scholarly journals Initiation of simian virus 40 DNA synthesis in vitro.

1991 ◽  
Vol 11 (5) ◽  
pp. 2350-2361 ◽  
Author(s):  
P A Bullock ◽  
Y S Seo ◽  
J Hurwitz
Keyword(s):  
Dna Synthesis ◽  
Hela Cells ◽  
Simian Virus 40 ◽  
Simian Virus ◽  
T Antigen ◽  
Nuclear Antigen ◽  
Present Evidence ◽  
Crude Extracts ◽  
Sv40 Origin

Simian virus 40 (SV40) T antigen can efficiently initiate SV40 origin-dependent DNA synthesis in crude extracts of HeLa cells. Therefore, initiation of SV40 DNA synthesis can be analyzed in detail. We present evidence that antibodies which neutralize proliferating cell nuclear antigen (PCNA) inhibit but do not abolish pulse-labeling of nascent DNA. The lengths of DNA products formed after a 5-s pulse in the absence and presence of anti-PCNA serum averaged 150 and 34 nucleotides, respectively. The small DNAs formed in the presence of anti-PCNA serum underwent little or no increase in size during further incubation periods. The addition of PCNA to reaction mixtures inhibited with anti-PCNA serum largely reversed the inhibitory effect of the antiserum. The small nascent DNAs formed in the presence or absence of anti-PCNA serum products arose from the replication of lagging strands. These results suggest that a PCNA-dependent elongation reaction participates in the synthesis of lagging strands as well as leading strands. We also present evidence that in crude extracts of HeLa cells, DNA synthesis generally does not initiate within the core origin. Initiation of DNA synthesis outside of a genetically defined origin region has not been previously described in a eukaryotic replication system but appears to be a common feature of initiation events in many prokaryotic organisms. Additional results presented indicate that in the absence of nucleoside triphosphates other than ATP, the preinitiation complex remains within or close to the SV40 origin.

Initiation of simian virus 40 DNA synthesis in vitro

1991 ◽  
Vol 11 (5) ◽  
pp. 2350-2361
Author(s):  
P A Bullock ◽  
Y S Seo ◽  
J Hurwitz
Keyword(s):  
Dna Synthesis ◽  
Hela Cells ◽  
Simian Virus 40 ◽  
Simian Virus ◽  
T Antigen ◽  
Nuclear Antigen ◽  
Present Evidence ◽  
Crude Extracts ◽  
Sv40 Origin

Simian virus 40 (SV40) T antigen can efficiently initiate SV40 origin-dependent DNA synthesis in crude extracts of HeLa cells. Therefore, initiation of SV40 DNA synthesis can be analyzed in detail. We present evidence that antibodies which neutralize proliferating cell nuclear antigen (PCNA) inhibit but do not abolish pulse-labeling of nascent DNA. The lengths of DNA products formed after a 5-s pulse in the absence and presence of anti-PCNA serum averaged 150 and 34 nucleotides, respectively. The small DNAs formed in the presence of anti-PCNA serum underwent little or no increase in size during further incubation periods. The addition of PCNA to reaction mixtures inhibited with anti-PCNA serum largely reversed the inhibitory effect of the antiserum. The small nascent DNAs formed in the presence or absence of anti-PCNA serum products arose from the replication of lagging strands. These results suggest that a PCNA-dependent elongation reaction participates in the synthesis of lagging strands as well as leading strands. We also present evidence that in crude extracts of HeLa cells, DNA synthesis generally does not initiate within the core origin. Initiation of DNA synthesis outside of a genetically defined origin region has not been previously described in a eukaryotic replication system but appears to be a common feature of initiation events in many prokaryotic organisms. Additional results presented indicate that in the absence of nucleoside triphosphates other than ATP, the preinitiation complex remains within or close to the SV40 origin.

