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At least 505 records · Page 28Linked to original sources
PubMed1992

Tip-radius-induced artifacts in AFM images of protamine-complexed DNA fibers.

Isolated DNA fibers complexed with protamine (the chromosomal protein that packages DNA in mammalian sperm) have been produced by partially decondensing the highly compacted mouse sperm chromatin particle on a glass coverslip. These DNA fibers were then scanned with the atomic force microscope (AFM). While the smallest of the fibers appear in AFM images as ribbon-like structures 250-350 A wide and 10-25 A high, experiments indicate that these images are the result of a convolution of the imaging-tip's shape with the object's actual shape. In such convolutions the height of the object is affected only by the compressibility of the object, while the width is affected in addition by the sharpness of the tip. Images of polyamidoamine particles also appear to show this artifact. We have also deduced the tip's radius of curvature from images of sharp steps and attempt to demonstrate the artifacts associated with a relatively large imaging tip.

Animals↗
PubMed2002

The effect of salts on the stability of the H2A-H2B histone dimer.

The core nucleosome, which comprises an H3-H4 tetramer and two H2A-H2B dimers, is not a static DNA packaging structure. The nucleosome is a dynamic protein-DNA complex, and the modulation of its structure is an important component of transcriptional regulation. To begin to understand the molecular details of nucleosome dynamics, we have investigated the stability of the isolated H2A-H2B dimer. The urea-induced equilibrium responses of the heterodimer have been examined by far-UV circular dichroism and intrinsic tyrosine fluorescence. The two spectroscopic probes yielded coincident transitions, and global fitting of the reversible urea-induced unfolding further demonstrated that H2A-H2B unfolds by a two-state equilibrium response. At physiological ionic strengths, the free energy of unfolding in the absence of urea of H2A-H2B is 11.8 +/- 0.3 kcal mol(-)(1), moderate stability for a dimer of 26.4 kDa. The m value, or sensitivity of the unfolding to urea, is 2.9 +/- 0.1 kcal mol(-)(1) M(-)(1). This value is significantly larger than would be predicted for the unfolding of the dimerization motif alone ( approximately 2 kcal mol(-)(1) M(-)(1)), suggesting that the N-terminal tails may adopt a collapsed, solvent-excluding structure that undergoes an unfolding transition. The efficacies of several potassium salts and three chloride salts to stabilize the H2A-H2B dimer were determined. The salt-dependent stabilization of the H2A-H2B dimer shows that the Hofmeister effect is the predominant mode of stabilization. However, studies employing multiple salts suggest that there is a component of stabilization that must arise from screening of electrostatic repulsion in the highly basic heterodimer. The most highly charged regions of the dimer are the N-terminal tails, sites of posttranslational modifications such as acetylation and phosphorylation. These modifications, which alter the charge density of the tails, are involved in regulation of nucleosome dynamics.

Ammonium Chloride↗
PubMed2002

Archaeal genome organization and stress responses: implications for the origin and evolution of cellular life.

For DNA to be used as an informational molecule it must exist in the cell on the edge of stability because all genomic processes require local controlled melting. This presents mechanistic opportunities and problems for genomic DNA from hyperthermophilic organisms, whose unpackaged DNA could melt at optimal temperatures for growth. Hyperthermophiles are suggested to employ the novel positively supercoiling topoisomerase enzyme reverse gyrase (RG) to form positively supercoiled DNA that is intrinsically resistant to thermal denaturation. RG is presently the only archaeal gene that is uniquely found in hyperthermophiles and therefore is central to hypotheses suggesting a hypothermophilic origin of life. However, the suggestion that RG has evolved by the fusion of two pre-existing enzymes has led to hypotheses for a lower temperature for the origin of life. In addition to the action of topoisomerases, DNA packaging and the intracellular ionic environment can also manipulate DNA topology significantly. In the Euryarchaeota, nucleosomes containing minimal histones can adopt two alternate DNA topologies in a salt-dependent manner. From this we hypothesize that since internal salt concentrations are increased following an increase in temperature, the genomic effects of temperature fluctuations could also be accommodated by changes in nucleosome organization. In addition, stress-induced changes in the nucleoid proteins could also play a role in maintaining the genome in the optimal topological state in changing environments. The function of these systems could therefore be central to temperature adaptation and thus be implicated in origin of life scenarios involving hyperthermophiles.

Archaea↗
PubMed1982

Characterization of a temperature-sensitive fiber mutant of type 5 adenovirus and effect of the mutation on virion assembly.

