Interaction of DNA with DNA binding proteins. III. Infectivity of protein-complexed phage fd DNA in Escherichia coli spheroplasts.

Complex formation of circular, single-stranded phage fd DNA with Escherichia coli DNA binding protein HD or phage fd gene 5 protein keeps infection of E. coli spheroplasts at the level of free phage DNA, whereas complexes of this DNA with E. coli DNA unwinding protein show a strongly reduced efficiency of transfection. Displacement of the unwinding protein by HD protein or gene 5 protein also maintains the poor adsorption of the complexes to spheroplasts. Free E. coli DNA unwinding protein and residual amounts of this protein bound to the DNA may interfere with the adsorption and the uptake of the phage genome.     

Interaction of DNA with DNA binding proteins. II. Displacement of Escherichia coli DNA unwinding protein and the condensed structure of DNA complexed with protein HD.

Three DNA binding proteins from Escherichia coli cells have been complexed with single-stranded phage fd DNA. Electron microscopy reveals granular substructures in the complexes formed with protein HD. In complexes of DNA unwinding protein with fd DNA both protein HD and phage-coded gene 5 protein partially displace the unwinding protein which results in the formation of structures characteristic for the DNA complexes formed with either protein HD or gene 5 protein alone. Combination of protein HD with double-stranded phage T7 DNA leads to a progressive folding and condensing of the genome. The structures observed are discussed in relation to current concepts of the packing of DNA in protein complexes.     

Functional aspects of Escherichia coli rep helicase in unwinding and replication of DNA

The gene for Escherichia coli rep helicase (rep protein) was subcloned in a pBR plasmid and the protein overproduced in cells transformed with the hybrid DNA. The effect of purified enzyme on strand unwinding and DNA replication was investigated by electron microscopy. The templates used were partial duplexes of viral DNA from bacteriophage fd::Tn5 and reannealed DNA from bacteriophage Mu. The experiments with the two DNA species show DNA unwinding uncoupled from replication. The single-stranded phage fd::Tn5 DNA with the inverted repeat of transposon Tn5 could be completely replicated in the presence of the E. coli enzymes rep helicase, DNA binding protein I, RNA polymerase and DNA polymerase III holoenzyme. A block in the unwinding step increases secondary initiation events in single-stranded parts of the template, as DNA polymerase III holoenzyme cannot switch across the stem structure of the transposon.     

Studies on invitro DNA synthesis: II. Isolation of a protein which stimulates deoxynucleotide incorporation catalyzed by DNA polymerases of E.coli

Purified RNA polymerase, DNA polymerase III and unwinding protein of $\text{Escherichia}\text{coli}$ catalyze limited rifampicin sensitive fd or ØX 174 DNA-dependent DNA synthesis. A protein has been partially purified from $\text{E.}\text{coli}$ which stimulates rifampicin sensitive dXMP incorporation in this system 20 to 30 fold. This protein also stimulates DNA synthesis catalyzed by DNA polymerases I and II; the stimulation occurs in reactions primed with natural and synthetic DNAs as well as RNA-DNA hybrids. The protein is not a product of the known dna genes. In contrast to the above system of purified enzymes, rifampicin sensitive dXMP incorporation in crude extracts of $\text{E.}\text{coli}$ is specifically dependent on fd but not ØX 174 DNA. An additional factor has been isolated from extracts of $\text{E.}\text{coli}$ which restores specificity to the purified rifampicin sensitive system by preventing ØX 174 DNA from serving as a template.     

Interaction between the gene 5 protein, gene 5 protein/single stranded fd DNA complex and gene 8 protein of the filamentous phage fd

An affinity column consisting of gene 8 protein, the major coat protein of fd phage, bound to Sepharose was prepared. Isolated gene 5 protein/single stranded fd DNA complex was found to bind to this column and was eluted with fd phage single stranded fd DNA. pH changes, and 1 M CaCl2 were not effective in eluting the protein from the affinity column. Gene 5 protein/single stranded fd DNA complex from the crude extracts of fd-infected E. coli also bound to the column, as did isolated gene 5 protein; whereas fd single stranded DNA alone did not. These results may be relevant for the illucidation of the molecular events occurring in the early stages of fd phage assembly.     

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.     

