Intracellular Forms of Adenovirus DNA III. Integration of the DNA of Adenovirus Type 2 into Host DNA in Productively Infected Cells

KB cells productively infected with human adenovirus type 2 contain an alkalistable class of viral DNA sedimenting in a broad zone between 50 and 90S as compared to 34S for virion DNA. This type of DNA is characterized as viral by DNA-DNA hybridization. It is extremely sensitive to shear fragmentation. Extensive control experiments demonstrate that the fast-sedimenting viral DNA is not due to artifactual drag of viral DNA mechanically trapped in cellular DNA or to association of viral DNA with protein or RNA. Furthermore, the fast-sedimenting DNA is found after infection with multiplicities between 1 and 1,000 PFU/cell and from 6 to 8 h postinfection until very late in infection (24 h). Analysis in dye-buoyant density gradients eliminates the possibility that the fast-sedimenting viral DNA represents supercoiled circular molecules. Upon equilibrium centrifugation in alkaline CsCl density gradients, the fast-sedimenting viral DNA bands in a density stratum intermediate between that of cellular and viral DNA. In contrast, the 34S virion DNA isolated and treated in the same manner as the fast-sedimenting DNA cobands with viral marker DNA. After ultrasonic treatment of the fast-sedimenting viral DNA, it shifts to the density positions of viral DNA and to a lesser extent to that of cellular DNA. The evidence presented here demonstrates that the 50 to 90S viral DNA represents adenovirus DNA covalently integrated into cell DNA.     

Rates of formation and thermal stabilities of RNA:DNA and DNA:DNA duplexes at high concentrations of formamide.

The thermal stabilities of RNA:DNA hybrids are substantially greater than those of DNA:DNA duplexes in aqueous electrolyte solutions containing high concentrations of formamide. Association rates to form DNA:DNA duplexes and DNA:RNA hybrids have been measured in these solvents. There is a temperature range in which DNA:DNA rates are negligible and RNA:DNA rates close to optimal.     

Comparative effect of microwaves and boiling on the denaturation of DNA

The effect of heat and microwave denaturation of small volumes of double-stranded plasmid DNA has been compared. Samples of intact plasmid DNA had plasmid DNA linearized by digestion with EcoRI were conventionally denatured in a boiling water bath or denatured by 2450 MHz of microwave energy for 0-300 s. Heat denaturation for periods longer than 120 s caused breakdown of linearized plasmid DNA; however, microwave denaturation for 10-300 s caused no apparent degradation of linearized DNA. Breakdown of DNA forms II and III was noted in plasmid DNA subjected to 300 s of either heat or microwave denaturation but breakdown of forms II and III occurred more quickly with heat than with microwave treatment. Microwave treatment was also found to be better than heat to denature 32P-labeled DNA probes subsequently used to detect homologous DNA samples immobilized on nitrocellulose filters. A microwave-treated 32P-labeled DNA probe was able to hybridize to DNA samples 20 times more dilute than a heat-treated 32P-labeled DNA probe. Depending on the form of DNA to be analyzed, these results indicate that small volumes of DNA solutions and radiolabeled DNA probes can be effectively denatured in a conventional microwave oven.     

Theoretical analysis of DNA branch migration in the presence of a slow reversible initiation step

Branched DNA structures include several DNA regions connected by three- or four-way DNA junctions. Branched DNAs can be intermediates in DNA replication and recombination in living organisms and in sequence-specific DNA targeting in vitro. Branched DNA structures are usually metastable and irreversibly dissociate to non-branched products via a DNA strand exchange process commonly known as DNA branch migration. The key parameter in the DNA dissociation process is its characteristic time, which depends on the length of the dissociating DNA structure. Here, we predict that the presence of a slow reversible initiation step, which precedes DNA branch migration, can alter, to almost linear dependence, the "classic" quadratic dependence of the dissociation time on the length of the dissociating DNA structure. This prediction can be applied to dissociation of Y-like DNA structures and double D-loop DNA hybrids, which are DNA structures similar to replication bubbles. In addition, the slow initiation step can increase the effect of DNA sequence heterologies within the structure on its kinetic stability. Applications of our analysis for genetic manipulations with branched DNA structures are discussed.     

Involvement of eucaryotic deoxyribonucleic acid polymerases alpha and gamma in the replication of cellular and viral deoxyribonucleic acid.

