Synthesis of simian virus 40 DNA in isolated nuclei

The presence or absence of calcium ions during the isolation of nuclei from SV40-infected African green monkey kidney cells significantly affects the size of SV40 DNA synthesized $\text{in vitro}$. When Ca⁺⁺ is present during the nuclear isolation procedure, the 3–7S fragments of SV40 DNA synthesized $\text{in vitro}$ mature into long chains; in the absence of Ca⁺⁺ they do not.     

Self-annealing of 4 S strands from replicating simian virus 40 DNA

The nascent short strands (4 S) isolated from replicating Simian, virus 40 DNA hybridize specifically with denatured SV40 DNA and self-anneal extensively (70 to 92%) when incubated at 68 °C in 1 m-NaCl. Since complementary genetic sequences are present in the 4 S strands, both growing chains of SV40 DNA appear to be synthesized discontinuously at each replication fork.     

Superhelix density of replicating simian virus 40 DNA molecules

Simian virus 40 replicating DNA molecules were isolated and fractionated according to the extent of replication by isopynic centrifugation in ethidium bromide-CsCl. Electron microscopic examination of the replicating molecules in the presence of ethidium bromide revealed that the sense of the superhelix in replicating molecules is the same as that of simian virus 40 DNA I. Replicating DNA molecules of differing extents of replication were also analyzed by sedimentation in varying concentrations of ethidium bromide. It was observed that the superhelix density of the unreplicated portion of replicating molecules was greater than that of DNA I and that it increased as the degree of replication increased. In contrast with the increase in superhelix density that was related to the extent of replication, all replicating molecules contained a rather constant number (2 to 5) of additional superhelical turns per molecule, irrespective of the extent of replication. This suggests that a region (or regions) of about 20 to 50 nucleotides may exist in a denatured state in replicating molecules, presumably at the replicating forks of the molecule.     

Maturation of replicating simian virus 40 DNA molecules in isolated nuclei by continued bidirectional replication to the normal termination region.

Mature SV40 DNA synthesized for different periods of time either in isolated nuclei or in intact cells was highly purified and then digested with restriction endonucleases in order to relate the time of synthesis of newly replicated viral DNA to its location in the genome. Replication in nuclei supplemented with a cytosol fraction from uninfected cells was a faithful continuation of the bidirectional process observed in intact cells, but did not exhibit significant initiation of new replicons. SV40 DNA replication in cells at 37 degrees C proceeded at about 145 nucleotides/min per replication fork. In the absence of cytosol, when DNA synthesis was limited and joining of Okazaki fragments was retarded, bidirectional SV40 DNA replication continued into the normal region where separation yeilded circular duplex DNA molecules containing one or more interruptions in the nascent DNA strands. In the presence of cytosol, this type of viral DNA was shown to be a precursor of covalently closed, superhelical SV40 DNA, the mature from of viral DNA.     

DNA synthesis by partially purified replicating simian virus 40 chromosomes.

We have partially purified replicating simian virus 40 (SV40) chromosomes in a form which allows continued DNA synthesis in vitro. We first prepare a soluble DNA-synthesizing system from SV40-infected monkey cells and then sediment the components through a neutral sucrose gradient of extremely low ionic strength. Replicating SV40 chromosomes isolated from such gradients are capable of continuing DNA synthesis in vitro in the same manner as two crude subnuclear systems we have previously described (4). This indicates that the enzymes and other proteins required for in vitro DNA synthesis are bound to the replicating chromosomes.     

Association of simian virus 40 T antigen with replicating nucleoprotein complexes of simian virus 40.

An immunoprecipitation assay was established for simian virus 40 T-antigen-bound nucleoprotein complexes by means of precipitation with sera from hamsters bearing simian virus 40-induced tumors. About 80% of simian virus 40 replicating nucleoprotein complexes in various stages of replication were immunoprecipitated. In contrast, less than 21% of mature nucleoprotein complexes were immunoprecipitated. Pulse-chase experiments showed that T antigen was lost from most of the nucleoprotein complexes concurrently with completion of DNA replication. T antigen induced by dl-940, a mutant with a deletion in the region coding for small T antigen, was also associated with most of the replicating nucleoprotein complexes. Once bound with replicating nucleoprotein complexes at the permissive temperature, thermolabile T antigen induced by tsA900 remained associated with the complexes during elongation of the replicating DNA chain at the restrictive temperature. These results suggest that simian virus 40 T antigen (probably large T antigen) associates with nucleoprotein complexes at or before initiation of DNA replication and that the majority of the T antigen dissociates from the nucleoprotein complexes simultaneously with completion of DNA replication.     

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.     

In vitro DNA synthesis by an alpha-like DNA polymerase bound to replicating simian virus 40 chromosomes.

Simian virus 40 chromosomes carry out replicative DNA synthesis in vitro which is sensitive to aphidicolin and to N-ethylmaleimide, resistant to 2',3'-dideoxythymidine-5'-triphosphate, and proportional to the amount of chromosome-associated alpha-like polymerase. Thus, an alpha-like DNA polymerase (alpha polymerase or delta polymerase) is responsible for in vitro DNA synthesis.     

DNA binding properties of simian virus 40 T-antigens synthesized in vivo and in vitro.

