Thermus thermophilus bacteriophage phiYS40 genome and proteomic characterization of virions

We determined the sequence of the 152,372 bp genome of phiYS40, a lytic tailed bacteriophage of Thermus thermophilus. The genome contains 170 putative open reading frames and three tRNA genes. Functions for 25% of phiYS40 gene products were predicted on the basis of similarity to proteins of known function from diverse phages and bacteria. phiYS40 encodes a cluster of proteins involved in nucleotide salvage, such as flavin-dependent thymidylate synthase, thymidylate kinase, ribonucleotide reductase, and deoxycytidylate deaminase, and in DNA replication, such as DNA primase, helicase, type A DNA polymerase, and predicted terminal protein involved in initiation of DNA synthesis. The structural genes of phiYS40, most of which have no similarity to sequences in public databases, were identified by mass spectrometric analysis of purified virions. Various phiYS40 proteins have different phylogenetic neighbors, including myovirus, podovirus, and siphovirus gene products, bacterial genes and, in one case, a dUTPase from a eukaryotic virus. phiYS40 has apparently arisen through multiple acts of recombination between different phage genomes as well as through acquisition of bacterial genes.     

The ATPase domain of the large terminase protein, gp17, from bacteriophage T4 binds DNA: implications to the DNA packaging mechanism

Translocation of double-stranded DNA into a preformed capsid by tailed bacteriophages is driven by powerful motors assembled at the special portal vertex. The motor is thought to drive processive cycles of DNA binding, movement, and release to package the viral genome. In phage T4, there is evidence that the large terminase protein, gene product 17 (gp17), assembles into a multisubunit motor and translocates DNA by an inchworm mechanism. gp17 consists of two domains; an N-terminal ATPase domain (amino acids 1-360) that powers translocation of DNA, and a C-terminal nuclease domain (amino acids 361-610) that cuts concatemeric DNA to generate a headful-size viral genome. While the functional motifs of ATPase and nuclease have been well defined and the ATPase atomic structure has been solved, the DNA binding motif(s) responsible for viral DNA recognition, cutting, and translocation are unknown. Here we report the first evidence for the presence of a double-stranded DNA binding activity in the gp17 ATPase domain. Binding to DNA is sensitive to Mg²⁺ and salt, but not the type of DNA used. DNA fragments as short as 20 bp can bind to the ATPase but preferential binding was observed to DNA greater than 1 kb. A high molecular weight ATPase-DNA complex was isolated by gel filtration, suggesting oligomerization of ATPase following DNA interaction. DNA binding was not observed with the full-length gp17, or the C-terminal nuclease domain. The small terminase protein, gp16, inhibited DNA binding, which was further accentuated by ATP. The presence of a DNA binding site in the ATPase domain and its binding properties implicate a role in the DNA packaging mechanism.     

Assembly architecture and DNA binding of the bacteriophage P22 terminase small subunit

Morphogenesis of bacteriophage P22 involves the packaging of double-stranded DNA into a preassembled procapsid. DNA is translocated by a powerful virally encoded molecular motor called terminase, which comprises large (gp2, 499 residues) and small (gp3, 162 residues) subunits. While gp2 contains the phosphohydrolase and endonuclease activities of terminase, the function of gp3 may be to regulate specific and nonspecific modes of DNA recognition as well as the enzymatic activities of gp2. Electron microscopy shows that wild-type gp3 self-assembles into a stable and monodisperse nonameric ring. A three-dimensional reconstruction at 18 Å resolution provides the first glimpse of P22 terminase architecture and implies two distinct modes of interaction with DNA—involving a central channel of 20 Å diameter and radial spikes separated by 34 Å. Electromobility shift assays indicate that the gp3 ring binds double-stranded DNA nonspecifically in vitro via electrostatic interactions between the positively charged C-terminus of gp3 (residues 143-152) and phosphates of the DNA backbone. Raman spectra show that nonameric rings formed by subunits truncated at residue 142 retain the subunit fold despite the loss of DNA-binding activity. Difference density maps between gp3 rings containing full-length and C-terminally truncated subunits are consistent with localization of residues 143-152 along the central channel of the nonameric ring. The results suggest a plausible molecular mechanism for gp3 function in DNA recognition and translocation.     