In an effort to identify the deoxyribonucleic acid (DNA) polymerase activities responsible for mammalian viral and cellular DNA replication, the effect of DNA synthesis inhibitors on isolated DNA polymerases was compared with their effects on viral and cellular DNA replication in vitro. DNA polymerase alpha, simian virus 40 (SV40) DNA replication in nuclear extracts, and CV-1 cell (the host for SV40) DNA replication in isolated nuclei all responded to DNA synthesis inhibitors in a quantitatively similar manner: they were relatively insensitive to 2',3'-dideoxythymidine 5'-triphosphate (d2TTP), but completely inhibited by aphidicolin, 1-beta-D-arabinofuranosylcytosine 5'-triphosphate (araCTP), and N-ethylmaleimide. In comparison, DNA polymerases beta and gamma were inhibited by d2TTP but insensitive to aphidicolin and 20--30 times less sensitive to araCTP than DNA polymerase alpha. Herpes simplex virus type 1 (HSV-1) DNA polymerase and DNA polymerase alpha were the only enzymes tested that were relatively insensitive to d2TTP; DNA polymerases beta and gamma, phage T4 and T7 DNA polymerases, and Escherichia coli DNA polymerase I were 100--250 times more sensitive. The results with d2TTP were independent of enzyme concentration, primer-template concentration, primer-template choice, and the labeled dNTP. A specific requirement for DNA polymerase alpha in the replication of SV40 DNA was demonstrated by the fact that DNA polymerase alpha was required, in addition to other cytosol proteins, to reconstitute SV40 DNA replication activity in N-ethylmaleimide-inactivated nuclear extracts containing replicating SV40 chromosomes. DNA polymerases beta and gamma did not substitute for DNA polymerase alpha. In contrast to SV40 and CV-1 DNA replication, adenovirus type 2 (Ad-2) DNA replication in isolated nuclei was inhibited by d2TTP to the same extent as gamma-polymerase. Ad-2 DNA replication was also inhibited by aphidicolin to the same extent as alpha-polymerase. Synthesis of CV-1 DNA, SV40 DNA, and HSV-1 DNA in intact CV-1 cells was inhibited by aphidicolin. Ad-2 DNA replication was also inhibited, but only at a 100-fold higher concentration. We found no effect of 2'-3'-dideoxythymidine (d2Thd) on cellular or viral DNA replication in spite of the fact that Ad-2 DNA replication in isolated nuclei was inhibited 50% by a ratio of d2TTP/dTTP of 0.02. This was due to the inability of CV-1 and Hela cells to phosphorylate d2Thd to d2TTP. These data are consistent with the hypothesis that DNA polymerase alpha is the only DNA polymerase involved in replicating SV40 DNA and CV-1 DNA and that Ad-2 DNA replication involves both DNA polymerases gamma and alpha.     

Characterization of a stable, major DNA polymerase alpha species devoid of DNA primase activity.

We have purified from Xenopus laevis ovaries a major DNA polymerase alpha species that lacked DNA primase activity. This primase-devoid DNA polymerase alpha species exhibited the same sensitivity as the DNA polymerase DNA primase alpha to BuAdATP and BuPdGTP, nucleotide analogs capable of distinguishing between DNA polymerase delta and DNA polymerase DNA primase alpha. The primase-devoid DNA polymerase alpha species also lacked significant nuclease activity indicative of the alpha-like (rather than delta-like) nature of the DNA polymerase. Using a poly(dT) template, the primase-devoid DNA polymerase alpha species elongated an oligo(rA10) primer up to 51-fold more effectively than an oligo(dA10) primer. In direct contrast, the DNA polymerase DNA primase alpha complex showed only a 4.6-fold preference for oligoribonucleotide primers at the same template/primer ratio. The catalytic differences between the two DNA polymerase alpha species were most dramatic at a template/primer ratio of 300. The primase-devoid DNA polymerase alpha species was found at high levels throughout oocyte and embryonic development. This suggests that the primase-devoid DNA polymerase alpha species could play a physiological role during DNA chain elongation in vivo, even if it is chemically related to DNA polymerase DNA primase alpha.     

Intracellular Replication of Mycoplasmavirus MVL51

The intracellular replication of MVL51, a group L1 mycoplasmavirus, was investigated. The single-stranded parental DNA was found to enter the cell and become converted to double-stranded DNA. This replicated to yield additional double-stranded DNA molecules. The parental viral DNA was found to leave the replication complex and become associated with large molecular weight DNA not involved with viral replication. Progeny viral DNA formed from the double-stranded DNA and an intracellular accumulation of virus chromosome size DNA was observed. The interpretation of this data and a suggested model for the viral replication are discussed and compared to viral DNA replication models for other single-stranded DNA viruses.     

