Comparison of the interactions of the adenovirus type 2 major core protein and its precursor with DNA.

The interactions of the major core protein of adenovirus type 2 (Ad2) protein VII, and its precursor, protein pre-VII, with viral DNA, were studied using UV light induced crosslinking of 32P-labelled oligonucleotides to the proteins. Proteolytic fragments of these two proteins that contain DNA-binding domains were identified by virtue of their covalently attached, alkali-resistant 32P-radioactivity. The overall efficiency of crosslinking of protein pre-VII to DNA, in H2ts1 virions assembled at 39 degrees C, was comparable to that of the crosslinking of protein VII to DNA in Ad2 virions. However, a protease V8 fragment comprising the N-terminal half of protein pre-VII crosslinked to DNA at least ten times more efficiently than the corresponding N-terminal fragment of protein VII, which is truncated by the removal of 23 amino acids from the N-terminus of protein pre-VII during virion maturation.     

Adenoviral protein VII packages intracellular viral DNA throughout the early phase of infection.

The proteins associated with parental, adenoviral DNA in productively-infected HeLa cells have been examined both directly and indirectly. HeLa cells infected with 32P-labelled Ad2 were irradiated with u.v. light at various points in the infectious cycle. Following degradation of the DNA, nuclear proteins carrying cross-linked nucleotides, or oligonucleotides, were distinguished from virion phosphoproteins by the resistance of their 32P radioactivity to 1 M NaOH. The major core protein of the virion, protein VII, was found to be associated with viral DNA throughout infection, even when cells were infected at a multiplicity of 0.14. Micrococcal nuclease digestion of intranuclear viral DNA 4 h after infection liberated two nucleoprotein particles containing viral DNA, neither of which co-migrated with HeLa cell mononucleosomes. These results indicate that core protein VII remains associated with parental adenoviral DNA during productive infections. The observation that protein VII can be cross-linked to DNA in cells infected at very low multiplicity, together with the results of a comparison of proteins cross-linkable to viral DNA in cells infected by wild-type virus and a non-infectious mutant containing the precursor to protein VII, suggest that nucleoproteins comprising viral DNA and protein VII must be the templates for expression of pre-early and early viral genes.     

Adenovirus protein VII functions throughout early phase and interacts with cellular proteins SET and pp32

Adenovirus protein VII is the major component of the viral nucleoprotein core. It is a highly basic nonspecific DNA-binding protein that condenses viral DNA inside the capsid. We have investigated the fate and function of protein VII during infection. "Input" protein VII persisted in the nucleus throughout early phase and the beginning of DNA replication. Chromatin immunoprecipitation revealed that input protein VII remained associated with viral DNA during this period. Two cellular proteins, SET and pp32, also associated with viral DNA during early phase. They are components of two multiprotein complexes, the SET and INHAT complexes, implicated in chromatin-related activities. Protein VII associated with SET and pp32 in vitro and distinct domains of protein VII were responsible for binding to the two proteins. Interestingly, protein VII was found in novel nuclear dot structures as visualized by immunofluorescence. The dots likely represent individual infectious genomes in association with protein VII. They appeared within 30 min after infection and localized in the nucleus with a peak of intensity between 4 and 10 h postinfection. After this, their intensity decreased and they disappeared between 16 and 24 h postinfection. Interestingly, disappearance of the dots required ongoing RNA synthesis but not DNA synthesis. Taken together these data indicate that protein VII has an ongoing role during early phase and the beginning of DNA replication.     

DNA-binding properties of an adenovirus 289R E1A protein.

An adenovirus 2 289 amino acid (289R) E1A protein purified from Escherichia coli has been shown to interact with DNA by two independent methods. UV-crosslinking of complexes containing unmodified, uniformly 32P-labelled DNA and purified E1A protein induced efficient labelling of the protein with covalently attached oligonucleotides, indicating that the E1A protein itself contacts DNA. Discrete nucleoprotein species were also observed when E1A protein--DNA complexes were analysed by gel electrophoresis. Although the 289R E1A protein exhibited no significant binding to single-stranded DNA or to RNA, no evidence for its sequence-specific binding to double-stranded DNA was obtained with either assay. Identification of the sites of covalent attachment of 32P-labelled oligonucleotides by partial proteolysis of the crosslinked E1A protein indicated that the interaction of this protein with DNA is mediated via domain(s) in the C-terminal half of the protein. Such previously unrecognized DNA-binding activity is likely to contribute to the regulatory activities of this important adenoviral protein.     

