Hotspot sites for acridine-induced frameshift mutations in bacteriophage T4 correspond to sites of action of the T4 type II topoisomerase

Ripley, Lynn S.; Dubins, Jeffrey S.; deBoer, Johan G.; DeMarini, David M.; Bogerd, Anne M.

doi:10.1016/0022-2836(88)90479-2

Cited by 67 publications

(39 citation statements)

References 30 publications

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“…In summary, we have shown that the T4-encoded type II DNA topoisomerase is the physiological target of m-AMSA in T4-infected E. coli. This assignment is further substantiated by the finding that T4 topoisomerase is involved in the mechanism of m-AMSA-induced frameshift mutagenesis in T4-infected E. coli (44). These results, coupled with highly suggestive evidence from mammalian systems, strongly support the proposal that the type II DNA topoisomerase is also the specific intracellular target of antitumor drug action in mammalian cells.…”

Section: Discussionsupporting

confidence: 70%

Bacteriophage T4 DNA topoisomerase is the target of antitumor agent 4'-(9-acridinylamino)methanesulfon-m-anisidide (m-AMSA) in T4-infected Escherichia coli.

Huff

Leatherwood

Kreuzer

1989

Proc. Natl. Acad. Sci. U.S.A.

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The mammalian type II DNA topoisomerase has been proposed to be the intracellular target of a variety of antitumor agents, including m-AMSA [4'-(9-acridinylamino)-methanesulfon-m-anisidide]. Because the bacteriophage T4-encoded topoisomerase resembles the mammalian enzyme, we are using T4 as a simple model system to investigate the mechanism of action of m-AMSA. A mutation that renders T4 growth m-AMSA-resistant is closely linked to an amber mutation in T4 gene 39, which encodes one of the topoisomerase subunits. In addition, the gene 39 subunit from the m-AMSAresistant mutant phage has an altered net charge, strongly indicating that the drug-resistance mutation is within gene 39. Topoisomerase purified from mutant phage-infected Escherichia coli exhibits drug-insensitive DNA relaxation and DNA cleavage activities. Because a single mutation results in both drug-resistant phage growth and a drug-insensitive viral topoisomerase, we conclude that the T4-encoded type II DNA topoisomerase is the physiological target of m-AMSA in phageinfected E. coli.The mammalian type II DNA topoisomerase has been proposed to be the intracellular target of several classes of antitumor agents, including acridine derivatives, anthracyclines, and certain epipodophyllotoxins (for review, see ref.1). These agents block topoisomerase activity in vitro and cause the accumulation of covalent complexes consisting of topoisomerase attached to each 5' terminus of double-strand DNA breaks (2, 3). Because the type II topoisomerase catalyzes passage of one duplex DNA segment through a transient double-strand break in a second DNA segment, the drug-induced covalent complexes are thought to be identical to an intermediate in the normal strand-passage reaction (3). The accumulation of covalent complexes in the presence of these agents suggests that the drugs block the resealing step of the topoisomerase reaction cycle.Although it is clear that topoisomerase is inhibited by these various antitumor agents, a definitive assignment of the specific intracellular target has not been made. Some of these agents inhibit other important cellular enzymes, such as DNA polymerase (4,5). In addition, the quantity of protein-linked DNA breaks does not always correlate with cytotoxicity (6, 7), and protein-free DNA breaks have been detected in drug-treated cells (4, 7). Although studies using drugresistant mammalian cell lines are consistent with the model that topoisomerase is the target of drug action (8-12), in no case has a single mutation been shown to confer both drug-resistant cell growth and drug-resistant topoisomerase activity.To circumvent problems encountered in complex mammalian systems, we used the simple bacteriophage T4 (13) as a model system to investigate the mechanism of action of the antitumor agent 4'-(9-acridinylamino)methanesulfon-manisidide (m-AMSA). Many of the properties of the T4 topoisomerase are similar to those of the mammalian, but not the bacterial, type II topoisomerase (14, 15). Most importantly, both the mammalian and phag...

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Section: Discussionsupporting

confidence: 70%

Bacteriophage T4 DNA topoisomerase is the target of antitumor agent 4'-(9-acridinylamino)methanesulfon-m-anisidide (m-AMSA) in T4-infected Escherichia coli.

Huff

Leatherwood

Kreuzer

1989

Proc. Natl. Acad. Sci. U.S.A.

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“…One model to explain the additional sites of m-AMSA-induced cleavage in the replicated (modified) DNA is that the T4 topoisomerase recognizes additional sites when DNA contains modified cytosine residues (35,56). If this is true, these additional sites should not be recognized when the replicated DNA is generated during infections by T4 dC, a multiple-mutant phage strain which replicates with unmodified cytosine residues (40).…”

Section: Resultsmentioning

confidence: 99%

An Antitumor Drug-Induced Topoisomerase Cleavage Complex Blocks a Bacteriophage T4 Replication Fork In Vivo

