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PubMed1985

Influence of embedding media on DNA structure in herpes simplex virus type 1.

The organization of encapsidated herpes simplex viral DNA in situ was examined by use of the osmium-amine stain specific for DNA. After either formaldehyde or glutaraldehyde fixation the DNA is packaged in a compact toroid without inner structure with Epon or GMA embedment but revealed a complex inner structure with Lowicryl K4M embedment. In the latter there was an inner cylindrical core, 50 X 80 nm, around which were apposed one or more thick filaments of 5-8 nm diameter. Thinner DNA filaments of 3-4 nm diameter form a cage of loose coils around the core with an intervening space of approximately 15 nm. Lowicryl embedding may be considered as a tool to investigate the packaging of viral DNA in virions.

Acrylic Resins↗
PubMed1999

Modulation of DNA damage and DNA repair in chromatin.

DNA is packaged in the highly compact and dynamic structure of chromatin in eukaryotic cells. It is generally accepted that DNA processing events in the nucleus, such as transcription, replication, recombination, and repair, are restricted by this packaging. For some processes (e.g., transcription), the chromatin fiber is "preset" in a more open structure to allow access of proteins to specific regions of DNA within this structural hierarchy. These regions contain modified nucleosomes that accommodate a less compact state of chromatin and allow access to specific regions of DNA. DNA repair proteins, however, must access DNA lesions in all structural domains of chromatin after sudden insult to the genome. Damaged DNA must be recognized, removed, and replaced by repair enzymes at all levels of chromatin packaging. Therefore, the modulation of DNA damage and its repair in chromatin is crucial to our understanding of the fate of potential mutagenic and carcinogenic lesions in DNA. In this review, we discuss the modulation of DNA damage and DNA repair by chromatin structure, and the modulation of chromatin structure by these events.

Animals↗
PubMed1990

Involvement of topoisomerases in replication, transcription, and packaging of the linear adenovirus genome.

The role of topoisomerases in the replication of human adenovirus type 5 was investigated with topoisomerase inhibitors. Both topoisomerase I and topoisomerase II inhibitors blocked adenovirus replication when added at the time of infection. Both types of inhibitors induced strand cleavages at specific sites in the adenovirus early templates. The cleavage sites were mapped near the 5' and 3' ends of the genes transcribed early during infection. At late times after infection, camptothecin, a topoisomerase I inhibitor, inhibited adenovirus DNA replication and induced the formation of single- and double-stranded fragments with breakpoints located at defined regions of the viral genome. The topoisomerase II inhibitors, VP-16 (etoposide) and ellipticine, did not block adenovirus DNA replication and did not induce an appreciable amount of double-strand cleavages in the newly synthesized DNA. On the other hand, VP-16 promoted double-strand cleavages at specific sites of nonreplicating adenovirus DNA. The packaging of adenovirus DNA into virus particles, which contain supercoiled adenovirus DNA (M.-L. Wong and M.-T. Hsu, Nucleic Acids Res. 17:3535-3550, 1989), was inhibited by the topoisomerase II inhibitors. Transcription of adenovirus major late genes was inhibited by both topoisomerase I and topoisomerase II inhibitors. In addition, camptothecin caused a premature termination of major late transcription. Electron microscopic analysis showed that adenovirus templates late after infection were arranged in topologically constrained loop domains. Together, these data provide evidence for the requirement of topoisomerase activities in the replication, transcription, and packaging of the linear adenovirus genome.

Adenoviruses, Human↗
PubMed2004

IHF and HU: flexible architects of bent DNA.

The energetic cost of bending short segments of DNA is very high. This bending is critical for the packaging of DNA and is exploited to regulate many cellular processes. In prokaryotes, IHF and HU are key architectural proteins present at high concentrations. New protein-DNA co-crystal structures, and the adaptation of advanced biophysical and biochemical techniques have led to an improved understanding of how these proteins interact with DNA. These techniques include time-resolved synchrotron X-ray footprinting, differential scanning calorimetry, isothermal titration calorimetry and single-molecule experiments.

