The initiation of SV40 DNA synthesis is not unique to the replication origin

Replicative intermediates of SV40 were isolated, digested with the restriction endonuclease Bgl I and examined by electron microscopy. Over 98% of the replicative intermediates isolated following infection with wild-type virions at 33 degrees, 37 degrees or 40 degrees C or with tsA209 at 33 degrees C had initiated replication about 35 nucleotides to one side of the Bgl I site. Approximately 1% of the molecules had initiated replication about 2400 nucleotides from the Bgl I site. The remaining molecules may have initiated at other sites. When tsA209 virion-infected cultures were shifted to 40.5 degrees C for 90 min, the relative rate of thymidine incorporation into superhelical viral DNA dropped by more than 97%. The remaining incorporation was not due to "leakiness." The label incorporated into mature superhelical molecules during brief pulses was not preferentially incorporated near the terminus of replication as it was at 33 degrees C. Approximately 33% of the incorporated label represented repair synthesis. Electron microscopy revealed that half of the replicative intermediates formed under these conditions appear to have been initiated randomly around the SV40 genome. Rolling circle molecules contaminated all the preparations of replicative intermediates.     

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

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.     

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.

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.     

Replicative intermediates in UV-irradiated simian virus 40

We have used Simian virus 40 (SV40) as a probe to study the replication of UV-damaged DNA in mammalian cells. Viral DNA replication in infected monkey kidney cells was synchronized by incubating a mutant of SV40 (tsA58) temperature-sensitive for the initiation of DNA synthesis at the restrictive temperature and then adding aphidicolin to temporarily inhibit DNA synthesis at the permissive temperature while permitting pre-replicative events to occur. After removal of the drug, the infected cells were irradiated at 100 J/m2 (254 nm) to produce 6-7 pyrimidine dimers per SV40 genome, and returned to the restrictive temperature to prevent reinitiation of replication from the SV40 origin. Replicative intermediates (RI) were labeled with [3H]thymidine, and isolated by centrifugation in CsCl/ethidium bromide gradients followed by BND-cellulose chromatography. The size distribution of daughter DNA strands in RI isolated shortly after irradiation was skewed towards lengths less than the interdimer spacing in parental DNA; this bias persisted for at least 1 h after irradiation, but disappeared within 3 h, by which time the size of the newly-synthesized DNA exceeded the interdimer distance. No significant excision of dimers from parental strands in either replicative intermediates or Form I (closed circular) DNA molecules was detected. These data are consistent with the hypothesis that replication forks are temporarily blocked by dimers encountered on the leading strand side of the fork, but that daughter strand continuity opposite dimers is eventually established. Evidence was obtained for the generation at late times after irradiation, of Form I molecules in which the daughter DNA strands contain dimers. Thus DNA strand exchange as well as trans-dimer synthesis may be involved in the generation of supercoiled Form I DNA from UV-damaged SV40 replicative intermediates.     

Intermediate in SV40 DNA Chain Growth

PREVIOUS studies of the DNA replication of simian virus 40 (SV40), an oncogenic member of the papoyavirus group, have been concerned with separation and characterization of replicative intermediates^1–4. Circular replicating intermediates have been identified for SV40^1–3, as well as for the similar replication system of polyoma viral DNA5,6. The replicative intermediates of SV40 DNA have been observed by electron microscopy to contain two forks, three branches and no free ends^1–3 as is the case for the circular replicating molecules of polyoma, bacteriophage λ⁷, Escherichia coli⁸ and colicin E1 in mini-cells^9,10. An important property of replicative intermediates of SV40 DNA that has also been observed in replicating molecules of colicin E1¹⁰ is that most molecules contain a superhelical region in the unreplicated portion of the molecule¹.     

In vitro initiation of DNA replication in simian virus 40 chromosomes

A soluble system has been developed that can initiate DNA replication de novo in simian virus 40 (SV40) chromatin isolated from virus-infected monkey cells as well as in circular plasmid DNA containing a functional SV40 origin of replication (ori). Initiation of DNA replication in SV40 chromatin required the soluble fraction from a high-salt nuclear extract of SV40-infected cells, a low-salt cytosol fraction, polyethylene glycol, and a buffered salts solution containing all four standard deoxyribonucleoside triphosphates. Purified SV40 large tumor antigen (T-ag) partially substituted for the high-salt nucleosol, and monoclonal antibodies directed against SV40 T-ag inhibited DNA replication. Replication began at ori and proceeded bidirectionally to generate replicating DNA intermediates in which the parental strands remained covalently closed, as observed in vivo. Partial inhibition of DNA synthesis by aphidicolin resulted in accumulation of newly initiated replicating intermediates in this system, a phenomenon not observed under conditions that supported completion of replication only. However, conditions that were optimal for initiation of replication repressed conversion of late-replicating intermediates into circular DNA monomers. Most surprising was the observation that p-n-butylphenyl-dGTP, a potent and specific inhibitor of DNA polymerase-alpha, failed to inhibit replication of SV40 chromatin under conditions that completely inhibited replication of plasmid DNA containing the SV40 ori and either purified or endogenous DNA polymerase-alpha activity. In contrast, all of these DNA synthesis activities were inhibited equally by aphidicolin. Therefore, DNA replication in mammalian cells is carried out either by DNA polymerase-alpha that bears a unique association with chromatin or by a different enzyme such as DNA polymerase-delta.     