Replication of cloned DNA containing the Alu family sequence during cell extract-promoting simian virus 40 DNA synthesis

1984 ◽  
Vol 4 (8) ◽  
pp. 1476-1482
Author(s):  
H Ariga
Keyword(s):  
Dna Replication ◽  
Dna Synthesis ◽  
Rna Polymerase Iii ◽  
Simian Virus 40 ◽  
Cell Extract ◽  
Simian Virus ◽  
T Antigen ◽  
Sv40 T Antigen ◽  
Sv40 Dna

The replicating activity of several cloned DNAs containing putative origin sequences was examined in a cell-free extract that absolutely depends on simian virus 40 (SV40) T antigen promoting initiation of SV40 DNA replication in vitro. Of the three DNAs containing the human Alu family sequence (BLUR8), the origin of (Saccharomyces cerevisiae plasmid 2 micron DNA (pJD29), and the yeast autonomous replicating sequence (YRp7), only BLUR8 was active as a template. Replication in a reaction mixture with BLUR8 as a template was semiconservative and not primed by a putative RNA polymerase III transcript synthesized on the Alu family sequence in vitro. Pulse-chase experiments showed that the small-sized DNA produced in a short-term incubation was converted to full-length closed circular and open circular DNAs in alkaline sucrose gradients. DNA synthesis in extracts began in a region of the Alu family sequence and was inhibited 80% by the addition of anti-T serum. Furthermore, partially purified T antigen bound the Alu family sequence in BLUR8 by the DNA-binding immunoassay. These results suggest that SV40 T antigen recognizes the Alu family sequence, similar to the origin sequence of SV40 DNA, and initiates semiconservative DNA replication in vitro.

scholarly journals Initiation of simian virus 40 DNA replication in vitro: aphidicolin causes accumulation of early-replicating intermediates and allows determination of the initial direction of DNA synthesis.

1986 ◽  
Vol 6 (11) ◽  
pp. 3815-3825 ◽  
Author(s):  
R S Decker ◽  
M Yamaguchi ◽  
R Possenti ◽  
M L DePamphilis
Keyword(s):  
Dna Replication ◽  
Dna Synthesis ◽  
Dna Polymerase ◽  
Simian Virus 40 ◽  
Simian Virus ◽  
T Antigen ◽  
Early Gene ◽  
Initial Direction ◽  
Dna Polymerase Alpha

Aphidicolin, a specific inhibitor of DNA polymerase alpha, provided a novel method for distinguishing between initiation of DNA synthesis at the simian virus 40 (SV40) origin of replication (ori) and continuation of replication beyond ori. In the presence of sufficient aphidicolin to inhibit total DNA synthesis by 50%, initiation of DNA replication in SV40 chromosomes or ori-containing plasmids continued in vitro, whereas DNA synthesis in the bulk of SV40 replicative intermediate DNA (RI) that had initiated replication in vivo was rapidly inhibited. This resulted in accumulation of early RI in which most nascent DNA was localized within a 600- to 700-base-pair region centered at ori. Accumulation of early RI was observed only under conditions that permitted initiation of SV40 ori-dependent, T-antigen-dependent DNA replication and only when aphidicolin was added to the in vitro system. Increasing aphidicolin concentrations revealed that DNA synthesis in the ori region was not completely resistant to aphidicolin but simply less sensitive than DNA synthesis at forks that were farther away. Since DNA synthesized in the presence of aphidicolin was concentrated in the 300 base pairs on the early gene side of ori, we conclude that the initial direction of DNA synthesis was the same as that of early mRNA synthesis, consistent with the model proposed by Hay and DePamphilis (Cell 28:767-779, 1982). The data were also consistent with initiation of the first DNA chains in ori by CV-1 cell DNA primase-DNA polymerase alpha. Synthesis of pppA/G(pN)6-8(pdN)21-23 chains on a single-stranded DNA template by a purified preparation of this enzyme was completely resistant to aphidicolin, and further incorporation of deoxynucleotide monophosphates was inhibited. Therefore, in the presence of aphidicolin, this enzyme could initiate RNA-primed DNA synthesis at ori first in the early gene direction and then in the late gene direction, but could not continue DNA synthesis for an extended distance.

scholarly journals Simian virus 40 DNA replication in vitro: identification of multiple stages of initiation.