Bypass of a nick by the replisome of bacteriophage T7

DNA polymerase and DNA helicase are essential components of DNA replication. The helicase unwinds duplex DNA to provide single-stranded templates for DNA synthesis by the DNA polymerase. In bacteriophage T7, movement of either the DNA helicase or the DNA polymerase alone terminates upon encountering a nick in duplex DNA. Using a minicircular DNA, we show that the helicase · polymerase complex can bypass a nick, albeit at reduced efficiency of 7%, on the non-template strand to continue rolling circle DNA synthesis. A gap in the non-template strand cannot be bypassed. The efficiency of bypass synthesis depends on the DNA sequence downstream of the nick. A nick on the template strand cannot be bypassed. Addition of T7 single-stranded DNA-binding protein to the complex stimulates nick bypass 2-fold. We propose that the association of helicase with the polymerase prevents dissociation of the helicase upon encountering a nick, allowing the helicase to continue unwinding of the duplex downstream of the nick.     

Interactions of Polyoma and Mouse DNAs. IV. Time Course and Extent of Integration of Polyoma DNA into Mouse DNA During Lytic Infection

The time course of covalent binding of polyoma viral DNA to mouse DNA was followed in mouse embryo cells that had been grown prior to infection in the presence of 5-bromodeoxyuridine. Density-labeled (HL) mouse DNA was separated from free polyoma DNA by CsCl isopycnic centrifugation. Polyoma DNA sequences present in HL mouse DNA were detected by hybridization with radioactive cRNA synthesized in vitro. In reconstruction experiments, the limit of detection was found to be, on the average, about 0.5 genome equivalent (g.e.) of polyoma DNA per cell. To find conditions for the isolation of HL mouse DNA and for its complete separation from free polyoma DNA, cultures infected at 4 degrees C were used. HL mouse DNA extracted with sodium dodecyl sulfate and high salt concentrations (5 to 6 M CsCl) and then purified by three consecutive CsCl density gradient centrifugations was free from detectable amounts of polyoma DNA, whereas HL mouse DNA extracted with chloroform and phenol and purified in the same way always contained contaminating, noncovalently bound polyoma DNA. In lytically infected bromodeoxyuridine-prelabeled mouse embryo cultures, polyoma DNA bound to HL mouse DNA that had been extracted by the sodium dodecyl sulfate-CsCl procedure was first detected in small amounts (1 to 2 g.e. per cell) at 10 h after infection. In cultures incubated with medium containing thymidine (5 mug/ml), 4 to 6 g.e. of polyoma DNA per cell was detected at 14 and 18 h after infection. In these samples, practically all viral DNA was bound to high-molecular-weight HL mouse DNA. In cultures incubated with normal medium (no additions) and extracted between 17 and 20 h after infection, 20 to 350 g.e. of polyoma DNA per cell banded with HL mouse DNA. However, when DNA of one of these samples was subfractionated by sodium dodecyl sulfate-salt precipitation prior to isolation of HL mouse DNA, about 80% of the viral DNA banding at increased density was present in the low-molecular-weight DNA fraction. This observation suggests that in normal medium some progeny viral DNA of increased density was synthesized. Covalent binding of polyoma DNA to density-labeled mouse DNA was demonstrated by alkaline CsCl density gradient centrifugation: nearly equal amounts of polyoma DNA were found in the H and L strands, respectively, as is expected for linear integration of viral DNA. The results lead to the conclusions that (i) early polyoma mRNA is transcribed from free parental viral DNA; (ii) covalent linear integration is first detectable at the time when tumor (T)-antigen is synthesized; and (iii) only few copies (<10 g.e./cell) become integrated between 10 and 18 h after infection, i.e., during the period when cellular and viral DNA replication starts in individual cells.     

Denaturation of mouse satellite DNA upon melting of chromatin in solution

The denaturation of mouse satellite DNA upon melting of chromatin in solution of low ionic strength has been studied. A procedure for preparation of partially denaturated chromatin was developed which enabled the isolation of double-stranded (non-denatured) DNA sequences according to their thermal stability in chromatin. The content of mouse satellite DNA in these DNA sequences was determined by hybridization with RNA, complementary to satellite DNA in order to find the temperature interval of denaturation of satellite DNA. It was found that the melting temperature of satellite DNA in chromatin was lower than that of the total DNA. The results are discussed in relation to previously reported anomalous behaviour of satellite DNA upon melting of chromatin on hydroxyapatite.     

