DNAaseI-hypersensitive minichromosomes of SV40 possess an elastic torsional strain in DNA.

Previously, we have shown that DNA in a small fraction (2-5%) of SV40 minichromosomes was torsionally strained and could be relaxed by treating minichromosomes with topoisomerase I. This fraction was enriched with endogeneous RNA polymerase II (Luchnik et al., 1982, EMBO J., 1, 1353). Here we show that one and the same fraction of SV40 minichromosomes is hypersensitive to DNAase I and is relaxable by topoisomerase I. Moreover, this fraction completely loses its hypersensitivity to DNAase I upon relaxation. The possibility that this fraction of minichromosomes can be represented by naked DNA is ruled out by the results of studying the kinetics of minichromosome digestion by DNAase I in comparison to digestion of pure SV40 DNA and by measuring the buoyant density of SV40 chromatin in equilibrium CsCl gradient. Our data obtained with SV40 minichromosomes may be relevant to the mechanism responsible for DNAase I hypersensitivity in the loops or domains of cellular chromatin.     

Simian virus 40 minichromosomes contain torsionally strained DNA molecules.

Sundin and Varshavsky (J. Mol. Biol. 132:535-546, 1979) found that nearly two-thirds of simian virus 40 (SV40) minichromosomes obtained from nuclei of SV40-infected cells become singly nicked or cleaved across both strands after digestion with staphylococcal nuclease at 0 degrees C. The same treatment of SV40 DNA causes complete digestion rather than the limited cleavages produced in minichromosomal DNA. We have explored this novel behavior of the minichromosome and found that the nuclease sensitivity is dependent upon the topology of the DNA. Thus, if minichromosomes are pretreated with wheat germ DNA topoisomerase I, the minichromosomal DNA is completely resistant to subsequent digestion with staphylococcal nuclease at 0 degrees C. If the minichromosome-associated topoisomerase is removed, virtually all of the minichromosomes are cleaved to nicked or linear structures by the nuclease treatment. The cleavage sites are nonrandomly located; instead they occur at discrete loci throughout the SV40 genome. SV40 minichromosomal DNA is also cleaved to nicked circles and full-length linear fragments after treatment with the single strand-specific endonuclease S1; this cleavage is also inhibited by pretreatment with topoisomerase I. Thus, it may be that the nuclease sensitivity of minichromosomes is due to the transient or permanent unwinding of discrete regions of their DNA. Direct comparisons of the extent of negative supercoiling of native and topoisomerase-treated SV40 minichromosomes revealed that approximately two superhelical turns were removed by the topoisomerase treatment. The loss of these extra negative supercoils from the DNA probably accounts for the resistance of the topoisomerase-treated minichromosomes to the staphylococcal and S1 nucleases. These findings suggest that the DNA in SV40 intranuclear minichromosomes is torsionally strained. The functional significance of this finding is discussed.     

Effect of X-ray induced DNA damage on DNAase I hypersensitivity of SV40 chromatin: relation to elastic torsional strain in DNA.

The effect of X-irradiation on DNAase I hypersensitivity of SV40 minichromosomes within nuclei or free in solution was investigated. The susceptibility of the specific DNA sites in the control region of minichromosomes to DNAase I decreased in a dose dependent manner after irradiation of isolated nuclei. On the other hand, the irradiation of minichromosomes extracted from nuclei in 0.1 M NaCl-containing buffer almost did not affect the level of their hypersensitivity to DNAase I. This suggests that DNAase I hypersensitivity may be determined by two different mechanisms. One of them may be connected with elastic torsional strain within a fraction of minichromosomes and another seems to be determined by nucleosome free region. The first mechanism may be primarily responsible for the hypersensitivity of minichromosomes within nuclei. After irradiation of the intact cells, DNAase I hypersensitivity tested in nuclei substantially increased. This was connected with activation of endogeneous nucleases by X-irradiation which led to accumulation of single- and double-strand breaks superimposed to DNAase I induced breaks in the control region of SV40 DNA.     

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.     

