Replicative Hybrid of T4 Bacteriophage DNA

Hybrid density replicative T4 DNA was isolated from CsCl, sheared, and reanalyzed in CsCl. The results rule out a branched model for T4 DNA replication and confirm that T4 DNA replicates to a conventional, semiconservative, colinear hybrid.     

Membrane Interaction of the Portal Protein gp20 of Bacteriophage T4

Assembly of the bacteriophage T4 head structure occurs at the cytoplasmic face of the inner membrane of Escherichia coli with the formation of proheads. The proheads contain an internal scaffolding core that determines the size and the structure of the capsid. In a mutant where the major shell protein gp23 was compromised, core structures without a shell had been detected. Such core structures were also found in the mutant T4am20am23. Since the mutation in gene 20 is at the N terminus of gp20, it was assumed that these core structures assemble in the absence of gp20. However, sequencing showed that the mutation introduces a new ribosome binding site that leads to a restart at codon 15. Although the mutant protein gp20s lacks the very N-terminal sequence, we found that it still binds to the membrane of the host cell and can initiate prohead assembly. This explains its activity to allow the assembly of core structures and proheads at the membrane surface. With a cross-linking approach, we show here that gp20 and gp20s are escorted by the chaperones DnaK, trigger factor, and GroEL and dock on the membrane at the membrane protein YidC.     

In Vitro Packaging of UV Radiation-Damaged DNA from Bacteriophage T7

When DNA from bacteriophage T7 is irradiated with UV light, the efficiency with which this DNA can be packaged in vitro to form viable phage particles is reduced. A comparison between irradiated DNA packaged in vitro and irradiated intact phage particles shows almost identical survival as a function of UV dose when Escherichia coli wild type or polA or uvrA mutants are used as the host. Although uvrA mutants perform less host cell reactivation, the polA strains are identical with wild type in their ability to support the growth of irradiated T7 phage or irradiated T7 DNA packaged in vitro into complete phage. An examination of in vitro repair performed by extracts of T7-infected E.coli suggests that T7 DNA polymerase may substitute for E. coli DNA polymerase I in the resynthesis step of excision repair. Also tested was the ability of a similar in vitro repair system that used extracts from uninfected cells to restore biological activity of irradiated DNA. When T7 DNA damaged by UV irradiation was treated with an endonuclease from Micrococcus luteus that is specific for pyrimidine dimers and then was incubated with an extract of uninfected E. coli capable of removing pyrimidine dimers and restoring the DNA of its original (whole genome size) molecular weight, this DNA showed a higher packaging efficiency than untreated DNA, thus demonstrating that the in vitro repair system partially restored the biological activity of UV-damaged DNA.     

Involvement of a Replicative DNA Helicase of Bacteriophage T4 in DNA Recombination

Bacteriophage T4 gene 41 encodes a replicative DNA helicase that is a subunit of the primosome which is essential for lagging-strand DNA synthesis. A mutation, rrh, was generated and selected in the helicase gene on the basis of limited DNA replication that ceases early. The survival of ultraviolet-irradiated phage and the frequency of genetic recombination are reduced by rrh. In addition, rrh diminishes the production of concatemeric DNA. These results strongly suggest that the gene 41 replicative helicase participates in DNA recombination.     

Mechanochemistry of a Viral DNA Packaging Motor

The pentameric ATPase motor gp16 packages double-stranded DNA into the bacteriophage ?29 virus capsid. On the basis of the results of single-molecule experimental studies, we propose a push and roll mechanism to explain how the packaging motor translocates the DNA in bursts of four 2.5 bp power strokes, while rotating the DNA. In this mechanism, each power stroke accompanies P_i release after ATP hydrolysis. Since the high-resolution structure of the gp16 motor is not available, we borrowed characterized features from the P4 RNA packaging motor in bacteriophage ?12. For each power stroke, a lumenal lever from a single subunit is electrostatically steered to the DNA backbone. The lever then pushes sterically, orthogonal to the backbone axis, such that the right-handed DNA helix is translocated and rotated in a left-handed direction. The electrostatic association allows tight coupling between the lever and the DNA and prevents DNA from slipping back. The lever affinity for DNA decreases towards the end of the power stroke and the DNA rolls to the lever on the next subunit. Each power stroke facilitates ATP hydrolysis in the next catalytic site by inserting an Arg -finger into the site, as captured in ?12-P4. At the end of every four power strokes, ADP release happens slowly, so the cycle pauses constituting a dwell phase during which four ATPs are loaded into the catalytic sites. The next burst phase of four power strokes starts once spontaneous ATP hydrolysis takes place in the fifth site without insertion of an Arg finger. The push and roll model provides a new perspective on how a multimeric ATPase transports DNA, and it might apply to other ring motors as well.     

