Herpes simplex virus DNA synthesis in a partially purified soluble extract from infected cells.

HEp-2 cells were infected with herpes simplex virus type 1 and extracted with 0.25% Triton X-100 and 0.1 M NaCl. The extract was sedimented on sucrose gradients, and the fractions containing the endogenous DNA polymerizing activity (replication complex) were collected. The properties of the replication complex were partially characterized. Under optimal conditions 375 pmol of dTMP per micrograms of DNA was incorporated, which corresponds to about 50% replication of preexisting viral DNA. The replication complex was shown to contain only DNA of viral origin by its density in CsCl. By using specific assays for DNA polymerases alpha, beta, gamma, and herpes simplex virus, we found that only the viral DNA polymerase copurified with the replication complex.     

Deoxyribonucleic Acid Synthesis in Permeabilized Spheroplasts of Saccharomyces cerevisiae

Osmotically shocked spheroplasts from Saccharomyces cerevisiae incorporated deoxynucleoside triphosphates specifically into double-stranded nuclear and mitochondrial deoxyribonucleic acid (DNA). Results with this in vitro system for cells with and without mitochondrial DNA were compared. Strains lacking mitochondrial DNA were used to study nuclear DNA replication. With a temperature-sensitive mutant defective in DNA replication in vivo, DNA synthesis in vitro was temperature sensitive as well. The product of synthesis with all strains after very short labeling times consisted principally of short fragments that sedimented at approximately 4S in alkali; with longer pulse times or a chase with unlabeled nucleotides, they grew to a more heterogenous size, with an average of 6 to 8S and a maximum of 15S. There was little, if any, integration of these DNA fragments into the high-molecular-weight nuclear DNA. Analysis by CsCl density gradient centrifugation after incorporation of bromodeoxyuridine triphosphate showed that most of the product consisted of chains containing both preexisting and newly synthesized material, but there was also a small fraction (ca. 20%) in which the strands were fully synthesized in vitro. (32)P-label transfer ("nearest-neighbor") experiments demonstrated that at least a part of the material synthesized in vitro contained ribonucleic acid-DNA junctions. DNA pulse-labeled in vivo in a mutant capable of taking up thymidine 5'-monophosphate, sedimented in alkali at 4S, as in the case of the in vitro experiments.     

The Werner syndrome gene product co-purifies with the DNA replication complex and interacts with PCNA and topoisomerase I

Werner syndrome (WS) is a recessive disorder characterized by genomic instability and by the premature onset of a number of age-related diseases. To understand the molecular basis of this disease, we deleted a segment of the murine Wrn gene and created Wrn-deficient embryonic stem (ES) cells. At the molecular level, wild type-but not mutant-WS protein co-purifies through a series of centrifugation, chromatography, and sucrose gradient steps with the well characterized 17 S multiprotein DNA replication complex. Furthermore, wild type WS protein co-immunoprecipitates with a prominent component of the multiprotein replication complex, proliferating cell nuclear antigen (PCNA). In vitro studies also indicate that PCNA binds to a region in the N terminus portion of the WS protein containing a potential 3'-5' exonuclease domain. Finally, human WS protein also co-immunoprecipitates with both PCNA and topoisomerase I. These results suggest that the WS protein interacts with several components of the DNA replication fork.     

In vitro system that synthesizes circular viral DNA of bacteriophage phi X 174.

An extract prepared from Escherichia coli cells infected with phi chi 174 bacteriophage was capable of incorporating dTTP into phage-specific DNAs in vitro. The synthesized DNAs were associated with proteins and sedimented with S values of 20, 50, and 90 in a sucrose gradient sedimentation. DNA isolated from 20S material was open circular replicative form (RF), DNA in 50S material was replicative-form DNA with an extended single-stranded viral DNA that ranged up to one genome in length, and DNA in 90S material consisted of circular and linear single-stranded viral DNA of full genome length and single-stranded viral DNA shorter than full genome length. Pulse and pulse-chase experiments indicated that 90S material derived from 50S material.     

Adenovirus deoxyribonucleic acid replication. II. Synthesis of viral deoxyribonucleic acid in vitro by a nuclear membrane fraction from infected KB cells.