A temperature-sensitive, fiber-minus mutant of type 5 adenovirus, H5ts142, was biochemically and genetically characterized. Genetic studies revealed that H5ts142 was a member of one of the three apparent fiber complementation groups which were detected owing to intracistronic complementation. Recombination analyses showed that it occupied a unique locus at the right end of the adenovirus genetic map. At the nonpermissive temperature, the mutant made stable polypeptides, but they were not glycosylated like wild-type fiber polypeptides. Sedimentation studies of extracts of H5ts142-infected cells cultured and labeled at 39.5 degrees C indicated that a limited number of the fiber polypeptides made at the nonpermissive temperature could assemble into a form having a sedimentation value of 6S (i.e., similar to the trimeric wild-type fiber), but that this 6S structure was not immunologically reactive. When H5ts142-infected cells were shifted to the permissive temperature, 32 degrees C, fiber polypeptides synthesized at 39.5 degrees C were as capable of being assembled into virions as fibers synthesized in wild type-infected cells; de novo protein synthesis was not required to allow this virion assembly. In H5ts142-infected cells incubated at 39.5 degrees C, viral proteins accumulated and aggregated into particles having physical characteristics of empty capsids. These particles did not contain DNA or its associated core proteins. However, when the infected culture was shifted to 32 degrees C, DNA appeared to enter the empty particles and complete virions developed. The intermediate particles obtained had the morphology of adenoviruses, but they contained less than unit-length viral genomes as measured by their buoyant density in a CsCl density gradient and the size of their DNA as determined in both neutral and alkaline sucrose gradients. The reduced size of the intermediate particle DNA was demonstrated to be the result of incompletely packaged DNA molecules being fragmented during the preparative procedures. Hybridization of labeled DNA extracted from the intermediate particles to filters containing restriction fragments of the adenovirus genome indicated that the molecular left end of the viral genome preferentially entered these particles.

Adenoviruses, Human↗
PubMed1980

DNA is replicated at the nuclear cage.

Structures resembling nuclei are released when HeLa cells are lysed in a detergent and 2 M salt. These nucleoids, which lack any organized membrane structure, contain all the nuclear DNA packaged within a cage of RNA and protein. Their DNA is supercoiled so that the linear DNA must remain unbroken and looped during lysis. Following digestion with the restriction endonuclease, EcoRI, cages and associated DNA were filtered free of unattached DNA. Pulse-labelled (i.e. newly synthesized) DNA remains preferentially associated with the cages. This association has been confirmed by autoradiography. When nucleoids are prepared for electron microscopy by the Kleinschmidt procedure the DNA spills out to form a skirt around the flattened cage. Labelling, which is restricted to the region of the cage after short pulses, extends out into the skirt as the labelling time increases. A model, based on the premise that replication takes place at the nuclear cage, is presented in the Appendix. The results of the biochemical experiments and electron microscopy both indicate that the average size of the unit of replication is approximately 2 micrometer. This is about one-quarter the size of the average structural unit - the loop. Therefore sequences in the loop must become attached to the nuclear cage prior to the initiation of DNA synthesis.

Autoradiography↗
PubMed2004

Residues within the B' motif are critical for DNA binding by the superfamily 3 helicase Rep40 of adeno-associated virus type 2.

We have recently published the crystal structure of the adeno-associated virus type 2 superfamily 3 (SF3) helicase Rep40. Although based on its biochemical properties it is unlikely that Rep40 plays a central role as a replicative helicase the involvement of this motor protein in DNA packaging has recently been demonstrated. Here we focused our attention on residues that fall within and adjacent to the B' motif of SF3 helicases that directly interact with single-stranded DNA during translocation of the motor protein. In vitro, alanine substitution at positions Lys-404 or Lys-406 abrogated the ability of the protein to interact with single-stranded DNA as demonstrated by electrophoretic mobility shift assay and fluorescence anisotropy, and accordingly these mutants could not unwind a partially duplex DNA substrate. Despite this loss of helicase activity, basal ATPase activity in these mutants remained intact. However, unlike the wild-type protein, K404A and K406A ATPase activity was not stimulated by DNA. As predicted, disruption of motor activity through interference with DNA binding resulted in an inability of Rep40 to package adeno-associated virus DNA in a tissue culture-based assay. Taken together, we characterized, for the first time in an SF3 helicase family member, residues that are directly involved in single-stranded DNA binding and that are critical for the Rep motor activity. Based on our findings we propose B' as the signature motif of SF3 helicases that is responsible for the complex interactions required for the coupling of DNA binding and ATP hydrolysis.

