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.     

Global effects of DNA replication and DNA replication origin activity on eukaryotic gene expression

This report provides a global view of how gene expression is affected by DNA replication. We analyzed synchronized cultures of Saccharomyces cerevisiae under conditions that prevent DNA replication initiation without delaying cell cycle progression. We use a higher‐order singular value decomposition to integrate the global mRNA expression measured in the multiple time courses, detect and remove experimental artifacts and identify significant combinations of patterns of expression variation across the genes, time points and conditions. We find that, first, ～88% of the global mRNA expression is independent of DNA replication. Second, the requirement of DNA replication for efficient histone gene expression is independent of conditions that elicit DNA damage checkpoint responses. Third, origin licensing decreases the expression of genes with origins near their 3′ ends, revealing that downstream origins can regulate the expression of upstream genes. This confirms previous predictions from mathematical modeling of a global causal coordination between DNA replication origin activity and mRNA expression, and shows that mathematical modeling of DNA microarray data can be used to correctly predict previously unknown biological modes of regulation.     

Diminishing adenovirus gene expression and viral replication by promoter replacement.

The adenovirus E4 promoter was replaced by a synthetic promoter composed of a minimal TATA box and five consensus 17-mer yeast GAL4-binding-site elements. The viral vectors, which also contained human factor IX (hFIX) cDNA driven by Rous sarcoma virus long terminal repeat in the E1 region, were then constructed and expanded in 293 cells permanently expressing GAL4/VP16 fusion protein. Viral replication and expression of adenovirus E4 genes and late genes (hexon and fiber) were evaluated in vitro in the human lung carcinoma cell line H1299. Viral replication and viral gene expression were dramatically reduced in the cells transduced by vectors with a replaced E4 promoter compared to the levels in the cells transduced by vectors with the wild-type E4 promoter. The levels of transgene (hFIX) expression remained similar between vectors with or without E4 promoter replacement. These results indicate that diminution of viral gene expression and viral replication is achievable by promoter replacement.     

Late gene function in bacteriophage T4 in the absence of phage DNA replication

Under certain conditions the late genes of coliphage T4 may function in the absence of phage DNA replication. Quasi-late gene function is the function of certain late genes in the absence of both phage DNA replication and the product of the maturation gene 55. It does not depend on how phage DNA synthesis is prevented. Replication-uncoupled late gene function is late gene function from unreplicated DNA in the absence of phage ligase, and is still under the control of gene 55. It is most efficient if phage DNA replication is prevented by a mutation in the phage gene (43) for DNA polymerase. Both quasi-late gene function and replication-uncoupled late gene function are enhanced by the presence of mutations controlling a phage exonuclease (gene 46 or 47).     

Plasticity of the gene functions for DNA replication in the T4-like phages

We have completely sequenced and annotated the genomes of several relatives of the bacteriophage T4, including three coliphages (RB43, RB49 and RB69), three Aeromonas salmonicida phages (44RR2.8t, 25 and 31) and one Aeromonas hydrophila phage (Aeh1). In addition, we have partially sequenced and annotated the T4-like genomes of coliphage RB16 (a close relative of RB43), A. salmonicida phage 65, Acinetobacter johnsonii phage 133 and Vibrio natriegens phage nt-1. Each of these phage genomes exhibited a unique sequence that distinguished it from its relatives, although there were examples of genomes that are very similar to each other. As a group the phages compared here diverge from one another by several criteria, including (a) host range, (b) genome size in the range between approximately 160 kb and approximately 250 kb, (c) content and genetic organization of their T4-like genes for DNA metabolism, (d) mutational drift of the predicted T4-like gene products and their regulatory sites and (e) content of open-reading frames that have no counterparts in T4 or other known organisms (novel ORFs). We have observed a number of DNA rearrangements of the T4 genome type, some exhibiting proximity to putative homing endonuclease genes. Also, we cite and discuss examples of sequence divergence in the predicted sites for protein-protein and protein-nucleic acid interactions of homologues of the T4 DNA replication proteins, with emphasis on the diversity in sequence, molecular form and regulation of the phage-encoded DNA polymerase, gp43. Five of the sequenced phage genomes are predicted to encode split forms of this polymerase. Our studies suggest that the modular construction and plasticity of the T4 genome type and several of its replication proteins may offer resilience to mutation, including DNA rearrangements, and facilitate the adaptation of T4-like phages to different bacterial hosts in nature.     

