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At least 991 records · Page 55Linked to original sources
PubMed1990

Structure of the bacteriophage lambda cohesive end site: bent DNA on both sides of the site, cosN, at which terminase introduces nicks during chromosome maturation.

Packaging of lambda DNA is mediated by the phage-encoded enzyme, terminase, which acts at a site termed cos. cos consists of cosB, the site where terminase binds lambda DNA, and cosN, the site where nicks are introduced to generate the cohesive ends of virion DNA. cos contains multiple binding sites for gpNu1, the small subunit of terminase, and integration host factor (IHF), an Escherichia coli DNA binding protein. Polyacrylamide gel electrophoresis of circularly permuted segments of cos DNA has been used to locate major bend loci in cos. Two major bends have been located; one bend is ca 146 bp to the left of cosN while the second major bend is located ca 92 bp to the right of cosN. The major bend at 92 coincides roughly with I1, the strongest IHF binding site in cos. The possible roles of static bending in DNA packaging are discussed.

Bacteriophage lambda↗
PubMed1983

Formation of oligomeric structures from plasmid DNA carrying cos lambda that is packaged into bacteriophage lambda heads.

Plasmids that carry cos lambda, the region necessary for lambda phage packaging and that are as small as four kilobases in size can be packaged into lambda phage heads in head-to-tail tandem oligomeric structures. Multimeric oligomers as large as undecamers have been detected. Oligomer formation depends upon the products of red and gam of lambda, and the general recombination occurs between different plasmids that share homologous DNA regions. The packaging efficiency of plasmids depends on its copy number in cells and its genome size. Upon injection into a cell, the DNA establishes itself as a plasmid in a tandem structure. When such a plasmid in a high oligomeric structure is used as the source of packaging DNA, the packaging efficiency of the plasmids is elevated. The oligomers are stable in recA cells, whereas they drift toward lower oligomers in recA+ cells.

Bacteriophage lambda↗
PubMed1985

A model of lambda DNA arrangement in the viral particle.

The icosahedral shape of the lambda head suggests a 12-subunit structure of the collapsed DNA inside. The internal space of an icosahedron can optimally be filled by 12 geometrical figures each of which is a combination of a cone and more than half of a sphere. Such a pear-like geometrical figure is, in fact, formed spontaneously by DNA collapsed under certain conditions in vitro (Eickbush & Moudrianakis, 1978). It is proposed that a pear-like structure formed by about 4000 bp is the fundamental structural subunit of packaged lambda DNA. A possible arrangement of the 12 subunits inside the phage head relative to the tail is discussed. We hypothesize that lambda DNA is packaged into proheads in its condensed form. A driving force promoting the DNA translocation could be an ATP-dependent activity of a DNA topoisomerase (gpA/gpNu1), which would induce further reduction in the linking number of the already strongly negatively supercoiled DNA by rotation of one DNA strand around the other. The additional strain accumulated at the end of DNA molecule bound by the topoisomerase beyond a critical value would lead to regional collapse of the viral genome into a pear-like structure.

Animals↗
PubMed2003

Trimethylated lysine 9 of histone H3 is a mark for DNA methylation in Neurospora crassa.

Besides serving to package nuclear DNA, histones carry information in the form of a diverse array of post-translational modifications. Methylation of histones H3 and H4 has been implicated in long-term epigenetic 'memory'. Dimethylation or trimethylation of Lys4 of histone H3 (H3 Lys4) has been found in expressible euchromatin of yeasts and mammals. In contrast, methylation of Lys9 of histone H3 (H3 Lys9) has been implicated in establishing and maintaining the largely quiescent heterochromatin of mammals, yeasts, Drosophila melanogaster and plants. We have previously shown that a DNA methylation mutant of Neurospora crassa, dim-5 (defective in methylation), has a nonsense mutation in the SET domain of an H3-specific histone methyltransferase and that substitutions of H3 Lys9 cause gross hypomethylation of DNA. Similarly, the KRYPTONITE histone methyltransferase is required for full DNA methylation in Arabidopsis thaliana. We used biochemical, genetic and immunological methods to investigate the specific mark for DNA methylation in N. crassa. Here we show that trimethylated H3 Lys9, but not dimethylated H3 Lys9, marks chromatin regions for cytosine methylation and that DIM-5 specifically creates this mark.

DNA Methylation↗
PubMed2002

Oxidative charge transport through DNA in nucleosome core particles.

