Structure of the RNA claw of the DNA packaging motor of bacteriophage ?29

Bacteriophage DNA packaging motors translocate their genomic DNA into viral heads, compacting it to near-crystalline density. The Bacillus subtilis phage ϕ29 has a unique ring of RNA (pRNA) that is an essential component of its motor, serving as a scaffold for the packaging ATPase. Previously, deletion of a three-base bulge (18-CCA-20) in the pRNA A-helix was shown to abolish packaging activity. Here, we solved the structure of this crucial bulge by nuclear magnetic resonance (NMR) using a 27mer RNA fragment containing the bulge (27b). The bulge actually involves five nucleotides (17-UCCA-20 and A100), as U17 and A100 are not base paired as predicted. Mutational analysis showed these newly identified bulge residues are important for DNA packaging. The bulge introduces a 33–35° bend in the helical axis, and inter-helical motion around this bend appears to be restricted. A model of the functional 120b pRNA was generated using a 27b NMR structure and the crystal structure of the 66b prohead-binding domain. Fitting this model into a cryo-EM map generated a pentameric pRNA structure; five helices projecting from the pRNA ring resemble an RNA claw. Biochemical analysis suggested that this shape is important for coordinated motor action required for DNA translocation.     

Role of the CCA bulge of prohead RNA of bacteriophage ø29 in DNA packaging

The oligomeric ring of prohead RNA (pRNA) is an essential component of the ATP-driven DNA packaging motor of bacteriophage ?29. The A-helix of pRNA binds the DNA translocating ATPase gp16 (gene product 16) and the CCA bulge in this helix is essential for DNA packaging in vitro. Mutation of the bulge by base substitution or deletion showed that the size of the bulge, rather than its sequence, is primary in DNA packaging activity. Proheads reconstituted with CCA bulge mutant pRNAs bound the packaging ATPase gp16 and the packaging substrate DNA-gp3, although DNA translocation was not detected with several mutants. Prohead/bulge-mutant pRNA complexes with low packaging activity had a higher rate of ATP hydrolysis per base pair of DNA packaged than proheads with wild-type pRNA. Cryoelectron microscopy three-dimensional reconstruction of proheads reconstituted with a CCA deletion pRNA showed that the protruding pRNA spokes of the motor occupy a different position relative to the head when compared to particles with wild-type pRNA. Therefore, the CCA bulge seems to dictate the orientation of the pRNA spokes. The conformational changes observed for this mutant pRNA may affect gp16 conformation and/or subsequent ATPase-DNA interaction and, consequently, explain the decreased packaging activity observed for CCA mutants.     

Stimulation of autolysis by adsorption of bacteriophage ØX174 to isolated cell walls

Adsorption of ØX174 to cell wall fragments fromE. coli labeled with³H-diaminopimel ic acid results in limited degradation of murein due to stimulation of autolysis. Pure murein was not degraded by ØX174.     

The nucleotide sequence of bacteriophage φX174

The complete nucleotide sequence of the DNA of bacteriophage φX174 has been determined. The provisional sequence (Sanger et al., 1977a) deduced largely by the plus and minus method, has been completed and confirmed, predominantly using the terminator method (Sanger et al., 1977b). About 30 revisions were found to be necessary in the 5386-nucleotide sequence. The amino acid sequences of the ten proteins for which the DNA codes have also been deduced.     

Is the injection of DNA enough to cause bacteriophage P22-induced changes in the cellular transport process of Salmonella typhimurium?

It was demonstrated earlier in this laboratory that phage P22 induces a transient depression in the cellular transport processes of the host Salmonella typhimurium immediately after infection and that an effective injection process is enough to cause the depression. By using defective phage particles that contain host DNA instead of phage DNA for infection, it has been demonstrated that the injection of phage-specific DNA is essential for this. The defective particles adsorbed to the host and injected their DNA, but the cellular transport processes of the host were not altered. Thus, the injection of host DNA by the phage fails to affect the transport process. Insensitivity of the phage DNA-induced depression in transport to chloramphenicol rules out the involvement of newly synthesized protein in this change and indirectly suggests the possible role of phage DNA-associated internal proteins of P22.     

