scholarly journals Accommodation of pyrimidine dimers during replication of UV-damaged simian virus 40 DNA.

1983 ◽  
Vol 3 (8) ◽  
pp. 1403-1411 ◽  
Author(s):  
P C Stacks ◽  
J H White ◽  
K Dixon
Keyword(s):  
Uv Irradiation ◽  
Simian Virus 40 ◽  
Simian Virus ◽  
Pyrimidine Dimer ◽  
Progressive Decrease ◽  
Replication Machinery ◽  
Pyrimidine Dimers ◽  
Infected Cells ◽  
Biophysical Techniques ◽  
Cellular Replication

UV irradiation of simian virus 40-infected cells at fluences between 20 and 60 J/m2, which yield one to three pyrimidine dimers per simian virus 40 genome, leads to a fluence-dependent progressive decrease in simian virus 40 DNA replication as assayed by incorporation of [3H]deoxyribosylthymine into viral DNA. We used a variety of biochemical and biophysical techniques to show that this decrease is due to a block in the progression of replicative-intermediate molecules to completed form I molecules, with a concomitant decrease in the entry of molecules into the replicating pool. Despite this UV-induced inhibition of replication, some pyrimidine dimer-containing molecules become fully replicated after UV irradiation. The fraction of completed molecules containing dimers goes up with time such that by 3 h after a UV fluence of 40 J/m2, more than 50% of completed molecules contain pyrimidine dimers. We postulate that the cellular replication machinery can accommodate limited amounts of UV-induced damage and that the progressive decrease in simian virus 40 DNA synthesis after UV irradiation is due to the accumulation in the replication pool of blocked molecules containing levels of damage greater than that which can be tolerated.

Accommodation of pyrimidine dimers during replication of UV-damaged simian virus 40 DNA

1983 ◽  
Vol 3 (8) ◽  
pp. 1403-1411
Author(s):  
P C Stacks ◽  
J H White ◽  
K Dixon
Keyword(s):  
Uv Irradiation ◽  
Simian Virus 40 ◽  
Simian Virus ◽  
Pyrimidine Dimer ◽  
Progressive Decrease ◽  
Replication Machinery ◽  
Pyrimidine Dimers ◽  
Infected Cells ◽  
Biophysical Techniques ◽  
Cellular Replication

UV irradiation of simian virus 40-infected cells at fluences between 20 and 60 J/m2, which yield one to three pyrimidine dimers per simian virus 40 genome, leads to a fluence-dependent progressive decrease in simian virus 40 DNA replication as assayed by incorporation of [3H]deoxyribosylthymine into viral DNA. We used a variety of biochemical and biophysical techniques to show that this decrease is due to a block in the progression of replicative-intermediate molecules to completed form I molecules, with a concomitant decrease in the entry of molecules into the replicating pool. Despite this UV-induced inhibition of replication, some pyrimidine dimer-containing molecules become fully replicated after UV irradiation. The fraction of completed molecules containing dimers goes up with time such that by 3 h after a UV fluence of 40 J/m2, more than 50% of completed molecules contain pyrimidine dimers. We postulate that the cellular replication machinery can accommodate limited amounts of UV-induced damage and that the progressive decrease in simian virus 40 DNA synthesis after UV irradiation is due to the accumulation in the replication pool of blocked molecules containing levels of damage greater than that which can be tolerated.

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.

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.

scholarly journals UV Irradiation induces an activity which stimulates Simian virus 40 rescue upon cell fusion.

1984 ◽  
Vol 4 (6) ◽  
pp. 1159-1162 ◽  
Author(s):  
S Nomura ◽  
M Oishi
Keyword(s):  
Cell Fusion ◽  
Uv Irradiation ◽  
Syrian Hamster ◽  
Simian Virus 40 ◽  
Simian Virus ◽  
African Green Monkey ◽  
Green Monkey ◽  
African Green Monkey Cells ◽  
Stimulation Of

UV irradiation of African green monkey cells greatly stimulated efficiency of simian virus 40 induction from simian virus 40-transformed Syrian hamster cells after cell fusion. The maximum inducing activity was observed at 15 to 20 h after irradiation but remained only transiently. The addition of cycloheximide after UV irradiation eliminated the stimulation of the activity.

scholarly journals Induction of cellular thymidine kinase occurs at the mRNA level.