Amino Acid Motifs↗
PubMed1985

Essential interaction between lambdoid phage 21 terminase and the Escherichia coli integrative host factor.

Lambdoid phage 21 requires the Escherichia coli integrative host factor (IHF) for growth. lambda-21 hybrids that have 21 DNA packaging specificity also require IHF. IHF-independent (her) mutants have been isolated. her mutations map in the amino-terminal half of the 21 1 gene. The 1 gene encodes the small subunit of the 21 terminase, and the amino-terminal half of the 1 polypeptide is a functional domain for specifically binding 21 DNA. Hence changes in the DNA-binding domain of terminase, her mutations, render 21 terminase able to function in the absence of IHF. Three of four her mutations studied are trans-dominant. An in vitro system was used to show that packaging of 21 DNA is IHF-dependent. IHF is directly required during the early, terminase-dependent steps of assembly. It is concluded that IHF is a host factor required for function of the 21 terminase. It is proposed, in analogy to the role of IHF in lambda integration, that IHF facilitates proper binding of 21 terminase to phage DNA. Consistent with this proposal, possible IHF-binding sites are present in the 21 cohesive end site.

Bacterial Proteins↗
PubMed1987

Characterization of ATPase activity of a defined in vitro system for packaging of bacteriophage T3 DNA.

We have developed a defined in vitro system for packaging phage T3 DNA which is composed of purified proheads and the noncapsid proteins gp18 and gp19, products of genes 18 and 19 (K. Hamada, H. Fujisawa, and T. Minagawa, 1986, Virology 151, 119-123). The in vitro system displayed an ATPase activity. The requirements for ATPase activity were the same as those for DNA packaging. ATPase was inhibited by a nonhydrolyzable ATP analog, adenosine-5'-O-(3'-thiotriphosphate) (ATP-gamma-S). ATPase activity did not display specificity for T3 DNA. A reaction mixture containing 8- proheads, proheads deficient in gp8, a portal protein for DNA entrance, or mature heads had no gp18- gp19-dependent ATPase activity. gp8 itself had no ATPase activity and did not complement 8- proheads for ATPase activity. Photoaffinity labeling of proheads, gp18 and gp19 with 8-azidoadenosine-5'-[alpha-32P]triphosphate([32P]8-N3ATP) resulted in preferential labeling of gp19. Protection from incorporation of [32P]8-N3ATP was afforded by ATP but not by AMP and ADP. From these results, it is concluded that gp19 has an ATP binding site(s). A conserved sequence of ATP-binding site containing Gly-X-Gly-X-X-Gly-X-Val is found in gp19.

Adenosine Triphosphatases↗
PubMed2002

Damage control of DNA in nucleosome core particles: when a histone's loving, protective embrace is just not good enough.

Packaging DNA into nucleosome core particles generally offers protection from damage by molecules diffusing in solution. However, on page 403 of this issue, Barton and coworkers report that although noncovalently bound, activated Rh (Rhodium) does not readily bind within nucleosomal DNA, activated Rh that is covalently tethered to the 5' terminus of a histone-associated oligonucleotide oxidizes guanine bases from a distance of up to 24 base pairs, demonstrating that histones do not protect DNA from long-range damage from the transport of charge through stacked bases. This implies that oxidative damage generated on DNA in vivo may spread from an initially damaged site to distal sites. Once created, such sites may persist and be resistant to repair because of the protective packaging by histones; they thus may result in permanent mutations.

Animals↗
PubMed2003

Role of the UL28 homologue of bovine herpesvirus 1 in viral DNA cleavage and packaging.

In the aim to study the function of the bovine herpesvirus 1 (BoHV-1) UL28 protein during the replicative cycle, we characterized a UL28 deletion mutant of BoHV-1, BoHV-1 Delta UL28. Productive growth of BoHV-1 Delta UL28 was only observed in a specifically engineered complementing cell line expressing the native UL28 protein, demonstrating that UL28 is essential for virus replication. UL28 deficiency did not compromised viral protein synthesis of the late class as shown by the detection of the viral alpha gene trans-inducing factor protein encoded by UL48, a gene of the gamma2 class. Southern blotting analyses of total DNA extracted from BoHV-1 Delta UL28-infected normal cells revealed that viral DNA replication was not compromised but the process of cleavage of the newly synthesized DNA was defective. Transmission electron microscopy of non-complementing BoHV-1 Delta UL28-infected cells revealed an accumulation of capsids devoid of DNA, suggesting that the DNA packaging was impaired. We conclude that the BoHV-1 UL28 protein is essential for viral replication and is necessary for the formation of mature capsid.