1989 ◽  
Vol 9 (9) ◽  
pp. 3839-3849 ◽  
Author(s):  
T Tsurimoto ◽  
M P Fairman ◽  
B Stillman
Keyword(s):  
Dna Replication ◽  
Dna Synthesis ◽  
Time Lag ◽  
Simian Virus 40 ◽  
Simian Virus ◽  
T Antigen ◽  
Nuclear Antigen ◽  
Replication Factor ◽  
Cellular Fraction ◽  
Template Dna

A cell-free DNA replication system dependent upon five purified cellular proteins, one crude cellular fraction, and the simian virus 40 (SV40)-encoded large tumor antigen (T antigen) initiated and completed replication of plasmids containing the SV40 origin sequence. DNA synthesis initiated at or near the origin sequence after a time lag of approximately 10 min and then proceeded bidirectionally from the origin to yield covalently closed, monomer daughter molecules. The time lag could be completely eliminated by a preincubation of SV40 ori DNA in the presence of T antigen, a eucaryotic single-stranded DNA-binding protein (replication factor A [RF-A]), and topoisomerases I and II. In contrast, if T antigen and the template DNA were incubated alone, the time lag was only partially decreased. Kinetic analyses of origin recognition by T antigen, origin unwinding, and DNA synthesis suggest that the time lag in replication was due to the formation of a complex between T antigen and DNA called the T complex, followed by formation of a second complex called the unwound complex. Formation of the unwound complex required RF-A. When origin unwinding was coupled to DNA replication by the addition of a partially purified cellular fraction (IIA), DNA synthesis initiated at the ori sequence, but the template DNA was not completely replicated. Complete DNA replication in this system required the proliferating-cell nuclear antigen and another cellular replication factor, RF-C, during the elongation stage. In a less fractionated system, another cellular fraction, SSI, was previously shown to be necessary for reconstitution of DNA replication. The SSI fraction was required in the less purified system to antagonize the inhibitory action of another cellular protein(s). This inhibitor specifically blocked the earliest stage of DNA replication, but not the later stages. The implications of these results for the mechanisms of initiation and elongation of DNA replication are discussed.

scholarly journals WAF1 retards S-phase progression primarily by inhibition of cyclin-dependent kinases.

1997 ◽  
Vol 17 (8) ◽  
pp. 4877-4882 ◽  
Author(s):  
V V Ogryzko ◽  
P Wong ◽  
B H Howard
Keyword(s):  
Mammalian Cells ◽  
Simian Virus 40 ◽  
Simian Virus ◽  
T Antigen ◽  
S Phase ◽  
In Vivo Studies ◽  
Nuclear Antigen ◽  
Phase Progression

The p21(WAF1/CIP1/sdi1) gene product (WAF1) inhibits DNA replication in vitro (J. Chen, P. Jackson, M. Kirschner, and A. Dutta, Nature 374:386-388, 1995; S. Waga, G. Hannon, D. Beach, and B. Stillman, Nature 369:574-578, 1994), but in vivo studies on the antiproliferative activity of WAF1 have not resolved G1-phase arrest from potential inhibition of S-phase progression. Here, we demonstrate that elevated WAF1 expression can retard replicative DNA synthesis in vivo. The WAF1-mediated inhibitory effect could be antagonized by cyclin A, cyclin E, or the simian virus 40 small-t antigen with no decrease in the levels of WAF1 protein in transfected cells. Proliferating-cell nuclear antigen (PCNA) overexpression was neither necessary nor sufficient to antagonize WAF1 action. Expression of the N-terminal domain of WAF1, responsible for cyclin-dependent kinase (CDK) interaction, had the same effect as full-length WAF1, while the PCNA binding C terminus exhibited modest activity. We conclude that S-phase progression in mammalian cells is dependent on continuing cyclin and CDK activity and that WAF1 affects S phase primarily through cyclin- and CDK-dependent pathways.

scholarly journals Primer-DNA formation during simian virus 40 DNA replication in vitro.

1993 ◽  
Vol 13 (5) ◽  
pp. 2882-2890 ◽  
Author(s):  
D Denis ◽  
P A Bullock
Keyword(s):  
Dna Replication ◽  
Proliferating Cell Nuclear Antigen ◽  
Simian Virus 40 ◽  
Simian Virus ◽  
T Antigen ◽  
Nuclear Antigen ◽  
Origin Region ◽  
Sv40 Dna ◽  
Proliferating Cell

