Exposure to camptothecin breaks leading and lagging strand simian virus 40 DNA replication forks

We have developed a method which allows determination of the direction in which replication forks move through segments of chromosomal DNA for which cloned probes are available. The method is based on the facts that DNA restriction fragments containing replication forks migrate more slowly through agarose gels than do non-fork-containing fragments and that the extent of retardation of the fork-containing fragments is a function of the extent of replication. The procedure allows the identification of DNA replication origins as sites from which replication forks diverge. In this paper we demonstrate the feasibility of this procedure, with simian virus 40 DNA as a model, and we discuss its applicability to other systems.

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Synthetic DNA Replication Bubbles Bound and Unwound with Twofold Symmetry by a Simian Virus 40 T-Antigen Double Hexamer

Journal of Virology ◽

10.1128/jvi.72.11.8676-8681.1998 ◽

1998 ◽

Vol 72 (11) ◽

pp. 8676-8681 ◽

Cited By ~ 21

Author(s):

Natalia V. Smelkova ◽

James A. Borowiec

Keyword(s):

Dna Replication ◽

Simian Virus 40 ◽

Simian Virus ◽

Dna Helicase ◽

T Antigen ◽

Dna Unwinding ◽

Replication Forks ◽

Helicase Activity ◽

Synthetic Dna ◽

Stimulation Of

ABSTRACT Dimerization of simian virus 40 T-antigen hexamers (TAgH) into double hexamers (TAgDH) on model DNA replication forks has been found to greatly stimulate T-antigen DNA helicase activity. To explore the interaction of TAgDH with DNA during unwinding, we examined the binding of TAgDH to synthetic DNA replication bubbles. Tests of replication bubble substrates containing different single-stranded DNA (ssDNA) lengths indicated that efficient formation of a TAgDH requires ≥40 nucleotides (nt) of ssDNA. DNase I probing of a substrate containing a 60-nt ssDNA bubble complexed with a TAgDH revealed that T antigen bound the substrate with twofold symmetry. The strongest protection was observed over the 5′ junction on each strand, with 5 bp of duplex DNA and ∼17 nt of adjacent ssDNA protected from nuclease cleavage. Stimulation of the T-antigen DNA helicase activity by an increase in ATP concentration caused the protection to extend in the 5′ direction into the duplex region, while resulting in no significant changes to the 3′ edge of strongest protection. Our data indicate that each TAgH encircles one ssDNA strand, with a different strand bound at each junction. The process of DNA unwinding results in each TAgH interacting with a greater length of DNA than was initially bound, suggesting the generation of a more highly processive helicase complex.

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Camptothecin, a specific inhibitor of type I DNA topoisomerase, induces DNA breakage at replication forks.

Molecular and Cellular Biology ◽

10.1128/mcb.8.8.3026 ◽

1988 ◽

Vol 8 (8) ◽

pp. 3026-3034 ◽

Cited By ~ 140

Author(s):

K Avemann ◽

R Knippers ◽

T Koller ◽

J M Sogo

Keyword(s):

Specific Inhibitor ◽

Simian Virus 40 ◽

Simian Virus ◽

Type I ◽

Replication Forks ◽

Electron Microscopic ◽

Dna Topoisomerase ◽

Infected Cells ◽

Replicative Intermediates ◽

Lagging Strand

The structure of replicating simian virus 40 minichromosomes, extracted from camptothecin-treated infected cells, was investigated by biochemical and electron microscopic methods. We found that camptothecin frequently induced breaks at replication forks close to the replicative growth points. Replication branches were disrupted at about equal frequencies at the leading and the lagging strand sides of the fork. Since camptothecin is known to be a specific inhibitor of type I DNA topoisomerase, we suggest that this enzyme is acting very near the replication forks. This conclusion was supported by experiments with aphidicolin, a drug that blocks replicative fork movement, but did not prevent the camptothecin-induced breakage of replication forks. The drug teniposide, an inhibitor of type II DNA topoisomerase, had only minor effects on the structure of these replicative intermediates.

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Topoisomerase inhibitors can selectively interfere with different stages of simian virus 40 DNA replication

Molecular and Cellular Biology ◽

10.1128/mcb.6.12.4221-4227.1986 ◽

1986 ◽

Vol 6 (12) ◽

pp. 4221-4227

Author(s):

R M Snapka

Keyword(s):