Molecular Interactions and Residues Involved in Force Generation in the T4 Viral DNA Packaging Motor

Many viruses utilize molecular motors to package their genomes into preformed capsids. A striking feature of these motors is their ability to generate large forces to drive DNA translocation against entropic, electrostatic, and bending forces resisting DNA confinement. A model based on recently resolved structures of the bacteriophage T4 motor protein gp17 suggests that this motor generates large forces by undergoing a conformational change from an extended to a compact state. This transition is proposed to be driven by electrostatic interactions between complementarily charged residues across the interface between the N- and C-terminal domains of gp17. Here we use atomistic molecular dynamics simulations to investigate in detail the molecular interactions and residues involved in such a compaction transition of gp17. We find that although electrostatic interactions between charged residues contribute significantly to the overall free energy change of compaction, interactions mediated by the uncharged residues are equally if not more important. We identify five charged residues and six uncharged residues at the interface that play a dominant role in the compaction transition and also reveal salt bridging, van der Waals, and solvent hydrogen-bonding interactions mediated by these residues in stabilizing the compact form of gp17. The formation of a salt bridge between Glu309 and Arg494 is found to be particularly crucial, consistent with experiments showing complete abrogation in packaging upon Glu309Lys mutation. The computed contributions of several other residues are also found to correlate well with single-molecule measurements of impairments in DNA translocation activity caused by site-directed mutations.     

The DNA-Delay Mutants of Bacteriophage T4

Mutants of phage T4 defective in genes 39, 52, 58-61, and 60 (the DNA delay or DD genes) are characterized by a delay in phage DNA synthesis during infection of a nonpermissive Escherichia coli host. Amber (am) mutants defective in these genes yield burst sizes varying from 30 to 110 at 37 C in E. coli lacking an am suppressor. It was found that when DD am mutants are grown on a non-permissive host at 25 C, rather than at 37 C, phage yield is reduced on the average 61-fold. At 25 C incorporation of labeled thymidine into phage DNA is also reduced to 3 to 10% of wild-type levels. Mutants defective in the DD genes were found to promote increased recombination as well as increased base substitution and addition-deletion mutation. These observations indicate that the products of the DD genes are necessary for normal DNA synthesis. The multiplication of the DD am mutants on an Su⁻ host at 37 C is about 50-fold inhibited if prior to infection the host cells were grown at 25 C. This suggests that a compensating host function allows multiplication of DD am mutants at 37 C in the Su⁻ host, and that this function is active in cells grown at 37 C prior to infection, but is inactive when the prior growth is at 25 C. Further results are described which suggest that the products of genes 52, 60, and 39 as well as a host product interact with each other.     

The Interaction of cos with Chi Is Separable from DNA Packaging in recA- recBC-Mediated Recombination of Bacteriophage Lambda

Chi (5'-GCTGGTGG) is a recombinator in RecA-RecBC-mediated recombination in Escherichia coli. In bacteriophage lambda vegetative recombination, Chi is fully active only when it is correctly oriented with respect to cos, the site that defines the ends of the packaged chromosome. Here we demonstrate that packaging from cos is not necessary for this cos-Chi interaction. Our evidence suggests that correctly oriented cos is an activator of Chi. cos, as an activator, is (1) dominant over cos-, (2) active opposite an extensive heterology, (3) able to interact with Chi only when on the same (cis) chromosome, and (4) able to interact with Chi at distances as far as greater than or equal to 20 kb. Thus, cos and Chi form a two-component recombinator system for general recombination. cos may serve as an asymmetric entry site for a recombination enzyme that recognizes Chi in an asymmetric way.     