Amino Acid Sequence↗
PubMed1997

High resolution microanalysis and three-dimensional nucleosome structure associated with transcribing chromatin.

The nucleosome is the ubiquitous and fundamental DNA-protein complex of the eukaryotic chromosome, participating in the packaging of DNA and in the regulation of gene expression. Biophysical studies have implicated changes in nucleosome structure from chromatin that is quiescent to active in transcription. Since DNA within the nucleosome contains a high concentration of phosphorus whereas histone proteins do not, the nucleosome structure is amenable to microanalytical electron energy loss mapping of phosphorus to delineate the DNA within the protein-nucleic acid particle. Nucleosomes associated with transcriptionally active genes were separated from nucleosomes associated with quiescent genes using mercury-affinity chromatography. The three-dimensional image reconstruction methods for the total nucleosome structure and for the 3D DNA-phosphorus distribution combined quaternion-assisted angular reconstitution of sets of single particles at random orientations and electron spectroscopic imaging. The structure of the active nucleosome has the conformation of an open clam-shell, C- or U-shaped in one view, elongated in another, and exhibits a protein asymmetry. A three-dimensional phosphorus map reveals a conformational change in nucleosomal DNA compared to DNA in the canonical nucleosome structure. It indicates an altered superhelicity and is consistent with unfolding of the particle. The results address conformational changes of the nucleosome and provide a direct structural linkage to biochemical and physiological changes which parallel gene expression.

Chromatin↗
PubMed1981

An unusual symmetric recombinant between adenovirus type 12 DNA and human cell DNA.

On purification of human adenovirus type 12 (Ad12) by equilibrium sedimentation in CsCl density gradients, two bands of particles, Ad12-3 and Ad12-3a, are observed. The particles from band Ad12-3a contain a recombinant of human host cell DNA and of Ad12 DNA. The human cell DNA sequences contain repetitive DNA recurring 200 to 500 times in cellular DNA. Ad12 DNA and the recombinant genomes exhibit the same or similar lengths. This finding suggests that a constant amount of DNA is packaged into complete Ad12 particles. On cleavage of KB cellular DNA with EcoRI, BamHI, HinfI, Msp I, Mbo I Pst I, or Bgl II, the (32)P-labeled cellular DNA from Ad12-3a particles hybridizes on Southern blots to distinct bands of KB DNA. There is also less-specific background hybridization that is not observed in the control. The cellular DNA from Ad12-3a particles is not methylated, whereas the same cellular sequences in KB cell DNA appear to be extensively methylated. On denaturation and renaturation, the recombinant DNA molecules are converted to molecules half as long as Ad12 DNA, as determined by gel electrophoresis and electron microscopy. The recombinant DNA molecules were terminally labeled by exonuclease III treatment and subsequent refilling of the depleted segments with [(32)P]dNTPs by using DNA polymerase I (Klenow fragment). When these molecules were cleaved with EcoRI, BamHI, Msp I, or Pst I, only one terminal DNA fragment was found to be labeled. The results of partial digestion experiments using Msp I, HinfI, or Mbo I are consistent with a model in which 700-1150 base pairs from the left terminus of Ad12 DNA are linked to host cell DNA containing repetitious sequences, and this structure is symmetrically duplicated as a large inverted repeat of the type ABCDD'C'B'A'. The Ad12 DNA sequences are flanking the entire molecule, which consists mainly of human KB cell DNA. The recombinants appear to be stable on serial passage of the virus preparation for many years, although variations in the sequence of the recombinants occur. These symmetric recombinant (SYREC) molecules suggest a way to use adenovirus DNA as a eukaryotic vector. Their occurrence provides further evidence for the generation of virus-host DNA recombinants and may help elucidate the role this interaction may have in adenovirus replication and oncogenesis.

Adenoviruses, Human↗
PubMed1996

Isolation and characterization of herpes simplex virus type 1 mutants defective in the UL6 gene.

