pro mutations in the E. coli enzyme result in quinolone resistance [58]. Antitumor drug resistance mutations in the eukaryotic topoisomerase have also been found in this general region, although somewhat more spread out than in the case of the quinolone-resistance mutations in bacterial DNA gyrase [59]. Recently, a mutation of Ser741 CTrp in yeast topoisomerase was shown to alter sensitivity to etoposide [60]. Consistent with the importance of this region in inhibitor sensitivity, we found that three substitutions of T4 gp52 Asn78 caused an increased sensitivity to m-AMSA, while two substitutions at Ser79 caused resistance to the antitumor drug (these ¢ve were the only substitutions tested at these two positions) [9]. The drug sensitivities of these mutants
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were tested in vivo; no experiments have yet been done with enzymes puri¢ed from these mutants. A beautiful correlation emerged when the three-dimensional structure of the yeast topoisomerase was determined [54]. These two residues are placed almost precisely at the broken 3P end of the DNA in a model which was generated for the complex of enzyme and broken DNA (the structure of a DNA^enzyme co-crystal has not been solved, but rather the broken DNA was modeled into the structure of the enzyme based on both the location of the active site tyrosine and a structural homology to the CAP and histone H5 DNA binding region). The correlation relates to the inhibitor crosslinking results described above ^ those studies placed the inhibitor in the immediate vicinity of the broken 3P end of the DNA. Additional insights emerged from the substitutions at Asn78 and Ser79 of T4 gp52 [9]. First, the mutations that a¡ected sensitivity to m-AMSA also generally a¡ected sensitivity to the quinolone oxolinic acid, although sometimes in opposite ways. Second, the mutations of Asn78 displayed a provocative parallel to bacterial DNA gyrase with respect to quinolone sensitivity. The T4 topoisomerase is intrinsically much less sensitive to quinolones than is DNA gyrase. However, mutation of gp52 Asn78 to Ser, which is the corresponding residue in gyrase (Ser83 ), conferred a strong quinolone sensitivity to the T4 topoisomerase. Furthermore, substitution of Asn78 with Trp caused quinolone resistance, just as the Ser83 CTrp substitution does in bacterial DNA gyrase. This single residue is therefore critical in determining the level of sensitivity to the antibacterial quinolones. It is a somewhat sobering thought that a single amino acid substitution in the mammalian enzyme might also lead to strong sensitivity to commonly used antibacterials. In a more global sense, the simultaneous alteration of sensitivity to quinolones and antitumor drugs provides additional evidence for common, or at least overlapping, inhibitor binding sites for the antitumor and antibacterial agents (also see [5]). In summary, utilization of the phage T4 model system has provided important insights into the molecular mechanism of topoisomerase inhibition, as well as the physiological events involved in toxicity, repair and mutationally derived drug resistance. The insight gained from this and other simple model sys-
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tems should help guide experiments in the less tractable, but more clinically relevant, mammalian tumor cell systems. Acknowledgements I thank my colleagues, whose work is cited herein, for many stimulating discussions. Research in my laboratory is supported by Grant CA60836 from the National Cancer Institute.
