Search PubMed⌕ Search

SEARCH · Search PubMed

Results for “DNA Packaging”

Search indexed PubMed citations on genomics, clinical trials, systematic reviews and public health. Explore titles, authors and supplied subject terms, then open the PubMed record.

Quote a phrase for an exact phrase match. Source license links do not imply unrestricted reuse.

At least 253 records · Page 14Linked to original sources
PubMed2004

Structure of the DNA-SspC complex: implications for DNA packaging, protection, and repair in bacterial spores.

Bacterial spores have long been recognized as the sturdiest known life forms on earth, revealing extraordinary resistance to a broad range of environmental assaults. A family of highly conserved spore-specific DNA-binding proteins, termed alpha/beta-type small, acid-soluble spore proteins (SASP), plays a major role in mediating spore resistance. The mechanism by which these proteins exert their protective activity remains poorly understood, in part due to the lack of structural data on the DNA-SASP complex. By using cryoelectron microscopy, we have determined the structure of the helical complex formed between DNA and SspC, a characteristic member of the alpha/beta-type SASP family. The protein is found to fully coat the DNA, forming distinct protruding domains, and to modify DNA structure such that it adopts a 3.2-nm pitch. The protruding SspC motifs allow for interdigitation of adjacent DNA-SspC filaments into a tightly packed assembly of nucleoprotein helices. By effectively sequestering DNA molecules, this dense assembly of filaments is proposed to enhance and complement DNA protection obtained by DNA saturation with the alpha/beta-type SASP.

Bacillus subtilis↗
PubMed1990

Quantized viral DNA packaging revealed by rotating gel electrophoresis.

Two classes of missense mutations in the bacteriophage T4 gene coding for the major head protein produce phage with different length heads. The pt (petite) mutations produce phage with normal, intermediate, and isometric heads, whereas ptg (petite and giant) mutations also produce greatly elongated (giant) heads. DNA from petite, normal, and giant particles was clearly resolved by discontinuous rotating gel electrophoresis, and several new species of headful length DNA were found. These results confirm the idea that the major stop points for head length regulation are at Q = 13, 17, and 21, and also show that minor stop points exist at Q = 16, 18 and 20. The existence of these well-defined classes of DNA that correlate with capsid structure suggest that a structural relationship between the scaffold protein and the capsid protein determines head length and thus DNA length.

DNA, Viral↗
PubMed1999

Domain structure of gpNu1, a phage lambda DNA packaging protein.

The terminase enzyme from bacteriophage lambda is responsible for the insertion of a dsDNA genome into the confines of the viral capsid. The holoenzyme is composed of gpA and gpNu1 subunits in a gpA(1) x gpNu1(2) stoichiometry. While genetic studies have described regions within the two proteins responsible for DNA binding, capsid binding, and subunit interactions in the holoenzyme complex, biochemical characterization of these domains is limited. We have previously described the cloning, expression, and biochemical characterization of a soluble DNA binding domain of the terminase gpNu1 subunit (Met1 to Lys100) and suggested that the hydrophobic region spanning Lys100 to Pro141 defines a domain responsible for self-association interactions, and that is important for cooperative DNA binding [Yang et al. (1999) Biochemistry 38, 465-477]. We further suggested that the genetically defined gpA-interactive domain in the C-terminal half of the protein is limited to the C-terminal approximately 40 amino acids of gpNu1. Here we describe the cloning, expression, and biochemical characterization of gpNu1DeltaP141, a deletion mutant of gpNu1 that comprises the DNA binding domain and the putative hydrophobic self-assembly domain of the full-length protein. Purified gpNu1DeltaP141 shows a strong tendency to aggregate in solution; However, the protein remains soluble in 0.4 M guanidine hydrochloride, and circular dichroism (CD) and fluorescence spectroscopic studies demonstrate that the protein is folded under these conditions. Moreover, CD spectroscopy and thermally induced unfolding studies suggest that the DNA binding domain and the self-association domain represent independent folding domains of gpNu1DeltaP141. The mutant protein interacts weakly with the gpA subunit, but does not form a catalytically competent holoenzyme complex, suggesting that the C-terminal 40 residues are important for appropriate subunit interactions. Importantly, gpNu1DeltaP141 binds DNA tightly, but with less specificity than does full-length protein, and the data suggest that the C-terminal residues are further required for specific DNA binding activity. The implications of these results in the assembly of a functional holoenzyme complex are discussed.

Bacteriophage lambda↗
PubMed1996

The testis-specific high-mobility-group protein, a phosphorylation-dependent DNA-packaging factor of elongating and condensing spermatids.