Differences in susceptibility to restriction by E. coli B between various heteroduplex molecules of bacteriophage FD DNA

Fragments of B-modified bacteriophage fd sB1^o sB2 RF DNA were prepared with the help of purified endonuclease R from Haemophilus parainfluenzae (Hpa II). These were hybridized with unmodified circular single stranded fd DNA. The resulting partial heteroduplex molecules were assayed for infectivity on competent cells of B-restricting and non restricting strains of E. coli. There of such heteroduplexes originating from neighbouring fragments on the physical map of fd RF DNA were shown to be more resistant to Eco B restriction than six others and the unmodified control. It is suggested that the three corresponding vicinal fragments contain essential parts of the Eco B recognition site on this phage DNA.     

A specific DNA orientation in the filamentous bacteriophage fd as probed by psoralen crosslinking and electron microscopy.

The molecular structure of the single-stranded fd DNA inside its filamentous virion has been stabilized by the photochemical reaction with a psoralen derivative and examined in the electron microscope. The results support the notion that the 6389 nucleotide-long DNA molecule is folded back on itself inside the 1 μm-long protein coat. At one end of the virion, there exists a DNA hairpin region 200±50 base-pairs long. This “end hairpin” is mapped on the fd genome to the site of the replication origin. The most stable in vitro hairpin of fd DNA has been mapped previously to this same site. This unique duplex region of fd DNA may play an important role in the formation of specific protein-DNA complexes which are crucial to stages of the fd life cycle: the adsorption of the phage to the bacteria, the initiation of replication of the single-stranded DNA, and the assembly of newly synthesized DNA strands into the filamentous virions.     

Transfection of Escherichia coli spheroplasts. VI. Transfection of nonpermissive spheroplasts by T5 and BF23 bacteriophage DNA carrying amber mutations in DNA transfer genes.

DNA was extracted from T5 and BF23 phage carrying amber mutations in genes A2, A1, or D9 and tested for its ability to transfect su minus spheroplasts. DNA from T5 am231, defective in gene A2, transfects Escherichia coli su minus recB minus spheroplasts with an efficiency of 16% of that of wild-type T5 DNA, whereas DNA from T5 am16d or BF23 am57, both defective in gene A1 or its equivalent, transfects E. coli su minus recB minus spheroplasts with an efficiency of 1.4% of that of wild-type T5 DNA, provided E. coli su+ bacteria is used as the indicator in all cases. More than 95% of the progeny from the am231, am16d, and am57 DNA that transfects su minus recB minus spheroplasts is still amber mutant. From these efficiencies of transfection we conclude that the product of gene A2 functions mainly in the mechanism of transfer of phage DNA to intact host cells, and that this function is not essential for transfection of spheroplasts. We also conclude that gene A1 controls functions in addition to DNA transfer, in agreement with previous studies which show that mutations in gene A1 have a pleiotropic effect. Apparently, the absence of these additional functions controlled by gene A1 leads to a high frequency of abortive infection. DNA from amber mutants defective in either gene A1 or A2 does not appreciably transfect su minus rec+ spheroplasts, indicating that the products of these two genes may both be needed to protect T5 DNA from the very active rec BC nuclease in spheroplasts.     

Gyrase-dependent initiation of bacteriophage T4 DNA replication: interactions of Escherichia coli gyrase with novobiocin, coumermycin and phage DNA-delay gene products.

Wild-type bacteriophage T4 and DNA-delay am mutants defective in genes 39, 52, 60 and 58–61 were tested for intracellular sensitivity to the antibiotics coumermycin and novobiocin, drugs which inhibit the DNA gyrase of Escherichia coli. Treatment with these antibiotics drastically reduced the characteristic growth of gene 39, 52 and 60 DNA-delay am mutants in E. coli lacking an amber suppressor (su^?). Wild-type phage-infected cells were unaffected by the drugs while the burst size of a gene 58–61 mutant was affected to an intermediate extent. A su^?E. coli strain which is resistant to coumermycin due to an altered gyrase permitted growth of the DNA-delay am mutants in the presence of the drug. Thus, the characteristic growth of the DNA-delay am mutants in an su^? host apparently depends on the host gyrase. An E. coli himB mutant is defective in the coumermycin-sensitive subunit of gyrase (H. I. Miller, personal communication). Growth of the gene 39, 52 and 60 am mutants was inhibited in the himB mutant while the gene 58–61 mutant and wild-type T4 showed small reductions in burst size in this host. Experiments with nalidixic acid-sensitive and resistant strains of E. coli show that wild-type phage T4 requires a functional nalA protein for growth.Novobiocin and coumermycin inhibit phage DNA synthesis in DNA-delay mutant-infected su^?E. coli if added during the early logarithmic phase of phage DNA synthesis. The gene 58–61 mutant showed the smallest inhibition of DNA synthesis in the presence of the drugs. Addition of the drugs during the late linear phase of phage DNA synthesis had no effect on further synthesis in DNA-delay mutant-infected cells. Coumermycin and novobiocin had no effect on DNA synthesis in wild-type-infected cells regardless of the time of addition of the antibiotics. Models are considered in which the DNA-delay gene products either form an autonomous phage gyrase or interact with the host gyrase and adapt it for proper initiation of phage DNA replication.     

