Structural state of newly replicated closed circular simian virus 40 DNA.

We have studied the early transition of newly replicated, segregated daughter molecules of simian virus 40 (SV40) into their mature, fully supercoiled state. The DNA of SV40 replicating in African green monkey kidney CV1 cells was chronically labeled with [14C]thymidine and pulse-labeled with [3H]thymidine. The cells were lysed and the viral DNA was isolated. Density gradient centrifugation of viral DNA in cesium chloride revealed that the pulse-labeled, newly synthesized, closed circular supercoiled DNA molecules banded at a slightly higher density (delta sigma = 0.0025) than the chronically labeled DNA, suggesting that the newly completed molecules were in a different structural state. Electrophoresis of DNA in agarose gels at appropriate chloroquine concentrations demonstrated that the mobility of the pulse-labeled closed, superhelical DNA was retarded relative to that of the chronically labeled DNA. These observations indicated that the newly completed SV40 DNA molecules existed in a structural state more relaxed than that of mature DNA by one or two linking numbers.     

Interaction of covalently closed circular PM-2 DNA and hedamycin.

The interaction of hedamycin with covalently closed circular PM-2 DNA was examined. Hedamycin produced strand breakage detectable in alkaline sucrose gradients. Under neutral conditions hedamycin inhibited ethidium bromide binding and induced conformational changes in PM-2 DNA.     

Specific association of simian virus 40 tumor antigen with simian virus 40 chromatin

Simian virus 40 tumor antigen (SV40 T antigen) was bound to both replicating and fully replicated SV40 chromatin extracted with a low-salt buffer from the nuclei of infected cells, and at least a part of the association was tight specific. T antigen cosedimented on sucrose gradients with SV40 chromatin, and T antigen-chromatin complexes could be precipitated from the nuclear extract specifically with anti-T serum. From 10 to 20% of viral DNA labeled to steady state with [3H]thymidine for 12 h late in infection or 40 to 50% of replicating viral DNA pulse-labeled for 5 min was associated with T antigen in such immunoprecipitates. After reaction with antibody, most of the T antigen-chromatin complex was stable to washing with 0.5 M NaCl, but only about 20% of the DNA label remained in the precipitate after washing with 0.5 M NaCl-0.4% Sarkosyl. This tightly bound class of T antigen was associated preferentially with a subfraction of pulse-labeled replicating DNA which comigrated with an SV40 form I marker. A tight binding site for T antigen was identified tentatively by removing the histones with dextran sulfate and heparin from immunoprecipitated chromatin labeled with [32P]phosphate to steady state and then digesting the DNA with restriction endonucleases HinfI and HpaII. The site was within the fragment spanning the origin of replication, 0.641 to 0.725 on the SV40 map.     

DNA replication and chromatin structure of simian virus 40 insertion mutants.

Insertion of DNA segments into the nuclease-sensitive region of simian virus 40 alters both replication efficiency and chromatin structure. Mutants containing large insertions between the simian virus 40 origin of replication (ori site) and the 21-base-pair repeated sequences replicated poorly when assayed by transfection into COS-1 cells. Replication of mutants with shorter insertions was moderately reduced. This effect was cis-acting and independent of the nucleotide sequence of the insert. The nuclease-sensitive chromatin structure was retained in these mutants, but the pattern of cleavage sites was displaced in the late direction from the ori site. New cleavage sites appeared within the inserted sequences, suggesting that information specifying the nuclease-sensitive chromatin structure is located on the late side of the inserts. Accessibility to BglI (which cleaves within the ori site) was reduced in the larger insertion mutants. These results support the conclusion that efficient function of the viral origin of replication is correlated with its proximity to an altered chromatin structure.     

Structure and formation of circular dimers of simian virus 40 DNA.

Most of the viral DNA extracted from simian virus 40 (SV40)-infected African green monkey kidney cells consists of circular molecules about 5.3 kilobases in contour length. However, about 1% of the viral DNA was found to occur as closed circular dimers that appeared to be formed, preferentially, late in infection. The monomeric units of dimers were organized in a head-to-tail, tandem arrangement; moreover, the monomeric units were not defective; i.e., they lacked deletions or other rearrangements. After infections with dimer DNA, nondefective monomers were formed. These findings suggest that dimers are not intermediates in the production of defective SV40 genomes. The majority of the dimers formed in mixed infections with two mutants were homodimers, but about 5% of the circular dimers were heterodimers and must have arisen by intermolecular recombination.     

Action of S1 nuclease on nicked circular simian virus 40 DNA.

SV40 DNA form II (FO II) containing on average more than one single strand nick per molecule was treated with S₁ nuclease. Linear duplex molecules of unit length (FO III) were generated at enzyme concentrations sufficient to achieve 95% hydrolysis of at least 100 times the amount of single-stranded DNA. Therefore, S₁ nuclease introduces under the described conditions only one double strand break per molecule despite the presence of several single strand nicks.     

Salt-stable association of simian virus 40 capsid with simian virus 40 DNA

In 8 M CsCl, a fraction of the wild-type previrions and tsB228 nucleoprotein complexes lose their core histones but retain their capsid. These histone-depleted complexes appear in the electron microscope as a protein shell attached to supercoiled DNA. Consistent with this result, we find that in 1 M NaCl, the wild-type previrions dissociate into two populations of nucleoprotein complexes. One population sediments between 50 and 140 S and morphologically resembles the shell-DNA complexes isolated in CsCl gradients. The other population is comprised primarily of nucleoproteins which sediment at 40 S.     