Mammalian spermiogenesis is characterized by a striking restructuring of the spermatid chromatin caused by the replacement of nucleohistones with transition proteins and their subsequent replacement with nucleoprotamines. The onset of nuclear elongation and chromatin condensation in spermatids is accompanied by a general decrease in the transcriptional activity of the DNA. A recently identified testis-specific high-mobility-group (tsHMG) protein, similar to the human mitochondrial transcription factor I and to the linker-associated protein delta of Tetrahymena thermophila micronuclei, is thought to play a structural role in this process. We confirm by immunoblot analysis of fractionated germ cells that the presence of tsHMG is restricted to transcriptionally quiescent elongating and condensing spermatids. Purified recombinant tsHMG protein displays preferential binding to supercoiled plasmid DNA, which reversibly protects the DNA against the DNA-relaxing activity of eukaryotic topoisomerase I and also impairs the transcriptional activity of this template when assayed in vitro. The tsHMG protein can also introduce negative supercoils into a relaxed plasmid substrate in a topoisomerase I-dependent manner. We also show that the tsHMG protein is the substrate of a Ca2+-phospholipid-dependent protein kinase (protein kinase C) present in testis extracts of adult mice and demonstrate that phosphorylation by protein kinase C is required for both the DNA-binding and the topoisomerase I-dependent supercoiling activities of tsHMG. Our results support the hypothesis that the spermatid tsHMG protein is a topological factor (transition protein) that can modulate the activity of topoisomerase I. This activity could contribute to the important transition in chromatin structure which leads to the decrease in DNA metabolism observed at the early stages of spermatid elongation.

Animals↗
PubMed2005

The herpes simplex virus type 1 DNA packaging protein UL17 is a virion protein that is present in both the capsid and the tegument compartments.

The UL17 protein of herpes simplex virus type 1 is essential for packaging the viral genome into the procapsid, a spherical assembly intermediate, and is present in the mature virus particle. We have examined the distribution of UL17 in various assembly products and virions to determine which component of the virus particle UL17 is associated with and at what stage in capsid assembly UL17 is required. UL17 was present in the procapsid, in the DNA-containing angularized C capsid, and in two other angularized capsid forms, A and B, that lack DNA and are thought to be dead-end products. The results suggest that UL17 is a minor capsid protein which is incorporated into the procapsid during assembly of the particle. UL17 was also found in virions and in noninfectious structures known as light (L) particles, which possess a tegument and envelope but lack a capsid. The level of UL17 in these particles was much greater than the amount that could be attributed to capsid contamination of the purified L-particle preparation, suggesting that UL17 is also a tegument protein. The finding that virions contain approximately twofold more UL17 than do C capsids provided further support for the idea that UL17 is present in two different structural components within the mature virion. The UL25 packaging protein, which is also present in virions, was not found in significant amounts in L particles, indicating that it is associated only with the capsid. UL6, the third virion-associated packaging protein, was present in slightly increased levels in L particles.

Animals↗
PubMedDate not supplied

Nanoparticulate DNA packaging using terpolymers of poly(lysine-g-(lactide-b-ethylene glycol)).

Terpolymers of poly(lysine-g-(lactide-b-ethylene glycol)) (pK-pLL-pEG) were synthesized by using ring-opening polymerization and functional end-group grafting. Synthesis was characterized with gel permeation chromatography, proton nuclear magnetic resonance spectroscopy, and a trinitrobenzene sulfonic acid binding assay. Polymer association behavior with DNA was investigated using an ethidium bromide exclusion assay, static light scattering, and scanning electron microscopy. Polylactide molecular weight was varied to investigate its impact on DNA association and resulting complex characteristics. Polylysine ( = 8800, DP = 42) modified with either 7400 or 10 870 pLL-pEG reduced the minimum amount of primary amines necessary for complete condensation by 23% and 48%, respectively, compared to unmodified polylysine (pK42). Complexes formed with the highest molecular weight terpolymer demonstrated significantly (p < 0.1) greater resistance to DNase I than lyophilized pK42-DNA particles. This study suggests that modification of pK42 with pLL-pEG diblock copolymers impacts polylysine's associative and binding behavior to DNA and resulting particle characteristics. Modulation of terpolymer composition in complexes can enable control over intracellular plasmid dissociation rates to improve transfection efficiency.

DNA↗
PubMed2003

Mechanics of DNA packaging in viruses.

