DNA packaging by the double-stranded DNA bacteriophages.
Explore the source record for details and available documents.
Adenosine Triphosphate↗
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.
Herpes simplex virus DNA packaging sequences adopt novel structures that are specifically recognized by a component of the cleavage and packaging machinery.
The product of the herpes simplex virus type 1 U(L)28 gene is essential for cleavage of concatemeric viral DNA into genome-length units and packaging of this DNA into viral procapsids. To address the role of U(L)28 in this process, purified U(L)28 protein was assayed for the ability to recognize conserved herpesvirus DNA packaging sequences. We report that DNA fragments containing the pac1 DNA packaging motif can be induced by heat treatment to adopt novel DNA conformations that migrate faster than the corresponding duplex in nondenaturing gels. Surprisingly, these novel DNA structures are high-affinity substrates for U(L)28 protein binding, whereas double-stranded DNA of identical sequence composition is not recognized by U(L)28 protein. We demonstrate that only one strand of the pac1 motif is responsible for the formation of novel DNA structures that are bound tightly and specifically by U(L)28 protein. To determine the relevance of the observed U(L)28 protein-pac1 interaction to the cleavage and packaging process, we have analyzed the binding affinity of U(L)28 protein for pac1 mutants previously shown to be deficient in cleavage and packaging in vivo. Each of the pac1 mutants exhibited a decrease in DNA binding by U(L)28 protein that correlated directly with the reported reduction in cleavage and packaging efficiency, thereby supporting a role for the U(L)28 protein-pac1 interaction in vivo. These data therefore suggest that the formation of novel DNA structures by the pac1 motif confers added specificity on recognition of DNA packaging sequences by the U(L)28-encoded component of the herpesvirus cleavage and packaging machinery.
Base Sequence↗
Self-association properties of the bacteriophage lambda terminase holoenzyme: implications for the DNA packaging motor.
Terminases are enzymes common to complex double-stranded DNA viruses and are required for packaging of viral DNA into a protective capsid. Bacteriophage lambda terminase holoenzyme is a hetero-oligomer composed of the A and Nu1 lambda gene products; however, the self-association properties of the holoenzyme have not been investigated systematically. Here, we report the results of sedimentation velocity, sedimentation equilibrium, and gel-filtration experiments studying the self-association properties of the holoenzyme. We find that purified, recombinant lambda terminase forms a homogeneous, heterotrimeric structure, consisting of one gpA molecule associated with two gpNu1 molecules (114.2 kDa). We further show that lambda terminase adopts a heterogeneous mixture of higher-order structures, with an average molecular mass of 528(+/-34) kDa. Both the heterotrimer and the higher-order species possess site-specific cos cleavage activity, as well as DNA packaging activity; however, the heterotrimer is dependent upon Escherichia coli integration host factor (IHF) for these activities. Furthermore, the ATPase activity of the higher-order species is approximately 1000-fold greater than that of the heterotrimer. These data suggest that IHF bending of the duplex at the cos site in viral DNA promotes the assembly of the heterotrimer into a biologically active, higher-order packaging motor. We propose that a single, higher-order hetero-oligomer of gpA and gpNu1 functions throughout lambda development.
Adenosine Triphosphatases↗
Bacteriophage lambda DNA packaging: a mutant terminase that is independent of integration host factor.
Lambda+ is able to grow in Escherichia coli cells lacking integration host factor (IHF), producing a burst of approximately 25% that produced in IHF+ cells. In vitro, however, we find that the lambda DNA packaging enzyme terminase is strongly dependent on IHF in both cos cleavage reactions and DNA packaging reactions. The cos59 mutation renders lambda dependent on IHF in vivo. The cos59 mutation is a deletion of 3 base pairs at the XmnI site in the cohesive end site (cos) of lambda. Variants of lambda cos59 that were able to grow in the absence of IHF were isolated and found to carry a mutation, called ms1, in the Nu1 gene, which codes for the small subunit of terminase. The Nu1ms1 mutation results in a change of the 40th amino acid of the Nu1 gene product from leucine to phenylalanine. The Nu1ms1 terminase was independent of IHF in packaging reactions in vitro. The results indicate that the mutation either renders terminase: (1) able to utilize some host protein other than IHF, or (2) totally independent of host factors.