DNA packaging motor assembly intermediate of bacteriophage phi29

Unraveling the structure and assembly of the DNA packaging ATPases of the tailed double-stranded DNA bacteriophages is integral to understanding the mechanism of DNA translocation. Here, the bacteriophage phi29 packaging ATPase gene product 16 (gp16) was overexpressed in soluble form in Bacillus subtilis (pSAC), purified to near homogeneity, and assembled to the phi29 precursor capsid (prohead) to produce a packaging motor intermediate that was fully active in in vitro DNA packaging. The formation of higher oligomers of the gp16 from monomers was concentration dependent and was characterized by analytical ultracentrifugation, gel filtration, and electron microscopy. The binding of multiple copies of gp16 to the prohead was dependent on the presence of an oligomer of 174- or 120-base prohead RNA (pRNA) fixed to the head-tail connector at the unique portal vertex of the prohead. The use of mutant pRNAs demonstrated that gp16 bound specifically to the A-helix of pRNA, and ribonuclease footprinting of gp16 on pRNA showed that gp16 protected the CC residues of the CCA bulge (residues 18-20) of the A-helix. The binding of gp16 to the prohead/pRNA to constitute the complete and active packaging motor was confirmed by cryo-electron microscopy three-dimensional reconstruction of the prohead/pRNA/gp16 complex. The complex was capable of supercoiling DNA-gp3 as observed previously for gp16 alone; therefore, the binding of gp16 to the prohead, rather than first to DNA-gp3, represents an alternative packaging motor assembly pathway.     

Multifunctional roles of a bacteriophage phi 29 morphogenetic factor in assembly and infection

Low copy number proteins within macromolecular complexes, such as viruses, can be critical to biological function while comprising a minimal mass fraction of the complex. The Bacillus subtilis double-stranded DNA bacteriophage phi 29 gene 13 product (gp13), previously undetected in the virion, was identified and localized to the distal tip of the tail knob. Western blots and immuno-electron microscopy detected a few copies of gp13 in phi 29, DNA-free particles, purified tails, and defective particles produced in suppressor-sensitive (sus) mutant sus13(330) infections. Particles assembled in the absence of intact gp13 (sus13(342) and sus13(330)) had the gross morphology of phi 29 but were not infectious. gp13 has predicted structural homology and sequence similarity to the M23 metalloprotease LytM. Poised at the tip of the phi 29 tail knob, gp13 may serve as a plug to help restrain the highly pressurized packaged genome. Also, in this position, gp13 may be the first virion protein to contact the cell wall in infection, acting as a pilot protein to depolymerize the cell wall. gp13 may facilitate juxtaposition of the tail knob onto the cytoplasmic membrane and the triggering of genome injection.     

Genetic Insertions and Diversification of the PolB-Type DNA Polymerase (gp43) of T4-Related Phages

In Escherichia coli phage T4 and many of its phylogenetic relatives, gene 43 consists of a single cistron that encodes a PolB family (PolB-type) DNA polymerase. We describe the divergence of this phage gene and its protein product (gp43) (gene product 43) among 26 phylogenetic relatives of T4 and discuss our observations in the context of diversity among the widely distributed PolB enzymes in nature. In two T4 relatives that grow in Aeromonas salmonicida phages 44RR and 25, gene 43 is fragmented by different combinations of three distinct types of DNA insertion elements: (a) a short intercistronic untranslated sequence (IC-UTS) that splits the polymerase gene into two cistrons, 43A and 43B, corresponding to N-terminal (gp43A) and C-terminal (gp43B) protein products; (b) a freestanding homing endonuclease gene (HEG) inserted between the IC-UTS and the 43B cistron; and (c) a group I intron in the 43B cistron. Phage 25 has all three elements, whereas phage 44RR has only the IC-UTS. We present evidence that (a) the split gene of phage 44RR encodes a split DNA polymerase consisting of a complex between gp43A and gp43B subunits; (b) the putative HEG encodes a double-stranded DNA endonuclease that specifically cleaves intron-free homologues of the intron-bearing 43B site; and (c) the group I intron is a self-splicing RNA. Our results suggest that some freestanding HEGs can mediate the homing of introns that do not encode their own homing enzymes. The results also suggest that different insertion elements can converge on a polB gene and evolve into a single integrated system for lateral transfer of polB genetic material. We discuss the possible pathways for the importation of such insertion elements into the genomes of T4-related phages.     