Eukaryotic DNA is packaged into nucleosomes, made up of 146 bp of DNA wrapped around a core of histone proteins. We used photoexcited rhodium intercalators to explore DNA charge transport within these assemblies. Although histone proteins inhibit intercalation of the rhodium complex within the core particle, they do not prevent 5'-GG-3' oxidation, the signature of oxidative charge transport through DNA. Moreover, using rhodium intercalators tethered to the 5' terminus of the DNA, we found that guanine bases within the nucleosome can be oxidized from a distance of 24 bp. Histone binding did not affect the pattern and extent of this oxidation. Therefore, although the structure of the nucleosome core particle generally protects DNA from damage by solution-borne molecules, packaging within the nucleosome does not protect DNA from charge transfer damage through the base pair stack.

2,2'-Dipyridyl↗
PubMed1987

Gene 18 protein of bacteriophage T7. Overproduction, purification, and characterization.

Genetic and physical analyses indicate that gene 18 protein of bacteriophage T7 is essential for packaging of T7 DNA. T7 DNA is replicated via linear intermediates, culminating in the formation of concatemers many genomes in length which are then packaged into capsids. In infections with phage carrying amber mutations in gene 18, development is blocked at the concatemer stage. Biochemical studies on the role of gene 18 protein in concatemer processing and DNA packaging have been hampered by its low level of expression of gene 18 during T7 infections. We have cloned gene 18 on a plasmid downstream from the bacteriophage lambda PL promoter controlled by the temperature-sensitive lambda repressor encoded by c 1857. Thermal induction leads to the expression of the 10,000-Da gene 18 protein to the extent of approximately 10% of the total protein after 2 h. The overexpressed gene 18 protein is susceptible to proteolytic degradation, a condition that can be alleviated by expression in an Escherichia coli strain carrying the lon100 deletion which reduces production of protease La. Extracts of induced cells will complement an extract of T7-infected cells lacking gene 18 protein for packaging of exogenous T7 DNA. The assay has been used to monitor the purification of gene 18 protein to essential homogeneity. The identity of the purified protein has been confirmed by sequencing of the N terminus. Gel filtration analysis suggests that the native protein is an octomer. Treatment of gene 18 protein with 3 M guanidine hydrochloride denatures it to a monomer. Removal of the denaturing agent by dialysis regenerates the octomeric structure and the ability to complement packaging extracts.

Chromatography, Gel↗
PubMed1998

Mechanisms of DNA bending.

Genome packaging and gene regulation require DNA bending. Recent developments in the elucidation of the mechanisms involved in DNA bending include new X-ray structures (most notably that of the mammalian nucleosome) wherein DNA is bent, controversy surrounding interpretation of DNA-bending experiments with basic-leucine zipper proteins, studies of electrostatic effects in DNA bending, and the design of artificial DNA-bending ligands.

Animals↗
PubMed1998

The herpes simplex virus type 1 cleavage/packaging protein, UL32, is involved in efficient localization of capsids to replication compartments.

Six genes, including UL32, have been implicated in the cleavage and packaging of herpesvirus DNA into preassembled capsids. We have isolated a UL32 insertion mutant which is capable of near-wild-type levels of viral DNA synthesis; however, the mutant virus is unable to cleave and package viral DNA, consistent with the phenotype of a previously isolated temperature-sensitive herpes simplex virus type 1 mutant, tsN20 (P. A. Schaffer, G. M. Aron, N. Biswal, and M. Benyesh-Melnick, Virology 52:57-71, 1973). A polyclonal antibody which recognizes UL32 was previously used by Chang et al. (Y. E. Chang, A. P. Poon, and B. Roizman, J. Virol. 70:3938-3946, 1996) to demonstrate that UL32 accumulates predominantly in the cytoplasm of infected cells. In this report, a functional epitope-tagged version of UL32 showed that while UL32 is predominantly cytoplasmic, some nuclear staining which colocalizes with the major DNA binding protein (ICP8, UL29) in replication compartments can be detected. We have also used a monoclonal antibody (5C) specific for the hexon form of major capsid protein VP5 to study the distribution of capsids during infection. In cells infected with wild-type KOS (6 and 8 h postinfection), 5C staining patterns indicate that capsids are present in nuclei within replication compartments. These results suggest that cleavage and packaging occur in replication compartments at least at 6 and 8 h postinfection. Cells infected with the UL32 mutant exhibit a hexon staining pattern which is more diffusely distributed throughout the nucleus and which is not restricted to replication compartments. We propose that UL32 may play a role in "bringing" preassembled capsids to the sites of DNA packaging and that the failure to localize to replication compartments may explain the cleavage/packaging defect exhibited by this mutant. These results suggest that the UL32 protein is required at a step distinct from those at which other cleavage and packaging proteins are required and may be involved in the correct localization of capsids within infected cells.