Bacterial Proteins↗
The role of cosB, the binding site for terminase, the DNA packaging enzyme of bacteriophage lambda, in the nicking reaction.
cosB is the binding site for terminase, the DNA packaging enzyme of ai-12581mbda, and cosN is the adjacent site at which terminase gm-07228es staggered nicks to generate mature lambda DNA molecules. There are three binding sites (R3, R2 and R1) within cosB for gpNu1, the small subunit of terminase. A particular transition mutation of R1, known to weaken binding of gpNu1 to R1, has been introduced into the other R sites, and in the present work the effects of R site mutations on nicking of cosN have been examined. Nicking experiments performed in the presence of ATP suggest that the most profound cosB mutation tested (the R3-R2-R1- mutation) would, at most, reduce cos nicking to congruent to 30% of the level observed for the wild-type substrate. In the presence of ATP, the R3-R2-R1- mutation had no significant effect on terminase nicking of the 1 strand and reduced r-strand nicking to 35% of the wild-type level. The other cosB mutations had no effect on the nicking of either DNA strand when nucleotide was added, but in the absence of ATP, most of the cos mutations resulted in some form of cosN nicking defect; the nicking defects, however, are milder than the in vivo packaging defects that result from the mutations. Quantitatively, only the effect of the R3-R2-R1- mutation on in vitro cosN nicking is reflective of the growth defect exhibited by a R3-R2-R1- phage but the nicking defect is only observed when ATP is omitted from the reaction. The proposal that the cosB mutations primarily affect DNA packaging rather than cosN nicking is discussed. All of the cosB mutations affect r-strand nicking to a greater extent than 1-strand nicking, implying that the interaction of terminase with the left half of cosN occurs via the direct recognition of cosNL by terminase. The level of DNA substrate required for half-maximal cos nicking is approximately equivalent for reactions performed in the presence or absence of ATP, indicating that ATP does not increase the affinity of terminase for cosB. ATP does accelerate the rate of cos nicking, suggesting that the role of ATP in promoting nicking of the cosB- DNAs is primarily to increase the rate of conversion of a cosN-terminase complex into product. A possible fourth R site, R4, is located on the other side of cosN from cosB.(ABSTRACT TRUNCATED AT 400 WORDS)
Adenosine Triphosphate↗
Nucleotide sequence of the DNA packaging and capsid synthesis genes of bacteriophage P2.
Overlapping DNA fragments containing the DNA packaging and capsid synthesis gene region of bacteriophage P2 were cloned and sequenced. In this report we present the complete nucleotide sequence of this 6550 bp region. Each of six open reading frames found in the interval was assigned to one of the essential genes (Q, P, O, N, M and L) by correlating genetic, physical and mutational data with DNA and protein sequence information. Polypeptides predicted were: a capsid completion protein, gpL; the major capsid precursor, gpN; the presumed capsid scaffolding protein; gpO; the ATPase and proposed endonuclease subunits of terminase, gpP and gpM, respectively; and a candidate for the portal protein, gpQ. These gene and protein sequences exhibited no homology to analogous genes or proteins of other bacteriophages. Expression of gene Q in E. coli from a plasmid caused production of a Mr 39,000 Da protein that restored Qam34 growth. This sequence analysis found only genes previously known from analysis of conditional-lethal mutations. No new capsid genes were found.
Adenosine Triphosphatases↗
The structure of an infectious P22 virion shows the signal for headful DNA packaging.
Bacteriophages, herpesviruses, and other large double-stranded DNA (dsDNA) viruses contain molecular machines that pump DNA into preassembled procapsids, generating internal capsid pressures exceeding, by 10-fold, that of bottled champagne. A 17 angstrom resolution asymmetric reconstruction of the infectious P22 virion reveals that tightly spooled DNA about the portal dodecamer forces a conformation that is significantly different from that observed in isolated portals assembled from ectopically expressed protein. We propose that the tight dsDNA spooling activates the switch that signals the headful chromosome packing density to the particle exterior.
Bacteriophage P22↗
The terminase enzyme from bacteriophage lambda: a DNA-packaging machine.