Effects of VM26, a specific inhibitor of type II DNA topoisomerase, on SV40 chromatin replication in vitro.

We have examined the influence of VM26 (teniposide), a specific inhibitor of mammalian type II DNA topoisomerase, on the replication of SV40 minichromosomes in vitro. The replication system we used consists of replicative intermediate SV40 chromatin as substrate which is converted to mature SV40 chromatin in the presence of ATP, deoxynucleotides and a protein extract from uninfected cells. The addition of 100 microM VM26 to this system reduces DNA synthesis to 70 to 80 percent of the control and leads to an accumulation of 'late replicative intermediates'. The VM26 induced block of replication was not released by the addition of large quantities of type I DNA topoisomerase. We conclude, that type II DNA topoisomerase is essential for the final replication steps leading from late Cairns structures of replicative intermediates to monomeric minichromosomes. It appears that type I DNA topoisomerase can function as a swivelase during most of the replicative elongation phase, but must later be replaced by type II DNA topoisomerase.     

Visualization of novel simian virus 40 DNA recombination intermediates induced by ultraviolet light irradiation.

Electron microscopic technique was used to examine the structures of SV40 DNA recombination intermediates induced by ultraviolet irradiation as an approach for understanding recombination mechanisms in animal cells. Putative recombination intermediate with the characteristic Holliday junction was observed in both SV40 and CV-1 monkey kidney cell DNA. These results suggest that Holliday recombination intermediate is a common intermediate in eukaryotic as well as prokaryotic recombination pathways. In UV irradiated cells, putative SV40 DNA recombination intermediates with multiple recombining partners were observed. In addition, UV irradiation induced two types of novel joint molecules of SV40 DNA. The first type contains replication intermediates as one of the joint molecules with the putative recombination junction located in the newly replicated DNA arms. The second type of novel joint molecules is represented by of the 'dumbbell' structures with two circular SV40 DNA linked by a linear DNA of varying lengths. The structures of these novel recombination intermediates suggest a strand-invasion mechanism for UV-induced DNA recombination.     

Effects of VM26 (teniposide), a specific inhibitor of type II DNA topoisomerase, on SV40 DNA replication in vivo.

Simian Virus 40 (SV40) infected cells were pulse labeled with (3H) thymidine and chased either in the absence or in the presence of the cytotoxic drug VM26 (teniposide). We investigated the structure of labeled SV40 DNA and found that VM26 had no significant effect on replicative chain elongation but strongly inhibited the conversion of late replication intermediates to mature DNA daughter molecules. The late replicative SV40 DNA intermediates which accumulate in VM26 treated cells contained essentially full length labeled DNA strands. These newly synthesized strands were not part of two catenated interlocked SV40 monomers suggesting that the block occurred prior to the final ligation reaction. Since VM26 is known to be a specific inhibitor of DNA topoisomerase II we conclude that this enzyme is dispensable for the chain elongation of replicating SV40 DNA, but that it is essential for the termination of SV40 DNA replication cycles.     

Arrest of segregation leads to accumulation of highly intertwined catenated dimers: dissection of the final stages of SV40 DNA replication

When SV40-infected cells are placed into hypertonic medium, newly synthesized DNA accumulates as form C catenated dimers. These molecules consist of two supercoiled monomer circles of SV40 DNA interlocked by one or more topological inter-twinings and are seen as transiently labeled inter-mediates during normal replication. Form C catenated dimers represent pure segregation intermediates, replicative DNA structures in which DNA synthesis is complete but which still require topological separation of the two daughter circles. Hypertonic shock seems to block selectively a type II topoisomerase activity involved in disentangling the two circles. This is reflected in the fact that form C catenated dimers that accumulate during the block are highly intertwined with catenation linkage numbers up to C(L) = 20. While initiation of replication is also inhibited by hypertonic treatment, ongoing SV40 DNA synthesis is not affected, and replication is free to proceed from the earliest cairns structure through to form C catenated dimers. The block to segregation is rapidly and completely released by shifting the cells back to normal medium. A much slower recovery of DNA segregation takes place on prolonged incubation in hypertonic medium, perhaps because of some cellular homeostatic mechanism. The results of this work lead to a detailed view of the final stages of SV40 DNA replication.     