Cloning of linear DNAs in vivo by overexpressed T4 DNA ligase: construction of a T4 phage hoc gene display vector.

A method was developed to clone linear DNAs by overexpressing T4 phage DNA ligase in vivo, based upon recombination deficient E. coli derivatives that carry a plasmid containing an inducible T4 DNA ligase gene. Integration of this ligase-plasmid into the chromosome of such E. coli allows standard plasmid isolation following linear DNA transformation of the strains containing high levels of T4 DNA ligase. Intramolecular ligation allows high efficiency recircularization of cohesive and blunt-end terminated linear plasmid DNAs following transformation. Recombinant plasmids could be constructed in vivo by co-transformation with linearized vector plus insert DNAs, followed by intermolecular ligation in the T4 ligase strains to yield clones without deletions or rearrangements. Thus, in vitro packaged lox-site terminated plasmid DNAs injected from phage T4 were recircularized by T4 ligase in vivo with an efficiency comparable to CRE recombinase. Clones that expressed a capsid-binding 14-aa N-terminal peptide extension derivative of the HOC (highly antigenic outer capsid) protein for T4 phage hoc gene display were constructed by co-transformation with a linearized vector and a PCR-synthesized hoc gene. Therefore, the T4 DNA ligase strains are useful for cloning linear DNAs in vivo by transformation or transduction of DNAs with nonsequence-specific but compatible DNA ends.     

Studies on the mechanism of enzymatic DNA elongation by Escherichia coli DNA polymerase II.

The mechanism of enzymatic elongation by Escherichia coli DNA polymerase II of a DNA primer, which is annealed to a unique position on the bacteriophage fd viral DNA, has been studied. The enzyme is found to dissociate from the substrate at specific positions on the genome which act as “barriers” to further primer extension. It is believed these are sites of secondary structure in the DNA. When the template is complexed with E. coli DNA binding protein many of these barriers are eliminated and the enzyme remains associated with the same primer-template molecule during extensive intervals of DNA synthesis. Despite the presence of E. coli DNA binding protein, at least one barrier on the fd genome remains rate-limiting to chain extension and disturbs the otherwise processive mechanism of DNA synthesis. This barrier is overcome by increasing the concentration of enzyme.In contrast, it is found that DNA polymerase I is not rate-limited by structural barriers in the template, however, it exhibits a non-processive mechanism of elongation.These findings provide a framework for understanding the necessity for participation of proteins other than a DNA polymerase in chain extension during chromosomal replication.     

Rapid Detection of Escherichia coli O157:H7 by Using Green Fluorescent Protein-Labeled PP01 Bacteriophage

A previously isolated T-even-type PP01 bacteriophage was used to detect its host cell, Escherichia coli O157:H7. The phage small outer capsid (SOC) protein was used as a platform to present a marker protein, green fluorescent protein (GFP), on the phage capsid. The DNA fragment around soc was amplified by PCR and sequenced. The gene alignment of soc and its upstream region was g56-soc.2-soc.1-soc, which is the same as that for T2 phage. GFP was introduced into the C- and N-terminal regions of SOC to produce recombinant phages PP01-GFP/SOC and PP01-SOC/GFP, respectively. Fusion of GFP to SOC did not change the host range of PP01. On the contrary, the binding affinity of the recombinant phages to the host cell increased. However, the stability of the recombinant phages in alkaline solution decreased. Adsorption of the GFP-labeled PP01 phages to the E. coli cell surface enabled visualization of cells under a fluorescence microscope. GFP-labeled PP01 phage was not only adsorbed on culturable E. coli cells but also on viable but nonculturable or pasteurized cells. The coexistence of insensitive E. coli K-12 (W3110) cells did not influence the specificity and affinity of GFP-labeled PP01 adsorption on E. coli O157:H7. After a 10-min incubation with GFP-labeled PP01 phage at a multiplicity of infection of 1,000 at 4°C, E. coli O157:H7 cells could be visualized by fluorescence microscopy. The GFP-labeled PP01 phage could be a rapid and sensitive tool for E. coli O157:H7 detection.     