A new generation of single-molecule experiments has opened up the possibility of reexamining many of the fundamental processes of biochemistry and molecular biology from a unique and quantitative perspective. One technique producing a host of intriguing results is the use of optical tweezers to measure the mechanical forces exerted by molecular motors during key processes such as the transcription of DNA or the packing of a viral genome into its capsid. The objective of the current article is to respond to such measurements on viruses and to use the theory of elasticity and a simple model of charge and hydration forces to derive the force required to pack DNA into a viral capsid as a function of the fraction of the viral genome that has been packed. The results are found to be in excellent accord with recent measurements and complement previous theoretical work. Because the packing of DNA in viral capsids occurs under circumstances of high internal pressure, we also compute how much pressure a capsid can sustain without rupture.

Biomechanical Phenomena↗
PubMed1999

Oligonucleotide and plasmid DNA packaging into polyoma VP1 virus-like particles expressed in Escherichia coli.

The drug delivery system described here is based on the properties of the capsoid or capsid-like structure resulting from the assembly of polyoma virus capsid protein VP1 expressed in Escherichia coli. The capsid protein VP1 was expressed as a fusion protein with a completely removable N-terminal His6 affinity tag. The pentameric morphology of the recombinant VP1 protein was confirmed by electron microscopy after affinity chromatography and factor Xa cleavage under conditions of low ionic strength. The self-assembly of VP1 capsoids can be induced from purified VP1 pentamers by increasing the ionic strength with (NH4)2SO4. These VP1 capsoid particles were packed in vitro with anti-sense oligonucleotides and plasmid DNA. The loading with DNA was pH-dependent. We observed the highest efficiency at pH 5. DNase I treatment of particles with encapsidated material showed that 37-55% of the bound oligonucleotides and fragments of 1.5-1.8 kb double-stranded DNA were protected against degradation.

Base Sequence↗
PubMed1984

DNA packaging in mouse spermatids. Synthesis of protamine variants and four transition proteins.

A comparison of the protein compositions of mouse late-step spermatids and cauda epididymal sperm has revealed that the relative distribution of the two amino acid sequence variants of mouse protamine differ markedly in spermatids and sperm. Sonication-resistant spermatids contain the two variants in a ratio of 1:1, while the ratio of these two proteins in cauda epididymal sperm is approx. 2:1. Labeling studies in vivo have shown that this difference is due, in part, to an asynchrony in the time of synthesis of the two protamine variants. Both proteins are synthesized in late-step spermatids, but synthesis of the tyrosine variant in sperm chromatin begins approximately one day before synthesis of the more predominant histidine variant. Analyses of the time of synthesis of protamine and the four transition proteins in late-step spermatids allowed us to estimate the spermatid stage in which these proteins are deposited on DNA and relate these events to the onset of sonication resistance in maturing spermatids. These results indicate that: (1) synthesis and deposition of protamine begins coincident with the onset of sonication resistance in early step 12 spermatids; (2) protamine deposition is complete by mid-step 15; and (3) synthesis of the transition proteins occurs coincident with protamine synthesis.

Animals↗
PubMed1983

The interaction of cos with Chi is separable from DNA packaging in recA-recBC-mediated recombination of bacteriophage lambda.

Chi (5'-GCTGGTGG) is a recombinator in RecA-RecBC-mediated recombination in Escherichia coli. In bacteriophage lambda vegetative recombination, Chi is fully active only when it is correctly oriented with respect to cos, the site that defines the ends of the packaged chromosome. Here we demonstrate that packaging from cos is not necessary for this cos-Chi interaction. Our evidence suggests that correctly oriented cos is an activator of Chi. cos, as an activator, is (1) dominant over cos-, (2) active opposite an extensive heterology, (3) able to interact with Chi only when on the same (cis) chromosome, and (4) able to interact with Chi at distances as far as greater than or equal to 20 kb. Thus, cos and Chi form a two-component recombinator system for general recombination. cos may serve as an asymmetric entry site for a recombination enzyme that recognizes Chi in an asymmetric way.

Bacteriophage lambda↗
PubMed2006

Herpes simplex virus 1 DNA packaging proteins encoded by UL6, UL15, UL17, UL28, and UL33 are located on the external surface of the viral capsid.

Studies to localize the herpes simplex virus 1 portal protein encoded by UL6, the putative terminase components encoded by UL15, UL 28, and UL33, the minor capsid proteins encoded by UL17, and the major scaffold protein ICP35 were conducted. ICP35 in B capsids was more resistant to trypsin digestion of intact capsids than pUL6, pUL15, pUL17, pUL28, or pUL33. ICP35 required sectioning of otherwise intact embedded capsids for immunoreactivity, whereas embedding and/or sectioning decreased the immunoreactivities of pUL6, pUL17, pUL28, and pUL33. Epitopes of pUL15 were recognized roughly equally well in both sectioned and unsectioned capsids. These data indicate that pUL6, pUL17, pUL28, pUL33, and at least some portion of pUL15 are located at the external surface of the capsid.