The structure of nucleoprotein cores released from adenovirions.

The morphology, protein composition and DNA organization of nucleoprotein core complexes isolated from type 5 adenovirions have been examined by electron microscopy and biochemical techniques. The morphology of such core structures is in some ways strikingly similar to that exhibited by cellular chromatin. 'Native' core preparations contain compact and less highly-folded forms: the latter appear as thick fibres, 150-300A in diameter. Upon exposure to 0.4M NaCl, adenovirus cores undergo a transition to a beaded string form, reminiscent of nucleosomes. Of the three arginine-rich proteins, polypeptides V, VII and mu present in 'native' cores, only polypeptide VII remains associated with viral DNA in the presence of 0.4M NaCl. We therefore conclude that the nucleosome-like beads are constructed solely of polypeptide VII. The results of micrococcal nuclease digestion experiments suggest that polypeptide VII is sufficient to protect some 100-300bp of adenoviral DNA.     

A method for detecting protein-DNA interactions at sites of chromatin replication

Two versions of an approach to identify DNA-protein interactions at sites of DNA replication in HeLa cell nuclei are described. In this procedure, newly replicated DNA chains are first labeled and photosensitized in vitro by the incorporation of [α-³²P]dCTP and bromodeoxyuridine triphosphate, respectively. Irradiation with ultraviolet light is then used to covalently crosslink the proteins that are adjacent to the photosensitized and isotopically labeled strands of newly replicated DNA. After the bulk of the DNA is digested with nucleases, the crosslinked proteins—marked by short covalently linked radioactive DNA tags—are fractionated by electrophoresis in sodium dodecyl sulfate-polyacrylamide gels and detected by autoradiography. With this technology, certain proteins have been shown to associate selectively with newly replicated DNA. The method appears adaptable for application to a variety of problems involving DNA-protein association.     

Protein kinase in HeLA nucleosomes: a reevaluation of the interactions of histomes with the ends of core particle DNA.

HeLa chromatin core particles contain a protein kinase which transfers phosphate from ATP to both nonhistone proteins and histones. The enzyme preferentially modifies H3 among the histones; about 7% of the H3 molecules in the nucleoprotein are modified at saturation. Activity of this kinase likely contributed to earlier results using crosslinking methodology to study which histones interact with the ends of core particle DNA. When the kinase is largely removed by sedimentation of core particles through sucrose gradients containing 0.45 M NaCl, crosslinking of the 5'-terminal label on DNA is observed only to histone H3. The overall efficiency of the crosslinking reaction is about 15%. The origin of the 5'-terminal 32P previously assigned as crosslinked to H4 is not explained by the current experiments.     

Stable association of viral protein VP1 with simian virus 40 DNA.

Mild dissociation of simian virus 40 particles releases a 110S virion core nucleoprotein complex containing histones and the three viral proteins VP1, VP2, and VP3. The association of viral protein VP1 within this nucleoprotein complex is mediated at least partially through a strong interaction with the viral DNA. Treatment of the virion-derived 110S nucleoprotein complex with 0.25% Sarkosyl dissociated VP2, VP3, and histones, leaving a stable VP1-DNA complex. The VP1-DNA complex had a sedimentation value of 30S and a density of 1.460 g/cm3. The calculated molecular weight of the complex was 7.9 x 10(6), with an average of 100 VP1 molecules per DNA. Agarose gel electrophoresis of the VP1-DNA complex demonstrated that VP1 is associated not only with form I and form II simian virus 40 DNAs but also with form III simian virus 40 DNA generated by cleavage with EcoRI.     

Binding of 4'-aminomethyl 4,5',8-trimethyl psoralen to DNA, RNA and protein in HeLa cells and Drosophila cells.

