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

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.

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

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.

Molecular analysis of enhanced replication of UV-damaged simian virus 40 DNA in UV-treated mammalian cells

1988 ◽  
Vol 8 (6) ◽  
pp. 2428-2434
Author(s):  
J M Treger ◽  
J Hauser ◽  
K Dixon
Keyword(s):  
Uv Radiation ◽  
Uv Irradiation ◽  
Simian Virus 40 ◽  
Simian Virus ◽  
Viral Dna ◽  
Repair Capacity ◽  
Pyrimidine Dimers ◽  
Infected Cells ◽  
Replicative Intermediates ◽  
Kinetics Of

Irradiation of simian virus 40 (SV40)-infected cells with low fluences of UV light (20 to 60 J/m2, inducing one to three pyrimidine dimers per SV40 genome) causes a dramatic inhibition of viral DNA replication. However, treatment of cells with UV radiation (20 J/m2) before infection with SV40 virus enhances the replication of UV-damaged viral DNA. To investigate the mechanism of this enhancement of replication, we analyzed the kinetics of synthesis and interconversion of viral replicative intermediates synthesized after UV irradiation of SV40-infected cells that had been pretreated with UV radiation. This enhancement did not appear to be due to an expansion of the size of the pool of replicative intermediates after irradiation of pretreated infected cells; the kinetics of incorporation of labeled thymidine into replicative intermediates were very similar after irradiation of infected control and pretreated cells. The major products of replication of SV40 DNA after UV irradiation at the low UV fluences used here were form II molecules with single-stranded gaps (relaxed circular intermediates). There did not appear to be a change in the proportion of these molecules synthesized when cells were pretreated with UV radiation. Thus, it is unlikely that a substantial amount of DNA synthesis occurs past pyrimidine dimers without leaving gaps. This conclusion is supported by the observation that the proportion of newly synthesized SV40 form I molecules that contain pyrimidine dimers was not increased in pretreated cells. Pulse-chase experiments suggested that there is a more efficient conversion of replicative intermediates into form I molecules in pretreated cells. This could be due to more efficient gap filling in relaxed circular intermediate molecules or to the release of blocked replication forks. Alternatively, the enhanced replication observed here may be due to an increase in the excision repair capacity of the pretreated cells.

scholarly journals Molecular analysis of enhanced replication of UV-damaged simian virus 40 DNA in UV-treated mammalian cells.

1988 ◽  
Vol 8 (6) ◽  
pp. 2428-2434 ◽  
Author(s):  
J M Treger ◽  
J Hauser ◽  
K Dixon
Keyword(s):  
Uv Radiation ◽  
Uv Irradiation ◽  
Simian Virus 40 ◽  
Simian Virus ◽  
Viral Dna ◽  
Repair Capacity ◽  
Pyrimidine Dimers ◽  
Infected Cells ◽  
Replicative Intermediates ◽  
Kinetics Of

Irradiation of simian virus 40 (SV40)-infected cells with low fluences of UV light (20 to 60 J/m2, inducing one to three pyrimidine dimers per SV40 genome) causes a dramatic inhibition of viral DNA replication. However, treatment of cells with UV radiation (20 J/m2) before infection with SV40 virus enhances the replication of UV-damaged viral DNA. To investigate the mechanism of this enhancement of replication, we analyzed the kinetics of synthesis and interconversion of viral replicative intermediates synthesized after UV irradiation of SV40-infected cells that had been pretreated with UV radiation. This enhancement did not appear to be due to an expansion of the size of the pool of replicative intermediates after irradiation of pretreated infected cells; the kinetics of incorporation of labeled thymidine into replicative intermediates were very similar after irradiation of infected control and pretreated cells. The major products of replication of SV40 DNA after UV irradiation at the low UV fluences used here were form II molecules with single-stranded gaps (relaxed circular intermediates). There did not appear to be a change in the proportion of these molecules synthesized when cells were pretreated with UV radiation. Thus, it is unlikely that a substantial amount of DNA synthesis occurs past pyrimidine dimers without leaving gaps. This conclusion is supported by the observation that the proportion of newly synthesized SV40 form I molecules that contain pyrimidine dimers was not increased in pretreated cells. Pulse-chase experiments suggested that there is a more efficient conversion of replicative intermediates into form I molecules in pretreated cells. This could be due to more efficient gap filling in relaxed circular intermediate molecules or to the release of blocked replication forks. Alternatively, the enhanced replication observed here may be due to an increase in the excision repair capacity of the pretreated cells.