Dna Replication ◽

Sister Chromatid ◽

Simian Virus 40 ◽

Simian Virus ◽

Sister Chromatid Exchanges ◽

Replication Forks ◽

Dna Breaks ◽

Rapid Reversal ◽

Chromatid Exchanges

I have found that antineoplastic drugs which are known to be inhibitors of mammalian DNA topoisomerases have pronounced and selective effects on simian virus 40 DNA replication. Ellipticine, 4'-(9-acridinylamino)methanesulfon-m-aniside, and Adriamycin blocked decatenation of newly replicated simian virus 40 daughter chromosomes in vivo. The arrested decatenation intermediates produced by these drugs contained single-strand DNA breaks. Ellipticine in particular produced these catenated dimers rapidly and efficiently. Removal of the drug resulted in rapid reversal of the block and completion of decatenation. The demonstration that these drugs interfere with decatenation suggests that they may exert their cytotoxic and antineoplastic effects by preventing the separation of newly replicated cellular chromosomes. Camptothecin rapidly breaks replication forks in growing Cairns structures. It is likely that the target of camptothecin is the "swivel" topoisomerase required for DNA replication and that it is located at or very near the replication fork in vivo. Evidence is presented that many of the broken Cairns structures are in fact half-completed sister chromatid exchanges. One pathway for the resolution of these structures is completion of the sister chromatid exchange to produce a circular head-to-tail dimer.

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Topoisomerase inhibitors can selectively interfere with different stages of simian virus 40 DNA replication.

Molecular and Cellular Biology ◽

10.1128/mcb.6.12.4221 ◽

1986 ◽

Vol 6 (12) ◽

pp. 4221-4227 ◽

Cited By ~ 86

Author(s):

R M Snapka

Keyword(s):

Dna Replication ◽

Sister Chromatid ◽

Simian Virus 40 ◽

Simian Virus ◽

Sister Chromatid Exchanges ◽

Replication Forks ◽

Dna Breaks ◽

Rapid Reversal ◽

Chromatid Exchanges

I have found that antineoplastic drugs which are known to be inhibitors of mammalian DNA topoisomerases have pronounced and selective effects on simian virus 40 DNA replication. Ellipticine, 4'-(9-acridinylamino)methanesulfon-m-aniside, and Adriamycin blocked decatenation of newly replicated simian virus 40 daughter chromosomes in vivo. The arrested decatenation intermediates produced by these drugs contained single-strand DNA breaks. Ellipticine in particular produced these catenated dimers rapidly and efficiently. Removal of the drug resulted in rapid reversal of the block and completion of decatenation. The demonstration that these drugs interfere with decatenation suggests that they may exert their cytotoxic and antineoplastic effects by preventing the separation of newly replicated cellular chromosomes. Camptothecin rapidly breaks replication forks in growing Cairns structures. It is likely that the target of camptothecin is the "swivel" topoisomerase required for DNA replication and that it is located at or very near the replication fork in vivo. Evidence is presented that many of the broken Cairns structures are in fact half-completed sister chromatid exchanges. One pathway for the resolution of these structures is completion of the sister chromatid exchange to produce a circular head-to-tail dimer.

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Camptothecin, a specific inhibitor of type I DNA topoisomerase, induces DNA breakage at replication forks

Molecular and Cellular Biology ◽

10.1128/mcb.8.8.3026-3034.1988 ◽

1988 ◽

Vol 8 (8) ◽

pp. 3026-3034

Author(s):

K Avemann ◽

R Knippers ◽

T Koller ◽

J M Sogo

Keyword(s):

Specific Inhibitor ◽

Simian Virus 40 ◽

Simian Virus ◽

Type I ◽

Replication Forks ◽

Electron Microscopic ◽

Dna Topoisomerase ◽

Infected Cells ◽

Replicative Intermediates ◽

Lagging Strand

The structure of replicating simian virus 40 minichromosomes, extracted from camptothecin-treated infected cells, was investigated by biochemical and electron microscopic methods. We found that camptothecin frequently induced breaks at replication forks close to the replicative growth points. Replication branches were disrupted at about equal frequencies at the leading and the lagging strand sides of the fork. Since camptothecin is known to be a specific inhibitor of type I DNA topoisomerase, we suggest that this enzyme is acting very near the replication forks. This conclusion was supported by experiments with aphidicolin, a drug that blocks replicative fork movement, but did not prevent the camptothecin-induced breakage of replication forks. The drug teniposide, an inhibitor of type II DNA topoisomerase, had only minor effects on the structure of these replicative intermediates.

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Method of mapping DNA replication origins.

Molecular and Cellular Biology ◽

10.1128/mcb.5.1.85 ◽

1985 ◽

Vol 5 (1) ◽

pp. 85-92 ◽

Cited By ~ 7

Author(s):

L D Spotila ◽

J A Huberman

Keyword(s):

Dna Replication ◽

Simian Virus 40 ◽

Simian Virus ◽

Replication Forks ◽

Replication Origins ◽

Chromosomal Dna ◽

Dna Restriction Fragments ◽

Dna Restriction ◽

Dna Replication Origins

We have developed a method which allows determination of the direction in which replication forks move through segments of chromosomal DNA for which cloned probes are available. The method is based on the facts that DNA restriction fragments containing replication forks migrate more slowly through agarose gels than do non-fork-containing fragments and that the extent of retardation of the fork-containing fragments is a function of the extent of replication. The procedure allows the identification of DNA replication origins as sites from which replication forks diverge. In this paper we demonstrate the feasibility of this procedure, with simian virus 40 DNA as a model, and we discuss its applicability to other systems.

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