Animals↗
PubMed2001

Fluorescence microscopy of single viral capsids.

To build a foundation for the single-molecule fluorescence microscopy of protein complexes, the present study achieved fluorescence microscopy of single, nucleic acid-free protein capsids of bacteriophage T7. The capsids were stained with Alexa 488 (green emission). Manipulation of the capsids' thermal motion was achieved in three dimensions. The procedure for manipulation included embedding the capsids in an agarose gel. The data indicate that the thermal motion of capsids is reduced by the sieving of the gel. The thermal motion can be reduced to any desired level. A semilogarithmic plot of an effective diffusion constant as a function of gel concentration is linear. Single, diffusing T7 capsids were also visualized in the presence of single DNA molecules that had been both stretched and immobilized by gel-embedding. The DNA molecules were stained with ethidium (orange emission). This study shows that single-molecule (protein and DNA) analysis is possible for both packaging of DNA in a bacteriophage capsid and other events of DNA metabolism. The major problem is the maintenance of biochemical activity.

Bacteriophage T7↗
PubMed2006

Evolutionary genomics of archaeal viruses: unique viral genomes in the third domain of life.

In terms of virion morphology, the known viruses of archaea fall into two distinct classes: viruses of mesophilic and moderately thermophilic Eueryarchaeota closely resemble head-and-tail bacteriophages whereas viruses of hyperthermophilic Crenarchaeota show a variety of unique morphotypes. In accord with this distinction, the sequenced genomes of euryarchaeal viruses encode many proteins homologous to bacteriophage capsid proteins. In contrast, initial analysis of the crenarchaeal viral genomes revealed no relationships with bacteriophages and, generally, very few proteins with detectable homologs. Here we describe a re-analysis of the proteins encoded by archaeal viruses, with an emphasis on comparative genomics of the unique viruses of Crenarchaeota. Detailed examination of conserved domains and motifs uncovered a significant number of previously unnoticed homologous relationships among the proteins of crenarchaeal viruses and between viral proteins and those from cellular life forms and allowed functional predictions for some of these conserved genes. A small pool of genes is shared by overlapping subsets of crenarchaeal viruses, in a general analogy with the metagenome structure of bacteriophages. The proteins encoded by the genes belonging to this pool include predicted transcription regulators, ATPases implicated in viral DNA replication and packaging, enzymes of DNA precursor metabolism, RNA modification enzymes, and glycosylases. In addition, each of the crenarchaeal viruses encodes several proteins with prokaryotic but not viral homologs, some of which, predictably, seem to have been scavenged from the crenarchaeal hosts, but others might have been acquired from bacteria. We conclude that crenarchaeal viruses are, in general, evolutionarily unrelated to other known viruses and, probably, evolved via independent accretion of genes derived from the hosts and, through more complex routes of horizontal gene transfer, from other prokaryotes.

Amino Acid Sequence↗
PubMed1999

Differences in the DNA-stainability of spermatozoa from fertile and suspected infertile men.

The aim of this work was to determine whether it is possible to distinguish between fertile (control group, already fathers) and infertile men (suspected infertility), by comparing the fluorescence intensity of the sperm-DNA after incubation with appropriate dyes. First we examined two different DNA-specific dyes (DAPI and YOYO-1) using bull spermatozoa. Based on good results in immunohistochemical applications, YOYO-1 was chosen for further work. The fluorescence-intensity of 200 single, morphologically normal spermatozoa in each semen sample were measured in a cytophotometer, means + SD determined and histograms delineated. Of 20 samples from the control group, 17 had markedly higher fluorescence-intensity than did 7/15 of the suspected infertile men. It is concluded that the DNA of the latter seven samples was less accessible to the dye than was the DNA of the control group. There are cases of infertility known in which there is loss of one or more of the DNA-binding proteins, which in spermatozoa are mainly (85%) protamines. The relationship between the stainability of the sperm-DNA and the packaging with DNA-binding proteins is discussed. Two of the histograms showed abnormalities in the distribution of the fluorescence-intensities, one sample was extremely fragile and most of the sperm lysed during the staining-procedure. Five samples showed normal histograms in comparison with the control group.