DNA topoisomerase activity associated with simian virus 40 nucleoprotein complexes

1994 ◽  
Vol 72 (5-6) ◽  
pp. 195-201 ◽  
Author(s):  
Claude Hamelin ◽  
Benoit D'Amours ◽  
Christian Page ◽  
Young Sup Chung
Keyword(s):  
Protein Complexes ◽  
Polyacrylamide Gel Electrophoresis ◽  
Simian Virus 40 ◽  
Simian Virus ◽  
Dna Topoisomerase ◽  
Infected Monkey ◽  
Before And After ◽  
Catalytically Active ◽  
Nucleoprotein Complexes ◽  
Sv40 Dna

Simian virus 40 (SV40) chromatin extracted from nuclei of infected monkey cells (CV1) was sedimented in neutral sucrose gradients, before and after digestion with bovine pancreatic RNase I-A or DNase I. DNA topoisomerase (TI) activity was found associated with RNase-resistant, DNase-sensitive SV40 nucleoprotein complexes. After polyacrylamide gel electrophoresis, a number of proteins with a molecular mass between 40 and 70 kDa were seen at the level of viral DNA peaks, some of which may represent catalytically active breakdown products of the TI enzyme. Large protein complexes were observed under the electron microscope in association with the viral chromosomes and appear to correspond to the SV40 DNA replication complex, including TI. Our results suggest that TI activity is indeed associated with the viral minichromosomes undergoing replication in vivo.Key words: deoxyribonucleoproteins, DNA topoisomerase, minichromosomes, ribonucleoproteins, simian virus 40, viral chromatin.

scholarly journals trans-activation of the simian virus 40 enhancer by a pX product of human T-cell leukemia virus type I.

1988 ◽  
Vol 62 (2) ◽  
pp. 644-648 ◽  
Author(s):  
S Saito ◽  
M Nakamura ◽  
K Ohtani ◽  
M Ichijo ◽  
K Sugamura ◽  
...  
Keyword(s):  
T Cell ◽  
Virus Type ◽  
Leukemia Virus ◽  
Simian Virus 40 ◽  
Simian Virus ◽  
Type I ◽  
Cell Leukemia ◽  
Cell Leukemia Virus Type ◽  
Human T Cell ◽  
T Cell Leukemia Virus

Method of mapping DNA replication origins

1985 ◽  
Vol 5 (1) ◽  
pp. 85-92
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.

Pyrimidine dimers block simian virus 40 replication forks

1986 ◽  
Vol 6 (10) ◽  
pp. 3443-3450
Author(s):  
C A Berger ◽  
H J Edenberg
Keyword(s):  
Mammalian Cells ◽  
Replication Fork ◽  
Uv Light ◽  
Large Fraction ◽  
Simian Virus 40 ◽  
Simian Virus ◽  
Replication Forks ◽  
Pyrimidine Dimers ◽  
Leading Strand ◽  
Uv Lesions

UV light produces lesions, predominantly pyrimidine dimers, which inhibit DNA replication in mammalian cells. The mechanism of inhibition is controversial: is synthesis of a daughter strand halted at a lesion while the replication fork moves on and reinitiates downstream, or is fork progression itself blocked for some time at the site of a lesion? We directly addressed this question by using electron microscopy to examine the distances of replication forks from the origin in unirradiated and UV-irradiated simian virus 40 chromosomes. If UV lesions block replication fork progression, the forks should be asymmetrically located in a large fraction of the irradiated molecules; if replication forks move rapidly past lesions, the forks should be symmetrically located. A large fraction of the simian virus 40 replication forks in irradiated molecules were asymmetrically located, demonstrating that UV lesions present at the frequency of pyrimidine dimers block replication forks. As a mechanism for this fork blockage, we propose that polymerization of the leading strand makes a significant contribution to the energetics of fork movement, so any lesion in the template for the leading strand which blocks polymerization should also block fork movement.

Evaluation of Topoisomerase Inhibitors as Potential Antiviral Agents

1993 ◽  
Vol 4 (2) ◽  
pp. 85-91 ◽  
Author(s):  
B. Maschera ◽  
E. Ferrazzi ◽  
M. Rassu ◽  
M. Toni ◽  
G. Palù
Keyword(s):  
Nalidixic Acid ◽  
Topoisomerase Ii ◽  
Antiviral Agents ◽  
Simian Virus 40 ◽  
Simian Virus ◽  
Etoposide Treatment ◽  
Cross Links ◽  
Infected Cells ◽  
Cellular Dna ◽  
Sv40 Dna

Anti-eukaryotic topoisomerase drugs, Camptothecin and Etoposide, were tested for their ability of selectively interfering with the replication of simian virus 40 (SV40) DNA. Nalidixic acid was also assayed for a comparison, since the compound has been previously reported to affect papoyavirus growth. Our results indicate that anti-eukaryotic topoisomerase drugs significantly inhibit viral DNA replication but at concentrations that are also toxic for uninfected cells. Etoposide treatment produced a relatively higher number of DNA-protein cross-links in virus-infected cells as compared to uninfected control cells. Nalidixic acid displayed some degree of selectivity for inhibiting SV40 DNA synthesis more effectively than synthesis of cellular DNA without appreciable reduction of cell growth. This activity does not appear to depend on DNA damage or interference with topoisomerase II and deserves further evaluation.

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