Heat induced capsid disassembly and DNA release of bacteriophage λ

Successive structural changes of bacteriophage λ upon heating were characterized with quantitative experimental methods. In the commonly used Tris-Mg buffer, differential scanning calorimetry measurements first established that the protein capsid of λ phage melts at 87 °C and its genomic DNA melts at 91 °C. Interestingly, prior to the capsid melting, λDNA was found to escape out of the capsid and subject to DNase digestion above ~68 °C, as concluded from light scattering, UV absorption, and electron microscopy studies. Further investigations indicated distinct temperature-dependent behaviors of the three phage proteins. Around 68 °C, disruption of the tail first occurs and leads to the escape of λ DNA; above the capsid melting temperature of 87 °C, the auxiliary protein gpD of the phage head remains soluble in solution and resists centrifugal sedimentation, whereas the major capsid protein gpE is easily precipitated and likely exists as aggregates.     

The process of infection with bacteriophage φX174: XXXIX. The structure of a DNA form with restricted binding of intercalating dyes observed during synthesis of φX single-stranded DNA

A DNA form with restricted binding of intercalating dyes (propidium iodide or ethidium bromide) has been found in bacteriophage φX-infected cells during the period of single-stranded DNA synthesis. In the electron microscope, this DNA form is seen to be a double-stranded DNA ring with two single-stranded DNA tails protruding from the same portion of the ring; it is composed of a linear φX DNA strand, longer than one φX genome, and a single-stranded ring complementary to φX DNA. Base-pairing of these two tails in partially complementary regions restricts unwinding of the double-stranded DNA ring and consequently intercalation and binding of the dyes. It is postulated that these molecules originate from a previously reported precursor of φX DNA, namely a double-stranded ring with a single-stranded tail, by branch migration.     

Ordered distribution of α-putrescinylthymine in the DNA of bacteriophage φW-14

In the DNA of bacteriophage W-14, half the thymine is replaced by a -putrescinylthymine (putThy). Analysis of monopyrimidine tracts shows that putThy and thymine are distributed nonrandomly in W-14 DNA: The sequence purine-putThy-purine occurs more than twice as frequently as the sequence purine-thymine-purine, which means that the post-replicative modification of W-14 DNA is sequence-specific.     

Alanine scanning and Fe-BABE probing of the bacteriophage ø29 prohead RNA-connector interaction

The DNA packaging motor of the Bacillus subtilis bacteriophage ?29 prohead is comprised in part of an oligomeric ring of 174 base RNA molecules (pRNA) positioned near the N termini of subunits of the dodecameric head-tail connector. Deletion and alanine substitution mutants in the connector protein (gp10) N terminus were assembled into proheads in Escherichia coli and the particles tested for pRNA binding and DNA-gp3 packaging in vitro. The basic amino acid residues RKR at positions 3-5 of the gp10 N terminus were central to pRNA binding during assembly of an active DNA packaging motor. Conjugation of iron(S)-1-(p-bromoacetamidobenzyl) ethylenediaminetetraacetate (Fe-BABE) to residue S170C in the narrow end of the connector, near the N terminus, permitted hydroxyl radical probing of bound [(32)P]pRNA and identified two discrete sites proximal to this residue: the C-helix at the junction of the A, C and D helices, and the E helix and the CE loop/D loop of the intermolecular base pairing site.     

The mechanism of inactivation of S-adenosylhomocysteinase by 2′-deoxyadenosine

S-Adenosylhomocysteinase (SAHase) is irreversibly inactivated by 2′-deoxyadenosine (Hirshfield, M.S. (1979) J. Biol. Chem. $\text{254}$, 22–25). In the course of this inactivation, 2′dAd becomes tightly bound to the enzyme, i.e., cannot be removed by gel filtration or dialysis. Inactivation is accompanied by reduction of the enzyme bound NAD. When the inactivated enzyme is denatured, no 2′dAd is recovered. Adenine equivalent to about 80% of the bound 2′dAd is isolated. It is proposed that 2′-deoxyadenosine is first oxidized to 3′-keto-2-deoxyadenosine by enzyme bound NAD. The 3′keto group activates the hydrogen at C-2′ and facilitates elimination of adenine.     

Electron microscopic study of the repressor of bacteriophage λ and its interaction with operator DNA

The structure of purified phage λ repressor has been examined by high resolution electron microscopy. The repressor molecule appears predominantly as a tetramer of about 95 Å × 120 Å. We have proposed a model to account for the variety of aspects seen on the electron micrographs. Spreading DNA without protein film and use of uranyl formate staining allowed the simultaneous visualization of the DNA and the structure of the repressor molecule bound to it. Mapping the positions of λ repressor bound to whole λ DNA shows preferential binding to the region containing the operators. At high resolution multiple binding of repressor to the operator can be demonstrated. Depending on the amount of repressor present, rows of one to four repressor tetramers are seen on the DNA, confirming the model of the operator containing four binding sites for repressor. The bound repressor can consequently protect against nuclease digestion of operator pieces of approximately 30, 57, 87 and 111 base-pairs. The isolated operator appears in the electron microscope as short double-stranded DNA fragments which can be shown to rebind repressor.     