Replication of simian virus 40 chromatin in vitro depends on the amount of DNA polymerase alpha associated with replicating chromatin

Replication of simian virus 40 (SV40) chromatin in vitro is inhibited by chloride but stimulated by acetate anions even at physiological concentrations of 100-200 mM. In a similar fashion DNA polymerase alpha is affected with respect to the activity with activated DNA as primer template. Furthermore, at concentrations of 100-200 mM acetate DNA polymerase alpha remains associated with replicating chromatin, whereas association is strongly reduced when chloride anions are used at the corresponding concentrations. Thus the salt behaviour of DNA polymerase alpha explains the salt sensitivity of the replication system. Our results confirm the importance of this enzyme for DNA replication.     

Ultraviolet irradiation inhibits encapsidation of simian virus 40 chromatin.

During normal maturation and majority of pulse-labeled simian virus 40 DNA progresses from chromatin to previrions and virions within 5 h. UV light inhibits this progression. In heavily irradiated cultures (108 J m-2) most of the simian virus 40 DNA synthesized immediately before irradiation remains as chromatin for at least 5 h. This inhibition of maturation seems to be a result of the inhibition of protein synthesis. The data suggest that the pool of proteins required for maturation is sufficient to convert one-third of the simian virus 40 DNA molecules labeled in a 10-min pulse (at 33 h postinfection) from chromatin to previrions and virions and is exhausted within 1 h.     

Chromatin assembly. Relationship of chromatin structure to DNA sequence during simian virus 40 replication

The accessibility of five specific DNA sequences to six different single site restriction endonucleases was evaluated in replicating and mature simian virus 40 chromosomes isolated by three different methods. Electron microscopic and gel electrophoretic analysis of the DNA digestion products demonstrated that DNA accessibility in chromatin was established within 400 base pairs of replication forks and remained essentially unchanged during production of mature chromosomes and their subsequent re-entry into the replication pool. Saturating amounts of each enzyme reproducibly cut a fraction of the chromosomes, ranging from 13 to 49%. This is consistent with a nearly random phasing of chromatin structure. Examples in which all chromosomes were either cleaved or intact were never observed. Although variation in the accessibility of DNA sites near the origin of replication could be interpreted as preferred phasing in about 25% of the chromosomes, the finding that two isoschizomers, Hpa II and Msp I, did not cut chromosomes to the same extent precludes an unambiguous interpretation of the extents of cleavage of individual restriction enzymes. Since the extent of DNA cleavage observed at each restriction site was essentially indistinguishable in replicating as compared to mature chromosomes, the accessibility of DNA sequences near the origin is not obviously related to replication. Furthermore, the accessibility of DNA sites on one arm of a single replication fork was the same as the homologous sites on the other arm, consistent with a nearly random phasing of chromatin structure on both arms. This suggests that chromatin assembly occurs independently on the 2 sibling molecules of a single replicating chromosome.     

Salt-resistant association of simian virus 40 T antigen with simian virus 40 DNA in nucleoprotein complexes.

Treatment of nucleoprotein complexes (NPCs) from simian virus 40 (SV40)-infected TC7 cells with NaCl (1 or 2 M) or guanidine-hydrochloride (1 or 2 M) resulted in a significant fraction of T antigen still associated with SV40 (I) DNA. Immunoprecipitation of the salt-treated NPCs with SV40 anti-T serum indicated that T antigen is preferentially associated with SV40 (I) DNA rather than with SV40 (II) DNA. Treatment of the NPCs with 4 M guanidine-hydrochloride, however, resulted in a substantial decrease in the amount of SV40 (I) and (II) DNA associated with T antigen. As the temperature was increased to 37 degrees C during incubation of NPCs with NaCl or guanidine-hydrochloride, there was a decrease in the amount of SV40 (I) and (II) DNA immunoprecipitated with SV40 anti-T serum. In the absence of salt, temperature had no effect on the association of T antigen with the SV40 DNA in the NPCs. Treatment of NPCs from SV40 wildtype or tsA58-infected cells grown at the permissive temperature with 1 or 2 M NaCl indicated that tsA T antigen has the same sensitivities as wild-type T antigen to high salt treatment when bound to DNA in NPCs. Characterization of the proteins associated with SV40 (I) DNA after high salt treatment revealed that, in addition to T antigen, a certain amount of viral capsid proteins VP1 and VP3 remained associated with the DNA. Complexes containing SV40 (I) DNA had a sedimentation value of 53S after 1 M NaCl treatment and 43S after 2 M NaCl treatment.     

Effect of pancreatic DNAse on DNA synthesis in Bacillus subtilis]

An addition of pancreatic DNAse to the cultural medium is found to stimulate DNA synthesis and proliferation of Bacillus subtilis cells. Pancreatic DNAse induces a single-stranded disruption of Bac. subtilis DNA, which may act as a mechanism of DNA synthesis increase and of the culture growth acceleration.     

Maturation of newly replicated chromatin of simian virus 40 and its host cell

The DNA in replicating simian virus 40 chromatin and cellular chromatin was labeled with short pulses of [³H]thymidine. The structure of pulse-labeled nucleoprotein complexes was studied by micrococcal nuclease digestion. It was found that in both newly replicated viral and cellular chromatin, a structural state appears which is characterized by an increased sensitivity to nuclease and a faster than usual rate of cleavage to DNA fragments of monomeric nucleosome size and smaller. Pulse-chase experiments show that each of these effects requires a characteristic time to disappear in both systems, suggesting the existence of different sub-processes of chromatin maturation. One of these processes, detectable by the reversion of the unusually fast production of subnucleosomal fragments, is delayed in SV40 chromatin replication.