Animals↗
PubMed2006

HP1 proteins are essential for a dynamic nuclear response that rescues the function of perturbed heterochromatin in primary human cells.

Cellular information is encoded genetically in the DNA nucleotide sequence and epigenetically by the "histone code," DNA methylation, and higher-order packaging of DNA into chromatin. Cells possess intricate mechanisms to sense and repair damage to DNA and the genetic code. However, nothing is known of the mechanisms, if any, that repair and/or compensate for damage to epigenetically encoded information, predicted to result from perturbation of DNA and histone modifications or other changes in chromatin structure. Here we show that primary human cells respond to a variety of small molecules that perturb DNA and histone modifications by recruiting HP1 proteins to sites of altered pericentromeric heterochromatin. This response is essential to maintain the HP1-binding kinetochore protein hMis12 at kinetochores and to suppress catastrophic mitotic defects. Recruitment of HP1 proteins to pericentromeres depends on histone H3.3 variant deposition, mediated by the HIRA histone chaperone. These data indicate that defects in pericentromeric epigenetic heterochromatin modifications initiate a dynamic HP1-dependent response that rescues pericentromeric heterochromatin function and is essential for viable progression through mitosis.

Cell Cycle Proteins↗
PubMed2001

Direct imaging of single-molecules: from dynamics of a single DNA chain to the study of complex DNA-protein interactions.

Recent years have seen significant advances in the characterization and manipulation of individual molecules. The combination of single-molecule fluorescence and micromanipulation enables one to study physical and biological systems at new length scales, to unravel qualitative mechanisms, and to measure kinetic parameters that cannot be addressed by traditional biochemistry. DNA is one of the most studied biomolecules. Imaging single DNA molecules eliminates important limitations of classical techniques and provides a new method for testing polymer dynamics and DNA-protein interactions. Here we review some applications of this new approach to physical and biological problems, focusing on videomicroscopy observations of individual DNA chains extended in a shear flow. We will first describe data obtained on the stretching, relaxation and dynamics of a single tethered polymer in a shear flow, to demonstrate that the deformation of sheared tethered chains is partially governed by the thermally driven fluctuations of the chain transverse to the flow direction. Next, we will show how single-molecule videomicroscopy can be used to study in real time DNA folding into chromatin, a complex association of DNA and proteins responsible for the packaging of DNA in the nucleus of an eukaryotic cell.

DNA↗
PubMed1995

Gel electrophoretic analysis of bacteriophage assembly intermediates in bacteriophage plaques.

To increase the efficiency with which the phenotype of bacteriophage mutants is determined by gel electrophoresis, procedures are developed here for the preparation of the contents of bacteriophage plaques for gel electrophoresis. During the formation of plaques, the plaque-supporting upper layer gel is changed from the traditional agar gel to a gel made of a mixture of low-melt agaroses; the lower layer gel is eliminated. To extract particles from plaques, the plaque-supporting gel is disintegrated by both shaking and raising the temperature to 39-43 degrees C. During shaking, the gel is broken to domains that are 5-30 microns in diameter. After extraction, the contents of plaques are subjected to two electrophoretic analyses: (1) Nondenaturing agarose gel electrophoresis is performed after treatment with DNase. This procedure reveals both mature bacteriophage and immature capsids. (2) Nondenaturing agarose gel electrophoresis is performed after release of DNA from DNase-treated capsids. This latter procedure reveals both completely packaged (mature length) DNA and incompletely packaged (shorter than mature length) DNA. The amount of mature length DNA released per 2-3 mm plaque is 10-60 ng. In agreement with results previously obtained in liquid culture, most incompletely packaged DNA has the right, but not the left, mature T7 DNA end.

Bacteriophage T3↗
PubMed1999

Improved titers for helper virus-free herpes simplex virus type 1 plasmid vectors by optimization of the packaging protocol and addition of noninfectious herpes simplex virus-related particles (previral DNA replication enveloped particles) to the packaging procedure.

