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.     

Enrichment and visualization of small replication units from cultured mammalian cells

DNA from cultured Chinese hamster cells has been fractionated to yield a population of DNA enriched for replicating molecules. Molecules containing replication structures were analyzed by electron microscopy, and replicon size was estimated. The enrichment procedure takes advantage of single-stranded regions characteristic of replicating molecules, and the greater affinity of mercuric ion for single-stranded rather than native DNA. After interaction with low concentrations of HgCl2, DNA with bound mercury is separated from the bulk of the DNA by virtue of its increased buoyant density in an isopycnic Cs2SO4 gradient. When DNA from cells labeled with [3H]thymidine for 45 s is interacted with HgCl2 and banded in Cs2SO4, the DNA with the highest specific activity is found in a dense region of the gradient. The high specific activity DNA behaves kinetically like nascent DNA since the radioactivity can be chased into main band if the cells are incubated for a further 2 h in excess unlabeled thymidine. Electron microscope analysis of the DNA in the enriched fraction confirmed that it contains a substantial fraction of molecules with replication structures. The level of enrichment is about 25-fold compared to unfractionated DNA or DNA taken from the main band of the Hg++/Cs2SO4 gradient. Of the replicating molecules visualized, 85% possessed a single replication structure. All molecules with multiple replication forms contained replicon sizes less than 5 micron, ranging from 0.2 to 4.5 micron. Replicon size was determined by measuring the distance from the center of one replication structure to the center of the adjacent replication structure on the same molecule. The replicons observed in this study are far smaller than can be detected by DNA fiber autoradiography and are in the same size range as the very small replication units reported in embryonic systems.     

Late replicative intermediates are accumulated during simian virus 40 DNA replication in vivo and in vitro.

Simian virus 40 (SV40) replicating chromosomes were extracted from nuclei of infected cells. The chromosomes in the extract were resolved on neutral sucrose gradients, and the extent of replication of the DNA in the chromosome peaks was determined. The extract, in combination with cytosol factors and the appropriate precursors, supports the continued replication of viral DNA. The products of the incubation were mature form I DNA and molecules (after deproteinization) with sedimentation coefficients, in neutral sucrose, of 22S and 29S. The results of our analysis of this system indicate the following. (i) The 22S molecule, which has been described by previous workers, is a relaxed, replicating molecule and is an artifact of the in vitro system. (ii) When the in vitro synthesis is performed at optimal ionic strength (150 mM potassium acetate), the artifactual 22S molecule does not appear. (iii) Late replicative intermediates do accumulate in vivo and in vitro. The major late form accumulated is 91% completed. (iv) The replicating chromosomes can be resolved into two distinct peaks on neutral sucrose gradients. The molecules in these peaks differ in extent of replication. (v) The nuclear extraction procedure preferentially extracts early replicating chromosomes. The relevance of these data to the problem of SV40 and cellular chromosome replication and termination is described.     

Some Properties of DNA from Phage-Infected Bacteria

Replicating T5 or λ phage DNA has been labeled by adding tritiated thymidine for short periods to cultures of phage-infected Escherichia coli before isolation of intracellular DNA. Two procedures are described for separating T5 replicating DNA from DNA of intracellular phage particles. Both T5 and λ replicating DNA had the same bouyant density in cesium chloride as DNA from phage particles but sedimented faster when centrifuged in sucrose density gradients. The fast sedimentation did not appear to be caused by DNA protein or DNA-RNA complexes or by aggregation of DNA, but is probably due to DNA molecules of unusual structure. Experiments involving hydrodynamic shear and sucrose density gradient centrifugation at alkaline pH have suggested that with λ the replicating form of DNA is a linear molecule considerably longer than the DNA molecules of λ-phage particles. The constituent polynucleotide chains of λ but not T5 replicating DNA also appear to be longer than those of phage DNA.     

Molecular structure of adeno-associated virus variant DNA

When lysates of human cells, infected jointly with the defective parvovirus, adeno-associated virus (AAV), and a helper adenovirus, are banded to equilibrium in CsCl buoyant density gradients, virus particles of various densities are obtained. Infectious AAV particles mainly band at a density of 1.41 g/cm3 with a minor component at 1.45 g/cm3. Noninfectious AAV particles band at densities between 1.41 and 1.32 g/cm3. We have analyzed, by mapping with site-specific endodeoxyribonucleases, the molecular structure of the variant AAV DNA molecules obtained from these light density particles. The size of variant DNA molecules ranged from 100 to 3% of genome length. In general, the variant DNAs are deleted for internal regions but retain the genome termini. Some of the variant DNAs appear to be cross-linked, spontaneously renaturing molecules having structures analogous to replicating forms of AAV DNA.     

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.     

