Regulation of the terminal stage of viral DNA development, DNA packaging, is poorly understood. A new phage T4 in vitro DNA packaging assay employed purified proheads, terminase (gp17 + gp16), and ...ATP to encapsidate DNA resistant to nuclease. Mature phage T4 DNA and linearized plasmid DNAs containing or lacking a cloned T4 gene were packaged with high (∼10%) efficiency. Supercoiled, relaxed covalently closed, and nicked circular plasmid DNAs were packaged inefficiently, if at all, by these components. However, efficient packaging is achieved for nicked circular plasmid DNA, but not covalently closed plasmid DNA, upon addition to packaging mixtures of the purified T4 late transcription-replication machinery proteins: gp45 (sliding clamp), gp44/gp62 (clamp loader complex), gp55 (late σ-factor), and gp33 (transcriptional co-activator). The small terminase subunit (gp16) is inhibitory for packaging linear DNAs, but enhances the transcription-replication protein packaging of nicked plasmid DNA. Taken together with genetic and biochemical evidence of a requirement for gp55 for concatemer packaging to assemble active wild-type phage particles (1), the plasmid packaging results show that initiation of phage T4 packaging on “endless” concatemeric DNA in vivo by terminase depends upon interaction with the DNA loaded gp45 coupled late transcription-replication machinery. The results suggest a close mechanistic connection in vivo between DNA packaging and developmentally concurrent replication-dependent late transcription.
The Escherichia coli CT596 prophage exclusion genes gmrS and gmrD were found to encode a novel type IV modification-dependent restriction nuclease that targets and digests glucosylated ...(glc)-hydroxymethylcytosine (HMC) DNAs. The protein products GmrS (36 kDa) and GmrD (27 kDa) were purified and found to be inactive separately, but together degraded several different glc-HMC modified DNAs (T4, T2 and T6). The GMR enzyme is able to degrade both α–glucosy-HMC T4 DNA and β–glucosyl-HMC T4 DNA, whereas no activity was observed against non-modified DNAs including unmodified T4 cytosine (C) DNA or non-glucosylated T4 HMC DNA. Enzyme activity requires NTP, favors UTP, is stimulated by calcium, and initially produces 4 kb DNA fragments that are further degraded to low molecular mass products. The enzyme is inhibited by the T4 phage internal protein I* (IPI*) to which it was found to bind. Overall activities of the purified GmrSD enzyme are in good agreement with the properties of the cloned gmr genes in vivo and suggest a restriction enzyme specific for sugar modified HMC DNAs. IPI* thus represents a third generation bacteriophage defense against restriction nucleases of the Gmr type.
Conserved bacteriophage ATP-based DNA translocation motors consist of a multimeric packaging terminase docked onto a unique procapsid vertex containing a portal ring. DNA is translocated into the ...empty procapsid through the portal ring channel to high density. In vivo the T4 phage packaging motor deals with Y- or X-structures in the replicative concatemer substrate by employing a portal-bound Holliday junction resolvase that trims and releases these DNA roadblocks to packaging. Here using dye-labeled packaging anchored 3.7-kb Y-DNAs or linear DNAs, we demonstrate FRET between the dye-labeled substrates and GFP portal-containing procapsids and between GFP portal and single dye-labeled terminases. We show using FRET-fluorescence correlation spectroscopy that purified T4 gp49 endonuclease VII resolvase can release DNA compression in vitro in prohead portal packaging motor anchored and arrested Y-DNA substrates. In addition, using active terminases labeled at the N- and C-terminal ends with a single dye molecule, we show by FRET distance of the N-terminal GFP-labeled portal protein containing prohead at 6.9 nm from the N terminus and at 5.7 nm from the C terminus of the terminase. Packaging with a C-terminal fluorescent terminase on a GFP portal prohead, FRET shows a reduction in distance to the GFP portal of 0.6 nm in the arrested Y-DNA as compared with linear DNA; the reduction is reversed by resolvase treatment. Conformational changes in both the motor proteins and the DNA substrate itself that are associated with the power stroke of the motor are consistent with a proposed linear motor employing a terminal-to-portal DNA grip-and-release mechanism.