Simian virus 40 large T- and small t-antigens have been shown previously to share immunological determinants and common sequences and to have roles in virus-induced cell transformation. However, only large T-antigen is a DNA binding protein. Under all conditions tested, small t-antigen did not interact with DNA. Large T-antigen synthesized in infected cells bound to both native calf thymus and simian virus 40 DNAs. As its binding efficiency was less than 100%, it is likely that there are different forms of T-antigen which vary in their affinity for DNA. Large T-antigen synthesized in cell-free protein-synthesizing systems primed by simian virus 40 mRNA also bound to DNA-cellulose, whereas small t-antigen similarly synthesized in vitro did not. An 82,000-molecular-weight T-antigen polypeptide synthesized in cell-free protein-synthesizing systems primed by simian virus 40 complementary RNA transcribed in vitro from simian virus 40 DNA by Escherichia coli RNA polymerase bound efficiently to simian virus 40 DNA. As this product did not share sequences with the small t-antigen, it can be concluded that the amino-terminal portion of the T-antigen is not required for some of its specific DNA binding properties.     

Swiveling and decatenation of replicating simian virus 40 genomes in vivo.

We have found that type II topoisomerase inhibitors have two effects on replicating simian virus 40 genomes in vivo: production of catenated dimers and slowed replication of the last 5% of the genome. This suggests that type II topoisomerase simultaneously decatenates and facilitates replication fork movement at this stage of DNA replication. On the basis of this observation, a detailed model is proposed for the roles of topoisomerases I and II in simian virus 40 DNA replication.     

Symmetric transcription of simian virus 40 DNA in the nuclei of transformed mouse cells.

Na⁺ channels from lobster nerve membranes stored frozen in sucrose were incorporated into artificial liposomes. Crude soybean phospholipids or mixtures of purified phospholipids were suitable for reconstitution provided the latter included phosphatidylserine or another acidic phospholipid. The ²²Na flux into the reconstituted vesicles was increased (2 to 3-fold) by veratridine (0.25 – 1 mM) or grayanotoxin I (50 –150 μM) and the increment was abolished by 10 nM tetrodotoxin (K_i = 2 nM). The reconstituted vesicles were inactivated after incubation for 15 min at 40° and exposure to 20 μM dicyclohexylcardobiimide inhibited by 80% the response to the drugs.     

Analysis of simian virus 40 chromosome-T-antigen complexes: T-antigen is preferentially associated with early replicating DNA intermediates

The fraction and DNA composition of simian virus 40 chromosomes that were complexed with large T-antigens (T-Ag) were determined at the peak of viral DNA replication. Simian virus 40 chromatin containing radiolabeled DNA was extracted by the hypotonic method of Su and DePamphilis (Proc. Natl. Acad. Sci. U.S.A. 73:3466-3470, 1976) and then fractionated by sucrose gradient sedimentation into replicating (90S) and mature (70S) chromosomes. Viral chromosomes containing T-Ag were isolated by immunoprecipitation with saturating amounts of either an anti-T-Ag monoclonal antibody or an anti—T-Ag hamster serum under conditions that specifically precipitated T-Ag protein from cytosol extracts. An average of 10% of the uniformly labeled DNA in the 90S pool and 7.5% in the 70S pool was specifically precipitated, demonstrating that under these conditions immunologically reactive T-Ag was tightly bound to only 8% of the total viral chromosomes. In contrast, simian virus 40 replicating intermediates (RI) represented only 1.2% of the viral DNA, but most of these molecules were associated with T-Ag. At the shortest pulse-labeling periods, an average of 72 ± 18% of the radiolabeled DNA in 90S chromosomes could be immunoprecipitated, and this value rapidly decreased as the labeling period was increased. Electron microscopic analysis of the DNA before and after precipitation revealed that about 55% of the 90S chromosomal RI and 72% of the total RI from both pools were specifically bound to T-Ag. Comparison of the extent of replication with the fraction of RI precipitated revealed a strong selection for early replicating DNA intermediates. Essentially all of the RI in the 70S chromosomes were less than 30% replicated and were precipitated with anti—T-Ag monoclonal antibody or hamster antiserum. An average of 88% of the 90S chromosomal RI which were from 5 to 75% replicated were immunoprecipitated, but the proportion of RI associated with T-Ag rapidly decreased as replication proceeded beyond 70% completion. By the time sibling chromosomes had separated, only 3% of the newly replicated catenated dimers in the 90S pool (<1% of the dimers in both pools) were associated with T-Ag. Measurements of the fraction of radiolabeled DNA in each quarter of the genome confirmed that T-Ag was preferentially associated with newly initiated molecules in which the nascent DNA was nearest the origin of replication. These results are consistent with a specific requirement for the binding of T-Ag to viral chromosomes to initiate DNA replication, and they also demonstrate that T-Ag does not immediately dissociate from chromosomes once replication begins. The biphasic relationship between the fraction of T-Ag—containing RI and the extent of DNA replication suggests either that 1 or 2 molecules of T-Ag remain stably bound until replication is about 70% completed or that 4 to 6 molecules of T-Ag are randomly released from each RI at a uniform rate throughout replication.     

In situ hybridization of repetitive monkey genome sequences isolated from defective simian virus 40 DNA.

The origin of a repetitive monkey DNA sequence that is incorporated into a defective simian virus 40 genome has been studied. A fragment (about 140 base pairs in length) containing essentially all the repetitive monkey DNA present in the defective and few, if any, SV40 sequences can be cleaved from the purified defective DNA by restriction endonucleases Hind(II and III). Radioactive cRNA prepared with the isolated fragment as template was hybridized in situ to African green monkey chromosomes. The results indicate that all or part of the sequence in question occurs at both centromeric and noncentromeric positions in many, but not all, chromosomes. Of the typical 60 chromosomes, between nine and eleven hybridize with the cRNA in noncentromeric regions.