Topoisomerase I is preferentially associated with isolated replicating simian virus 40 molecules after treatment of infected cells with camptothecin.

Detergent extraction of simian virus 40 (SV40) DNA from infected monkey CV-1 cells, after a brief exposure to the drug camptothecin, yields covalent complexes between topoisomerase I and DNA that band with reduced buoyant densities in CsCl. The following lines of evidence indicate that the enzyme is preferentially associated with SV40 replicative intermediates. First, the percentage of the isolated labeled viral DNA that exhibited a reduced buoyant density is inversely proportional to the length of the labeling period and approximately parallels the percentage of replicative intermediates for each labeling time (5 to 60 min). Second, after labeling for 60 min, the isolated low-density material was found to be enriched for replicative intermediates as measured by sedimentation in neutral sucrose. Third, analysis of extracted viral DNA by equilibrium centrifugation in CsCl-propidium diiodide gradients that separate replicating molecules from completed form I DNA revealed that camptothecin pretreatment specifically caused the linkage of topoisomerase I to replicating molecules. In addition, analysis of the low-density material obtained under conditions when only the newly synthesized strands of the replicative intermediates were labeled showed that the enzyme was associated almost exclusively with the parental strands. Taken together, these observations indicate that topoisomerase I is involved in DNA replication, and they are consistent with the hypothesis that the enzyme provides swivels to allow the helix to unwind. The observed bias in the distribution of topoisomerase I on intracellular SV40 DNA could be the result of rapid encapsidation of replicated molecules that precludes the association of topoisomerase I with the DNA or, alternatively, the result of a specific association of the enzyme with replicative intermediates.     

Synthesis of Superhelical Simian Virus 40 Deoxyribonucleic Acid in Cell Lysates*.

In vivo-labeled SV40 replicating DNA molecules can be converted into covalently closed superhelical SV40 DNA (SV40(I) using a lysate of sv40-infected monkey cells containing intact nuclei. Replication in vitro occurred at one-third the in vivo rate for 30 min at 30 degrees. After 1 hour of incubation, about 54% of the replicating molecules had been converted to SV40(I), 5% to nicked, circular molecules (SV40(II), 5% to covalently closed dimers; the remainder failed to complete replication although 75% of the prelabeled daughter strands had been elongated to one-genome length. Density labeling in vitro showed that all replicating molecules had participated during DNA synthesis in vitro. Velocity and equilibrium sedimentation analysis of pulse-chased and labeled DNA using radioactive and density labels suggested that SV40 DNA synthesis in vitro was a continuation of normal ongoing DNA synthesis. Initiation of new rounds of SV40 DNA replication was not detectable.     

Induction of Duplication Reversion in Human Fibroblasts, by Wild-Type and Mutated Sv40 T Antigen, Covaries with the Ability to Induce Host DNA Synthesis

Intrachromosomal homologous recombination, manifest as reversion of a 14-kbp duplication in the hypoxanthine phosphoribosyl transferase (HPRT) gene, is elevated in human cells either stably transformed or transiently transfected by the SV40 (simian virus 40) large T antigen gene. Following introduction of wild-type SV40, or any of several T-antigen point mutations in a constant SV40 background, we observed a strong correlation between the stimulation of chromosomal recombination and induction of host-cell DNA synthesis. Moreover, inhibitors of DNA replication (aphidicolin and hydroxyurea) suppress SV40-induced homologous recombination to the extent that they suppress DNA synthesis. Stable integration of plasmids encoding T antigen also augments homologous recombination, which is suppressed by aphidicolin. We infer that the mechanism by which T antigen stimulates homologous recombination in human fibroblasts involves DNA replicative synthesis.     

Infectious linear DNA sequences replicating in simian virus 40-infected cells.