Previous studies have shown that the protein encoded by herpes simplex virus type 1 (HSV-1) gene UL6 is required for processing and packaging of replicated viral DNA and is a minor component of virions and capsids. In this report, we describe the construction of UL6- HSV-1 mutants with a disrupted UL6 gene using complementing cells and show that they fail to synthesize the UL6 protein or produce infectious virus in noncomplementing cells. The mutants synthesized but failed to process and encapsidate viral DNA and accumulated only immature capsids which lacked the UL6 protein. Immunofluorescence analysis showed that the UL6 protein, when expressed transiently in transfected cells in the absence of other HSV-1 proteins, is localized exclusively to the nucleus. We also investigated an HSV-1 mutant with a defect in gene UL33, the product of which is also thought to be involved in viral DNA processing and packaging. The phenotype of this mutant on noncomplementing cells with regard to failure to process and encapsidate viral DNA, accumulation of immature capsids, and inability to produce infectious virus was the same as that of UL6- viruses. This mutant, however, produced capsids containing the UL6 protein, indicating that association of the UL6 protein with the capsid is independent of the UL33 protein.

Animals↗
PubMed1991

DNA attachment to the hamster sperm nuclear matrix and nuclear annulus.

Hamster sperm DNA is packaged so tightly that it is the most highly condensed eukaryotic DNA known. Nevertheless, the sperm genome is also organized in a very specific fashion by two nuclear structures, the nuclear matrix and the nuclear annulus. The nuclear matrix organizes sperm DNA into loop domains approximately 46 Kb in length by specific attachment sequences. When the sperm nucleus is induced to decondense, the nuclear matrix almost completely dissipates, leaving only the nuclear annulus, a structure located at the implantation fossa within the nucleus. All the DNA remains anchored to the annulus during decondensation. Rotary shadowing transmission electron microscopy of isolated nuclear annuli retaining only a small portion of this anchored DNA suggests that the DNA is anchored to the annulus in a series of mini-loops, 5-10 Kb in length. These data support our previous suggestions that sperm DNA is organized in a very specific manner.

Animals↗
PubMed1994

Systemic lupus erythematosus. Antibodies to DNA, DNA-binding proteins, and histones.

Pathogenic autoantibodies to DNA are frequently associated with autoantibodies to chromatin-associated proteins such as histones or the Ku (p70/p80) antigens. In view of the increasing evidence that autoantibody production is antigen-driven, and because DNA is packaged with proteins in the cell, we suggest that anti-DNA antibodies may arise in response to DNA-protein complexes rather than naked DNA. Recent studies of the specificities of autoantibodies directed against the components of nucleosomes and transcriptional complexes are consistent with this hypothesis. The possible clinical significance of immune recognition of various types of chromatin complexes in autoimmune disease is discussed.

Antibodies, Antinuclear↗
PubMed1972

Ultraviolet-induced cross-links in the deoxyribonucleic acid of single-stranded deoxyribonucleic acid viruses as a probe of deoxyribonucleic acid packaging.

Deoxyribonucleic acid (DNA) from ultraviolet (UV)-irradiated phiX174 sediments in alkali at rates up to 1.7 times that of unirradiated phiX174 DNA and is observed as a condensed, cross-linked structure when examined in the electron microscope by the formamide spreading technique. This structure appears to result from multiple cross-links induced in the tightly coiled DNA contained within the spherical phiX174 capsid. In contrast, the DNA extracted after UV irradiation of the filamentous bacteriophage M13 is not strikingly altered in its sedimentation properties and appears by electron microscopy to be rod-shaped as a result of side-to-side association of the circular DNA. The differences in these UV-induced structures reflect the differences in the packaging of the single-stranded DNA in the two virions.

Bacteriophages↗
PubMed1994

Selected mutations of the duck hepatitis B virus P gene RNase H domain affect both RNA packaging and priming of minus-strand DNA synthesis.