Hong

Kreuzer

2000

Molecular and Cellular Biology

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Many antitumor and antibacterial drugs inhibit DNA topoisomerases by trapping covalent enzyme-DNA cleavage complexes. Formation of cleavage complexes is important for cytotoxicity, but evidence suggests that cleavage complexes themselves are not sufficient to cause cell death. Rather, active cellular processes such as transcription and/or replication are probably necessary to transform cleavage complexes into cytotoxic lesions. Using defined plasmid substrates and two-dimensional agarose gel analysis, we examined the collision of an active replication fork with an antitumor drug-trapped cleavage complex. Discrete DNA molecules accumulated on the simple Y arc, with branch points very close to the topoisomerase cleavage site. Accumulation of the Y-form DNA required the presence of a topoisomerase cleavage site, the antitumor drug, the type II topoisomerase, and a T4 replication origin on the plasmid. Furthermore, all three arms of the Y-form DNA were replicated, arguing strongly that these are trapped replication intermediates. The Y-form DNA appeared even in the absence of two important phage recombination proteins, implying that Y-form DNA is the result of replication rather than recombination. This is the first direct evidence that a drug-induced topoisomerase cleavage complex blocks the replication fork in vivo. Surprisingly, these blocked replication forks do not contain DNA breaks at the topoisomerase cleavage site, implying that the replication complex was inactivated (at least temporarily) and that topoisomerase resealed the drug-induced DNA breaks. The replication fork may behave similarly at other types of DNA lesions, and thus cleavage complexes could represent a useful (site-specific) model for chemical-and radiation-induced DNA damage.Type II DNA topoisomerases are involved in diverse cellular processes such as replication, transcription, recombination, chromosome condensation, and the maintenance of genome stability. These enzymes transform the topological state of DNA by a strand passage reaction (for reviews, see references 6, 65, and 66). After binding to one segment of duplex DNA, a type II topoisomerase cleaves the duplex with a four-base stagger while covalently attaching to both 5Ј ends via phosphotyrosine linkages. This reaction intermediate, with the enzyme covalently linked to broken DNA, is referred to as the cleavage complex. After passage of a second duplex segment through the break, the topoisomerase reverses the phosphotyrosine linkages and rejoins the cleaved DNA ends.Mammalian type II topoisomerases are targets of many important antitumor drug classes, including aminoacridines, anthracenediones, anthracyclines, ellipticines, and epipodophyllotoxins (for reviews, see references 10, 13, and 55). In addition, bacterial type II topoisomerases are targets of clinically important antibacterial agents (quinolones and flouroquinolones) (17, 28). Remarkably, all these antitumor and antibacterial agents inhibit the enzyme by trapping the cleavage complex. The simplest model is that the drug, l...

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“…Topoisomerase inhibitors that induce the cleavage complex have been shown to stimulate homologous recombination, sister chromatid exchange, gross genetic rearrangements, and frameshift mutations at sites of cleavage complex formation (4,18,41). For each of these genetic events, the helicase-mediated generation of discrete DNA breaks from a cleavage complex would be a reasonable first step.…”

Section: Discussionmentioning

confidence: 99%

Disruption of a topoisomerase-DNA cleavage complex by a DNA helicase.

Howard

Neece

Matson

1994

Proc. Natl. Acad. Sci. U.S.A.

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The type I DNA topoisomerases are targets for a variety of chemotherapeutic agents, Includin the antibacterial quinolones and several famile of antitumor drugs. These agents stabilize an epzyme-DNA cleavage complex that consists of the topoisomerase covalently linked to the 5' phosphates of a double-stranded DNA break. Although the drugstabilized cleavage complex is readily reversible, it can result in cell death by a mechanism that remains uncertain. Here we demonstrate that the action of a DNA helicase can convert the cleavage complex into a nonreversible DNA break by displacing DNA strands from the complex. Formation of a nonreversible DNA break, induced by a DNA helicase, could explain the cytotoxicity of these topoisomerase poisons. The type II topoisomerases are a primary cellular target for many of the chemotherapeutic drugs described above. Studies on drug-resistant mammalian cell lines revealed alterations in topoisomerase levels or activity, suggesting that the enzyme is an important component of the cytotoxic response (6, 7). Genetic studies in Saccharomyces cerevisiae have demonstrated that a temperature-sensitive mutation in the type II topoisomerase gene greatly decreased drug sensitivity at the semipermissive temperature, presumably due to a reduction in topoisomerase activity (8). Perhaps the clearest evidence for a type II topoisomerase being the primary drug target comes from the T4 bacteriophage system. The properties of the T4 topoisomerase closely resemble those of the mammalian enzyme (1, 9). Most importantly, both are sensitive to the aminoacridine m-AMSA [4'-(9-acridinylamino)methanesulfon-m-anisidide] (10, 11). A point mutation in one of the genes encoding the bacteriophage T4 topoisomerase allows drug-resistant phage growth, and topoisomerase purified from the mutant phage has drug-resistant topoisomerase activity (12,13).Topoisomerase inhibitors convert the enzymes that they target into poisons by inducing formation of the cleavage complex, a form of DNA damage. This view was first articulated when it was found that bacteriophage 17 12031The publication costs of this article were defrayed in part by page charge payment. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. §1734 solely to indicate this fact.

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Hotspot sites for acridine-induced frameshift mutations in bacteriophage T4 correspond to sites of action of the T4 type II topoisomerase

Cited by 67 publications

References 30 publications

Bacteriophage T4 DNA topoisomerase is the target of antitumor agent 4'-(9-acridinylamino)methanesulfon-m-anisidide (m-AMSA) in T4-infected Escherichia coli.

Bacteriophage T4 DNA topoisomerase is the target of antitumor agent 4'-(9-acridinylamino)methanesulfon-m-anisidide (m-AMSA) in T4-infected Escherichia coli.

An Antitumor Drug-Induced Topoisomerase Cleavage Complex Blocks a Bacteriophage T4 Replication Fork In Vivo

Disruption of a topoisomerase-DNA cleavage complex by a DNA helicase.

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