A cell-free system for the study of viral DNA replications was developed by the isolation of a nuclear membrane fraction "DNA replication complex" from adenovirus 2-infected human KB cells late after infection. This complex which possesses both DNA polymerase activity and a virus-specific endonuclease synthesizes exclusively virus-specific DNA sequences in vitro by a semiconservative mechanism. Analysis by rate zonal sedimentation in alkaline sucrose gradients showed that the products of the reaction are small DNA chains approximately 6 to 9 S, presumably "Okazaki intermediates," that are not sealed under our in vitro conditions. Analysis by rate zonal sedimentation in neutral sucrose gradients showed that labeled viral DNA fragments are hydrogen bonded to viral 18 S DNA segments, 0.25 the size of the linear, viral 31 S DNA genome. The 18 S DNA is probably a specific cleavage product of the viral endonuclease found in the replication complex and could represent intermediates in viral DNA replication or degradation products.     

Viral DNA-synthesizing intermediate complex isolated during assembly of bacteriophage phi X174.

A DNA protein complex that is a precursor of mature phi X174 phage was isolated. The complex sedimented with an S value of 50 in a sucrose gradient and contained phage DNA consisting of a replicative form molecule with an extended tail of single-stranded viral DNA. The viral-strand DNA ranged from one to two genomes in length. Proteins coded on the phi X174 genome as well as the host genome were associated with the viral DNA in the 50S precursor complex. Our results indicated that both viral DNA synthesis and cleavage of the growing viral-strand DNA occurred in the 50S complex.     

A protein which facilitates assembly of nucleosome-like structures in vitro in mammalian cells

An activity which facilitates assembly of nucleosome-like structures in vitro at physiological ionic strength was detected both in human HeLa S3 cells and mouse FM3A cells. The assembly protein was purified from FM3A cells by fractionation with ammonium sulfate, DEAE-cellulose, phosphocellulose, Sephadex G-150 column chromatography, and sucrose gradient centrifugation. In the sucrose gradient, the activity was detected at 5S and the active fraction contained three peptides of 59,000, 65,000, and 102,000 daltons. When core histones were mixed with these peptides, the 59,000 peptide sedimented at the 6S and 10S positions, where the histones co-sedimented. The 6S fraction contained H2A, H2B, and A24 proteins, and the 10S fraction contained four kinds of core histones in equal amounts. Nucleosomes were formed by mixing DNA with the 10S fraction, but were not formed with the 6S fraction. The nucleosome structure assembled was assessed using the sensitivity to micrococcal nuclease.     

Nucleoprotein Complexes Containing Replicating Simian Virus 40 DNA: Comparison with Polyoma Nucleoprotein Complexes

Procedures for isolating nucleoprotein complexes containing replicating polyoma DNA from infected mouse cells were used to prepare short-lived nucleoprotein complexes (r-SV40 complexes) containing replicating simian virus 40 (SV40) DNA from infected monkey cells. Like the polyoma complexes, r-SV40 complexes were only partially released from nuclei by cell lysis but could be extracted from nuclei by prolonged treatment with solutions containing Triton X-100. r-SV40 complexes sedimented faster than complexes containing SV40 supercoiled DNA (SV40 complex) in sucrose gradients, and both types of SV40 nucleoprotein complexes sedimented ahead of polyoma complexes containing supercoiled polyoma DNA (py complex). The sedimentation rates of py complex and SV40 complex were 56 and 61S, respectively, based on the sedimentation rate of the mouse large ribosomal subunit as a marker. r-SV40 complexes sedimented as multiple peaks between 56 and 75S. Sedimentation and buoyant density measurements indicated that protein is bound to all forms of SV40 DNA at about the same ratio of protein to DNA (1-2/1) as was reported for polyoma nucleoproteins.     

Structural and functional characterization of the poliovirus replication complex.

Two populations of membrane-bound replication complexes were isolated from poliovirus-infected HEp-2 cells by sucrose gradient centrifugation. The two fractions show similar ultrastructural features: the replication complex is enclosed in a rosettelike shell of virus-induced vesicles and contains a very tightly packed second membrane system (compact membranes). The vesicular fraction, which bands in 30% sucrose, contains replicative intermediate (RI) and 36S RNA. The fraction banding in 45% sucrose contains only minute amounts of RI and contains mainly 36S RNA, two-thirds of which is encapsidated. In vitro, the two fractions show similar RNA synthesizing capacities and produce 36S plus-strand RNA. Dissolving the membranes within and around synthetically active replication complexes with sodium deoxycholate abolishes the completion of 36S RNA but still allows elongation in the RI. Our findings suggest an architecture of the replication complex that has the nascent plus strands on the RI enclosed in the compact membranes and the replication forks wrapped additionally in protein. Plus-strand RNA can be localized by in situ hybridization with a biotinylated riboprobe between the replication complex and the rosette of the virus-induced vesicles. It was found that the progeny RNA strands are set free soon after completion from the replication complex at the sites where the compact membranes within the replication complex are in close contact with the surrounding virus-induced vesicles.     