The DNA translocating ATPase of bacteriophage T4 packaging motor

In double-stranded DNA bacteriophages the viral DNA is translocated into an empty prohead shell by a powerful ATP-driven motor assembled at the unique portal vertex. Terminases consisting of two to three packaging-related ATPase sites are central to the packaging mechanism. But the nature of the key translocating ATPase, stoichiometry of packaging motor, and basic mechanism of DNA encapsidation are poorly understood. A defined phage T4 packaging system consisting of only two components, proheads and large terminase protein (gp17; 70 kDa), is constructed. Using the large expanded prohead, this system packages any linear double-stranded DNA, including the 171 kb T4 DNA. The small terminase protein, gp16 (18 kDa), is not only not required but also strongly inhibitory. An ATPase activity is stimulated when proheads, gp17, and DNA are actively engaged in the DNA packaging mode. No packaging ATPase was stimulated by the N-terminal gp17-ATPase mutants, K166G (Walker A), D255E (Walker B), E256Q (catalytic carboxylate), D255E-E256D and D255E-E256Q (Walker B and catalytic carboxylate), nor could these sponsor DNA encapsidation. Experiments with the two gp17 domains, N-terminal ATPase domain and C-terminal nuclease domain, suggest that terminase association with the prohead portal and communication between the domains are essential for ATPase stimulation. These data for the first time established an energetic linkage between packaging stimulation of N-terminal ATPase and DNA translocation. A core pathway for the assembly of functional DNA translocating motor is proposed. Since the catalytic motifs of the N-terminal ATPase are highly conserved among >200 large terminase sequences analyzed, these may represent common themes in phage and herpes viral DNA translocation.     

Snapshots of a Y-family DNA polymerase in replication: substrate-induced conformational transitions and implications for fidelity of Dpo4

Y-family DNA polymerases catalyze translesion DNA synthesis over damaged DNA. Each Y-family polymerase has a polymerase core consisting of a palm, finger and thumb domain in addition to a fourth domain known as a little finger domain. It is unclear how each domain moves during nucleotide incorporation and what type of conformational changes corresponds to the rate-limiting step previously reported in kinetic studies. Here, we present three crystal structures of the prototype Y-family polymerase: apo-Dpo4 at 1.9 Å resolution, Dpo4-DNA binary complex and Dpo4-DNA-dTMP ternary complex at 2.2 Å resolution. Dpo4 undergoes dramatic conformational changes from the apo to the binary structures with a 131° rotation of the little finger domain relative to the polymerase core upon DNA binding. This DNA-induced conformational change is verified in solution by our tryptophan fluorescence studies. In contrast, the polymerase core retains the same conformation in all three conformationally distinct states. Particularly, the finger domain which is responsible for checking base pairing between the template base and an incoming nucleotide retains a rigid conformation. The inflexibility of the polymerase core likely contributes to the low fidelity of Dpo4, in addition to its loose and solvent-accessible active site. Interestingly, while the binary and ternary complexes of Dpo4 retain an identical global conformation, the aromatic side chains of two conserved tyrosines at the nucleotide-binding site change orientations between the binary and ternary structures. Such local conformational changes may correspond to the rate-limiting step in the mechanism of nucleotide incorporation. Together, the global and local conformational transitions observed in our study provide a structural basis for the distinct kinetic steps of a catalytic cycle of DNA polymerization performed by a Y-family polymerase.     

The Crystal Structure of Bacteriophage HK97 gp6: Defining a Large Family of Head-Tail Connector Proteins

The final step in the morphogenesis of long-tailed double-stranded DNA bacteriophages is the joining of the DNA-filled head to the tail. The connector is a specialized structure of the head that serves as the interface for tail attachment and the point of egress for DNA from the head during infection. Here, we report the determination of a 2.1 Å crystal structure of gp6 of bacteriophage HK97. Through structural comparisons, functional studies, and bioinformatic analysis, gp6 has been determined to be a component of the connector of phage HK97 that is evolutionarily related to gp15, a well-characterized connector component of bacteriophage SPP1. Whereas the structure of gp15 was solved in a monomeric form, gp6 crystallized as an oligomeric ring with the dimensions expected for a connector protein. Although this ring is composed of 13 subunits, which does not match the symmetry of the connector within the phage, sequence conservation and modeling of this structure into the cryo-electron microscopy density of the SPP1 connector indicate that this oligomeric structure represents the arrangement of gp6 subunits within the mature phage particle. Through sequence searches and genomic position analysis, we determined that gp6 is a member of a large family of connector proteins that are present in long-tailed phages. We have also identified gp7 of HK97 as a homologue of gp16 of phage SPP1, which is the second component of the connector of this phage. These proteins are members of another large protein family involved in connector assembly.     