Studies of simian virus 40 (SV40) DNA replication in vitro have identified a small (approximately 30-nucleotide) RNA-DNA hybrid species termed primer-DNA. Initial experiments indicated that T antigen and the polymerase alpha-primase complex are required to form primer-DNA. Proliferating cell nuclear antigen, and presumably proliferating cell nuclear antigen-dependent polymerases, is not needed to form this species. Herein, we present an investigation of the stages at which primer-DNA functions during SV40 DNA replication in vitro. Hybridization studies indicate that primer-DNA is initially formed in the origin region and is subsequently synthesized in regions distal to the origin. At all time points, primer-DNA is synthesized from templates for lagging-strand DNA replication. These studies indicate that primer-DNA functions during both initiation and elongation stages of SV40 DNA synthesis. Results of additional experiments suggesting a precursor-product relationship between formation of primer-DNA and Okazaki fragments are presented.

Replication and supercoiling of simian virus 40 DNA in cell extracts from human cells

1985 ◽  
Vol 5 (8) ◽  
pp. 2051-2060
Author(s):  
B W Stillman ◽  
Y Gluzman
Keyword(s):  
Dna Synthesis ◽  
Simian Virus 40 ◽  
Nuclear Extract ◽  
Simian Virus ◽  
T Antigen ◽  
Cell Extracts ◽  
293 Cells ◽  
Dna Strands

Soluble extracts prepared from the nucleus and cytoplasm of human 293 cells are capable of efficient replication and supercoiling of added DNA templates that contain the origin of simian virus 40 replication. Extracts prepared from human HeLa cells are less active than similarly prepared extracts from 293 cells for initiation and elongation of nascent DNA strands. DNA synthesis is dependent on addition of purified simian virus 40 tumor (T) antigen, which is isolated by immunoaffinity chromatography of extracts from cells infected with an adenovirus modified to produce large quantities of this protein. In the presence of T antigen and the cytoplasmic extract, replication initiates at the origin and continues bidirectionally. Initiation is completely dependent on functional origin sequences; a plasmid DNA containing an origin mutation known to affect DNA replication in vivo fails to replicate in vitro. Multiple rounds of DNA synthesis occur, as shown by the appearance of heavy-heavy, bromodeoxyuridine-labeled DNA products. The products of this reaction are resolved, but are relaxed, covalently closed DNA circles. Addition of a nuclear extract during DNA synthesis promotes the negative supercoiling of the replicated DNA molecules.

scholarly journals Methylation of single sites within the herpes simplex virus tk coding region and the simian virus 40 T-antigen intron causes gene inactivation.

1994 ◽  
Vol 14 (3) ◽  
pp. 2004-2010 ◽  
Author(s):  
A Graessmann ◽  
G Sandberg ◽  
E Guhl ◽  
M Graessmann
Keyword(s):  
Gene Expression ◽  
Herpes Simplex Virus ◽  
Herpes Simplex ◽  
Dna Synthesis ◽  
Simian Virus 40 ◽  
Simian Virus ◽  
T Antigen ◽  
Coding Region ◽  
Simplex Virus

In order to determine whether partial methylation of the herpes simplex virus (HSV) tk gene prevents tk gene expression, the HSV tk gene was cloned as single-stranded DNA. By in vitro second-strand DNA synthesis, specific HSV tk gene segments were methylated, and the hemimethylated DNA molecules were microinjected into thymidine kinase-negative rat2 cells. Conversion of the hemimethylated DNA into symmetrical methylated DNA and integration into the host genome occurred early after gene transfer, before the cells entered into the S phase. HSV tk gene expression was inhibited either by promoter methylation or by methylation of the coding region. Using the HindIII-SphI HSV tk DNA fragment as a primer for in vitro DNA synthesis, all cytosine residues within the coding region, from +499 to +1309, were selectively methylated. This specific methylation pattern caused inactivation of the HSV tk gene, while methylation of the cytosine residues within the nucleotide sequence from +811 to +1309 had no effect on HSV tk gene activity. We also methylated single HpaII sites within the HSV tk gene using a specific methylated primer for in vitro DNA synthesis. We found that of the 16 HSV tk HpaII sites, methylation of 6 single sites caused HSV tk inactivation. All six of these "methylation-sensitive" sites are within the coding region, including the HpaII-6 site, which is 571 bp downstream from the transcription start site. The sites HpaII-7 to HpaII-16 were all methylation insensitive. We further inserted separately the methylation-sensitive HSV tk HpaII-6 site and the methylation-insensitive HpaII-13 site as DNA segments (32-mer) into the intron region of the simian virus 40 T antigen (TaqI site). Methylation of these HpaII sites caused inhibition of simian virus 40 T-antigen synthesis.