Role of Gene 52 in Bacteriophage T4 DNA Synthesis

In an attempt to elucidate the mechanism of delayed DNA synthesis in phage T4, Escherichia coli B cells were infected with H17 (an amber mutant defective in gene 52 possessing a "DNA-delay" phenotype). The fate of (14)C-labeled H17 parental DNA after infection was followed: we could show that this DNA sediments more slowly in neutral sucrose than wild-type DNA 3 min postinfection. In pulse-chase experiments progeny DNA was found to undergo detachment from the membrane at 12 min postinfection. Reattachment to the membrane was found to be related to an increase in rate of DNA synthesis. A nucleolytic activity that is absent from cells infected by wild-type phage and from uninfected cells could be detected in extracts prepared from mutant-infected cells. In contrast, degradation of host DNA was found to be less extensive in am H17 compared with wild-type infected cells. Addition of chloramphenicol to mutant-infected cells 10 min postinfection inhibited the appearance of a nuclease activity on one hand and suppressed the "DNA-delay" phenotype on the other hand. We conclude that the gene 52 product controls the activity of a nuclease in infected cells whose main function may be specific strand nicking in association with DNA replication. This gene product might directly attack both E. coli and phage T4 DNA, or indirectly determine their sensitivity to degradation by another nuclease.     

Genetic Mapping of the Amino-Terminal Domain of Bacteriophage T4 DNA Polymerase

The DNA polymerase of bacteriophage T4 is a multifunctional enzyme that harbors DNA-binding, DNA-synthesizing and exonucleolytic activities. We have cloned in bacterial plasmids about 99% of the structural gene for this enzyme (T4 gene 43). The gene was cloned in six contiguous 5'-terminal DNA fragments that defined seven intragenic mapping regions. Escherichia coli hosts harboring recombinant plasmids carrying the gene 43 subsegments were used in marker-rescue experiments that assigned a large number of ts and nonsense polymerase mutations to different physical domains of the structural gene. Conspicuously, only one missense mutation in a large collection of mutants mapped in the 5'-terminal 450 base-pair segment of the approximately 2700 base-pair gene. To test if this indicated a DNA polymerase domain that is relatively noncritical for biological activity, we mutagenized a recombinant plasmid carrying this 5'-terminal region and generated new conditional-lethal mutations that mapped therein. We identified five new ts sites, some having mutated at high frequency (nitrosoguanidine hot spots). New ts mutations were also isolated in phage genes 62 and 44, which map upstream of gene 43 on the T4 chromosome. A preliminary examination of physiological consequences of the ts gene 43 mutations showed that they exhibit effects similar to those of ts lesions that map in other gene 43 segments: some were mutators, some derepressed gene 43 protein synthesis and they varied in the severity of their effects on T4-induced DNA synthesis at nonpermissive temperatures. The availability of the gene 43 clones should make it possible to isolate a variety of lesions that affect different activities of the T4 DNA polymerase and help to define the different domains of this multifunctional protein.     

Ndd, the Bacteriophage T4 Protein That Disrupts the Escherichia coli Nucleoid, Has a DNA Binding Activity

Early in a bacteriophage T4 infection, the phage ndd gene causes the rapid destruction of the structure of the Escherichia coli nucleoid. Even at very low levels, the Ndd protein is extremely toxic to cells. In uninfected E. coli, overexpression of the cloned ndd gene induces disruption of the nucleoid that is indistinguishable from that observed after T4 infection. A preliminary characterization of this protein indicates that it has a double-stranded DNA binding activity with a preference for bacterial DNA rather than phage T4 DNA. The targets of Ndd action may be the chromosomal sequences that determine the structure of the nucleoid.     

Functional Analysis of the Bacteriophage T4 Rad50 Homolog (gp46) Coiled-coil Domain

Rad50 and Mre11 form a complex involved in the detection and processing of DNA double strand breaks. Rad50 contains an anti-parallel coiled-coil with two absolutely conserved cysteine residues at its apex. These cysteine residues serve as a dimerization domain and bind a Zn²⁺ cation in a tetrathiolate coordination complex known as the zinc-hook. Mutation of the zinc-hook in bacteriophage T4 is lethal, indicating the ability to bind Zn²⁺ is critical for the functioning of the MR complex. In vitro, we found that complex formation between Rad50 and a peptide corresponding to the C-terminal domain of Mre11 enhances the ATPase activity of Rad50, supporting the hypothesis that the coiled-coil is a major conduit for communication between Mre11 and Rad50. We constructed mutations to perturb this domain in the bacteriophage T4 Rad50 homolog. Deletion of the Rad50 coiled-coil and zinc-hook eliminates Mre11 binding and ATPase activation but does not affect its basal activity. Mutation of the zinc-hook or disruption of the coiled-coil does not affect Mre11 or DNA binding, but their activation of Rad50 ATPase activity is abolished. Although these mutants excise a single nucleotide at a normal rate, they lack processivity and have reduced repetitive exonuclease rates. Restricting the mobility of the coiled-coil eliminates ATPase activation and repetitive exonuclease activity, but the ability to support single nucleotide excision is retained. These results suggest that the coiled-coiled domain adopts at least two conformations throughout the ATPase/nuclease cycle, with one conformation supporting enhanced ATPase activity and processivity and the other supporting nucleotide excision.     