Modulation of T4 gene 32 protein DNA binding activity by the recombination mediator protein UvsY

Bacteriophage T4 UvsY is a recombination mediator protein that promotes assembly of the UvsX-ssDNA presynaptic filament. UvsY helps UvsX to displace T4 gene 32 protein (gp32) from ssDNA, a reaction necessary for proper formation of the presynaptic filament. Here we use DNA stretching to examine UvsY interactions with single DNA molecules in the presence and absence of gp32 and a gp32 C-terminal truncation (*I), and show that in both cases UvsY is able to destabilize gp32-ssDNA interactions. In these experiments UvsY binds more strongly to dsDNA than ssDNA due to its inability to wrap ssDNA at high forces. To support this hypothesis, we show that ssDNA created by exposure of stretched DNA to glyoxal is strongly wrapped by UvsY, but wrapping occurs only at low forces. Our results demonstrate that UvsY interacts strongly with stretched DNA in the absence of other proteins. In the presence of gp32 and *I, UvsY is capable of strongly destabilizing gp32-DNA complexes in order to facilitate ssDNA wrapping, which in turn prepares the ssDNA for presynaptic filament assembly in the presence of UvsX. Thus, UvsY mediates UvsX binding to ssDNA by converting rigid gp32-DNA filaments into a structure that can be strongly bound by UvsX.     

Delivery and processing of exogenous double-stranded DNA in mouse CD34 + hematopoietic progenitor cells and their cell cycle changes upon combined treatment with cyclophosphamide and double-stranded DNA

We previously reported that fragments of exogenous double-stranded DNA can be internalized by mouse bone marrow cells without any transfection. Our present analysis shows that only 2% of bone marrow cells take up the fragments of extracellular exogenous DNA. Of these, ~ 45% of the cells correspond to CD34 + hematopoietic stem cells. Taking into account that CD34 + stem cells constituted 2.5% of the total cell population in the bone marrow samples analyzed, these data indicate that as much as 40% of CD34 + cells readily internalize fragments of extracellular exogenous DNA. This suggests that internalization of fragmented dsDNA is a general feature of poorly differentiated cells, in particular CD34 + bone marrow cells.     

Visualization of bacteriophage T3 capsids with DNA incompletely packaged in vivo

The tightly packaged double-stranded DNA (dsDNA) genome in the mature particles of many tailed bacteriophages has been shown to form multiple concentric rings when reconstructed from cryo-electron micrographs. However, recent single-particle DNA packaging force measurements have suggested that incompletely packaged DNA (ipDNA) is less ordered when it is shorter than ∼ 25% of the full genome length. The study presented here initially achieves both the isolation and the ipDNA length-based fractionation of ipDNA-containing T3 phage capsids (ipDNA-capsids) produced by DNA packaging in vivo; some ipDNA has quantized lengths, as judged by high-resolution gel electrophoresis of expelled DNA. This is the first isolation of such particles among the tailed dsDNA bacteriophages. The ipDNA-capsids are a minor component (containing ∼ 10^− 4 of packaged DNA in all particles) and are initially detected by nondenaturing gel electrophoresis after partial purification by buoyant density centrifugation. The primary contaminants are aggregates of phage particles and empty capsids. This study then investigates ipDNA conformations by the first cryo-electron microscopy of ipDNA-capsids produced in vivo. The 3-D structures of DNA-free capsids, ipDNA-capsids with various lengths of ipDNA, and mature bacteriophage are reconstructed, which reveals the typical T = 7l icosahedral shell of many tailed dsDNA bacteriophages. Though the icosahedral shell structures of these capsids are indistinguishable at the current resolution for the protein shell (∼ 15 Å), the conformations of the DNA inside the shell are drastically different. T3 ipDNA-capsids with 10.6 kb or shorter dsDNA (< 28% of total genome) have an ipDNA conformation indistinguishable from random. However, T3 ipDNA-capsids with 22 kb DNA (58% of total genome) form a single DNA ring next to the inner surface of the capsid shell. In contrast, dsDNA fully packaged (38.2 kb) in mature T3 phage particles forms multiple concentric rings such as those seen in other tailed dsDNA bacteriophages. The distance between the icosahedral shell and the outermost DNA ring decreases in the mature, fully packaged phage structure. These results suggest that, in the early stage of DNA packaging, the dsDNA genome is randomly distributed inside the capsid, not preferentially packaged against the inner surface of the capsid shell, and that the multiple concentric dsDNA rings seen later are the results of pressure-driven close-packing.     

