Polyoma virus minichromosomes: associated enzyme activities.

Polyoma minichromosomes were isolated and fractionated on glycerol gradients as described by Gourlie et al. (J. Virol. 38:805-814, 1981). Specific assays for DNa polymerases alpha, beta, and gamma, DNA topoisomerase I, and RNase H were carried out on each fraction. The number of units of activity in each fraction was compared with the number of total polyoma and replicative intermediate DNA molecules in each fraction determined by quantitative electron microscopy (M. R. Krauss and R. M. Benbow, J. Virol. 38:815-825, 1981). DNA polymerase alpha cosedimented with polyoma replicative intermediate DNA molecules. DNA polymerase beta and DNA topoisomerase I activities sedimented with mature polyoma minichromosomes. Although the bulk of RNase H activity sedimented in the minichromosome region, the peak of activity was found one fraction behind the peak of mature minichromosomes. Virtually no DNA polymerase gamma activity cosedimented with polyoma minichromosomes.     

Hydroxyurea-Induced Accumulation of Short Fragments During Polyoma DNA Replication II. Behavior During Incubation of Isolated Nuclei

The synthesis of polyoma DNA was studied in isolated nuclei from hydroxyurea-inhibited 3T6 cells infected with polyoma virus. During incubation of nuclei under conditions suitable for polyoma DNA synthesis in vitro, the short DNA fragments with a sedimentation coefficient of 4S formed in vivo (hydroxyurea fragments) became associated with preformed, replicating DNA strands. Centrifugation in dye-buoyant density gradients showed that the fragments formed part of the structure of the replicative intermediate of polyoma DNA. The proportion of "young" replicative intermediates was larger after hydroxyurea inhibition than in uninhibited controls. Hydroxyurea fragments appear to be closely related to the 4S fragments formed as normal intermediates during discontinuous synthesis of polyoma DNA.     

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.     

Immunoprecipitation of SV40 replicating minichromosomes complexed with bacteriophage T4 gene 32 protein.

Simian Virus 40 (SV40) DNA replication is a useful model to study eukaryotic cell DNA replication because it encodes only one replication protein and its genome has a nucleoprotein structure ('minichromosome') indistinguishable from cellular chromatin. Late after infection SV40 replicating DNA molecules represent about 5% of total viral minichromosomes. Since gene 32 protein (P32) from bacteriophage T4 interacts with single-stranded DNA and SV40 replication complexes are expected to contain single-stranded regions at the replication forks, we asked whether P32 might be used to isolate replicating SV40 minichromosomes. When nuclear extracts from SV40 infected cells were treated sequentially with P32 and anti-P32 antibodies, pulse-labeled minichromosomes were selectively immunoprecipitated. Agarose gel electrophoresis analysis confirmed that immunoprecipitated material corresponded to SV40 replicative intermediates. Protein analysis of the pelleted material revealed several proteins of viral and cellular origin. Among them, T antigen and histones were found to be complexed with at least other three proteins from cellular origin, to the replicative complexes. Additionally, anti-P32 antibodies were able to detect three cellular proteins of approximately 70, 32 and 13 kDa in western blots. These proteins could correspond to those found as part of an eukaryotic multisubunit single-stranded DNA binding protein. The use of P32 and anti-P32 antibodies thus allows the separation of replicating from mature SV40 minichromosomes and can constitute a novel method to enrich and to study replicative active chromatin.     

Polyomavirus minichromosomes: associated DNA topoisomerase II and DNA ligase activities.

Polyomavirus minichromosomes were isolated and fractionated as described previously (B. B. Gourlie, M. R. Krauss, A. J. Buckler-White, R. M. Benbow, and V. Pigiet, J. Virol. 38:805-814, 1981). Specific assays for DNA topoisomerase II and DNA ligase activity were carried out on each fraction. The enzymatic activity in each fraction was determined by quantitative electron microscopy and compared with the number of replicative intermediate and total polyomavirus DNA molecules in each fraction. DNA topoisomerase II activity cosedimented with polyomavirus replicative intermediate minichromosomes. DNA ligase activity cosedimented with mature polyomavirus minichromosomes.     

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.     

Lack of repair of ultraviolet light damage in Mycoplasma gallisepticum

Molecules with single-stranded tails (rolling circles) were isolated as replicating intermediates in G4 progeny single-stranded DNA synthesis. Lysates from infected cells harvested late in infection during single-stranded DNA synthesis were not deproteinised but analysed directly in caesium chloride and propidium diiodide gradients. The gradient fractionated them on the basis of tail length. If the lysates were first deproteinised however, the tailed replicative intermediates banded as a peak at a density just greater than that of replicative form II DNA (RFII) and did not spread down the gradient. The origin of synthesis of the viral strand tail was mapped by electron microscopy as 55 to 60% away from the single EcoRI cleavage site. Termination molecules finishing a round of viral strand DNA synthesis have been identified as molecules consisting of a closed single-stranded DNA circle attached by a very small region to the parent double-stranded DNA circle.     