The genome of all hepadnaviruses has an open reading frame called the P gene, which encodes a polypeptide of 90 to 97 kDa. The product or products of this P gene are involved in multiple functions of the viral life cycle. These functions include a priming activity which initiates minus-strand DNA synthesis, a polymerase activity which synthesizes DNA by using either RNA or DNA templates (reverse transcriptase), a nuclease activity which degrades the RNA strand of RNA-DNA hybrids (RNase H), and involvement in packaging the RNA pregenome into nucleocapsids. In a previous study, we found that a single point mutation at position 711 in the duck hepatitis B virus (DHBV) P gene product RNase H domain prevented viral RNA packaging. In the present experiments, we have mutated additional conserved amino acids in the DHBV RNase H domain and examined the ability of viral genomes containing these mutations to package RNA and replicate viral DNA. Charged and sulfur group amino acids adjacent to Cys-711 were mutated. None of these mutants was defective in either RNA packaging or viral replication. We also tested a number of mutations on the basis of common elements in the crystal structures of Escherichia coli and human immunodeficiency virus reverse transcriptase RNase H enzymes and on the basis of the similarities of their amino acid sequences to those of the RNase H domains of DHBV and HBV. Our results revealed that the entire beta 4 strand and amino acids Leu-712, Leu-697, and Val-719 in the putative hydrophobic cores of the beta 4, alpha A, and alpha B regions, respectively, are involved in pregenomic RNA encapsidation. This suggests that the basic structure of the RNase H domain in the DHBV P gene product is required for viral RNA packaging. We used the in vitro DHBV minus-strand DNA priming system developed by Wang and Seeger (G.-H. Wang and C. Seeger, Cell 71:663-670, 1992) to test the effect of RNase H packaging mutations on P gene product enzymatic activity. While all packaging-defective mutants tested maintained DNA priming activity, levels were decreased 5- to 20-fold compared with that of the wild-type genome. This observation suggests that the hepadnavirus RNase H domain plays a role in optimizing priming of minus-strand DNA synthesis.

Amino Acid Sequence↗
PubMed2005

Binding of pRNA to the N-terminal 14 amino acids of connector protein of bacteriophage phi29.

During assembly, bacterial virus phi29 utilizes a motor to insert genomic DNA into a preformed protein shell called the procapsid. The motor contains one twelve-subunit connector with a 3.6 nm central channel for DNA transportation, six viral-encoded RNA (packaging RNA or pRNA) and a protein, gp16, with unknown stoichiometry. Recent DNA-packaging models proposed that the 5-fold procapsid vertexes and 12-fold connector (or the hexameric pRNA ring) represented a symmetry mismatch enabling production of a force to drive a rotation motor to translocate and compress DNA. There was a discrepancy regarding the location of the foothold for the pRNA. One model [C. Chen and P. Guo (1997) J. Virol., 71, 3864-3871] suggested that the foothold for pRNA was the connector and that the pRNA-connector complex was part of the rotor. However, one other model suggested that the foothold for pRNA was the 5-fold vertex of the capsid protein and that pRNA was the stator. To elucidate the mechanism of phi29 DNA packaging, it is critical to confirm whether pRNA binds to the 5-fold vertex of the capsid protein or to the 12-fold symmetrical connector. Here, we used both purified connector and purified procapsid for binding studies with in vitro transcribed pRNA. Specific binding of pRNA to the connector in the procapsid was found by photoaffinity crosslinking. Removal of the N-terminal 14 amino acids of the gp10 protein by proteolytic cleavage resulted in undetectable binding of pRNA to either the connector or the procapsid, as investigated by agarose gel electrophoresis, SDS-PAGE, sucrose gradient sedimentation and N-terminal peptide sequencing. It is therefore concluded that pRNA bound to the 12-fold symmetrical connector to form a pRNA-connector complex and that the foothold for pRNA is the connector but not the capsid protein.

Amino Acid Sequence↗
PubMed1997

Mutations in the terminase genes of bacteriophage lambda that bypass the necessity for FI.

