DNA packaging and cutting by phage terminases: Control in phage T4 by a synaptic mechanism

Phage DNA packaging occurs by DNA translocation into a prohead. Terminases are enzymes which initiate DNA packaging by cutting the DNA concatemer, and they are closely fitted structurally to the portal vertex of the prohead to form a ‘packasome’. Analysis among a number of phages supports an active role of the terminases in coupling ATP hydrolysis to DNA translocation through the portal. In phage T4 the small terminase subunit promotes a sequence-specific terminase gene amplification within the chromosome. This link between recombination and packaging suggests a DNA synapsis mechanism by the terminase to control packaging initiation, formally homologous to eukaryotic chromosome segregation.     

A base-flipping mechanism for the T4 phage beta-glucosyltransferase and identification of a transition-state analog

T4 phage beta-glucosyltransferase (BGT) modifies T4 DNA. We crystallized BGT with UDP-glucose and a 13mer DNA fragment containing an abasic site. We obtained two crystal structures of a ternary complex BGT-UDP-DNA at 1.8A and 2.5A resolution, one with a Tris molecule and the other with a metal ion at the active site. Both structures reveal a large distortion in the bound DNA. BGT flips the deoxyribose moiety at the abasic site to an extra-helical position and induces a 40 degrees bend in the DNA with a marked widening of the major groove. The Tris molecule mimics the glucose moiety in its transition state. The base-flipping mechanism, which has so far been observed only for glycosylases, methyltransferases and endonucleases, is now reported for a glucosyltransferase. BGT is unique in binding and inserting a loop into the DNA duplex through the major groove only. Furthermore, BGT compresses the backbone DNA one base further than the target base on the 3'-side.     

T4 bacteriophage as a phage display platform

Analysis of molecular events in T4-infected Escherichia coli has revealed some of the most important principles of biology, including relationships between structures of genes and their products, virus-induced acquisition of metabolic function, and morphogenesis of complex structures through sequential gene product interaction rather than sequential gene activation. T4 bacteriophages and related strains were applied in the first formulations of many fundamental biological concepts. These include the unambiguous recognition of nucleic acids as the genetic material, the definition of the gene by fine-structure mutation, recombinational and functional analyses, the demonstration that the genetic code is triplet, the discovery of mRNA, the importance of recombination and DNA replications, light-dependent and light-independent DNA repair mechanisms, restriction and modification of DNA, self-splicing of intron/exon arrangement in prokaryotes, translation bypassing and others. Bacteriophage T4 possesses unique features that make it a good tool for a multicomponent vaccine platform. Hoc/Soc-fused antigens can be assembled on the T4 capsid in vitro and in vivo. T4-based phage display combined with affinity chromatography can be applied as a new method for bacteriophage purification. The T4 phage display system can also be used as an attractive approach for cancer therapy. The data show the efficient display of both single and multiple HIV antigens on the phage T4 capsid and offer insights for designing novel particulate HIV or other vaccines that have not been demonstrated by other vector systems.     

Campylobacter jejuni group III phage CP81 contains many T4-like genes without belonging to the T4-type phage group: implications for the evolution of T4 phages

CP81 is a virulent Campylobacter group III phage whose linear genome comprises 132,454 bp. At the nucleotide level, CP81 differs from other phages. However, a number of its structural and replication/recombination proteins revealed a relationship to the group II Campylobacter phages CP220/CPt10 and to T4-type phages. Unlike the T4-related phages, the CP81 genome does not contain conserved replication and virion modules. Instead, the respective genes are scattered throughout the phage genome. Moreover, most genes for metabolic enzymes of CP220/CPt10 are lacking in CP81. On the other hand, the CP81 genome contains nine similar genes for homing endonucleases which may be involved in the attrition of the conserved gene order for the virion core genes of T4-type phages. The phage apparently possesses an unusual modification of C or G bases. Efficient cleavage of its DNA was only achieved with restriction enzymes recognizing pure A/T sites. Uncommonly, phenol extraction leads to a significant loss of CP81 DNA from the aqueous layer, a property not yet described for other phages belonging to the T4 superfamily.     