Bacteriophage T4 self-assembly: in vitro reconstitution of recombinant gp2 into infectious phage

T4 gene 2 mutants have a pleiotropic phenotype: degradation of injected phage DNA by exonuclease V (ExoV) in the recBCD(+) host cell cytoplasm and a low burst size due, at least in part, to a decreased ability for head-to-tail (H-T) joining. The more N terminal the mutation, the more pronounced is the H-T joining defect. We have overexpressed and purified the recombinant gene 2 product (rgp2) to homogeneity in order to test its role in H-T joining, during in vitro reconstitution. When we mix extracts of heads from a gp2(+) phage infection (H(+)) with tails from a gp2(+) or gp2(-) phage infection (T(+) or T(-)), the H-T joining is fast and all of the reconstituted phage grow equally well on cells with or without ExoV activity. When heads from gene 2 amber mutants (H(-)) are used, addition of rgp2 is required for H-T joining. In this case, H-T joining is slow and only about 10% of the reconstituted phage can form plaques on ExoV(+) cells. When extracts of heads with different gene 2 amber mutations are mixed with extracts of tails (with a gene 2 amber mutation) in the presence of rgp2, we find that the size of the gp2 amber peptide of the head extract is inversely related to the fraction of reconstituted phage with a 2(+) phenotype. We conclude that free rgp2 is biologically active and has a direct role in H-T joining but that the process is different from H-T joining promoted by natural gp2 that is incorporated into the head in vivo. Furthermore, it seems that gp2 has a domain which binds it to the head. Thus, the presence of the longer gp2am mutants (with this domain) inhibits their replacement by full-length rgp2.     

Nucleophosmin1 Is a Negative Regulator of the Small GTPase Rac1

The Rac1 GTPase is a critical regulator of cytoskeletal dynamics and controls many biological processes, such as cell migration, cell-cell contacts, cellular growth and cell division. These complex processes are controlled by Rac1 signaling through effector proteins. We have previously identified several effector proteins of Rac1 that also act as Rac1 regulatory proteins, including caveolin-1 and PACSIN2. Here, we report that Rac1 interacts through its C-terminus with nucleophosmin1 (NPM1), a multifunctional nucleo-cytoplasmic shuttling protein with oncogenic properties. We show that Rac1 controls NPM1 subcellular localization. In cells expressing active Rac1, NPM1 translocates from the nucleus to the cytoplasm. In addition, Rac1 regulates the localization of the phosphorylated pool of NPM1 as this pool translocated from the nucleus to the cytosol in cells expressing activated Rac1. Conversely, we found that expression of NPM1 limits Rac1 GTP loading and cell spreading. In conclusion, this study identifies NPM1 as a novel, negative regulator of Rac1.     

An Improved Cosmid Vector for the Nested Deletion Method Using the Bacteriophage T3 DNA Packaging System

We constructed a new cosmid vector suitable for the previouslydeveloped nested deletion method which used the in vitro DNApackaging system of bacteriophage T3. The first step of thismethod is linearization of a cosmid clone to be packaged, andwe previously introduced cleavage at the cos site using -Terminase,but optimization of the reaction conditions was required forcomplete digestion because of its instability. In the newlyconstructed vector, pAT5, the sites of 4 different restrictionenzymes, Sse8387I, Asc I, Fse I and Pme I, each of which recognizesan 8-bp sequence (8-base cutter) were introduced in the vicinityof the cos site. In addition, the species of restriction sitesfor cloning were increased to broaden its application. The cosmidclone constructed by this new vector could be linearized atone of the 8-base cutter sites which are assumed to rarely occurin the genome, and followed by in vitro packaging, nested deletionclones were successfully prepared.