DNA maturation in bacteriophage lambda is the process by which the concatemeric precursor DNA is cleaved at sites called cos to generate mature lambda DNA molecules. These DNA molecules are then packaged into procapsids, the empty capsid precursors. The enzyme that catalyses these events is lambda DNA terminase. It is composed of two subunits, made of 181 and 641 amino acids, the products of genes Nu1 and A, respectively. The product of the FI gene (gpFI) stimulates the formation of an intermediate in capsid assembly called complex II, which contains a procapsid, terminase and DNA. The mechanism of stimulation remains unknown. It has been suggested that gpFI may also stimulate terminase-mediated cos cleavage, in the absence of procapsids, by increasing enzyme turnover. Mutants in FI fail to mature and package DNA but, in comparison with other capsid gene mutants, FI mutants are leaky. Second site mutants of FI phages, called 'fin' (for FI independence), bypass the necessity for gpFI. These mutants were originally localized to the region of Nu1 and A and are of two classes: finA includes those that induce the synthesis of fourfold more gene A product (gpA) than wild-type phages, and finB includes those that produce normal amounts of gpA. Whereas all finA mutants analysed map to Nu1, finB mutants have been found both in E and in Nu1. The existence of E mutants able to bypass the necessity for gpFI in vivo shows that gpE and gpFI interact, directly or indirectly. Here we have analysed and sequenced two finA mutants and one finB mutant. All of these map in Nu1. Of the two finA mutants, one corresponds to an Ala163Ser change and the other is a silent mutation. It is likely that the finA mutations alter mRNA conformation in a manner that results in an increase in the efficiency of A mRNA translation. The fourfold increase in gpA synthesis translates into a 10-fold increase in terminase activity. These results show that terminase overproduction is sufficient to bypass the necessity for gpFI and that such an overproduction can be achieved by changes in the efficiency of translation of A due to subtle changes in the sequence upstream of the gene. The finBcs103 mutation is a His-87-->Tyr change in Nu1. Therefore, an alternative way in which to bypass the requirement for gpFI involves an alteration in the structure of gpNu1. It is likely that the altered gpNu1 would increase cleavage and packaging efficiency directly or indirectly. We have determined that DNA cleavage in vivo does not occur in the absence of gpFI. Therefore it seems that gpFI somehow facilitates an otherwise latent capacity of terminase to autoactivate its nucleolytic activity.

Amino Acid Sequence↗
PubMed2005

Some organotin compounds enhance histone acetyltransferase activity.

Eukaryotic DNA is packaged into chromatin, whose basic subunit is the nucleosome, which consists of DNA and a core histone octamer. Histone acetylation is important for the regulation of gene expression and is catalyzed by histone acetyltransferase (HAT). We observed the effects of suspected endocrine-disrupting chemicals (EDCs) on HAT activity. We showed that some organotin compounds--tributyltin (TBT) and triphenyltin (TPT)--enhanced HAT activity of core histones in a dose-dependent way and other EDCs did not affect HAT activity. Organotin compounds have various influences on physical function including the hormone and immune systems, embryogenesis, and development. Dibutyltin and diphenyltin, metabolites of TBT and TPT, respectively, also promoted HAT activity, but monobutyltin, monophenyltin, and inorganic tin had no effect. Further, TBT and TPT enhanced HAT activity when nucleosomal histones were used as substrates. These data indicate that the organotin compounds have unique effects on HATs independent of their EDC activities and suggest that the varied toxicities of the organotin compounds may be caused by aberrant gene expression following altered histone acetylation.

Acetylation↗
PubMed1987

Pathologic features of cytomegalovirus retinopathy after treatment with the antiviral agent ganciclovir.