T4 phage and T4 ghosts inhibit f2 phage replication by different mechanisms

Both T4 phage and DNA-free ghosts inhibit replication of RNA phage f2. Most but not all of the effects by T4 upon f2 growth can be blocked by the addition of rifampicin prior to T4 superinfection; by contrast, the inhibition of f2 synthesis by T4 ghosts cannot be blocked by rifampicin. This indicates that inhibition by intact T4 requires gene function, while inhibition by ghosts does not. There is a small, multiplicity-dependent inhibition by viable T4 on f2 growth in the presence of rifampicin which may be similar to the gene function-independent inhibition by T4 ghosts. With one viable T4 per cell, there appears to be no effect by viable T4 upon f2 growth which does not require T4 gene action. Moreover, increasing multiplicities of viable T4 appear to inhibit T4 replication as well.In the absence of rifampicin, pre-existing f2 single and double-stranded RNA are degraded after superinfection by viable T4, but remain stable after superinfection by ghosts. However, no new f2 RNA is synthesized after superinfection with either. In the presence of rifampicin, f2-specific protein synthesis is largely unaffected by viable T4, but is completely inhibited by ghosts. Both Escherichia coli, as well as f2-speciflc polysomes disappear in the presence of ghosts.We conclude that, at low multiplicities, T4 phage and T4 ghosts inhibit replication of f2 phage, and presumably host syntheses, by different mechanisms.     

Recombination apparatus of T4 phage

The substantial process of general DNA recombination consists of production of ssDNA, exchange of the ssDNA and its homologous strand in a duplex, and cleavage of branched DNA to maturate recombination intermediates. Ten genes of T4 phage are involved in general recombination and apparently encode all of the proteins required for its own recombination. Several proteins among them interact with each other in a highly specific manner based on a protein-protein affinity and constitute a multicomponent protein machine to create an ssDNA gap essential for production of recombinogenic ssDNA, a machine to supply recombinogenic ssDNA which has a free end, or a machine to transfer the recombinogenic single strand into a homologous duplex.     

Recombination-dependent DNA replication in phage T4

Studies in the 1960s implied that bacteriophage T4 tightly couples DNA replication to genetic recombination. This contradicted the prevailing wisdom of the time, which staunchly supported recombination as a simple cut-and-paste process. More-recent investigations have shown how recombination triggers DNA synthesis and why the coupling of these two processes is important. Results from T4 were instrumental in our understanding of many important replication and recombination proteins, including the newly recognized replication/recombination mediator proteins. Recombination-dependent DNA replication is crucial to the T4 life cycle as it is the major mode of DNA replication and is also central to the repair of DNA breaks and other damage.     

Adenine recognition is a key checkpoint in the energy release mechanism of phage T4 DNA packaging motor

ATP is the source of energy for numerous biochemical reactions in all organisms. Tailed bacteriophages use ATP to drive powerful packaging machines that translocate viral DNA into a procapsid and compact it to near-crystalline density. Here we report that a complex network of interactions dictates adenine recognition and ATP hydrolysis in the pentameric phage T4 large "terminase" (gp17) motor. The network includes residues that form hydrogen bonds at the edges of the adenine ring (Q138 and Q143), base-stacking interactions at the plane of the ring (I127 and R140), and cross-talking bonds between adenine, triphosphate, and Walker A P-loop (Y142, Q143, and R140). These interactions are conserved in other translocases such as type I/type III restriction enzymes and SF1/SF2 helicases. Perturbation of any of these interactions, even the loss of a single hydrogen bond, leads to multiple defects in motor functions. Adenine recognition is therefore a key checkpoint that ensures efficient ATP firing only when the fuel molecule is precisely engaged with the motor. This may be a common feature in the energy release mechanism of ATP-driven molecular machines that carry out numerous biomolecular reactions in biological systems.     

Comparative genomics of the T4-Like Escherichia coli phage JS98: implications for the evolution of T4 phages

About 130 kb of sequence information was obtained from the coliphage JS98 isolated from the stool of a pediatric diarrhea patient in Bangladesh. The DNA shared up to 81% base pair identity with phage T4. The most conserved regions between JS98 and T4 were the structural genes, but their degree of conservation was not uniform. The head genes showed the highest sequence conservation, followed by the tail, baseplate, and tail fiber genes. Many tail fiber genes shared only protein sequence identity. Except for the insertion of endonuclease genes in T4 and gene 24 duplication in JS98, the structural gene maps of the two phages were colinear. The receptor-recognizing tail fiber proteins gp37 and gp38 were only distantly related to T4, but shared up to 83% amino acid identity to other T6-like phages, suggesting lateral gene transfer. A greater degree of variability was seen between JS98 and T4 over DNA replication and DNA transaction genes. While most of these genes came in the same order and shared up to 76% protein sequence identity, a few rearrangements, insertions, and replacements of genes were observed. Many putative gene insertions in the DNA replication module of T4 were flanked by intron-related endonuclease genes, suggesting mobile DNA elements. A hotspot of genome diversification was located downstream of the DNA polymerase gene 43 and the DNA binding gene 32. Comparative genomics of 100-kb genome sequence revealed that T4-like phages diversify more by the accumulation of point mutations and occasional gene duplication events than by modular exchanges.     