In Drosophila cells and HeLa cells treated with 4'-aminomethyl trioxsalen and ultraviolet light, this compound binds covalently to DNA and RNA. The maximum number of molecules bound to 10(3) base pairs in DNA is 60 and in RNA it is 20. In nuclei treated likewise the number of molecules bound to 10(3) base pairs in DNA can be as high as 376. When cells are irradiated in the frozen state the number of 4'-aminomethyl trioxsalen molecules bound per 10(3) base pairs in DNA is about 40 and in RNA about 20. DNA molecules from cells or nuclei treated with 4'-aminomethyl trioxsalen and ultraviolet light are highly crosslinked and appear as loops interspersed by double stranded regions when analyzed in the electron microscope under denaturing conditions. The loop sizes are heterogeneous and the fraction of double stranded regions increases to almost complete double-strandedness at high degrees of reaction. No secondary structures could be found in ribosomal RNA from Drosophila cells or HeLa cells after treatment with 4'-aminomethyl trioxsalen and ultraviolet light. In cells treated with 4'-aminomethyl trioxsalen and ultraviolet light the RNAase activity is increased considerably suggesting a release of lysosomal enzymes. 4'-aminomethyl trioxsalen and its photodecomposition products bind strongly to cellular proteins.     

DNA-binding properties of the major core protein of adenovirus 2.

The major adenovirus core protein (P.VII) binds to various species of duplex and single-stranded DNA molecules as a linear function of P.VII concentration. P.VII progressively condenses 32S Ad2 DNA into rapidly sedimenting forms having an S value of around 2,280. P.VII does not coat DNA like cytochrome C, instead DNA-protein beads are visualized in the electron microscope at low protein concentration. These beads appear to interact forming larger structures and at high P.VII concentrations the DNA molecule becomes highly compacted. Analysis of DNA fragments formed after digestion of P.VII-DNA complexes and isolated cores with micrococcal nuclease suggest that the organization of the DNA in the two structures is essentially identical. The initial P.VII and DNA interaction is sensitive to both ionic and hydrophobic environments, whereas the in vitro DNA-P.VII complexes are extremely stable and are not disrupted in the presence of 3 M NaCl, 1% sarcosyl or 5% deoxycholate. Properties of these in vitro DNA-protein VII complexes share striking similarities to isolated viral core particles.     

Isolation and characterization of adenovirus core nucleoprotein subunits.

Digestion of adenovirus type 2 (Ad2) or Ad5 cores with micrococcal nuclease generated four nucleoprotein species that could be resolved by electrophoresis in low-ionic-strength polyacrylamide gels: these nucleoproteins displayed mobilities equivalent to those of DNA fragments of 900 to 1,025, 775 to 850, 650 to 725, and 525 to 600 base pairs (bp) and thus were readily distinguishable from HeLa cell mononucleosomes. The DNA fragments associated with the core nucleoprotein species were more than 250 to 90 bp long. Nucleoproteins containing 150, 120, or 90 bp of DNA were the most stable. Polypeptide VII was associated with each of the nucleoprotein species liberated from Ad2 cores. These data suggest that polypeptide VII and viral DNA of 90 to 150 bp comprise the unit particle of the Ad2 or Ad5 core nucleoproteins.     

Adenovirus core protein synthesis in the absence of viral DNA synthesis late in infection.

The acid extraction of the adenovirus type 5 core proteins V, VII, and pVII (the precursor to VII) from infected cells and the subsequent electrophoresis on a 15% acrylamide-2.5 M urea-0.9 N acetic acid (pH 2.7) gel, revealed that peptide VII has a similar electrophoretic mobility to that of histone H1. The core proteins, which are coded by late adenovirus mRNA, continued to be synthesized late in infection when viral DNA synthesis was inhibited either by cytosine arabinoside in wild-type infections or by shifting adenovirus H5 ts 125-infected cells to the nonpermissive temperature (40 degree C). Only the initiation, not the continuation, of viral DNA replication was essential for core protein synthesis. The synthesis of viral core proteins continued for over 8 h after the cassation of DNA synthesis. This was in contrast to the rapid shutdown of cellular histone synthesis in the absence of cellular DNA synthesis.     

Crosslinking of DNA to nuclear lamina proteins by UV irradiation in vivo

We have been able to demonstrate that a fraction of DNA becomes crosslinked to nuclear lamina shells isolated from Ehrlich ascites tumour cells irradiated with UV light. Terminal labeling of short DNA fragments covalently attached to proteins reveals that DNA has become crosslinked to all three lamins and to a protein comigrating with vimentin.     