Ganciclovir is a new antiviral compound (also called BW B759U, DHPG, BIOLF-62, and 2'NDG) that has been used for the treatment of cytomegalovirus (CMV) retinopathy in immunocompromised patients (bone marrow recipients or acquired immune deficiency syndrome [AIDS] victims). The authors studied the eyes of three AIDS patients with CMV retinopathy who died while receiving ganciclovir chemotherapy. Gross, microscopic, and ultrastructural studies of these cases showed varying degrees of retinal scarring and active CMV lesions at the margins of the scars. CMV antigens were localized in cells at all layers of retina at the border of the lesions and in isolated cells in a perivascular location within histologically normal appearing retina. These areas probably represent sites of recrudescence when the drug is discontinued. In situ hybridization using a cloned complementary DNA (cDNA) probe of human CMV corroborated the immunocytologic localization of the virus. Ultrastructural studies showed megalic syncytial cells containing mostly capsids exclusively in the cell nucleus. The cytoplasmic electron-dense membrane-bound bodies that have characterized untreated cases of CMV retinopathy were absent in the treated cases. An attempt to isolate CMV in tissue culture from the vitreous and retina of one of the cases yielded a negative result. Our results indicate that ganciclovir does not effectively eliminate CMV from the retina nor does it suppress expression of all viral genes. Ganciclovir appears to function by limiting viral DNA synthesis and subsequent packaging of viral DNA into infectious units, thereby acting as a virostatic chemotherapeutic agent.

Acquired Immunodeficiency Syndrome↗
PubMed1999

Role of covalent modifications of histones in regulating gene expression.

DNA is organized into a hierarchy of structures, resulting in the level of compaction required to pack 2m of DNA into a nucleus with a diameter of 10 micrometer. The orderly packaging of DNA in the nucleus plays an important role in the functional aspects of gene regulation. A small percentage of chromatin is made available to transcription factors and the transcription machinery, while the remainder of the genome is in a state that is essentially invisible to the RNA polymerases. Modification of histones has a key role in altering chromatin higher order structure and function. In this review, we will present the latest developments in the study of histone modifications (ubiquitination, acetylation, methylation, and phosphorylation) and the enzymes involved in these processes.

Acetylation↗
PubMed2001

Haploinsufficiency of protamine-1 or -2 causes infertility in mice.

Protamines are the major DNA-binding proteins in the nucleus of sperm in most vertebrates and package the DNA in a volume less than 5% of a somatic cell nucleus. Many mammals have one protamine, but a few species, including humans and mice, have two. Here we use gene targeting to determine if the second protamine provides redundancy to an essential process, or if both protamines are necessary. We disrupted the coding sequence of one allele of either Prm1 or Prm2 in embryonic stem (ES) cells derived from 129-strain mice, and injected them into blastocysts from C57BL/6-strain mice. Male chimeras produced 129-genotype sperm with disrupted Prm1 or Prm2 alleles, but failed to sire offspring carrying the 129 genome. We also found that a decrease in the amount of either protamine disrupts nuclear formation, processing of protamine-2 and normal sperm function. Our studies show that both protamines are essential and that haploinsufficiency caused by a mutation in one allele of Prm1 or Prm2 prevents genetic transmission of both mutant and wild-type alleles.

Animals↗
PubMed2026

Multivalent cations stabilize DNA duplexes beyond charge neutralization.

Multivalent cations are abundant in cells and play essential roles in DNA duplex stability, genome packaging, and DNA-protein interactions. They can also condense DNA, making it challenging to determine their influence on DNA duplex stability. To overcome this challenge, we studied DNA unpeeling at equilibrium under high tension using magnetic tweezers, thereby preventing condensation. Experiments show that DNA duplex stability first increases and then decreases as cation concentration increases and the maximum DNA duplex stability increases with cation valence. The maximum free energy change of DNA was 3.33 k B T/bp for Na+ and increased to 3.98 k B T/bp for protamine, which is a small arginine-rich protein with a highly positive charge (≈21 for salmon sperm), corresponding to a relative increase of 19.5%. Consistently, all-atom molecular dynamics simulations show that higher-valent cations preferentially embed in the minor groove of DNA and clamp the minor groove, in contrast to the major-groove clamping reported for RNA, thereby stabilizing the helix more efficiently. These findings establish a single-molecule framework for quantifying DNA thermodynamics in complex ionic environments, which contributes to understanding ionic control of genome stability and to designing ion-tunable DNA-based nanostructures and delivery systems.

Journal Article↗