Molecular Weight of Deoxyribonucleic Acid Synthesized During Initiation of Chromosome Replication in Escherichia coli

Alkaline sucrose gradients were used to study the molecular weight of deoxyribonucleic acid (DNA) synthesized during the initiation of chromosome replication in Escherichia coli 15 TAU-bar. The experiments were conducted to determine whether newly synthesized, replication origin DNA is attached to higher-molecular-weight parental DNA. Little of the DNA synthesized after readdition of required amino acids to cells previously deprived of the amino acids was present in DNA with a molecular weight comparable to that of the parental DNA. The newly synthesized, low-molecular-weight DNA rapidly appeared in higher-molecular-weight material, but there was an upper limit to the size of this intermediate-molecular-weight DNA. This limit was not observed when exponentially growing cells converted newly synthesized DNA to higher-molecular-weight material. The size of the intermediate-molecular-weight DNA was related to the age of the replication forks, and the size increased as the replication forks moved further from the replication origin. The results indicate that the newly synthesized replication origin DNA is not attached to parental DNA, but it is rapidly attached to the growing strands that extend from the replication fork to the replication origin, or to the other replication fork if replication is bidirectional. Experiments are reported which demonstrate that the DNA investigated was from the vicinity of the replication origin and was not plasmid DNA or DNA from random positions on the chromosome.     

Influence of template inactivators on the binding of DNA polymerase to DNA

The agents daunomycin, ethidium bromide, distamycin A and cytochrome c inhibit DNA dependent DNA polymerase I (E. coli) reaction competitively to DNA. The influence of these template inactivators on the binding of DNA polymerase to native as well as denatured DNA has been determined by affinity chromatography. Cytochrome c blocks the binding of the enzyme to double-stranded and to single-stranded DNA Sepharose. In contrast to these results daunomycin, ethidium bromide or distamycin A reduce the binding affinity only with denatured DNA Sepharose as matrix. These data are discussed with respect to the modification by template inactivators of the affinity of DNA to the different binding sites of the DNA polymerase.     

Stable integration of foreign DNA into the chromosome of the cyanobacterium Synechococcus R2

The blue-green alga, Synechococcus R2, is transformed to antibiotic resistance by chimeric DNA molecules consisting of Synechococcus R2 chromosomal DNA linked to antibiotic-resistance genes from Escherichia coli. Chimeric DNA integrates into the Synechococcus R2 chromosome by homologous recombination. The efficiency of transformation, as well as the stability of integrated foreign DNA, depends on the position of the foreign genes relative to Synechococcus R2 DNA in the chimeric molecule. When the Synechococcus R2 DNA fragment is interrupted by foreign DNA, integration occurs through replacement of chromosomal DNA by homologous chimeric DNA containing the foreign insert; transformation is efficient and the foreign gene is stable. Mutagenesis in some cases attends integration, depending on the site of insertion. Foreign DNA linked to the ends of Synechococcus R2 DNA in a circular molecule, however, integrates less efficiently. Integration results in duplicate copies of Synechococcus R2 DNA flanking the foreign gene and the foreign DNA is unstable. Transformation in Synechococcus R2 can be exploited to modify precisely and extensively the genome of this photosynthetic microorganism.     

Uptake of circular deoxyribonucleic acid and mechanism of deoxyribonucleic acid transport in genetic transformation of Streptococcus pneumoniae.

Deoxyribonucleic acid (DNA) from the covalently closed circular DNA molecules of Pseudomonas phage PM2 was found to enter normally transformable cells of Streptococcus pneumoniae as readily as linear bacterial DNA. In a mutant of S. pneumoniae that lacks a membrane nuclease and is defective in DNA entry, as many molecules of PM2 DNA as of linear DNA were bound on the outside of cells at equivalent DNA concentrations. Bound DNA suffered single-strand breaks, but circular DNA with preexisting breaks was bound no better than closed circles. In the presence of divalent cations, DNA bound to cells of a leaky nuclease mutant showed double-strand breaks. At least the majority of PM2 DNA that entered normal cells was single stranded. These results are consistent with a mechanism for DNA entry in which DNA is first nicked on binding, then a double-strand break is formed by cleavage of the complementary strand, and continued processive action of the membrane nuclease facilitates entry of the originally nicked strand. Although the bulk of circular donor DNA appeared to enter in this way, the results do not exclude entry of a small amount of donor DNA in an intact form.     