This review focuses on the biochemical, biophysical, and catalytic properties of terminase, an enzyme involved in bacteriophage lambda genome packaging. The holoenzyme possesses ATPase, DNA strand-separation, and site-specific nuclease activities that work in concert to insert a viral genome into the confines of a performed capsid. Moreover, the terminase subunits are part of a series of nucleoprotein complexes involved in genome packaging, including remarkably stable intermediates that transition to a highly mobile DNA packaging 'machine.' Models for the assembly and interconversion of these complexes are presented. Interactions between the catalytic sites in the enzyme complex, and modulation of these catalytic activities as it relates to the assembly and relative stability of the packaging intermediates are discussed. This ordered progression of nucleoprotein intermediates is a common theme in biology as demonstrated by mechanistic similarities between viral DNA packaging, the initiation of chromosomal replication, and the initiation of transcription. Terminase is thus part of a growing number of examples of biological 'machines' or molecular 'motors.'
Adenosine Triphosphatases↗
Cryo-EM asymmetric reconstruction of bacteriophage P22 reveals organization of its DNA packaging and infecting machinery.
The mechanisms by which most double-stranded DNA viruses package and release their genomic DNA are not fully understood. Single particle cryo-electron microscopy and asymmetric 3D reconstruction reveal the organization of the complete bacteriophage P22 virion, including the protein channel through which DNA is first packaged and later ejected. This channel is formed by a dodecamer of portal proteins and sealed by a tail hub consisting of two stacked barrels capped by a protein needle. Six trimeric tailspikes attached around this tail hub are kinked, suggesting a functional hinge that may be used to trigger DNA release. Inside the capsid, the portal's central channel is plugged by densities interpreted as pilot/injection proteins. A short rod-like density near these proteins may be the terminal segment of the dsDNA genome. The coaxially packed DNA genome is encapsidated by the icosahedral shell. This complete structure unifies various biochemical, genetic, and crystallographic data of its components from the past several decades.
Bacteriophage P22↗
Alterations of the portal protein, gpB, of bacteriophage lambda suppress mutations in cosQ, the site required for termination of DNA packaging.
The cosQ site of bacteriophage lambda is required for DNA packaging termination. Previous studies have shown that cosQ mutations can be suppressed in three ways: by a local suppressor within cosQ, an increase in the length of the lambda chromosome, and missense mutations affecting the prohead's portal protein, gpB. In the present work, revertants of a set of lethal cosQ mutants were screened for suppressors. Seven new cosQ suppressors affected gene B, which encodes the portal protein of the prohead. All seven were allele-nonspecific suppressors of cosQ mutations. Experiments with several phages having two cosQ suppressors showed that the suppression effects were additive. Furthermore, these double suppressors had minimal effects on the growth of cosQ(+) phages. These trans-acting suppressors affecting the portal protein are proposed to allow the mutant cosQ site to be more efficiently recognized, due to the slowing of the rate of translocation.
Alleles↗
Methods for structural and functional analysis of an RNA hexamer of bacterial virus phi29 DNA packaging motor.
During multiplication and maturation, the lengthy genomic DNA of dsDNA viruses is translocated with remarkable velocity into a limited space within the procapsid and packaged to crystalline density. A viral DNA-packaging motor accomplishes this energy consuming motion task. An RNA molecule of bacterial virus phi29 has been found to be a vital component of the DNA-packaging motor. Six pRNAs form a hexagonal complex to gear the DNA translocating machine using a mechanism similar to the driving of a bolt with a hex nut. Sequential action of six RNA molecules to drive the motor is similar to the consecutive firing of six cylinders of a car engine. This article reviews the structure of pRNA to demonstrate that its structure plays a vital role in its function, and focuses on methods and unique approaches that lead to the elucidation of pRNA structure.
Bacillus Phages↗
The products of herpes simplex virus type 1 gene UL26 which are involved in DNA packaging are strongly associated with empty but not with full capsids.