Animals↗
PubMed1997

Cloning with Mud-P22 hybrid prophages: mapping of IS200 elements on the chromosome of Salmonella typhimurium LT2.

Specific DNA fragments from the chromosome of Salmonella typhimurium LT2 were packaged in P22 capsids by induction of "locked-in" Mud-P22 hybrid prophages. High yields of the packaged DNA were obtained upon capsid disruption. DNA hybridization using a fragment of insertion sequence IS200 as probe permitted physical mapping of IS200 elements on the chromosome of S. typhimurium LT2 within +/- 1 centisome (CS). IS200 copies were found at the following locations: CS 24 (copy VI), CS 53 (copy V), CS 63 (copy I), CS 80 (copy II) and CS 93 (copy III). Copy IV, previously mapped near fliA (CS 42), was not included in our study.

Bacteriophage P22↗
PubMed1998

The intrinsic curvature of a 51 bp K-DNA fragment of Leishmania tarentolae: a molecular model.

DNA intrinsic structure and curvature is a subject of debate because of the importance of these attributes in processes such as DNA packaging, transcription, and gene regulation. X-ray crystallography of DNA single crystals has provided a wealth of information about the local, short range conformational features of DNA. On the other hand, gel electrophoresis analysis of DNA has not only uncovered the macroscopic curvature of DNA but it also provides most of the available data on DNA intrinsic curvature. However, gel electrophoresis can not identify features of DNA structure at the nucleotide or atomic level. In order to address the problem of DNA intrinsic curvature in an attempt to bridge the gap between X-ray crystallography and gel electrophoresis, we use the computational method of molecular dynamics (MD). In this study, we report the results of 2.0 ns MD simulations on a 51 bp fragment of the K-DNA of Leishmania tarentolae containing several A-tracts. The K-DNA double helix is very stable and remains in an intermediate state between the canonical A and B forms of the duplex. The magnitude of global curvature (75 degrees) agrees well with the experimental estimate (72 degrees) available. Analysis of local (every base triplet) and sublocal (every helix turn) curvature shows that the 51 bp K-DNA fragment has curvature features also present in the Wedge, Junction and Calladine's models of DNA intrinsic curvature. We further characterize the flexibility of individual nucleotides in the molecule and find the sugar flexibility within the A-tracts to be strongly correlated with the pattern of A-tract cleavage by the hydroxyl radical. Differential curvature and flexibility at the 5' and 3'junctions between A-tracts and general-sequence DNA are found to modulate the global curvature of the K-DNA fragment.

Animals↗
PubMed1977

Bacteriophage T4D head morphogenesis. VIII. DNA-protein associations in intermediate head structures that accumulate in gene 49--mutant-infected cells.

We have utilized the gene 49(-) mutant-infected cells of bacteriophage T4D to accumulate large numbers of nucleic acid-protein intermediate head structures. These heads were used as substrates for experiments in the investigations of the mechanism of DNA packaging. Specifically, we have examined: (i) the susceptibility of the DNA in these structures to digestion by a variety of nucleases after a series of increasing temperature pulses from 25 to 100 degrees C, (ii) the physicochemical characteristics of the DNA inside these heads, and (iii) the mechanism by which proteins are displaced from the interior of the head after treatment with basic proteins. We isolated DNA from these gene 49(-) heads by use of gradient centrifugation procedures. The DNA had a molecular weight of 8 x 10(6) and a density of 1.697 +/- 0.005 g/cm(3), and it contained a short resistant fraction (SRF) which, when associated with the gene 49(-) heads, exhibited AT-protected regions that were not susceptible to micrococcal nuclease digestion. Such a fraction may contain pieces which are important in the initial association of the DNA with the prohead. Exposure of the gene 49(-) intermediate capsid structures to basic proteins, such as bovine trypsin inhibitor, lysozyme, and l-polylysine-70, caused a displacement of an amorphous-appearing structure which may be a complex of the gene 49(-) DNA and interior components of the capsid (e.g., internal proteins, polyamines). Our general conclusion is that in the gene 49(-) intermediate head structures which are only partly filled with DNA, this DNA is held inside the head by strong electrostatic linkages with interior polypeptides and polyamines.

Capsid↗
PubMed2004

Fluorescently labeled adenovirus with pIX-EGFP for vector detection.