Summary
Encased within the 280 kb genome in the capsid of the giant myovirus φKZ is an unusual cylindrical proteinaceous ‘inner body’ of highly ordered structure. We present here mass spectrometry, ...bioinformatic and biochemical studies that reveal novel information about the φKZ head and the complex inner body. The identification of 39 cleavage sites in 19 φKZ head proteins indicates cleavage of many prohead proteins forms a major morphogenetic step in φKZ head maturation. The φKZ head protease, gp175, is newly identified here by a bioinformatics approach, as confirmed by a protein expression assay. Gp175 is distantly related to T4 gp21 and recognizes and cleaves head precursors at related but distinct S/A/G‐X‐E recognition sites. Within the φKZ head there are six high‐copy‐number proteins that are probable major components of the inner body. The molecular weights of five of these proteins are reduced 35–65% by cleavages making their mature form similar (26–31 kDa), while their precursors are dissimilar (36–88 kDa). Together the six abundant proteins sum to the estimated mass of the inner body (15–20 MDa). The identification of these proteins is important for future studies on the composition and function of the inner body.
Linear DNAs of any sequence can be packaged into empty viral procapsids by the phage T4 terminase with high efficiency
in vitro. Packaging substrates of 5 kbp and 50 kbp, terminated by energy ...transfer dye pairs, were constructed from plasmid and λ phage DNAs. Nuclease and fluorescence correlation spectroscopy (FCS) assays showed that ∼
20% of the substrate DNA was packaged and that the DNA dye ends of the packaged DNA were protected from nuclease digestion. Upon packaging, both 5-kbp and 50-kbp DNAs produced comparable fluorescence resonance energy transfer (FRET) between Cy5 and Cy5.5 double-dye terminated DNAs. Single-molecule FRET (sm-FRET) and photobleaching analysis shows that FRET is intramolecular rather than intermolecular upon packaging of most procapsids and demonstrates that single-molecule detection allows mechanistic analysis of packaging
in vitro. FRET-FCS and sm-FRET measurements are comparable and show that both the 5-kbp and the 50-kbp packaged DNA ends are held within 8–9 nm of each other, within the dimensions of the long axis of the procapsid portal. The calculated distribution of FRET distances is relatively narrow for both FRET-FCS and sm-FRET, suggesting that the two packaged DNA ends are held at the same fixed distance relative to each other in most capsids. Because one DNA end is known to be positioned for ejection through the portal, it can be inferred that both DNAs ends are held in proximity to the portal entrance and ejection channel. The analysis suggests that a DNA loop, rather than a DNA end, is translocated by the packaging motor to fill the procapsid.
The first reported Type IV restriction endonuclease (REase) GmrSD consists of GmrS and GmrD subunits. In most bacteria, however, the gmrS and gmrD genes are fused together to encode a single-chain ...protein. The fused coding sequence for ECSTEC94C_1402 from E. coli strain STEC_94C was expressed in T7 Express. The protein designated as Eco94GmrSD displays modification-dependent ATP-stimulated REase activity on T4 DNA with glucosyl-5-hydroxymethyl-cytosines (glc-5hmC) and T4gt DNA with 5-hydroxymethyl-cytosines (5hmC). A C-terminal 6xHis-tagged protein was purified by two-column chromatography. The enzyme is active in Mg(2+) and Mn(2+) buffer. It prefers to cleave large glc-5hmC- or 5hmC-modified DNA. In phage restriction assays, Eco94GmrSD weakly restricted T4 and T4gt, whereas T4 IPI*-deficient phage (Δip1) were restricted more than 10(6)-fold, consistent with IPI* protection of E. coli DH10B from lethal expression of the closely homologous E. coli CT596 GmrSD. Eco94GmrSD is proposed to belong to the His-Asn-His (HNH)-nuclease family by the identification of a putative C-terminal REase catalytic site D507-H508-N522. Supporting this, GmrSD variants D507A, H508A, and N522A displayed no endonuclease activity. The presence of a large number of fused GmrSD homologs suggests that GmrSD is an effective phage exclusion protein that provides a mechanism to thwart T-even phage infection.