We report on the properties of a family of related herpes simplex virus type 1 polypeptides (designated p40) of Mr around 40,000. The intracellular localization of these polypeptides has been examined using monoclonal antibodies and their association with viral capsids within the nuclei of infected cells has been demonstrated directly by immunoelectron microscopy. Specific DNA staining and the use of mutants defective for DNA packaging has revealed, in contrast to earlier findings, that p40 is present in empty capsids. Protein p40 is not present as a major component of full capsids or of mature virions indicating that it is transiently associated with capsids and that its removal from capsids is linked with the process of DNA packaging.
Capsid↗
Changes of the DNA packaging mode during boar sperm maturation.
Changes in the mode of DNA packaging in nuclei during spermatogenesis were studied by measuring of the fluorescence anisotropy decay of an ethidium dye intercalated in the DNA in whole nuclei. The nuclei were isolated from boar spermatid or sperm cells at three distinct stages of spermatogenesis: just before the completion of a maturation process in the testis (late spermatid), immediately after a subsequent transformation into spermatozoa (caput spermatozoon), and after full maturation (cauda spermatozoon). Although these three kinds of nucleus were morphologically indistinguishable from each other, the anisotropy decay detected a clear difference. In the late spermatid nuclei, in which the replacement of histones by protamine was still in progress, the anisotropy decayed extensively. The decay suggested that the DNA in the spermatid nuclei contained very flexible regions, in which the interaction of the DNA and proteins may be weak. The rapid and extensive anisotropy decay was absent in the caput and cauda nuclei. The flexible portions must have turned into very rigid structure during transformation from the late spermatid into the caput spermatozoon.
Animals↗
DNA packaging and organization in mammalian spermatozoa: comparison with somatic cells.
Mammalian sperm DNA is the most tightly compacted eukaryotic DNA, being at least sixfold more highly condensed than the DNA in mitotic chromosomes. To achieve this high degree of packaging, sperm DNA interacts with protamines to form linear, side-by-side arrays of chromatin. This differs markedly from the bulkier DNA packaging of somatic cell nuclei and mitotic chromosomes, in which the DNA is coiled around histone octamers to form nucleosomes. The overall organization of mammalian sperm DNA, however, resembles that of somatic cells in that both the linear arrays of sperm chromatin and the 30-nm solenoid filaments of somatic cell chromatin are organized into loop domains attached at their bases to a nuclear matrix. In addition to the sperm nuclear matrix, sperm nuclei contain a unique structure termed the sperm nuclear annulus to which the entire complement of DNA appears to be anchored when the nuclear matrix is disrupted during decondensation. In somatic cells, proper function of DNA is dependent upon the structural organization of the DNA by the nuclear matrix, and the structural organization of sperm DNA is likely to be just as vital to the proper functioning of the spermatozoa.
Animals↗
Confirmation of the helical structure of the 5'/3' termini of the essential DNA packaging pRNA of phage phi 29.
Bacteriophage phi 29 is typical of double-stranded DNA viruses in that its genome is packaged into a preformed procapsid during viral assembly. An intriguing feature of phi 29 is the presence of a 120-base virus-encoded RNA (pRNA) that is indispensable for DNA packaging. Phylogenetic comparison of similar RNAs in numerous phages has revealed that the secondary structure of the pRNA is well conserved. Computer analysis predicts the presence of an extensive segment of helix with three single-base bulges generated by the pairing of the 5' and 3' ends. The desire to understand the role played by the pRNA in DNA packaging has led to a mutational analysis of the 5'-/3'-terminal region, which is believed to be important in DNA translocation. Deletion of 3 bases from the 3' end of the RNA, shortening the pRNA from 120 to 117 bases, was tolerated without loss of activity, but additional deletion of the base 117 resulted in 100-fold less activity, and a 115-base pRNA was virtually nonfunctional. Additionally, the three unpaired one-base bulges within the helical stretches of the paired proximate ends were nonessential for pRNA activity, as demonstrated by deletion of the bulge individually. An extensive series of helix disruptions by single- and multiple-base substitution almost invariably led to the loss of DNA packaging activity. Additional mutations that restored predicted base pairings rescued pRNA activity. This second site suppression confirmed that the 5'- and 3'-end region was paired and was indeed a helical stretch. The secondary structure was of greater importance than the primary sequence, with the exception of the requirement of an adenine at either the third or fourth position. The specific requirement of an adenine in phi 29 pRNA at this position, as well as conservation of this position in other phage pRNAs, implicates that this base may play a special role in either the DNA-packaging reaction or the maintenance of the pRNA tertiary structure.