A new class of linear duplex DNA structures that contain simian virus 40 (SV40) DNA sequences and that are replicated during productive infection of cells with SV40 is described. These structures comprise up to 35% of the radioactively labeled DNA molecules that can be isolated by selective extraction. These molecules represent a unique size class corresponding to the length of an open SV40 DNA molecule (FO III), and they contain a heterogeneous population of DNA sequences either of host or of viral origin, as shown by restriction endonuclease analysis and nucleic acid hybridization. Part of the FO III DNA molecules contain viral-host DNA sequences covalently linked with each other. They start to replicate with the onset of SV40 superhelix replication 1 day after infection. Their rate of synthesis is most pronounced 3 days after infection when superhelix replication is already declining. Furthermore, they cannot be chased into other structures. At least a fraction of these molecules is infectious when administered together with DEAE-dextran to permissive cells. After intracellular circularization, superhelical DNA FO I with an aberrant cleavage pattern accumulates. In addition, tumor and viral capsid antigen are induced, and infectious viral progeny is obtained. Infection of cells with purified SV40 FO I DNA does not result in FO III DNA molecules in the infected cells or in the viral progeny. It is suggested, therefore, that these FO III DNA molecules are perpetuated within SV40 virus pools by encapsidation into pseudovirions.     

Replication of the origin region of simian virus 40 DNA in permeabilized monkey cells

Simian virus 40 (SV40) DNA replication was studied in monolayers of infected monkey CV-1 cells, permeabilized with lysolecithin, by incubation with [alpha-32P]dTTP, the other dNTPs and rNTPs and an ATP-regenerating system. Analysis of the labeled SV40 DNA by sedimentation in alkaline sucrose gradients showed that about 30% of the material synthesized by the permeable cells in the course of 60 min consisted of covalently closed circular SV40 DNA (form I), with the remainder sedimenting as relaxed circles (form II) and replicative intermediates between 18 S and 4 S. The synthesis of SV40 DNA in the permeabilized cell system required the presence of all four dNTPs and was completely inhibited by aphidicolin, consistent with the involvement of DNA polymerase alpha. A detailed analysis of the distribution of radioactivity in the DNA synthesized involved cleavage with BstNI restriction endonuclease, followed by polyacrylamide gel electrophoresis and radioautography. The extent of labeling of all restriction fragments was nearly proportional to their length, suggesting that the entire SV40 chromosome was being replicated. This was confirmed by the careful comparison of the rate of labeling of a DNA fragment which includes the replication origin, and a fragment which includes the replication terminus. Their labeling was proportional to their size, regardless of the time for which the labeling was carried out. This demonstrated that the replication of the entire SV40 chromosome occurred in a steady state and that the start and termination of replication continuously occurred throughout the labeling period. The availability of an in vitro system in which replication of SV40 DNA undergoes multiple replication cycles should be of considerable value in the analysis of the mechanism of replication of this viral genome.     

Distribution of Replicating Simian Virus 40 DNA in Intact Cells and Its Maturation in Isolated Nuclei

The maturation of replicating simian virus 40 (SV40) chromosomes into superhelical viral DNA monomers [SV40(I) DNA] was analyzed in both intact cells and isolated nuclei to investigate further the role of soluble cytosol factors in subcellular systems. Replicating intermediates [SV40(RI) DNA] were purified to avoid contamination by molecules broken at their replication forks, and the distribution of SV40(RI) DNA as a function of its extent of replication was analyzed by gel electrophoresis and electron microscopy. With virus-infected CV-1 cells, SV40(RI) DNA accumulated only when replication was 85 to 95% completed. These molecules [SV40(RI^*) DNA] were two to three times more prevalent than an equivalent sample of early replicating DNA, consistent with a rate-limiting step in the separation of sibling chromosomes. Nuclei isolated from infected cells permitted normal maturation of SV40(RI) DNA into SV40(I) DNA when the preparation was supplemented with cytosol. However, in the absence of cytosol, the extent of DNA synthesis was diminished three- to fivefold (regardless of the addition of ribonucleotide triphosphates), with little change in the rate of synthesis during the first minute; also, the joining of Okazaki fragments to long nascent DNA was inhibited, and SV40(I) DNA was not formed. The fraction of short-nascent DNA chains that may have resulted from dUTP incorporation was insignificant in nuclei with or without cytosol. Pulse-chase experiments revealed that joining, but not initiation, of Okazaki fragments required cytosol. Cessation of DNA synthesis in nuclei without cytosol could be explained by an increased probability for cleavage of replication forks. These broken molecules masqueraded during gel electrophoresis of replicating DNA as a peak of 80% completed SV40(RI) DNA. Failure to convert SV40(RI^*) DNA into SV40(I) DNA under these conditions could be explained by the requirement for cytosol to complete the gap-filling step in Okazaki fragment metabolism: circular monomers with their nascent DNA strands interrupted in the termination region [SV40(II^*) DNA] accumulated with unjoined Okazaki fragments. Thus, separation of sibling chromosomes still occurred, but gaps remained in the terminal portions of their daughter DNA strands. These and other data support a central role for SV40(RI^*) and SV40(II^*) DNAs in the completion of viral DNA replication.     