The pathway of recombination in phage T4

Summary A study has been made of the effect of modifying the products of the early T4 genes on the frequency with which haploid segregants are generated by recombination from a phage harbouring a standard genetic duplication. Alterations in the products of genes 32, 44, 46, 47 and 59 have been found to significantly decrease the segregation frequency and are, therefore, considered to be involved in the T4 recombination pathway.     

Gene function of heterozygotes in phage T4

Summary Gene function of various T4-heterozygotes was tested. About half of the HETs containing wild type and anam-mutation disappeared under non-permissive conditions, if theam-defect concerned early functions. The same was found when phages, heterozygous forr ⁺ and anrII-point-mutation, were adsorbed to K12 (). A much more extensive loss of HETs in K could be observed if anrIIA- and anrIIB-point-mutation (block-mutations showed different results) occurred together in a non-recombinant heterozygote. The findings provide evidence that one class of T4-heterozygotes has a heteroduplex DNA-structure.With 3 Figures in the Text     

The kinetic mechanism of phage T4 DNA-[N6-adenine]-methyltransferase

Kinetic analysis of methyl group transfer from S-adenosyl-L-methionine (SAM) to the GATC recognition site catalyzed by the phage T4 DNA-[N6-adenine]-methyltransferase (MTase) [EC 2.1.1.72] showed that the reverse reaction is at least 500 times slower than the direct one. The overall pattern of product inhibition corresponds to an ordered steady-state mechanism following the sequence SAM decreases DNA decreases metDNA increases SAH increases (S-adenosyl-L-homocysteine). Pronounced inhibition was observed at high concentrations of the 20-meric substrate duplex, which may be attributed to formation of a dead-end complex MTase-SAH-DNA. In contrast, high SAM concentrations proportionally accelerated the reaction. Thus, the reaction may include a stage whereby the binding of SAM and the release of SAH are united into one concerted event. Computer fitting of alternative kinetic schemes to the aggregate of experimental data revealed that the most plausible mechanism involves isomerization of the enzyme.     

Folding kinetics of phage T4 thioredoxin

The folding mechanism for bacteriophage T4 thioredoxin is best described by a four-state box mechanism, N----Uc----Ut----It----N, where N indicates native, Uc the unfolded form with the cis proline isomer, Ut unfolded with the trans proline isomer, and It a compact form with a trans proline isomer. Both manual mixing fluorescence and size-exclusion chromatography indicate that there is a cis-trans proline isomerization that is important to the folding pathway. Furthermore, the data suggest that the cis-trans isomerization can also occur in a compact nativelike state which is referred to as It. The slow phase seen in fluorescence seems to be monitoring the cis-trans isomerization in the compact form, not the isomerization which occurs in the denatured state.     

Radiation sensitive mutants of phage T4

Summary A comparative series of phage survival, multiplicity reactivation and radiation stability experiments have been performed with single and double combinations of the four UV-sensitive mutations v, x, y and 1206. These mutations appear to fall into two groups with v (which is associated with excision repair) in one, and x, y and 1206 (associated with what can be non-committally termed replication repair) in the other. The pattern of the results suggest that the extent of the initial shoulder exhibited by multiplication reactivation curves is determined by replication repair, while the slope of the subsequent linear portion depends on excision repair. The development of the characteristic radiation stability observed in Luria-Latarjet experiments is shown to depend to a major extent on replication repair and only in a minor way on excision repair.     

Hybrid transfer RNA genes in phage T4

We describe the isolation and characterization of two unusual amber suppressor forms of T4 tRNALeu. The sequences of the suppressor tRNAs can be described as hybrids of wild-type tRNALeu and suppressor tRNAGln molecules: the chain lengths and majority of the nucleotide residues corresponded to tRNALeu, but CUA anticodons flanked by 2-14 residues were identical to tRNAGln. The uncertainty as to the exact number of flanking residues correlated with tRNAGln is due to the similarity of the two tRNA sequences in this region. No evidence was found for changes in other T4 tRNAs. We propose that genes for the hybrid tRNAs were produced by mispairing of DNAs at anticodon segments of tRNALeu and tRNAGln with a double crossover flanking those segments.     

Caffeine as a comutagen for ethylmethanesulfonate in strains of phage T4

Summary The mutation frequency of DNA polymerase mutants of phage T4 treated with ethyl methanesulfonate (EMS) then incubated in the presence and absence of caffeine was studied using an rII reversion system. The DNA polymerase mutation is shown to be antimutagenic for EMS induction of reversions which occur by a GC to AT transition. Caffeine acts as a comutagen for the induction by EMS of mutant phages and produces a significant increase in the frequency of reversions from rII to r⁺. Caffeine is slightly mutagenic for the phage strain carrying the wild type polymerase and inhibits the action of the 35 exonuclease function of T₄ DNA polymerase as measured in vitro. These findings suggest that caffeine acts by directly influencing nucleotide selection or the editing function of the DNA polymerase.