References [1] E. Wycko¡, D. Natalie, J.M. Nolan, M. Lee, T. Hsieh, J. Mol. Biol. 205 (1989) 1^13. [2] K.N. Kreuzer, C.V. Jongeneel, Methods Enzymol. 100 (1983) 144^160. [3] T.C. Rowe, K.M. Tewey, L.F. Liu, J. Biol. Chem. 259 (1984) 9177^9181. [4] E.M. Nelson, K.M. Tewey, L.F. Liu, Proc. Natl. Acad. Sci. USA 81 (1984) 1361^1365. [5] A.C. Hu¡, K.N. Kreuzer, J. Biol. Chem. 265 (1990) 20496^ 20505. [6] A.C. Hu¡, J.K. Leatherwood, K.N. Kreuzer, Proc. Natl. Acad. Sci. USA 86 (1989) 1307^1311. [7] A.C. Hu¡, R.E. Ward IV, K.N. Kreuzer, Mol. Gen. Genet. 221 (1990) 27^32. [8] K.N. Kreuzer, S.H. Neece, in: M.-A. Bjornsti, N. Oshero¡ (Eds.), Methods in Molecular Biology: Protocols in DNA Topology and Topoisomerases, Humana Press, Totowa, NJ, 1998, pp. 171^177. [9] C.H. Freudenreich, C. Chang, K.N. Kreuzer, Cancer Res. 58 (1998) 1260^1267. [10] L.F. Liu, Annu. Rev. Biochem. 58 (1989) 351^375. [11] A.Y. Chen, L.F. Liu, Annu. Rev. Pharmacol. Toxicol. 34 (1994) 191^218. [12] K. Drlica, R.J. Franco, Biochemistry 27 (1988) 2253^2259. [13] S.H. Neece, K. Carles-Kinch, D.J. Tomso, K.N. Kreuzer, Mol. Microbiol. 20 (1996) 1145^1154. [14] H. Ikeda, Mol. Gen. Genet. 202 (1986) 518^520. [15] H. Ikeda, Proc. Natl. Acad. Sci. USA 83 (1986) 922^926. [16] M. Chiba, H. Shimizu, A. Fujimoto, H. Nashimoto, H. Ikeda, J. Biol. Chem. 264 (1989) 12785^12790. [17] L.S. Ripley, A. Clark, Proc. Natl. Acad. Sci. USA 83 (1986) 6954^6958. [18] L.S. Ripley, J.S. Dubins, J.G. deBoer, D.M. DeMarini, A.M. Bogerd, K.N. Kreuzer, J. Mol. Biol. 200 (1988) 665^ 680. [19] M. Masurekar, K.N. Kreuzer, L.S. Ripley, Genetics 127 (1991) 453^462.
[20] V.L. Kaiser, L.S. Ripley, Proc. Natl. Acad. Sci. USA 92 (1995) 2234^2238. [21] P. D'Arpa, L.F. Liu, Biochim. Biophys. Acta 989 (1989) 163^177. [22] C. Holm, J.M. Covey, D. Kerrigan, Y. Pommier, Cancer Res. 49 (1989) 6365^6368. [23] P. D'Arpa, C. Beardmore, L.F. Liu, Cancer Res. 50 (1990) 6919^6924. [24] S.N. Ebert, S.S. Shtrom, M.T. Muller, J. Virol. 64 (1990) 4059^4066. [25] M.T. Howard, S.H. Neece, S.W. Matson, K.N. Kreuzer, Proc. Natl. Acad. Sci. USA 91 (1994) 12031^12035. [26] J. Nitiss, J.C. Wang, Proc. Natl. Acad. Sci. USA 85 (1988) 7501^7505. [27] W.-K. Eng, L. Faucette, R.K. Johnson, R. Sternglanz, Mol. Pharmacol. 34 (1989) 755^760. [28] P.A. Jeggo, K. Caldecott, S. Pidsley, G.R. Banks, Cancer Res. 49 (1989) 7057^7063. [29] K. Caldecott, G. Banks, P. Jeggo, Cancer Res. 50 (1990) 5778^5783. [30] C. Bernstein, Microbiol. Rev. 45 (1981) 72^98. [31] C. Bernstein, S.S. Wallace, in: C.K. Mathews, E.M. Kutter, G. Mosig, P.B. Berget (Eds.), Bacteriophage T4, American Society for Microbiology, Washington, DC, 1983, pp. 138^ 151. [32] K.N. Kreuzer, J.W. Drake, in: J.D. Karam (Ed.), Molecular Biology of Bacteriophage T4, ASM Press, Washington, DC, 1994, pp. 89^97. [33] S. Luria, Proc. Natl. Acad. Sci. USA 33 (1947) 