Chromatin assembly during SV40 DNA replication in vitro

A cytosol extract from human 293 cells supports efficient replication of SV40 origin-containing plasmid DNA in the presence of the SV40 T antigen. Addition of a nuclear extract from the same cells promotes negative supercoiling of the replicated DNA but not the bulk of the unreplicated DNA. The level of superhelicity is affected by the concentrations of T antigen and nuclear extract factors and by the time of addition of the nuclear extract. The replicated DNA in isolated DNA-protein complexes resists relaxation by purified HeLa cell topoisomerase I. Micrococcal nuclease digestion, sucrose gradient sedimentation, and electron microscopy demonstrate that the negative supercoils result from assembly of the replicating DNA into a chromatin structure. These results suggest that, during DNA replication, the core histones can be assembled on both sides of the replication fork by an active, replication-linked mechanism that does not require a template of preexisting nucleosomes.     

Isolation of Z-DNA binding proteins from SV40 minichromosomes: evidence for binding to the viral control region

Proteins dissociated from SV40 minichromosomes by increasing NaCl concentration were tested for their binding to Z-DNA [Br-poly(dG-dC)] and B-DNA [poly (dG-dC)]. Z-DNA binding proteins are largely released in 0.2 M NaCl whereas most B-DNA binding proteins are not released until 0.6 M NaCl. Incubation of SV40 minichromosomes with Z-DNA-Sephadex in low salt solution results in proteins with Z-DNA binding activity (PZ proteins). These proteins bind to negatively supercoiled DNAs containing left-handed Z-DNA but not to relaxed DNAs. They compete with anti-Z-DNA antibodies in binding to negatively supercoiled DNAs. The binding is tighter to negatively supercoiled SV40 DNA than to other plasmids, suggesting sequence-specific Z-DNA binding. PZ proteins binding to negatively supercoiled SV40 DNA interfere with cleavage at the Sph I sites, within the 72 bp repeat sequences of the viral control region, but not with cleavage at the Bgl I site, at the origin of replication. Removal of PZ proteins also exposes the Sph I sites in the SV40 minichromosomes while addition of PZ proteins makes the sites inaccessible.     

Both DNA topoisomerases I and II relax 2 micron plasmid DNA in living yeast cells

Measurements at various temperatures of the linking number of yeast 2 microns plasmid DNA in wild-type cells and in cells bearing mutations in the DNA topoisomerase I and II genes show that bulk 2 microns plasmid minichromosome are maintained in a relaxed state by the combined action of topoisomerases I and II. Bulk 2 microns minichromosomes are not under torsional stress in vivo and are not substrates for a putative gyrase-like topoisomerase.     

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.     

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.     

A specific DNA unwinding activity associated with SV40 large T antigen

The incubation of highly purified large T antigen with relaxed, circular SV40 DNA in the presence of topoisomerase I (nicking closing enzyme) resulted in the introduction of negative superhelical turns in the DNA. ATP was not required for this reaction. A similar introduction of superhelical turns could also be obtained when a recombinant plasmid DNA (Y182), which contains sequences from both SV40 DNA and pBR322, was used. However, no effect was observed when relaxed pBR322 DNA, which does not contain SV40 DNA sequences, was incubated with T antigen in the presence of topoisomerase. These results are consistent with the hypothesis that large T antigen can recognize and unwind specific sequences on SV40 DNA.     

Structural requirements for simian virus 40 replication and virion maturation.

The nuclear matrix plays an important role in simian virus 40 (SV40) DNA replication in vivo, since functional replication complexes containing large T and replicating SV40 minichromosomes are anchored to this structure (R. Schirmbeck and W. Deppert, J. Virol. 65:2578-2588, 1991). In the present study, we have analyzed the course of events leading from nuclear matrix-associated replicating SV40 minichromosomes to fully replicated minichromosomes and, further, to their encapsidation into mature SV40 virions. Pulse-chase experiments revealed that newly replicated SV40 minichromosomes accumulated at the nuclear matrix and were directly encapsidated into DNase-resistant SV40 virions at this nuclear structure. Alternatively, a small fraction of newly replicated minichromosomes left the nuclear matrix to associate with the cellular chromatin. During the course of infection, progeny virions continuously were released from the nuclear matrix to the cellular chromatin and into the cytoplasm-nucleoplasm. The bulk of SV40 progeny virions, however, remained at the nuclear matrix until virus-induced cell lysis.