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Molecular and Cellular Biology, January 2000, p. 594-603, Vol. 20, No. 2
0270-7306/0/$04.00+0
Copyright © 2000, American Society for Microbiology. All rights reserved.

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

George Hong and Kenneth N. Kreuzer*

Department of Microbiology, Duke University Medical Center, Durham, North Carolina 27710

Received 23 August 1999/Returned for modification 21 September 1999/Accepted 20 October 1999

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.

* Corresponding author. Mailing address: Department of Microbiology, Box 3020, Duke University Medical Center, Durham, NC 27710. Phone: (919) 684-6466. Fax: (919) 681-8911. E-mail: kenneth.kreuzer{at}duke.edu.

Molecular and Cellular Biology, January 2000, p. 594-603, Vol. 20, No. 2
0270-7306/0/$04.00+0
Copyright © 2000, American Society for Microbiology. All rights reserved.


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