Unwinding of Parental Strands During Simian Virus 40 DNA Replication

Pools of young (less than 60% replicated) and mature (60-90% replicated) replicating molecules of simian virus 40 (SV40) DNA have been treated at pH 12.2 in order to dissociate growing chains from the parental strands. The molecules are neutralized so that the parental strands can reassociate and they have then been isolated. They are covalently closed structures which sediment rapidly in alkaline sucrose gradients; however, the sedimentation rates are less than the sedimentation rate of SV40 DNA I. Isopycnic banding in CsCl-ethidium bromide and sedimentation velocity studies in the presence of various amounts of ethidium bromide indicate that these structures contain negative superhelical turns and several-fold-higher superhelix densities than SV40 DNA I (the covalently closed DNA molecule). These structures are those that would be predicted if nicking, unwinding, and sealing of the parental strands occurred as replication proceeded. These experiments provide a direct demonstration that there is a progressive decrease in the topological winding number which accompanies SV40 DNA replication.     

Polyoma virus minichromosomes: associated DNA molecules

Electron microscopy was used to identify and quantitate DNA molecules associated with 3H-labeled polyoma minichromosomes which had been fractionated on a sucrose gradient. The percentage of replicating DNA molecules observed in the fractions of the gradient normally designated the replicative intermediate region was up to ninefold higher than in fractions from the mature region. Nevertheless, because of the higher overall concentration of polyoma DNA molecules in the mature region, nearly as many replicating DNA molecules were computed to be in the mature region as in the replicative intermediate region. The replicating molecules in the mature region was predominantly early replicative intermediates. Almost all late replicative intermediates were found in the replicative intermediate region. Under aqueous spreading conditions, a substantial fraction of the replicating DNA structures appeared to be asymmetrical or otherwise unusual, suggesting that extensive single-stranded regions may exist in replicating polyoma minichromosomes.     

Procedure for purification of intact DNA from vaccinia virus.

A procedure for the isolation of intact vaccinia DNA molecules by chromatography on hydroxyapatite in the presence of 6 M urea is described. When lysates of virions containing 0.5 to 10 microgram of DNA were employed, over 95% of the viral DNA could be recovered free of poteins. Vaccinia DNA molecules isolated in this manner sedimented at 68S in neutral sucrose gradients and had an average contour length of 62.3 micrometer when examined in an electron microscope, and the DNA could be cleaved with the restriction endonuclease EcoRI and BamHI. The results of these analyses showed that intact vaccinia DNA molecules of 120 X 10(6) to 130 X 10(6) molecular weight could be obtained by the procedures described.     

Size classes of replication units in DNA from sea urchin embryos

Sea urchin DNA containing replication structures was isolated from two to four cell stage and blastula stage embryos, and examined by electron microscopy. In addition to the expected eye forms, we also observed molecules with large internal single-stranded gaps. Such structures were not present in DNA devoid of replicating molecules such as that isolated from sea urchin sperm. When the size of eye forms and interbubble distances between the two stages were compared, there was no detectable difference. In both stages, we observed two distinct size classes of bubbles and of interbubble distances. In the case of bubble sizes, the smaller size class was comprised of clustered microbubbles that ranged from 200 base pairs to 1 Kilobase (kb) with a mean of 432 base pairs. The large eye forms measured 1--35 kb with a mean of 6.8 kb. Interbubble distances also yielded two distinct populations, with the smaller class ranging from 400 base pairs to 2.3 kb (mean = 1.1 kb) and the larger population ranging from 2.8 to 36 kb (mean = 10.9 kb). Although other possibilities cannot be entirely excluded, the data support the contention that a substantial fraction of the larger eye-form population arises from the fusion of the clustered microbubbles.     

Analysis of human cytomegalovirus nucleoprotein complexes

When chromatin was isolated from cells infected with human cytomegalovirus, the virus DNA remained with the chromatin fraction. If deproteinized virus DNA was added to either isolated nuclei or chromatin, the DNA was lost during the chromatin isolation. When isolated chromatin from cytomegalovirus-infected cells was banded in isopycnic metrizamide gradients, a single peak with a density of 1.18 g/cm3 was present. Analysis of this peak in isopycnic neutral CsCl gradients indicated that it contained both human cytomegalovirus and human embryonic lung cell DNAs. When infected nuclei were treated with micrococcal nuclease, 11S subunit particles which cosedimented with cell nucleosomes and contained virus DNA were isolated.     

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.     

DNA-histone interaction in the vicinity of replication points.

Chromatin replication was studied in isolated nuclei from Concanavalin A activated lymphocytes. Digestion with micrococcal nuclease revealed that the resistant fraction of in vitro replicated DNA is associated with nucleosomes. Earlier experiments had shown that the nuclease resistant fraction of nascent DNA is composed of fragments which are shorter than the nuclease resistant fragments of bulk DNA. In this communication we demonstrate that the short fragments of nascent DNA are differently bound to nucleosome like structures compared to bulk DNA. At 0.5 M NaCl a fraction of pulse labeled labeled DNA is released from these structures and appears as free double stranded DNA of about 140 base pair length (5S DNA) while the 185 pair fragments of mature replicated DNA remain attached to nucleosomes under these conditions. The experiments may indicate that the interaction of a fraction of replicating DNA with histones differs from that of bulk DNA.     