Animals↗
PubMed2002

Structure and function of phi29 hexameric RNA that drives the viral DNA packaging motor: review.

One notable feature of linear dsDNA viruses is that, during replication, their lengthy genome is squeezed with remarkable velocity into a preformed procapsid and packed into near crystalline density. A molecular motor using ATP as energy accomplishes this energetically unfavorable motion tack. In bacterial virus phi29, an RNA (pRNA) molecule is a vital component of this motor. This 120-base RNA has many novel and distinctive features. It contains strong secondary structure, is tightly folded, and unusually stable. Upon interaction with ion and proteins, it has a knack to adapt numerous conformations to perform versatile function. It can be easily manipulated to form stable homologous monomers, dimers, trimers and hexamers. As a result, many unknown properties of RNA have been and will be unfolded by the study of this extraordinary molecule. This article reviews the structure and function of this pRNA and focuses on novel methods and unique approaches that lead to the illumination of its structure and function.

Bacillus Phages↗
PubMed2003

Morphology of temperate bacteriophage SfV and characterisation of the DNA packaging and capsid genes: the structural genes evolved from two different phage families.

The entire genome of SfV, a temperate serotype-converting bacteriophage of Shigella flexneri, has recently been sequenced (Allison, G.E., Angeles, D., Tran-Dinh, N., Verma, N.K. 2002, J. Bacteriol. 184, 1974-1987). Based on the sequence analysis, we further characterised the SfV virion structure and morphogenesis. Electron microscopy indicated that SfV belongs to the Myoviridae morphology family. Analysis of the proteins encoded by orf1, orf2, and orf3 revealed that they were homologous to small and large terminase subunits, and portal proteins, respectively; the protein encoded by orf5 showed homology to capsid proteins. Western immunoblot of the phage with anti-SfV sera revealed two antigenic proteins, and the N-terminal amino acid sequence of the 32-kDa protein corresponded to amino acids 116 to 125 of the ORF5 protein, suggesting that the capsid may be processed. Functional analysis of orf4 showed that it encodes the phage capsid protease. The proteins encoded by orfs1, 2, 3, 4, and 5 are homologous to similar proteins in the Siphoviridae phage family of both gram-positive and gram-negative origin. The capsid and morphogenesis genes are upstream and adjacent to the genes encoding Myoviridae (Mu-like) tail proteins. The organisation of the structural genes of SfV is therefore unique as the head and tail genes originate from different morphology groups.

Amino Acid Sequence↗
PubMed1985

Packaged DNA. An elastic model.

We review and deepen a theory of elastic bending of DNA on a persistence length scale. In a regime of extensive charge neutralization the axis of the double helix is elastically unstable when straight. Its stable bent conformation allows nucleation of DNA toruses and in principle could direct the supercoiled (solenoid) form of a polynucleosome. The Euler theory of elastic instability of macroscopic rods gives a partial description of the intrinsic ability of DNA to form locally stable bends. A different, quasi-Eulerian theory can be based on what is probably the dominant bending mechanism of DNA in solution-flexible kinking at the sites of open base pairs. This predictive theory is in quantitative agreement with the observed value (about 16 nm) for the minimum radius of torus holes. Stability of the inner torus ring is achieved when DNA phosphate groups are about 90% neutralized by trivalent cations, another prediction that is consistent with the observed formation of toruses in these conditions. The predicted stable radius of curvature of charge-neutralized DNA is also equal to the radial dimension of a maximally contracted polynucleosome supercoil as measured by neutron scattering (17 nm), but further experimental investigation of the geometrical disposition of the spacer DNA regions in the solenoid will be necessary to rule out the possibility of accidental agreement for this complex system. We stress again the experimental reality and probable importance of open base pairs in the equilibrium solution conformation of DNA.

Adenine Nucleotides↗
PubMed2006

Specific short hairpin RNA-mediated inhibition of viral DNA packaging of human cytomegalovirus.

To clearly demonstrate the role of the human cytomegalovirus (HCMV) portal protein pUL104 RNA interference was applied. Expressing cell lines were constructed by transduction of shRNAs via the infection with retroviral vectors. After infection of these cells with HCMV AD169 the expression of pUL104 was reduced up to 80% for shRNA S1 and 54% for shRNA S2 at late times of infection compared to controls. In addition, the inhibitory effect was corresponding with a decrease in viral mRNA and plaque formations. Electron microscopic analysis demonstrated that infection of cells expressing pUL104-specific shRNAs resulted in the inhibition of formation of replicative particles.

Cell Line↗