Interaction of contractile proteins with DNA

The interaction of contractile proteins (myosin, actin, tropomyosin and troponin) with DNA was studied in vitro using a nitrocellulose filter binding technique. The data indicate a high affinity of myosin and troponin for DNA, a lesser interaction between DNA and tropomyosin and the absence of binding of actin to DNA. When binding to DNA was detected, the interaction was higher with single-stranded DNA than with RNA or double-stranded DNA, although in some conditions myosin binds equally as well to native as to denatured eukaryotic DNA. Myosin binds better to eukaryotic than to phage native DNA.     

Characterization of cytoplasmic DNA of mouse-myeloma cells.

Cytoplasmic DNA from mouse myeloma cells comprised between 1% and 2% of the total cellular DNA. Detergent-prepared cytoplasmic lysate consisted mainly of 8-S and 22-S species. While these DNA species were present in the 13000 times g pellet of the detergent-prepared cytoplasmic lysate, only the light DNA species was present in the 13000 times g supernatant fraction. In neutral CsCl gradients the DNA of both cytoplasmic fractions had a buoyant density of 1700 g/cm3, which is identical to that of nuclear DNA. The similarity between the cytoplasmic and nuclear DNA was also demonstrated by analysis on alkaline CsCl gradients. A small proportion of closed-circular DNA, presumably of mitochondrial origin, was demonstrated only in cytoplasmic fraction obtained from mechanically disrupted cells and not in detergent-prepared cytoplasmic lysate. It was found that poly (A)-containing mRNA and 28-S ribosomal RNA hybridized to about the same extent to the cytoplasmic DNA as compared to nuclear DNA. The results indicate that most of the cytoplasmic DNA in myeloma cells is similar to nuclear DNA and does not consist of mitochondrial DNA.     

Cisplatin-DNA adducts inhibit translocation of the Ku subunits of DNA-PK

We have determined the effect of cisplatin–DNA damage on the ability of the DNA-dependent protein kinase (DNA-PK) to interact with duplex DNA molecules in vitro. The Ku DNA binding subunits of DNA-PK display a reduced ability to translocate on duplex DNA containing cisplatin–DNA adducts compared to control, undamaged duplex DNA. The decreased rates of translocation resulted in a decrease in the association of the p460 catalytic subunit of DNA-PK (DNA-PKcs) with the Ku–DNA complex. In addition to a decrease in DNA-PKcs association, the DNA-PKcs that is bound with Ku at a DNA end containing cisplatin–DNA adducts has a reduced catalytic rate compared to heterotrimeric DNA-PK assembled on undamaged DNA. The position of the cisplatin–DNA lesion from the terminus also effects kinase activation, with maximal inhibition occurring when the lesion is closer to the terminus. These results are consistent with a model for DNA-PK activation where the Ku dimer translocates away from the DNA terminus and facilitates the association of DNA-PKcs which interacts with both Ku and DNA resulting in kinase activation. The presence of cisplatin adducts decreases the ability to translocate away from the terminus and results in the formation of inactive kinase complexes at the DNA terminus. The results are discussed with respect to the ability of cisplatin to sensitize cells to DNA damage induced by ionizing radiation and the ability to repair DNA double-strand breaks.     

Further biochemical characterization of wheat DNA primase: possible functional implication of copurification with DNA polymerase A.

DNA primase has been partially purified from wheat germ. This enzyme, like DNA primases characterized from many procaryotic and eucaryotic sources, catalyses the synthesis of primers involved in DNA replication. However, the wheat enzyme differs from animal DNA primase in that it is found partially associated with a DNA polymerase which differs greatly from DNA polymerase alpha. Moreover, the only wheat DNA polymerase able to initiate on a natural or synthetic RNA primer is DNA polymerase A. In this report we describe in greater detail the chromatographic behaviour of wheat DNA primase and its copurification with DNA polymerase A. Some biochemical properties of wheat DNA primase such as pH optimum, Mn + 2 or Mg + 2 optima, and temperature optimum have been determined. The enzyme is strongly inhibited by KCI, cordycepine triphosphate and dATP, and to a lesser extent by cAMP and formycine triphosphate. The primase product reaction is resistant to DNAse digestion and sensitive to RNAse digestion. Primase catalyses primer synthesis on M13 ssDNA as template allowing E.coli DNA polymerase I to replicate the primed M13 single-stranded DNA leading to double-stranded M13 DNA (RF). M13 replication experiments were performed with wheat DNA polymerases A, B, CI and CII purified in our laboratory. Only DNA polymerase A is able to recognize RNA-primed M13 ssDNA.