The DNA packaging machinery of bacteriophage T4 was studied in vitro using fluorescence correlation spectroscopy. The ATP-dependent translocation kinetics of labeled DNA from the bulk solution, to ...the phage interior, was measured by monitoring the accompanied decrease in DNA diffusibility. It was found that multiple short DNA fragments (100 basepairs) can be sequentially packaged by an individual phage prohead. Fluorescence resonance energy transfer between green fluorescent protein donors within the phage interior and acceptor-labeled DNA was used to confirm DNA packaging. Without ATP, no packaging was observed, and there was no evidence of substrate association with the prohead.
Condensed Genome Structure Black, Lindsay W.; Thomas, Julie A.
Advances in experimental medicine and biology,
2012, Letnik:
726
Book Chapter, Journal Article
Recenzirano
Odprti dostop
Large, tailed dsDNA-containing bacteriophage genomes are packaged to a conserved and high density (∼500 mg/ml), generally in ∼2.5-nm, duplex-to-duplex, spaced, organized DNA shells within icosahedral ...capsids. Phages with these condensate properties, however, differ markedly in their inner capsid structures: (1) those with a naked condensed DNA, (2) those with many dispersed unstructured proteins embedded within the DNA, (3) those with a small number of localized proteins, and (4) those with a reduced or DNA-free internal protein structure of substantial volume. The DNA is translocated and condensed by a high-force ATPase motor into a procapsid already containing the proteins that are to be ejected together with the DNA into the infected host. The condensed genome structure of a single-phage type is unlikely to be precisely determined and can change without loss of function to fit an altered capsid size or internal structure. Although no such single-phage condensed genome structure is known exactly, it is known that a single general structure is unlikely to apply to all such phages.
Phage T4 protects its DNA from the two-gene-encoded
gmrS/gmrD (
glucose-
modified hydroxymethylcytosine
restriction endonuclease) CT of pathogenic
Escherichia coli, CT596, by injecting several ...hundred copies of the 76-amino-acid-residue nuclease inhibitor, IPI*, into the infected host. Here, the three-dimensional solution structure of mature IPI* is reported as determined by nuclear magnetic resonance techniques using 1290 experimental nuclear Overhauser effect and dipolar coupling constraints (∼
17 constraints per residue). Close examination of this oblate-shaped protein structure reveals a novel fold consisting of two small β-sheets (β1: B1 and B2; β2: B3–B5) flanked at the N- and C-termini by α-helices (H1 and H2). Such a fold is very compact in shape and allows ejection of IPI* through the narrow 30-Å portal and tail tube apertures of the virion without unfolding. Structural and dynamic measurements identify an exposed hydrophobic knob that is a putative
gmrS/gmrD-binding site. A single gene from the uropathogenic
E. coli UT189, which codes for a gmrS/gmrD-like UT fusion enzyme (with ∼
90% identity to the heterodimeric CT enzyme), has evolved IPI* inhibitor immunity. Analysis of the
gmrS/gmrD restriction endonuclease enzyme family and its IPI* family phage antagonists reveals an evolutionary pathway that has elaborated a surprisingly diverse and specifically fitted set of coevolving attack and defense structures.
The Escherichia coli isolate CT596 excludes infection by the Myoviridae T4 ip1− phage that lacks the encapsidated IPI* protein normally injected into the host with the phage DNA. Screening of a CT596 ...genomic library identified adjacent genes responsible for this exclusion, gmrS (942 bp) and gmrD (708 bp) that are encoded by a cryptic prophage DNA. The two genes are necessary and sufficient to confer upon a host the ability to exclude infection by T4 ip1− phage and other glucosyl-hydroxymethylcytosine (glc-HMC) Tevens lacking the ip1 gene, yet allow infection by phages with non-glucoslyated cytosine (C) DNA that lack the ip1 gene. A plasmid expressing the ip1 gene product, IPI*, allows growth of Tevens lacking ip1 on E. coli strains carrying the cloned gmrS/gmrD genes. Members of the Teven family carry a diverse and, in some cases, expanded set of ip1 locus genes. In vivo analysis suggests a family of gmr genes that specifically target sugar-HMC modified DNA have evolved to exclude Teven phages, and these exclusion genes have in turn been countered by a family of injected exclusion inhibitors that likely help determine the host range of different glc-HMC phages.
PDF