In vivo methylation of replicating bacteriophage phi chi174 DNA

The pattern of DNA methylation during the infection of Escherichia coli C cells with bacteriophage φX174, has been studied. In vivo methylated DNA was isolated and analyzed using the following techniques: velocity sedimentation through neutral and alkaline sucrose gradients, isopycnic analysis, chromatography on benzoylated DBAE-cellulose columns and specific enzymatic digestion. All these analytical methods indicated that the DNA molecules that are methylated during the process of phage φX DNA replication are the replicating intermediates composed of a circular complementary strand and a viral strand larger than one genome length. It is concluded that methylation occurs on the nascent DNA strand of the replicating intermediates involved in the synthesis of progeny single-stranded DNA.     

Studies on the mechanism of DNA replication in Physarum polycephalum

The synthesis of single-stranded DNA subunits (4 × 10⁷ daltons) in Physarum polycephalum was studied by alkaline sucrose density gradient centrifugation. The results were compared with the synthesis of the double-stranded DNA molecules (2.3 × 10⁸ daltons) which they comprise, as determined from neutral sucrose density gradient centrifugation patterns. Although the initiation of synthesis of most double-stranded DNA molecules takes place relatively early in the S period, synthesis of the subunits within them is initiated throughout at least the first two hours of this period. Similarly, replicating (presumably forked) DNA molecules appear to split into daughter DNA molecules prior to the completion of synthesis of the subunits therein. The average rate of DNA chain elongation within subunits is 0.3 × 10⁶ daltons/minute. It is suggested that alkaline sucrose density gradient centrifugation may be a more sensitive method for determining the time required for the completion of replication than other methods based solely on the incorporation of radioactive DNA precursors into an acid-insoluble product.     

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.     

Computer modelling of DNA structures involved in chromosome maintenance.

Sequence-dependent DNA bending of synthetic and natural molecules was studied by computer analysis. Modelling of synthetic oligonucleotides and of 107 kb of natural sequences gave results which closely resembled published electrophoretic data, demonstrating the powerful predictive capacity of the procedure. The analysis was extended to the study of DNA structures involved in chromosome maintenance. Centromeric DNAs from yeast were found to have sequences in their functional elements which cause them to be unusually straight. Autonomous replicating sequences were found to have two structural domains, one consisting of unusually straight sequences surrounding the consensus and the other of bending elements in flanking DNA. In addition to a structural homology, centromeric and autonomous replicating sequences share common sequence elements. These observations show that computer modelling of natural sequences is a viable approach to the study of the biological implications of alternative DNA structures.     

DNA of a Human Hepatitis B Virus Candidate

Particles containing DNA polymerase (Dane particles) were purified from the plasma of chronic carriers of hepatitis B antigen. After a DNA polymerase reaction with purified Dane particle preparations treated with Nonidet P-40 detergent, Dane particle core structures containing radioactive DNA product were isolated by sedimentation in a sucrose density gradient. The radioactive DNA was extracted with sodium dodecyl sulfate and isolated by band sedimentation in a preformed CsCl gradient. Examination of the radioactive DNA band by electron microscopy revealed exclusively circular double-stranded DNA molecules approximately 0.78 mum in length. Identical circular molecules were observed when DNA was isolated by a similar procedure from particles that had not undergone a DNA polymerase reaction. The molecules were completely degraded by DNase 1. When Dane particle core structures were treated with DNase 1 before DNA extraction, only 0.78-mum circular DNA molecules were detected. Without DNase treatment of core structures, linear molecules with lengths between 0.5 and 12 mum, in addition to the 0.78-mum circles were found. These results suggest that the 0.78-mum circular molecules were in a protected position within Dane particle cores and the linear molecules were not within core structures. Length measurements on 225 circular molecules revealed a mean length of 0.78 +/- 0.09 mum which would correspond to a molecular weight of around 1.6 x 10(6). The circular molecules probably serve as primer-template for the DNA polymerase reaction carried out by Dane particle cores. Thermal denaturation and buoyant density measurements on the Dane particle DNA polymerase reaction product revealed a guanosine plus cytosine content of 48 to 49%.     

The secondary structure and nitrocellulose affinity of freshly replicated DNA from Ehrlich ascites cells (author's transl)]

Hydroxyapatite chromatography and isopycnic Cs2SO4 centrifugation normally yield no indications of single-stranded DNA when that fraction of replicating DNA from Ehrlich ascites cells which can be separated by nitrocellulose chromatography is analyzed. Single-stranded DNA is detected by both methods if the DNA is fragmented by ultrasound before the nitrocellulose chromatography. The digestion of this DNA fraction by single-strand-specific nucliase leads to a loss of its binding to nitrocellulose and of the indications of single-stranded DNA. The loss for the affinity to nitrocellulose is also observed when the corresponding fraction separated from unfragmented DNA is digested by endonuclease. It is suggested that replicating DNA is bound to nitrocellulose by means of single-stranded gaps on the replication fork. These gaps are apparently too small to be detected within large, otherwise entirely double-stranded molecules by hydroxyapatite chromatography and Cs2SO4 centrifugation. In the case of nitrocellulose-binding ultrasound fragments, this relation seems to be more favorable because of the separation of most of the residual double-stranded part. It is demonstrated that sonication of helical DNA also generates a small amount of fragments with some single-stranded character. The effects observed with replicating DNA could be distinguished from these artifacts.