Elongation of duplex DNA by recA protein

recA protein, which is essential for the recombination process in Escherichia coli, was incubated in the presence of 5′-γ-thiotriphosphate with circular plasmid pBR_βG containing small single-stranded gaps. Stable complexes were formed which appear in the electron microscope as fibres with a diameter about five times that of naked DNA. Complex formation appears to be a co-operative process whereby the average rise per base-pair with respect to the fibre axis increases from 3·39 ± 0·08 Å to 5·20 ± 0·18 Å. The elongation of DNA by about 50% is compatible with an unwinding of the double helix and an intercalating mode of binding of recA and/or 5′-γ-thiotriphosphate to DNA.     

Rapid single-molecule characterisation of enzymes involved in nucleic-acid metabolism

The activity of enzymes is traditionally characterised through bulk-phase biochemical methods that only report on population averages. Single-molecule methods are advantageous in elucidating kinetic and population heterogeneity but are often complicated, time consuming, and lack statistical power. We present a highly-generalisable and high-throughput single-molecule assay to rapidly characterise proteins involved in DNA metabolism. The assay exclusively relies on changes in total fluorescence intensity of surface-immobilised DNA templates as a result of DNA synthesis, unwinding or digestion. Combined with an automated data-analysis pipeline, our method provides enzymatic activity data of thousands of molecules in less than an hour. We demonstrate our method by characterising three fundamentally different enzyme activities: digestion by the phage λ exonuclease, synthesis by the phage Phi29 polymerase, and unwinding by the E. coli UvrD helicase. We observe the previously unknown activity of the UvrD helicase to remove neutravidin bound to 5′-, but not 3′-ends of biotinylated DNA.     

Some Properties of DNA from Phage-Infected Bacteria

Replicating T5 or λ phage DNA has been labeled by adding tritiated thymidine for short periods to cultures of phage-infected Escherichia coli before isolation of intracellular DNA. Two procedures are described for separating T5 replicating DNA from DNA of intracellular phage particles. Both T5 and λ replicating DNA had the same bouyant density in cesium chloride as DNA from phage particles but sedimented faster when centrifuged in sucrose density gradients. The fast sedimentation did not appear to be caused by DNA protein or DNA-RNA complexes or by aggregation of DNA, but is probably due to DNA molecules of unusual structure. Experiments involving hydrodynamic shear and sucrose density gradient centrifugation at alkaline pH have suggested that with λ the replicating form of DNA is a linear molecule considerably longer than the DNA molecules of λ-phage particles. The constituent polynucleotide chains of λ but not T5 replicating DNA also appear to be longer than those of phage DNA.     

Control of bacteriophage T4 DNA polymerase synthesis

Analysis of sodium dodecyl sulphate/acrylamide gels of ¹⁴C-labelled proteins from phage-infected bacteria suggests the existence of a self-regulatory control mechanism in bacteriophage T4.Infection of Escherichia coli with phage T4 carrying a mutation in gene 43 (which codes for the phage DNA polymerase) results in a greatly increased rate of synthesis of the gene 43 protein. Such overproduction of defective polymerase occurs in restrictive infections with all gene 43 amber and most gene 43 temperature-sensitive mutants tested. Gene 43 protein synthesis in gene 43⁺ infections or increased synthesis in gene 43^? infections appears to require no additional function of other phage proteins essential for DNA synthesis. Functional gene 43 protein is needed continuously to keep its own levels down to normal.     