Bacillus Phages↗
The chromosome of Shigella flexneri bacteriophage Sf6: complete nucleotide sequence, genetic mosaicism, and DNA packaging.
Shigella flexneri temperate bacteriophage Sf6 is of interest in part because its prophage expresses the oac gene that alters the antigenic properties of the surface O-antigen polysaccharide of its host bacterium. We have determined the complete sequence of its 39,044 bp genome. The sequence shows that Sf6 is a member of the canonical lambdoid phage group, and like other phages of this type has a highly mosaic genome. It has chromosomal regions that encode proteins >80% identical with at least 15 different previously characterized lambdoid phages and prophages, but 43% of the genome, including the virion assembly genes, is homologous to the genome of one phage, HK620. An analysis of the nucleotide differences between Sf6 and HK620 indicates that even these similar regions are highly mosaic. This mosaicism suggests ways in which the virion structural proteins might interact with each other. The Sf6 early operons are arranged like a typical lambdoid phage, with "boundary sequences" often found between functional modules in the "metabolic" genome domain. By virtue of high degree of similarity in the encoding genes and their DNA target sites, we predict that the integrase, early transcription anti-terminator, CI and Cro repressors, and CII protein of Sf6 have DNA binding specificities very similar to the homologous proteins encoded by phages HK620, lambda, 434 and P22, respectively. The late operon contains two tRNA genes. The Sf6 terminase genes are unusual. Analysis of in vivo initiation of the DNA packaging series showed that the Sf6 apparatus that recognizes DNA for packaging appears to cleave DNA for initiation of packaging series at many sites within a large region of about 1800 bp that includes a possible pac site. This is unlike previously characterized phage packaging mechanisms.
Bacteriophages↗
The functional asymmetry of cosN, the nicking site for bacteriophage lambda DNA packaging, is dependent on the terminase binding site, cosB.
cosN is the site at which terminase, the DNA packaging enzyme of phage lambda, introduces staggered nicks into viral concatemeric DNA to initiate genome packaging. Although the nick positions and many of the base pairs of cosN show 2-fold rotational symmetry, cosN is functionally asymmetric. That is, the cosN G2C mutation in the left half-site (cosNL) causes a strong virus growth defect whereas the symmetrically disposed cosN C11G mutation in the right half-site (cosNR) does not affect virus growth. The experiments reported here test the proposal that the genetic asymmetry of cosN results from terminase interactions with cosB, a binding site to the right of cosN. In the presence of cosB, the left half-site mutation, cosN G2C, strongly affected the cos cleavage reaction, while the symmetric right half-site mutation, cosN C11G, had little effect. In the absence of cosB, the two mutations moderately reduced the rate of cos cleavage by the same amount. The results indicated that the functional asymmetry of cosNdepends on the presence of cosB. A model is discussed in which terminase-cosN interactions in the nicking complex are assisted by anchoring of terminase to cosB.
Bacteriophage lambda↗
DNA packaging orders the membrane of bacteriophage PRD1.
Bacteriophage PRD1 contains a linear dsDNA genome enclosed by a lipid membrane lying within a protein coat. Determination of the structure of the detergent-treated particle to 2 nm by cryo-electron microscopy and three-dimensional reconstruction has defined the position of the major coat protein P3. The coat contains 240 copies of trimeric P3 packed into positions of local 6-fold symmetry on a T = 25 lattice. The three-dimensional structures of the PRD1 virion and a DNA packaging mutant to a resolution of 2.8 nm have revealed specific interactions between the coat and the underlying membrane. The membrane is clearly visible as two leaflets separated by 2 nm and spanned by transmembrane density. The size of the coat does not change upon DNA packaging. Instead, the number of interactions seen between the protein shell and the membrane and the order of the membrane components increase. Thus the membrane of PRD1 plays a role in assembly which is akin to that played by the nucleocapsid in other membrane viruses.
Bacteriophages↗