1985 ◽  
Vol 5 (6) ◽  
pp. 1490-1497 ◽  
Author(s):  
P Stuart ◽  
M Ito ◽  
C Stewart ◽  
S E Conrad
Keyword(s):  
Steady State ◽  
Thymidine Kinase ◽  
Cdna Clone ◽  
Blot Hybridization ◽  
Mrna Level ◽  
Simian Virus 40 ◽  
Simian Virus ◽  
Chromosomal Dna ◽  
Infected Cells ◽  
Steady State Levels

The thymidine kinase (TK) gene has been isolated from human genomic DNA. The gene was passaged twice by transfection of LTK- cells with human chromosomal DNA, and genomic libraries were made in lambda Charon 30 from a second-round TK+ transformant. When the library was screened with a human Alu probe, seven overlapping lambda clones from the human TK locus were obtained. None of the seven contained a functional TK gene as judged by transfection analysis, but several combinations of clones gave rise to TK+ colonies when cotransfected into TK- cells. A functional cDNA clone encoding the human TK gene has also been isolated. Using this cDNA clone as a probe in restriction enzyme/blot hybridization analyses, we have mapped the coding sequences and direction of transcription of the gene. We have also used a single-copy subclone from within the coding region to monitor steady-state levels of TK mRNA in serum-stimulated and simian virus 40-infected simian CV1 tissue culture cells. Our results indicate that the previously reported increase in TK enzyme levels seen after either treatment is paralleled by an equivalent increase in the steady-state levels of TK mRNA. In the case of simian virus 40-infected cells, the induction was delayed by 8 to 12 h, which is the length of time after infection required for early viral protein synthesis. In both cases, induction of TK mRNA coincides with the onset of DNA synthesis, but virally infected cells ultimately accumulate more TK mRNA than do serum-stimulated cells.

scholarly journals Evidence for transcriptional and post-transcriptional control of the cellular thymidine kinase gene.

1987 ◽  
Vol 7 (3) ◽  
pp. 1156-1163 ◽  
Author(s):  
C J Stewart ◽  
M Ito ◽  
S E Conrad
Keyword(s):  
Cell Cycle ◽  
Thymidine Kinase ◽  
Simian Virus 40 ◽  
Simian Virus ◽  
S Phase ◽  
The Body ◽  
Cdna Clones ◽  
Mrna Levels ◽  
Regulated Expression ◽  
Infected Cells

We have studied the cell cycle-regulated expression of the thymidine kinase (TK) gene in mammalian tissue culture cells. TK mRNA and enzyme levels are low in resting, G0-phase cells, but increase dramatically (10- to 20-fold) during the S phase in both serum-stimulated and simian virus 40-infected cells. To determine whether an increase in the rate of TK gene transcription is responsible for this induction, nuclear run-on transcription assays were performed at various times after serum stimulation or simian virus 40 infection of growth-arrested simian CV1 cells. When assays were performed at 12-h intervals, a small (two- to threefold) but reproducible increase in TK transcription was detected during the S phase. When time points were chosen to span the G1-S interface a larger (six- to sevenfold) increase in transcriptional activity was observed in serum-stimulated cells but not in simian virus 40-infected cells. The large increase in TK mRNA levels and the relatively small increase in transcription rates in growth-stimulated cells suggest that TK gene expression is controlled at both a transcriptional and post-transcriptional level during the mammalian cell cycle. To identify the DNA sequences required for cell cycle-regulated expression, several TK cDNA clones were transfected into Rat-3 TK- cells, and their expression was examined in resting and serum-stimulated cultures. These experiments indicated that the body of the TK cDNA is sufficient to insure cell cycle-regulated expression regardless of the promoter or polyadenylation signal used.

scholarly journals Variation in intracellular P1P4-bis(5′-adenosyl) tetraphosphate (Ap4A) in virus-infected cells

1990 ◽  
Vol 268 (3) ◽  
pp. 791-793 ◽  
Author(s):  
D J Johnston ◽  
C A Hart ◽  
A G McLennan
Keyword(s):  
Herpes Simplex Virus ◽  
Herpes Simplex ◽  
Virus Infection ◽  
Simian Virus 40 ◽  
Fold Increase ◽  
Vero Cells ◽  
Simian Virus ◽  
Infected Cells ◽  
Simplex Virus

The effect of virus infection on the intracellular concentration of the proposed stress alarmone P1P4-bis(5′-adenosyl) tetraphosphate (Ap4A) has been examined in Vero cells. Compared with exposure to 0.8 mM-Cd2+, which causes a 30-fold increase in Ap4A, infection with simian virus 40 and poliovirus causes only a 2-fold increase, whereas herpes simplex virus type 1 results in a decrease in Ap4A during the course of the infection.

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