A promiscuous antitoxin of bacteriophage T4 ensures successful viral replication

Bacteria are constantly threatened by predation from bacteriophage parasites and, in response, have evolved an array of resistance mechanisms. These resistance mechanisms then place greater selection pressure on the infecting bacteriophages, which develop counter-strategies in a perpetual 'arms race' between virus and host. Toxin-antitoxin (TA) loci are widespread in bacteria and can confer multiple benefits, including resistance to bacteriophages. The study by Otsuka and Yonesaki, published in this issue of Molecular Microbiology, describes a new plasmid-encoded TA system, lsoAB, which confers resistance to a dmd(-) mutant of bacteriophage T4 through the activity of the LsoA toxin. Infections with wild-type T4, however, are unaffected as the Dmd protein acts as an alternative antitoxin to LsoA, thus preventing its anti-bacteriophage activity. Dmd has also been shown to negate the activity of a related toxin, RnlA. This is a striking result indicating that Dmd can act as a promiscuous antitoxin, binding and inhibiting multiple toxin partners, when antitoxin activity is generally considered to be limited to a single cognate toxin. This study is an exciting addition to both the bacteriophage resistance and TA fields, and suggests a greater role for TA system-based resistance and counter-resistance in the world's oldest predator-prey relationship.     

The bacteriophage T4 dexA gene: sequence and analysis of a gene conditionally required for DNA replication

Summary We have cloned and sequenced a bacteriophage T4 EcoRI fragment that complements T4 del (39-56) infections of an optA defective Escherichia coli strain. Bacteria containing this recombinant plasmid synthesize two new proteins with molecular weights of 9 and 26 kilodaltons. We have identified the gene encoding the 26 kilodalton protein as essential for T4 infections of optA defective E. coli. Genetic and biochemical results are consistent with the identification of this protein as the product of the dexA gene, which encodes a 3 to 5 exonuclease.     

DNA replication and head assembly in bacteriophage T4.

Phage DNA was accumulated in cells of E. coli B, infected with the phage T4DtsLB3 (gene 42), without the synthesis of late proteins (in the presence of chloramphenicol). Then (stage II), chloramphenicol was removed and further replication of the phage DNA suppressed with hydroxyurea and by simultaneously raising the temperature to 40 degrees. The media M9 or M9 with 1% amino acid were used; the times of addition of chloramphenicol and the hydroxyurea concentration were also varied. It was also shown that in medium M9, at stage II, chiefly early proteins were synthesized. In the medium containing amino acids, at stage II the following was observed: 1) DNA synthesis was entirely suppressed and a degradation of DNA occurred; 2) both early and late proteins were synthesized, with a predominance of the latter; 3) an assembly of the elements of the phage tails and capsids occurred without the neck and flagellum, and a small number of phage particles were also found; 4) the capsids, isolated in a sucrose density gradient after lysis with chloroform, contained the proteins Palt, P20, P23, P24, several unidentified proteins, and did not contain Pwac, P23, and P22, 5) the yield of viable phage varied from 0.05 to 15% per cell. Thus, the entire morphogenesis of T4 phage can occur without accompanying replication of phage DNA.     

Crystallization of the gene 45 protein from the DNA replication fork of bacteriophage T4

The gene 45 protein from bacteriophage T4 has been purified and is crystallized. This protein is part of the T4 DNA replication complex. The crystallized protein is active in complementation assays. X-ray diffraction analysis is in progress; data are measured for the native and several heavy atom derivatives. The crystals diffract to about 3.5-A resolution.