Knotted single-stranded DNA rings: A novel topological isomer of circular single-stranded DNA formed by treatment with Escherichia coli ω protein

Treatment of single-stranded circular phage fd DNA with Escherichia coli ω protein yields a new species which sediments 1.2 to 1.5 times faster than the untreated DNA in an alkaline medium. The infectivity of this species in spheroplast assays, after purification of the DNA by zone sedimentation in an alkaline sucrose gradient, is only slightly lower than that of untreated fd DNA. The formation of this species requires Mg(II) and is strongly dependent on salt concentration and temperature. At 37 °C, over 85% of the input DNA can be converted to this form when incubation is carried out in media containing 0.15 to 0.25 m-salt. The yield decreases with increasing temperature or decreasing salt concentration. The increase in sedimentation coefficient of fd DNA in an alkaline medium following treatment with ω is not due to protein binding, as no change was observed upon treatment of the product with phenol or Pronase. Furthermore, neither the buoyant density of this new species in neutral CsCl nor its sedimentation coefficient in a neutral medium is significantly different from the corresponding properties of untreated fd DNA. Examination by electron microscopy shows that the new form has the appearance of a knotted ring of about the same contour length as an untreated monomeric single-stranded fd DNA. The new form can be converted to full-length linear fd DNA by treatment with pancreatic DNAase I. The rate of conversion is approximately the same as that of untreated circular fd DNA to the linear form. These results show that the new form of fd DNA is a novel topological isomer: a knotted single-stranded DNA ring. It is also found that further treatment of the knotted DNA rings with ω at low ionic strength can reverse the reaction, i.e. the knotted DNA rings can be converted back to simple DNA rings indistinguishable from fd DNA from the phage. At intermediate ionic strength the two forms are interconvertible and form an equilibrium mixture. Results similar to those obtained for fd DNA have also been observed for single-stranded circular φX174 DNA. A mechanism based on the known activity of ω protein on double-stranded DNA, the secondary structure of a single-stranded circular DNA, and the experimental results described here is proposed.     

Electron microscopic analysis of DNA forks generated by Escherichia coli DNA helicase II

T7 phage DNA eroded with lambda exonuclease (to create 3'-protruding strands) or exonuclease III (to create 5'-protruding strands) was treated under unwinding assay conditions with DNA helicase II. Single-stranded DNA-binding protein (of Escherichia coli or phage T4) was added to disentangle the denatured DNA and the complexes were examined in the electron microscope. DNA helicase II complexes filtered through a gel column before assay retain the ability to generate forks suggesting that DNA helicase II unwinds in a preformed complex by translocating along the bound DNA strand. The enzyme initiates preferentially at the ends of the lambda-exonuclease-treated duplexes and is found at a fork on the initially protruding strand. It also initiates at the ends of the exonuclease-III-treated duplexes where, as with approximately 5% of the forks traceable back to a single-stranded gap, it is found on the initially recessed strand. The results are consistent with the view that DNA helicase II unwinds in the 3'-5' direction relative to the bound strand. They also confirm that the enzyme can initiate at the end of a fully base-paired strand. At a fork, DNA helicase II is bound as a tract of molecules of approximately 110 nm in length. Tracts of enzyme assemble from non-cooperatively bound molecules in the presence of ATP. During unwinding, DNA helicase II apparently can translocate to the displaced strand which conceivably can deplete the leading strand of the enzyme. Continued adsorption of enzyme to DNA might replenish forks arrested by strand switch of the unwinding enzyme.     

Modulation of enzymatic activities of Escherichia coli DnaB helicase by single-stranded DNA-binding proteins

The modulation of enzymatic activities of Escherichia coli DnaB helicase by homologous and heterologous single-stranded DNA-binding proteins (SSBs) and its DNA substrates were analyzed. Although DnaB helicase can unwind a variety of DNA substrates possessing different fork-like structures, the rate of DNA unwinding was significantly diminished with substrates lacking a 3′ fork. A 5 nt fork appeared to be adequate to attain the maximum rate of DNA unwinding. Efficient helicase action of DnaB requires the participation of SSBs. Studies involving heterologous SSBs demonstrated that they can stimulate the helicase activity of DnaB protein under certain conditions. However, this stimulation occurs in a manner distinctly different from that observed with cognate E.coli SSB. The E.coli SSB was found to stimulate the helicase activity over a wide range of SSB concentrations and was unique in its strong inhibition of single-stranded DNA-dependent ATPase activity when uncoupled from the DNA helicase activity. In the presence of a helicase substrate, the ATPase activity of DnaB helicase remained uninhibited. Thus, E.coli SSB appears to coordinate and couple the ATPase activity to the DNA helicase activity by suppressing unproductive ATP hydrolysis by DnaB helicase.