A helper virus-free herpes simplex virus type 1 (HSV-1) plasmid vector system has potential for both gene therapy and physiological studies, but relatively low titers have complicated use of this system. In this article, the packaging efficiency was improved by optimizing the packaging protocol and by adding noninfectious HSV-1-related particles, i.e., previral DNA replication enveloped particles (PREPs), during the packaging procedure. PREPs contain many of the tegument proteins that are thought to enhance an HSV-1 infection. Use of both the optimized packaging protocol and the PREPs resulted in an approximately 50-fold increase in the titer, and five different HSV-1 vectors were packaged using this procedure. A purified vector stock (7.8x10(8) infectious vector particles/ml) was microinjected into the striatum, the rats were sacrificed 4 days after gene transfer, and the brains were found to contain an average of approximately 6740 X-Gal-positive striatal cells. This improved packaging procedure may augment use of this vector system.

Animals↗
PubMed1981

Non-nucleosomal packaging of a tandemly repeated DNA sequence at termini of extrachromosomal DNA coding for rRNA in Tetrahymena.

A tandemly repeated DNA hexanucleotide sequence, 5'C-C-C-C-A-A3', that occurs at the termini of extrachromosomal DNA molecules coding for rRNA (rDNA) in Tetrahymena macronuclei was examined to determine whether it is packaged as nucleosomes. This repeated DNA sequence comprises the terminal few hundred base pairs at each end of the linear rDNA molecules. Digestion of macronuclei with micrococcal nuclease showed that this DNA sequence is protected from digestion but is left, following digestion, as a single but broad size class of DNA fragments several hundred base pairs long, under conditions in which bulk macronuclear DNA and rDNA were digested to fragments that were multiples of approximately 200 base pairs in length. The repeated C-C-C-C-A-A was found protected as fragments longer than the bulk macronuclear DNA digestion products at all times during digestion. Together with putative associated protein(s), this protected DNA was soluble after lysis of micrococcal nuclease-digested macronuclei at low salt concentrations but was insoluble in 0.075--0.2 M KCl, regardless of the extend of digestion. The size and solubility properties of the repeated C-C-C-C-A-A DNA nucleoprotein complex after micrococcal nuclease digestion of macronuclei are clearly distinguishable from those of nucleosomes, and it is inferred that this DNA sequence in macronuclei is packaged in chromatin by proteins other than histones.

Cell Nucleus↗
PubMed2004

Initiation of intracellular offspring in Epulopiscium.

Epulopiscium spp. are the largest heterotrophic bacteria yet described. A distinguishing feature of the Epulopiscium group is their viviparous production of multiple, internal offspring as a means of cellular reproduction. Based on their phylogenetic position, among low G + C Gram-positive endospore-forming bacteria, and the remarkable morphological similarity between developing endospores and Epulopiscium offspring, we hypothesized that intracellular offspring production in Epulopiscium evolved from endospore formation. These observations also raise the possibility that a cell with the capacity to form multiple intracellular offspring was the ancestor of all contemporary endospore-forming bacteria. In an effort to characterize mechanisms common to both processes, we describe the earliest stages of offspring formation in Epulopiscium. First, in anticipation of polar division, some of the mother cell DNA coalesces at the cell poles. FtsZ then localizes in a bipolar pattern and the cell divides. A portion of the pole-associated DNA is trapped within the small cells formed by division at both poles. As development progresses, more pole-associated DNA is apparently packaged into the offspring primordia. These results illustrate three mechanisms, the reorganization of cellular DNA, asymmetric division and DNA packaging, that are common to both endospore formation in Bacillus subtilis and the production of active, intracellular offspring in Epulopiscium. Unlike most endospore formers, Epulopiscium partitions only a small proportion of mother cell DNA into the developing offspring.

Animals↗
PubMed1975

Influence of insertions on packaging of host sequences covalently linked to bacteriophage Mu DNA.

Insertions in bacteriophage Mu DNA have been identified. These insertions are responsible for at least seven X mutations, all of which eliminate essential Mu functions. The insertions are about 800 base pairs long and are located to the left of the cleavage site of restriction endonuclease EcoRI, near the immunity end of Mu DNA. We have found that such insertions cause a reduction in the length of nonhomologous terminal sequences which are seen as split ends in denatured and renatured Mu DNA molecules. These heterogeneous sequences apparently arise from packaging of host DNA from maturation precursors in which Mu and host DNA are covalently linked. We infer that a single Mu genome length is too short to be cut during morphogenesis, and thus some host DNA is packaged into mature virions. Since the insertions increase the length of Mu DNA, they decrease the amount of host DNA needed for packaging.

Chromosome Aberrations↗