The mechanism of C-banding: depurination and β-elimination

C-banding of chromosomes involves the differential solubilization of fragmented DNA from euchromatin by three sequential treatments: 1. Acid, 2. Mild base, 3. Hot salt. The data indicate solubilization is effected by 1) depurination, 2) DNA denaturation, 3) chain breakage of the depurinated sites respectively in the three treatments. Conditions were found wherein each treatment in proper sequence was necessary for C-banding and the appropriate chemical reactions were measured in these treatment conditions. The acid treatment (0.2 N HCl) depurinates chromosomal DNA at the rate of 0.26×10^–6 purines/dalton min to an alkaline molecular weight of 10⁵ daltons but does not break the depurinated sites. Bleomycin can substitute for acid as a base removing agent. Sodium borohydride, by reducing the depurinated sugar's aldehyde thereby preventing chain breakage by the -elimination reaction, reversibly inhibits DNA-extraction. Chain breakage at the DNA's apurinic sites occurs not in the 2 min mild alkali treatment where the half-life for breakage is 26 min but in the 18 h hot salt treatment where the half-life for chain breakage is 1–2 h. Most of the DNA extraction occurs in the hot salt as 10⁵ dalton fragments as measured in formamide gradients. Bleomycin is introduced as a substitute for HCl; it removes nitrogenous bases from DNA in situ while better preserving the morphology of the final C-banded chromosomes.     

Characterization of bacteriophage P1 library containing inserts of Drosophila DNA of 75–100 kilobase pairs

A multiple-hit bacteriophage P1 library containing DNA fragments from Drosophila melanogaster in the size range 75–100 kb was created and subjected to a preliminary evaluation for completeness, randomness, fidelity, and clone stability. This P1 library presently contains 3840 individual clones, or approximately two genome equivalents. The library was screened with a small set of unique-sequence test probes, and clones containing the sequences have been recovered. In situ hybridization with salivary gland chromosomes indicates that the clones originate from the site of the probe sequences in the genome, and filter hybridization of restriction digests suggests that the clones are not rearranged in comparison with the genomic sequences. Approximately 1.7% of the clones contain sequences that hybridize with ribosomal DNA. A small subset of these clones was tested for stability by examination of restriction fragments produced after repeated subculturing, and no evidence for instability was found. The P1 cloning system has general utility in molecular genetics and may provide an important intermediate level of resolution in physical mapping of the Drosophila genome.by W. Hennig     

The effect of DNA backbone on the triplet mechanism of UV-induced thymine-thymine (6–4) dimer formation

Density functional theory calculations were carried out to investigate the formation mechanism of the thymine-thymine (6–4) dimer ((6–4)TT), which is one of the main DNA lesions induced by ultraviolet radiation and is closely related to skin cancers. The DNA backbone was found to have nonnegligible effects on the triplet reaction pathway, particularly the reaction steps involving substantial base rotations. The mechanism for the isomerization from (6–4)TT to its Dewar valence isomer (DewarTT) was also explored, confirming the necessity of absorbing a second photon. In addition, the solvation effects were examined and showed considerable influence on the potential energy surface.

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Graphical Abstract DFT calculations on the influence of DNA backbone on the mechanism of UV-induced thymine-thymine (6–4) dimer formation.

     

The in situ formation of DNA · DNA duplexes of Drosophila virilis satellite DNA

The DNA of Drosophila virilis brains and imaginal discs was labeled in vitro to a specific activity of 6 X 10(-5) dpm/mug, using an organ culture medium. The DNA was fractionated on neutral and alkaline CsC1 gradients and the heavy strands of satellite I annealed in situ to denatured polytene chromosomes from squash preparations of larval salivary glands. Nuclease S1 from Aspergillus oryzae was used to digest the unpaired ssDNA, resulting in a distinct labeling of the alpha-heterochromatin in the chromocenter and a small amount of diffused labeling in the proximal beta-heterochromatic part of the X-Chromsome.