253^264. [34] G. Mosig, in: J.D. Karam (Ed.), Molecular Biology of Bacteriophage T4, ASM Press, Washington, DC, 1994, pp. 54^ 82. [35] K.N. Kreuzer, S.W. Morrical, in: J.D. Karam (Ed.), Molecular Biology of Bacteriophage T4, ASM Press, Washington, DC, 1994, pp. 28^42. [36] D.L. Woodworth, K.N. Kreuzer, Genetics 143 (1996) 1081^ 1090. [37] M.A. Resnick, J. Theor. Biol. 59 (1976) 97^106. [38] J.W. Szostak, T.L. Orr-Weaver, R.J. Rothstein, F.W. Stahl, Cell 33 (1983) 25^35. [39] S.W. Yang, A.B. Burgin Jr., B.N. Huizenga, C.A. Robertson, K.C. Yao, H.A. Nash, Proc. Natl. Acad. Sci. USA 93 (1996) 11534^11539. [40] J. Barry, B. Alberts, J. Biol. Chem. 269 (1994) 33049^33062. [41] S.W. Morrical, K. Hempstead, M.D. Morrical, J. Biol. Chem. 269 (1994) 33069^33081. [42] K. Carles-Kinch, J.W. George, K.N. Kreuzer, EMBO J. 16 (1997) 4142^4151. [43] J.W. George, K.N. Kreuzer, Genetics 143 (1996) 1507^1520. [44] J.E. Mueller, T. Clyman, Y.J. Huang, M.M. Parker, M. Belfort, Genes Dev. 10 (1996) 351^364. [45] W.E. Ross, T.C. Rowe, B.S. Glisson, J. Yalowich, L.F. Liu, Cancer Res. 44 (1984) 5857^5860. [46] K.M. Tewey, G.L. Chen, E.M. Nelson, L.F. Liu, J. Biol. Chem. 259 (1984) 9182^9187.
BBAEXP 93160 15-9-98
K.N. Kreuzer / Biochimica et Biophysica Acta 1400 (1998) 339^347 [47] K.M. Tewey, T.C. Rowe, L. Yang, B.D. Halligan, L.F. Liu, Science 226 (1984) 466^468. [48] T.C. Rowe, G.L. Chen, Y.-H. Hsiang, L.F. Liu, Cancer Res. 46 (1986) 2021^2026. [49] G. Capranico, F. Zunino, K.W. Kohn, Y. Pommier, Biochemistry 29 (1990) 562^569. [50] G. Capranico, K.W. Kohn, Y. Pommier, Nucleic Acids Res. 18 (1990) 6611^6619. [51] C.H. Freudenreich, K.N. Kreuzer, EMBO J. 12 (1993) 2085^ 2097. [52] C.H. Freudenreich, K.N. Kreuzer, Proc. Natl. Acad. Sci. USA 91 (1994) 11007^11011. [53] T.-L. Shieh, P. Hoyos, E. Kolodziej, J.G. Stowell, W.M. Baird, S.R. Byrn, J. Med. Chem. 33 (1990) 1225^1230. [54] J.M. Berger, S.J. Gamblin, S.C. Harrison, J.C. Wang, Nature 379 (1996) 225^232.
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[55] J.H. Morais Cabal, A.P. Jackson, C.V. Smith, N. Shikotra, A. Maxwell, R.C. Liddington, Nature 388 (1997) 903^905. [56] P.R. Caron, J.C. Wang, in: T. Andoh, H. Ikeda, M. Oguro (Eds.), Molecular Biology of DNA Topoisomerases and Its Application to Chemotherapy, CRC Press, Boca Raton, FL, 1993, pp. 1^18. [57] P.R. Caron, J.C. Wang, in: L.F. Liu (Ed.), DNA Topoisomerases: Topoisomerase-Targeting Drugs, Academic Press, San Diego, CA, 1994, pp. 271^297. [58] A. Maxwell, J. Antimicrob. Chemother. 30 (1992) 409^416. [59] W.T. Beck, M.K. Danks, J.S. Woverton, M. Chen, B. Granzen, R. Kim, D.P. Suttle, in: DNA Topoisomerases: Topoisomerase-Targeting Drugs, L.F. Liu (Ed.), Academic Press, San Diego, CA, 1994, pp. 145^169. [60] J.L. Nitiss, Y.-X. Liu, Y. Hsiung, Cancer Res. 53 (1993) 89^ 93.
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