Multiplication of parvovirus LuIII in a synchronized culture system. III. Replication of viral DNA.

The replication of the single-stranded DNA (ssDNA) of parvovirus LuIII was studied in synchronized HeLa cells. After infection of the cells in early S phase, synthesis of a replicative form (RF) DNA became detectable as early as 9 h postinfection, i.e., after display of the cellular helper function(s) indispensable for the replication of LuIII virus. According to digestion with nuclease S1, hybridization studies, and electron microscopy, RF DNA is a linear, double-stranded molecule comparable in length to mature ssDNA. It sedimented around 15S in neutral solution and banded at 1.714 g/ml in CsCl. Moreover, replication of LuIII DNA obviously includes a further replicative intermediate DNA which sedimented in front of RF DNA and bore single-stranded side-chains. Newly synthesized DNA disappeared from pools containing both RF DNA and replicative intermediate DNA within 5 min and reappeared in progeny virions only after 15 min. Intranuclear accumulation of significant amounts of progeny ssDNA could not be detected. It was postulated, therefore, that newly synthesized ssDNA is immediately enclosed in a stable maturation complex and resists extraction by the method of Hirt (1967).     

Functions of gene C and gene D products of bacteriophage phi X 174.

Phage-related materials existing in cells infected with various mutants of bacteriophage phi chi 174 were investigated. A novel species of replicative-form (RF) DNA was found in cells infected with a phage mutant of gene B, C, D, F, or G. This species, called RFI, sedimented at a position between RFI and RFII in a neutral sucrose gradient. It was converted to RFI upon denaturation in alkali, denaturation in formamide and subsequent renaturation, or RNase treatment at low ionic strength. In cells infected with a phage mutant of gene C, RFI was derived from pulse-labeled RFII after a short chase. TLLS INFECTED WITH A MUTANT OF GENE B, D, or F. A possible function of the C gene product of phi chi 174 could be to prevent the conversion of RFII to RFI, thereby maintaining the availability of RFII to act as the template for single-stranded viral DNA synthesis. A protein complex containing no DNA, which sedimented with an S value of 108 in a sucrose gradient and contained virion proteins F, G, and H, and nonvirion protein D, was found in cells infected with the gene C mutant. A possible function of protein D was considered as a scaffolding protein for assembly of phage structural proteins.     

Selective cleavage of AAVS1 substrates by the adeno-associated virus type 2 rep68 protein is dependent on topological and sequence constraints

The adeno-associated virus type 2 (AAV-2) Rep78 and Rep68 proteins are required for replication of the virus as well as its site-specific integration into a unique site, called AAVS1, of human chromosome 19. Rep78 and Rep68 initiate replication by binding to a Rep binding site (RBS) contained in the AAV-2 inverted terminal repeats (ITRs) and then specifically nicking at a nearby site called the terminal resolution site (trs). Similarly, Rep78 and Rep68 are postulated to trigger the integration process by binding and nicking RBS and trs homologues present in AAVS1. However, Rep78 and Rep68 cleave in vitro AAVS1 duplex-linear substrates much less efficiently than hairpinned ITRs. In this study, we show that the AAV-2 Rep68 endonuclease activity is affected by the topology of the substrates in that it efficiently cleaves in vitro in a site- and strand-specific manner the AAVS1 trs only if this sequence is in a supercoiled (SC) conformation. DNA sequence mutagenesis in the context of SC templates allowed us to elucidate for the first time the AAVS1 trs sequence and position requirements for Rep68-mediated cleavage. Interestingly, Rep68 did not cleave SC templates containing RBS from other sites of the human genome. These findings have intriguing implications for AAV-2 site-specific integration in vivo.     

Use of ethidium bromide fluorescence enhancement to detect duplex DNA and DNA bacteriophages during zone sedimentation in sucrose gradients: molecular weight of DNA as a function of sedimentation rate

Duplex DNA molecules and DNA bacteriophages have been sedimented through 5--25% sucrose gradients containing ethidium bromide. The location of DNA within the gradients has been determined by illuminating gradients with ultraviolet light and observing the ethidium bromide fluorescence enhancement induced by the DNA. The relative sedimentation rates of linear, duplex DNAs from bacteriophages T4, T5, T7 and an 8.3% T7 deletion mutant have been determined. The distances sedimented by DNA have been corrected, when necessary, for a progressive decrease in sedimentation rate that occurs after the DNA has traversed 40% of the sucrose gradient. The corrected distances sedimented by two DNA molecules, r1' and r2', are related to the DNA molecular weights, m1 and m2, by the equation: r1'/r2' = (m1/m2)0.38 when 0.025--0.70 microgram of each type of DNA is sedimented. Intact bacteriophages were also sedimented in ethidium bromide--sucrose gradients and detected by fluorescence enhancement.     