Structure of replicating DNA molecules of Bacillus subtilis bacteriophage phi 29.

We isolated phi 29 DNA replicative intermediates from extracts of phage-infected Bacillus subtilis, pulsed-labeled with [3H]thymidine, by velocity sedimentation in neutral sucrose followed by CsCl equilibrium density gradient centrifugation. During a chase, the DNA with a higher sedimentation coefficient in neutral sucrose and a lower sedimentation rate in alkaline sucrose than that of viral phi 29 DNA was converted into mature DNA. The material with a density higher than that of mature phi 29 DNA consisted of replicative intermediates, as analyzed with an electron microscope. We found two major types of molecules. One consisted of unit-length duplex DNA with one single-stranded branch at a random position. The length of the single-stranded branches was similar to that of one of the double-stranded regions. The other type of molecules was unit-length DNA with one double-stranded region and one single-stranded region extending a variable distance from one end. Partial denaturation of the latter molecules showed that replication was initiated with a similar frequency from either DNA end. These findings suggest that phi 29 DNA replication occurs by a mechanism of strand displacement and that replication starts non-simultaneously from either DNA end, as in the case of adenovirus.     

Effects of 2''-deoxy-2''-azidocytidine on polyoma virus DNA replication: evidence for rolling circle-type mechanism.

Rolling circle-type molecules were found in polyoma virus-infected cells after inhibition of DNA synthesis with 2'-deoxy-2'-azidocytidine. The circular DNA molecules were always relaxed and of polyoma length. Most of the attached tails were less than two times the length of the polyoma genome, but tails with a length of up to 4.75 times the genome were also found. After cleavage of the total pool of replicating molecules with either endo R.EcoRI or endo R.BamI, Y-shaped molecules with replicated portions of various lengths were generated from rolling circle-type molecules. Moreover, after cleavage, Y-shaped molecules with three unequal arms were found, which could be explained as derived from the tail in rolling circle-type molecules starting from the normal origin, i.e., 29% from the endo R.EcoRI cleavage site. Rolling circle-type molecules were also found during a normal, noninhibited infection cycle. In such cells, a relatively higher frequency of rolling circle-type molecules was observed late during infection. Compared with control cultures, cultures inhibited with 2'-deoxy-2'-azidocytidine showed a greater amount of rolling circle-type molecules relative to normal replicative intermediates. 2'-Deoxy-2'-azidocytidine has previously been shown to inhibit the initiation of new rounds of replication; thus, the result obtained here indicates that a rolling circle-type mechanism is independent of the reinitiation of DNA synthesis.     

Role of amino-terminal histone domains in chromatin replication.

Simian virus 40 minichromosomes were treated with trypsin to specifically remove the amino-terminal histone domains (tails). Trypsin treatment does not affect the spacing and the number of nucleosomes on minichromosomes but indices a more extended conformation, as shown by the reduced sedimentation coefficient of trypsinized minichromosomes compared with the untreated controls. Trypsinized minichromosomes replicate more efficiently than control minichromosomes in in vitro replication assays. The increased template efficiency appears to be due to higher rates of replicative fork movement. In vitro replication in the presence of protein-free competitor DNA shows that replicating trypsinized minichromosomes do not lose nucleosomes and replicating competitor DNA does not gain nucleosomes. This finding suggests that tailless nucleosomes are transferred from the unreplicated prefork stem to replicated DNA branches and excludes a participation of the basic histone domains in nucleosome transfer.     

Polyoma virus minichromosomes: a soluble in vitro replication system.

Polyoma virus minichromosomes were isolated from infected 3T6 cells by hypotonic extraction of isolated nuclei. The kinetics of in vitro DNA synthesis in the nuclear extract was similar to that observed with intact nuclei. The majority of the products of in vitro DNA synthesis sedimented with replicative intermediate (RI) minichromosomes and migrated as two bands (RI-a and RI-b) on 1.4% agarose gels. The kinetics of deoxynucleotide monophosphate incorporation into these species was consistent with the existence of several rate-limiting steps in in vitro replication by polyoma minichromosomes. Electron microscope analysis showed that the RI-a band consisted almost entirely of RI theta structures ranging from 46 to 87% replicated, with one-half of all theta structures 67 +/- 4% replicated. The RI-b material was more complex, consisting of sigma and alpha structures with tails ranging from 7 to 114% of polyoma genome length and, less frequently, of linked and multiple linked dimeric structures.     

Polyoma virus minichromosomes: characterization of the products of in vitro DNA synthesis.

A two-dimensional (neutral-alkali) agarose gel electrophoretic system was used to separate three families of replicative intermediate (RI) polyoma virus DNA molecules (form I, form II, and form III RIs). Two of these families, form II and III RIs, are the result of artifactual nicking of one of the parental strands of supercoiled RIs (form I RIs) during in vitro replication of soluble minichromosomes. Kinetic studies in vitro showed that the nicked RIs serve as templates for limited DNA synthesis. The nicked species are not converted into normal products, however. The nicking reaction, which appears to be specific for the parental strands, is dependent on magnesium ions and occurs concurrently with the in vitro synthesis of DNA.     