Entamoeba histolytica: construction and applications of subgenomic databases

Knowledge about the influence of environmental stress such as the action of chemotherapeutic agents on gene expression in Entamoeba histolytica is limited. We plan to use oligonucleotide microarray hybridization to approach these questions. As the basis for our array, sequence data from the genome project carried out by the Institute for Genomic Research (TIGR) and the Sanger Institute were used to annotate parts of the parasite genome. Three subgenomic databases containing enzymes, cytoskeleton genes, and stress genes were compiled with the help of the ExPASy proteomics website and the BLAST servers at the two genome project sites. The known sequences from reference species, mostly human and Escherichia coli, were searched against TIGR and Sanger E. histolytica sequence contigs and the homologs were copied into a Microsoft Access database. In a similar way, two additional databases of cytoskeletal genes and stress genes were generated. Metabolic pathways could be assembled from our enzyme database, but sometimes they were incomplete as is the case for the sterol biosynthesis pathway. The raw databases contained a significant number of duplicate entries which were merged to obtain curated non-redundant databases. This procedure revealed that some E. histolytica genes may have several putative functions. Representative examples such as the case of the delta-aminolevulinate synthase/serine palmitoyltransferase are discussed.     

Ultra-high resolution imaging of DNA and nucleosomes using non-contact atomic force microscopy

Visualisation of nano-scale biomolecules aids understanding and development in molecular biology and nanotechnology. Detailed structure of nucleosomes adsorbed to mica has been captured in the absence of chemical-anchoring techniques, demonstrating the usefulness of non-contact atomic force microscopy (NC-AFM) for ultra-high resolution biomolecular imaging. NC-AFM offers significant advantages in terms of resolution, speed and ease of sample preparation when compared to techniques such as cryo-electron microscopy and X-ray crystallography. In the absence of chemical modification, detailed structure of DNA deposited on a gold substrate was observed for the first time using NC-AFM, opening up possibilities for investigating the electrical properties of unmodified DNA.     

REDOR NMR characterization of DNA packaging in bacteriophage T4

Bacteriophage T4 is a large-tailed Escherichia coli virus whose capsid is 120 × 86 nm. ATP-driven DNA packaging of the T4 capsid results in the loading of a 171-kb genome in less than 5 min during viral infection. We have isolated 50-mg quantities of uniform ¹⁵N- and [ε-¹⁵N]lysine-labeled bacteriophage T4. We have also introduced ¹⁵NH₄⁺ into filled, unlabeled capsids from synthetic medium by exchange. We have examined lyo- and cryoprotected lyophilized T4 using ¹⁵N{³¹P} and ³¹P{¹⁵N} rotational-echo double resonance. The results of these experiments have shown that (i) packaged DNA is in an unperturbed duplex B-form conformation; (ii) the DNA phosphate negative charge is balanced by lysyl amines (3.2%), polyamines (5.8%), and monovalent cations (40%); and (iii) 11% of lysyl amines, 40% of -NH₂ groups of polyamines, and 80% of monovalent cations within the lyophilized T4 capsid are involved in the DNA charge balance. The NMR evidence suggests that DNA enters the T4 capsid in a charge-unbalanced state. We propose that electrostatic interactions may provide free energy to supplement the nanomotor-driven T4 DNA packaging.     

Determination of the cleavage site of the phage T4 prohead protease in gene product 68. Influence of protein secondary structure on cleavage specificity

The cleavage site of the T4 prohead protease in gene product 68 of bacteriophage T4 has been determined by direct protein sequencing. It is located close to the carboxy-terminal end of a predicted alpha-helix in the sequence Asn-Val-Glu-Ala between the Glu and Ala residues. Secondary structure seems to be more important in determining cleavage than the presence of an aliphatic amino acid three residues before the cleavage site that was proposed earlier. In this case, that position is occupied by Asn, a hydrophilic residue. A second potentially cleavable Glu-Ala is found five residues after the cleaved sequence and this is preceded by an Ile at the -3 position. Despite this, the sequences of the amino and carboxyl termini of the uncleaved protein are identical to those previously proposed from an analysis of the DNA sequence of the gene.