Purified presequence binding factor (PBF) forms an import-competent complex with a purified mitochondrial precursor protein.

In vitro mitochondrial import of the purified precursor form (pOTC) of rat ornithine carbamoyltransferase (OTC) is stimulated by a cytosolic factor(s) contained in rabbit reticulocyte lysate. A protein factor that binds to pOTC but not to mature OTC and was named presequence binding factor or PBF, was purified 91,000-fold from the lysate by affinity chromatography using pOTC-bound Sepharose, DEAE-5PW HPLC and sucrose gradient centrifugation. The purified PBF migrated as a single polypeptide of 50,000 daltons on SDS-PAGE. On sucrose gradients, urea-denatured pOTC sedimented to the bottom, whereas PBF sedimented with an S20,w value of 5.5S. When pOTC and PBF were centrifuged together, both polypeptides sedimented as a complex of 7.1S. Formation of the pOTC-PBF complex was inhibited by micromolar concentrations of the synthetic presequence of pOTC and those of other mitochondrial precursor proteins. The purified PBF markedly stimulated the import of purified or in vitro synthesized pOTC into the mitochondria. PBF-stimulated pOTC import was further enhanced by a 70 kd heat shock protein (hsp 70) purified from yeast; the hsp70 alone had little effect. Thus, PBF binds to the presequence portion of the precursors and may hold them in a transport-competent form in cooperation with hsp70.     

Properties of adenovirus messenger ribonucleic Acid synthesized in vitro

Adenovirus deoxyribonucleic acid (DNA) was used as template for the in vitro synthesis of viral-specific ribonucleic acid (RNA). When the kinetics of the reaction were compared by using native and heat-denatured DNA templates, the latter synthesized RNA at a slower rate. The fate of the DNA after acting as template and physical characteritstics of the RNA product were studied. The DNA template, according to its sedimentation rate, was not significantly degraded by the Micrococcus lysodeicticus RNA polymerase. The products of the RNA polymerase reaction had the following properties. (i) Hybridization experiments revealed a high degree of complementarity (50 to 70%) for its homologous DNA. (ii) A very low complementarity (6 to 7%) was found for its heterologous DNA. (iii) The sedimentation rate of the synthetic RNA in a sucrose gradient was 5 to 10S when native DNA was used as the template. When heat-denatured DNA was used, the resulting RNA product, free of the template, sedimented at a rate of 3 to 16S. A rapidly sedimenting (>30S) DNA-RNA complex resulted when denatured DNA was the template. The DNA moiety of the complex was sensitive to 125 μg of deoxyribonuclease per ml. The RNA of the complex, however, was fully refractory to 50 μg of ribonuclease per ml. When the adenovirus DNA was sonically treated and then used as template, the RNA product sedimented at 3 to 9S. The heat-denatured sonically treated DNA template yielded a DNA-RNA complex that also sedimented at an unusually fast rate (>18S).     

Adeno-associated virus DNA replication complexes in herpes simplex virus or adenovirus-infected cells.

A complex which is active in in vitro synthesis of adeno-associated virus (AAV) DNA was solubilized from Vero cells that were co-infected with AAV and either adenovirus (Ad5) or a herpes simplex virus type 1 (HSV-1) as the helper virus. The complexes from the Ad5 and HSV-1-infected cells sedimented at 23 S and 28 S, respectively. The optimal conditions for in vitro DNA synthesis for the two types of complex using the endogenous AAV template and the endogenous DNA polymerase, differed with respect to the effect of KCl and K2SO4 concentration. In addition the complex from HSV-1-infected cells, but not that from Ad5-infected cells, was inhibited by phosphonoacetic acid. Thus, the two complexes appear to contain different DNA polymerase activities. This was verified by phosphocellulose chromatography of the DNA polymerases solubilized from the isolated complexes. The major activity in the complex from HSV-1 infected cells was the HSV-induced DNA polymerase with lesser amounts of cellular DNA polymerase alpha and gamma or both. The complex from the Ad5-infected cells contained mainly a cellular DNA polymerase gamma.