scholarly journals Topoisomerase I Associates Specifically with Simian Virus 40 Large-T-Antigen Double Hexamer–Origin Complexes

2000 ◽  
Vol 74 (11) ◽  
pp. 5224-5232 ◽  
Author(s):  
Dahai Gai ◽  
Rupa Roy ◽  
Chunxiao Wu ◽  
Daniel T. Simmons
Keyword(s):  
Dna Replication ◽  
Topoisomerase I ◽  
Simian Virus 40 ◽  
Simian Virus ◽  
T Antigen ◽  
Replication Initiation ◽  
Initiation Complex ◽  
Initiation Of Replication ◽  
Sv40 Origin

ABSTRACT Topoisomerase I (topo I) is required for releasing torsional stress during simian virus 40 (SV40) DNA replication. Recently, it has been demonstrated that topo I participates in initiation of replication as well as in elongation. Although T antigen and topo I can bind to one another in vitro, there is no direct evidence that topo I is a component of the replication initiation complex. We demonstrate in this report that topo I associates with T-antigen double hexamers bound to SV40 origin DNA (TDH) but not to single hexamers. This association has the same nucleotide and DNA requirements as those for the formation of double hexamers on DNA. Interestingly, topo I prefers to bind to fully formed TDH complexes over other oligomerized forms of T antigen associated with the origin. High ratios of topo I to origin DNA destabilize TDH. The partial unwinding of a small-circular-DNA substrate is dependent on the presence of both T antigen and topo I but is inhibited at high topo I concentrations. Competition experiments with a topo I-binding fragment of T antigen indicate that an interaction between T antigen and topo I occurs during the unwinding reaction. We propose that topo I is recruited to the initiation complex after the assembly of TDH and before unwinding to facilitate DNA replication.

scholarly journals Simian virus 40 DNA replication in vitro: identification of multiple stages of initiation

1989 ◽  
Vol 9 (9) ◽  
pp. 3839-3849
Author(s):  
T Tsurimoto ◽  
M P Fairman ◽  
B Stillman
Keyword(s):  
Dna Replication ◽  
Dna Synthesis ◽  
Time Lag ◽  
Simian Virus 40 ◽  
Simian Virus ◽  
T Antigen ◽  
Nuclear Antigen ◽  
Replication Factor ◽  
Cellular Fraction ◽  
Template Dna

A cell-free DNA replication system dependent upon five purified cellular proteins, one crude cellular fraction, and the simian virus 40 (SV40)-encoded large tumor antigen (T antigen) initiated and completed replication of plasmids containing the SV40 origin sequence. DNA synthesis initiated at or near the origin sequence after a time lag of approximately 10 min and then proceeded bidirectionally from the origin to yield covalently closed, monomer daughter molecules. The time lag could be completely eliminated by a preincubation of SV40 ori DNA in the presence of T antigen, a eucaryotic single-stranded DNA-binding protein (replication factor A [RF-A]), and topoisomerases I and II. In contrast, if T antigen and the template DNA were incubated alone, the time lag was only partially decreased. Kinetic analyses of origin recognition by T antigen, origin unwinding, and DNA synthesis suggest that the time lag in replication was due to the formation of a complex between T antigen and DNA called the T complex, followed by formation of a second complex called the unwound complex. Formation of the unwound complex required RF-A. When origin unwinding was coupled to DNA replication by the addition of a partially purified cellular fraction (IIA), DNA synthesis initiated at the ori sequence, but the template DNA was not completely replicated. Complete DNA replication in this system required the proliferating-cell nuclear antigen and another cellular replication factor, RF-C, during the elongation stage. In a less fractionated system, another cellular fraction, SSI, was previously shown to be necessary for reconstitution of DNA replication. The SSI fraction was required in the less purified system to antagonize the inhibitory action of another cellular protein(s). This inhibitor specifically blocked the earliest stage of DNA replication, but not the later stages. The implications of these results for the mechanisms of initiation and elongation of DNA replication are discussed.

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