Adenoviruses are extensively studied in terms of their use as gene therapy vectors and pathogenesis. These vectors have been targeted on both transcriptional and transductional levels to achieve cell-specific gene delivery. Current detection strategies, including reporter gene expression, viral component detection, and vector labeling with fluorophores, have been applied to analyze adenoviral vectors; however, these methods are inadequate for assessing transductional targeting. As an alternative to conventional vector detection techniques, we developed a specific genetic labeling system whereby an adenoviral vector incorporates a fusion between capsid protein IX and EGFP. DNA packaging and thermostability were marginally hampered by the modification while DNA replication, cytopathic effect, and CAR-dependent binding were not affected. The fluorescent label was associated with the virus capsid and conferred a fluorescent property useful in detecting adenoviral particles in flow cytometry, tracking, and tissue sections. We believe our genetic adenovirus labeling system has important implications for vector development, detecting adenovirus vectors in targeting schemes, and studying adenovirus biology. In addition, this technique has potential utility for dynamic monitoring of adenovirus replication and spread.

Adenovirus E1 Proteins↗
PubMedDate not supplied

[Chromatin transcription].

Eukaryotic genome is organized in DNA--histone complexes called chromatin. The nucleosome core (approximately 150 bp DNA tightly wrapped around the histone octamer in two superhelical coils) is a minimal unit of chromatin organization. Chromatin is designed to allow both tight DNA packaging and proper functioning and regulation of enzymes such as DNA- and RNA-polymerases. The mechanisms allowing DNA to be accessible to enzymes of DNA metabolism in regulated manner are beginning to emerge. Thus the histone octamer has a remarkable ability to survive transcription by stepping around the transcribing polymerase without ever going free in solution. Moreover, nucleosomes can present a high barrier for transcribing polymerases that dramatically decreases the rate of transcription and probably is regulated in vivo. The mechanism of transcription through nucleosomal barrier will be discussed in detail below.

Animals↗
PubMed1981

Evidence for double replication of chromosomal DNA segments as a general consequence of DNA replication inhibition.

We have previously presented evidence that a transient inhibition of DNA synthesis by a pulse of 1-beta-D-arabinofuranosylcytosine (ara-C) results in a disruption of the pattern of replication of the chromosomal DNA of cultured human cells, resulting in some DNA segments being replicated more than once in a single S phase. Further evidence is presented in this paper that this effect is not a specific property of the ara-C molecule in that a similar effect is produced in cells by a pulse of 9-beta-D-arabinofuranosyladenine (ara-A) and also by a pulse of cycloheximide. The activated form of ara-A and ara-C (the triphosphates) both inhibit DNA synthesis at the level of the polymerase. Double replication following an ara-A pulse demonstrates that double replication after an ara-C pulse is not caused by some specific property of the ara-C molecule which might be unrelated to any effect on DNA synthesis. However, cycloheximide is an inhibitor of mammalian protein synthesis and inhibits DNA synthesis only indirectly, probably through a consequent deficiency of DNA-packaging proteins. Hence, the occurrence of double replication of chromosomal DNA segments following a pulse of cycloheximide is consistent with this phenomenon being a general and nonspecific consequence of the freezing of DNA replication forks.

Cell Line↗
PubMed2000

Interaction of the adenovirus IVa2 protein with viral packaging sequences.

We have demonstrated previously that the adenovirus L1 52/55-kDa protein binds to the viral IVa2 protein in infected cells. The significance of this interaction was unclear, however, based on the known functions of these two proteins: the 52/55-kDa protein is required for viral DNA packaging, while the IVa2 protein is a transactivator of the major late promoter (MLP). In this report, we have attempted to elucidate a role for each of the two proteins in the other's known function. There is no apparent effect of the 52/55-kDa protein on the interaction of the IVa2 protein with the MLP. Surprisingly, however, we found that the IVa2 protein can interact with the adenoviral packaging signal and that this interaction involves DNA sequences that have previously been demonstrated to be required for packaging.

Adenoviruses, Human↗
PubMed1988

Packaging of transducing DNA by bacteriophage P1.

P1 transduces bacterial chromosomal markers with widely differing frequencies. We use quantitative Southern hybridisations here to show that, despite this, most markers are packaged at similar levels. Exceptions are a group of markers near 2 min and another at 90 min which seem to be packaged at levels two- to threefold higher. We thus conclude that certain marker frequency variations in transduction can be explained by differences in packaging level, but that most cannot. The limited range in packaging levels suggests that P1 can initiate the packaging of chromosomal DNA from many sites. This idea is supported by our failure to find any chromosomal sequences with homology to the phage pac site and by the occurrence of hybridising bands which seem to suggest sequential packaging from a large number of specific sites. We eliminate the possibility that chromosomal DNA packaging is the result of endonucleolytic cutting by the P1 res enzyme.

Autoradiography↗