Physical and functional interaction between a nucleolar protein nucleophosmin/B23 and adenovirus basic core proteins

We identified nucleophosmin/B23 as a component of template-activating factor-III that stimulates the DNA replication from the adenovirus DNA complexed with viral basic core proteins. Here, we have studied the functional interaction of B23 with viral core proteins. We found that B23 interacts with viral basic core proteins, core protein V and precursor of core protein VII (pre-VII), in infected cells. Biochemical analyses demonstrated that B23 suppresses formation of aggregates between DNA and core proteins and transfers pre-VII to DNA. These results indicate that B23 functions as a chaperone in the viral chromatin assembly process in infected cells.     

Photochemical crosslinking of bacteriophage T4 single-stranded DNA-binding protein (gp32) to oligo-p(dT)8: identification of phenylalanine-183 as the site of crosslinking

Using ultraviolet light, both the 33,000-dalton single-stranded DNA-binding protein from T4 bacteriophage (gp32) as well as a 25,000-dalton limited trypsin cleavage product of gp32 (core gp32*) that retains high affinity for single-stranded DNA can be crosslinked to an oligodeoxynucleotide, p(dT)8. After photolysis, a single tryptic peptide crosslinked to p(dT)8 was isolated by anion-exchange high-performance liquid chromatography. Gas-phase sequencing of this modified peptide gave the following sequence: Gln-Val-Ser-Gly-(X)-Ser-Asn-Tyr-Asp-Glu-Ser-Lys, which corresponds to residues 179-190 in gp32. Based on the absence of the expected phenylthiohydantoin derivative of phenylalanine 183 at cycle 5 (X) we infer that crosslinking has occurred at this position and that phenylalanine 183 is at the interface of the gp32:p(dT)8 complex in an orientation that allows covalent bond formation with the thymine radical produced by ultraviolet irradiation.     

A comparison of the interactions of proflavine with DNA and with deoxyribonucleohistone using circular dichroism spectroscopy

Complexes of proflavine with DNA and deoxyribonucleohistone from calf thymus show different optical activity in the visible and the ultraviolet. Although the visible CD spectra of both complexes arise from interactions of dye molecules, the variation in the optical activity with the amount of dye bound suggests that a lesser conformational mobility exists in DNH. This is confirmed by the ultraviolet CD spectra of the complexes, and it is suggested that the conformation of DNA within nucleohistone is altered by separation of the base pairs by a greater extent than occurs in DNA in free solution. Even if the protein is unequally distributed along the DNA, the conformation of all of the DNA is altered by its incorporation into the nucleoprotein complex, since no evidence could be detected to show that DNA in a “free” conformation existed.     

Monoazido analog of ethidium as a chromatin probe: Binding to DNA

Two photoaffinity analogs of ethidium, 8-azido-3-amino, and 3-azido-8-amino-5-ethyl-6-phenylphenanthridinium chloride, have been used to probe the structure of mammalian chromatin and its interactions with the ethidium moiety. The monoazido analogs were established as suitable probes by comparing their interactions with chromatin and pure DNA prepared from chromatin to those of the parent ethidium bromide. Scatchard analysis of the binding data determined from spectrophotometric titrations showed that the analogs interacted with both nucleic acids in a manner similar to the parent compound. The effect of chromatin proteins on the interaction of the ethidium moiety with intact chromatin was investigated directly. By exposing the noncovalent complex to visible light, the monoazido analog was attached covalently in its interaction sites within chromatin, and the amount of drug bound covalently to DNA was determined for both protein-free DNA and chromatin. Using saturating concentrations of drug, DNA within intact chromatin was found to be associated with only half as much drug as DNA extracted from its protein prior to drug exposure. The distribution of drug bound within chromatin was determined following the attachment of the monoazido analog (by photoactivation) to chromatin that had undergone limited nuclease digestion. Several distinct populations isolated by size fractionation and quantitative measurements revealed that (1) both the core particles and the spacer-containing particles contained bound drug, reflecting high-affinity binding sites; and (2) chromatin particles containing 150 DNA base pairs (putatively nucleosome core structures) contained less total bound drug at high drug concentrations than those particles having intact spacer DNA.