Binding of anti-Z-DNA antibodies to negatively supercoiled SV40 DNA.

The binding of anti-Z-DNA antibody preparations to negatively supercoiled, protein-free SC40 DNA was analyzed. Covalent cross-linking with 0.1% glutaraldehyde followed by DNA restriction endonucleolytic fragmentation and nitrocellulose filtration allowed accurate mapping of antibody binding sites. The critical superhelical density necessary to allow antibody binding was -sigma = 0.056. The major region of antibody-DNA interaction was found within an SV40 segment spanning viral map positions 40 to 474. This region coincides with the nucleosome free region in SV40 minichromosomes and harbours the early and late promoter regions including the SV40 enhancer segment. Although it is unknown whether alternative, non-B-DNA conformations are generated in vivo within SV40 minichromosomes our results emphasize the high degree of DNA structural flexibility that can be realized under negative torsional stress.     

Structure of Replicating Simian Virus 40 Deoxyribonucleic Acid Molecules

Properties of replicating simian virus 40 (SV40) deoxyribonucleic acid (DNA) have been examined by sedimentation analysis and by direct observation during a lytic cycle of infection of African green monkey kidney cells. Two types of replicating DNA molecules were observed in the electron microscope. One was an open structure containing two branch points, three branches, and no free ends whose length measurements were consistent with those expected for replicating SV40 DNA molecules. A second species had the same features as the open structure, but in addition it contained a superhelix in the unreplicated portion of the molecule. Eighty to ninety per cent of the replicative intermediates (RI) were in this latter configuration, and length measurements of these molecules also were consistent with replicating SV40 DNA. Replicating DNA molecules with this configuration have not been described previously. RI, when examined in ethidium bromide-cesium chloride (EB-CsCl) isopycnic gradients, banded in a heterogeneous manner. A fraction of the RI banded at the same density as circular SV40 DNA containing one or more single-strand nicks (component II). The remaining radioactive RI banded at densities higher than that of component II, and material was present at all densities between that of supercoiled double-stranded DNA (component I) and component II. When RI that banded at different densities in EB-CsCl were examined in alkaline gradients, cosedimentation of parental DNA and newly replicated DNA did not occur. All newly replicated DNA sedimented more slowly than did intact single-stranded SV40 DNA, a finding that is inconsistent with the rolling circle model of DNA replication. An inverse correlation exists between the extent of replication of the SV40 DNA and the banding density in EB-CsCl. Under alkaline conditions, the parental DNA strands that were contained in the RI sedimented as covalently closed structures. The sedimentation rates in alkali of the covalently closed parental DNA decreased as replication progressed. Based on these observations, some possible models for replication of SV40 DNA are proposed.     

Dideoxynucleoside triphosphates inhibit a late stage of SV40 DNA replicationin vitro

Summary The role of DNA polymerases in the replication of SV40 DNA was studied using a T-antigen-dependent assay supplemented with a human KB cell extract. Inhibition of DNA polymerase α by addition of aphidicolin or monoclonal antibodies prevented DNA synthesis, confirming the requirement for this enzyme in replication. The replication process was unaffected by ddTTP at a concentration (5 μM) inhibitory to DNA polymerases β and γ, however, higher concentrations of ddTTP (200 μM) caused an apparent accumulation of relaxed circular plasmid with a concomitant decrease in DNA synthesis. An analysis of this replication intermediate indicated that it was formed during the replication reaction and that the replicative cycle was nearly complete. A kinetic study of ddTTP inhibition strongly suggested DNA polymerase ε (PCNA-independent DNA polymerase δ) was the target of the inhibitor and that this enzyme functions during the final stages of DNA replication.     

Identification of the replicative intermediates in SV40 DNA replication in vitro.

The soluble replication system is which the exogenously added simian virus 40 (SV40) DNA can be replicated semiconservatively in vitro, has been developed (Ariga and Sugano, J.Virol. 48, 481, 1983). This paper further characterized the in vitro products synthesized on the cloned DNA containing the origin of SV40 DNA replication. The time course and pluse-chase experiments showed that the in vitro products were converted from the open circle to closed circles having the various superhelical densities, and finally to the twisted formI DNA seen in vivo by the analysis of agarose gel electrophoresis, alkaline sucrose gradient centrifugation, and density-transfer in isopycnic centrifugation. The replicative intermediates isolated after the short term incubation had replicated strands of the size smaller than the full length, most of which correspond to that of the putative Okazaki fragment. These and the previous results indicate that this in vitro system should be useful to investigate the molecular mechanism of SV40 DNA replication.