Resolution of linear minichromosomes with hairpin ends from circular plasmids containing vaccinia virus concatemer junctions

The junctions, separating unit-length genomes in intracellular concatemeric forms of vaccinia virus DNA, are duplex copies of the hairpin loops that form the ends of mature DNA molecules present in infectious virus particles. Circular E. coli plasmids with palindromic junction fragments were replicated in vaccinia virus-infected cells and resolved into linear minichromosomes with vector DNA in the center and vaccinia virus DNA hairpins at the two ends. Resolution did not occur when the concatemer joint was less than 250 bp or when plasmids were transfected into uninfected cells, indicating requirements for a specific DNA structure and viral trans-acting factors. These studies indicate that concatemers can serve as replicative intermediates and account for the generation of flip-flop sequence variation of the hairpins at the ends of the mature vaccinia virus genome.     

Camptothecin, a specific inhibitor of type I DNA topoisomerase, induces DNA breakage at replication forks.

The structure of replicating simian virus 40 minichromosomes, extracted from camptothecin-treated infected cells, was investigated by biochemical and electron microscopic methods. We found that camptothecin frequently induced breaks at replication forks close to the replicative growth points. Replication branches were disrupted at about equal frequencies at the leading and the lagging strand sides of the fork. Since camptothecin is known to be a specific inhibitor of type I DNA topoisomerase, we suggest that this enzyme is acting very near the replication forks. This conclusion was supported by experiments with aphidicolin, a drug that blocks replicative fork movement, but did not prevent the camptothecin-induced breakage of replication forks. The drug teniposide, an inhibitor of type II DNA topoisomerase, had only minor effects on the structure of these replicative intermediates.     

A large-tumor-antigen-specific monoclonal antibody inhibits DNA replication of simian virus 40 minichromosomes in an in vitro elongation system.

In productively infected cells, a fraction of large-tumor antigen (T antigen) is tightly bound to replicating simian virus 40 (SV40) minichromosomes and does not dissociate at salt concentrations of greater than 1 M NaCl. We present electronmicrograms demonstrating the presence of T antigen on the replicated sections of replicating SV40 minichromosomes. We also show that the fraction of tightly bound T antigen is recognized by antibodies from mouse tumor serum and, more specifically, by a particular T-antigen-specific monoclonal antibody, PAb 1630. A second T-antigen-specific monoclonal antibody, PAb 101, does not react with the T-antigen fraction remaining on replicating SV40 chromatin at high salt concentrations. We used an in vitro replication system which allows, via semiconservative DNA replication, the completion of in vivo-initiated replicative intermediate DNA molecules. We show that monoclonal antibody PAb 1630, but not monoclonal antibody PAb 101, inhibits viral DNA replication. We discuss the possibility that SV40 T antigen may play a role in chain elongation during SV40 chromatin 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.     

Biosynthetic studies of the Y base in yeast phenylalanine tRNA. Incorporation of guanine

Replicating polyoma virus DNA, pulse-labeled with ³H-thymidine, was isolated from infected mouse embryo cells by velocity sedimentation in neutral sucrose and purified by benzoylated-naphthoylated DEAE-cellulose chromatography. Nascent strands, prepared by heat denaturation of purified replicative intermediate, banded at a slightly higher buoyant density in neutral cesium sulfate gradients than single strands derived from superhelical viral DNA. Treatment of nascent strands with a mixture of ribonucleases 1A and T1 shifted their buoyant density to that of single strands derived from superhelical viral DNA. These results indicate that an oligoribonucleotide component is covalently associated with replicating polyoma DNA strands.     

MITOCHONDRIAL DNA REPLICATION IN SEA URCHIN OOCYTES

Mitochondrial DNA (mtDNA) replicative intermediates from Strongylocentrotus purpuratus oocytes were isolated by ethidium bromide-CsCl density gradient centrifugation and examined by electron microscopy after formamide spreading. In some experiments, the mtDNA was radioactively labeled by exposing isolated oocytes to [³H]thymidine. Oocyte mtDNA replication appears to follow the displacement loop model outlined in mouse L cells. There are differences in detail. The frequency of D-loop DNA is much lower in oocytes, suggesting that the relative holding time at the D-loop stage is shorter. Duplex synthesis on the displaced strand occurs early and with multiple initiations. The frequency of totally duplex replicative forms, or Cairns' forms, is the highest reported for mtDNA. The differences may be related to the fact that oocyte mtDNA replication occurs in the absence of cell division and need not be coordinated with a cell cycle. Molecules with expanded D loops banded in the intermediate region between the lower and upper bands in an ethidium bromide-CsCl gradient, supporting the notion that displacement replication proceeds on a closed circular template which is subject to nicking-closing cycles. In mature sea urchin eggs, replicative forms are absent and virtually all the mtDNA is stored as clean circular duplexes. Some novel structural variants of superhelical circular DNA (molecules with denaturation loops and double branch-migrated replicative forms) are reported.     

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.