Forces controlling the rate of DNA ejection from phage lambda

The goal of this work was to investigate how internal and external forces acting on DNA affect the rate of genome ejection from bacteriophage lambda after the ejection is triggered in vitro by a lambda receptor. The rate of ejection was measured with time-resolved static and dynamic light scattering, while varying such parameters as temperature and packaged DNA length, as well as adding DNA-binding proteins to the host solution. We found that temperature has a strong effect on the ejection rate, with an exponential increase of the initial ejection rate as a function of temperature. This can possibly be explained by the temperature-induced conformational changes in the tail pore-forming proteins where the "open" conformation dominates over "closed", at elevated temperatures. The DNA length also had an effect on initial ejection rate, with a nearly linear dependence comparing the three different genomes (37.7, 45.7 and 48.5 kb DNA), with faster ejection rate for longer genomes. Since the initial rate of ejection increases in an almost direct relationship with the length of the genome, the total time needed to eject DNA completely appeared to be nearly constant for all three DNA length phage mutants. The increased initial rate of ejection with increasing DNA length is due to the increased DNA bending and inter-strand repulsion forces for the longer DNA chains. Finally, we also show that addition of non-specific DNA-binding proteins (HU and DNase I) increases the rate of ejection by exerting additional "pulling" forces on the DNA that is being ejected.     

Langevin dynamics simulation of DNA ejection from a phage

We have performed Langevin dynamics simulations of a coarse-grained model of ejection of dsDNA from Φ29 phage. Our simulation results show significant variations in the local ejection speed, consistent with experimental observations reported in the literature for both in vivo and in vitro systems. In efforts to understand the origin of such variations in the local speed of ejection, we have investigated the correlations between the local ejection kinetics and the packaged structures created at various motor forces and chain flexibility. At lower motor forces, the packaged DNA length is shorter with better organization. On the other hand, at higher motor forces typical of realistic situations, the DNA organization inside the capsid suffers from significant orientational disorder, but yet with long orientational correlation times. This in turn leads to lack of registry between the direction of the DNA segments just to be ejected and the direction of exit. As a result, a significant amount of momentum transfer is required locally for successful exit. Consequently, the DNA ejection temporarily slows down exhibiting pauses. This slowing down occurs at random times during the ejection process, completely determined by the particular starting conformation created by prescribed motor forces. In order to augment our inference, we have additionally investigated the ejection of chains with deliberately changed persistence length. For less inflexible chains, the demand on the occurrence of large momentum transfer for successful ejection is weaker, resulting in more uniform ejection kinetics. While being consistent with experimental observations, our results show the nonergodic nature of the ejection kinetics and call for better theoretical models to portray the kinetics of genome ejection from phages.     

Real-time imaging of DNA ejection from single phage particles

Infection by tailed dsDNA phages is initiated by release of the viral DNA from the capsid and its polarized injection into the host. The driving force for the genome transport remains poorly defined. Among many hypothesis [1], it has been proposed that the internal pressure built up during packaging of the DNA in the capsid is responsible for its injection [2-4]. Whether the energy stored during packaging is sufficient to cause full DNA ejection or only to initiate the process was tested on phage T5 whose DNA (121,400 bp) can be released in vitro by mere interaction of the phage with its E. coli membrane receptor FhuA [5-7]. We present a fluorescence microscopy study investigating in real time the dynamics of DNA ejection from single T5 phages adsorbed onto a microfluidic cell. The ejected DNA was fluorescently stained, and its length was measured at different stages of the ejection after being stretched in a hydrodynamic flow. We conclude that DNA release is not an all-or-none process but occurs in a stepwise fashion and at a rate reaching 75,000 bp/sec. The relevance of this stepwise ejection to the in vivo DNA transfer is discussed.     

Localization of low-melting regions in phage T7 DNA

Specific fragmentation of T7 DNA at glyoxal-fixed denatured regions by the S1 endonuclease followed by restriction analysis made it possible to localize four low-melting regions in phage T7 DNA. These regions have the following coordinates:0.5-1.2;14.8+/-0.3;46.3+/-0.5; 98.4+/-0.3 (in T7 DNA length units). The location of the low-melting regions was refined by means of electron-microscopic denaturation mapping and gel electrophoresis of partially denatured DNA. The obtained localization of the low-melting regions is consistent with the available data on the sequence of T7 DNA. The map of low-melting regions was compared with the genetic map of T7 DNA.Images     

Thioredoxin reductase-dependent insulin disulfide reduction by phage T7 DNA polymerase reflects dissociation of the enzyme into subunits

Phage T7 DNA polymerase contains Escherichia coli thioredoxin as a subunit and is a 1:1 complex with T7 gene 5 protein. The enzyme showed high thioredoxin activity in assays at 37 degrees C using reduction of insulin disulfides with NADPH and thioredoxin reductase, leading Randahl (Randahl, H. (1982) FEBS Lett. 150, 109-113) to propose that the thioredoxin dithiol active site is exposed in T7 DNA polymerase. However, T7 DNA polymerase and free thioredoxin differ in reactivity with iodoacetic acid after preincubation with dithiothreitol or incubation with insulin. Insulin reduction assays work at low temperatures even at 0 degrees C. The time and temperature dependence of the thioredoxin activity of T7 DNA polymerase demonstrated that dissociation into subunits at 25 or 37 degrees C accounts for the previously observed activity. Thus, T7 DNA polymerase contains the reduced form of thioredoxin with its active site SH groups masked by the subunit contact with the gene 5 protein in agreement with the results of Adler and Modrich (Adler, S., and Modrich, P. (1983) J. Biol. Chem. 258, 6956-6962). The subunit interaction of thioredoxin and gene 5 protein is salt-insensitive, but markedly temperature-dependent consistent with involvement of a hydrophobic surface area in reduced thioredoxin.     

Exploring the DNA mimicry of the Ocr protein of phage T7

DNA mimic proteins have evolved to control DNA-binding proteins by competing with the target DNA for binding to the protein. The Ocr protein of bacteriophage T7 is the most studied DNA mimic and functions to block the DNA-binding groove of Type I DNA restriction/modification enzymes. This binding prevents the enzyme from cleaving invading phage DNA. Each 116 amino acid monomer of the Ocr dimer has an unusual amino acid composition with 34 negatively charged side chains but only 6 positively charged side chains. Extensive mutagenesis of the charges of Ocr revealed a regression of Ocr activity from wild-type activity to partial activity then to variants inactive in antirestriction but deleterious for cell viability and lastly to totally inactive variants with no deleterious effect on cell viability. Throughout the mutagenesis the Ocr mutant proteins retained their folding. Our results show that the extreme bias in charged amino acids is not necessary for antirestriction activity but that less charged variants can affect cell viability by leading to restriction proficient but modification deficient cell phenotypes.     

Nitrous acid induced damage in T7 DNA and phage

T7 phage was exposed to 56 mM nitrous acid at pH 4.6 causing a 90% decrease in survival for each 10 min duration of exposure. The survival of phage made by encapsulating nitrous acid treated DNA into empty phage heads was nearly the same as the survival of phage exposed to nitrous acid in vivo. In contrast to previous reports, growth of SOS-induced wild-type E. coli showed no increase in survival. The survival of nitrous acid treated phage was not lowered when grown on E. coli strains deficient in DNA polymerase I, exonuclease III, and the uvrA component of the nucleotide excision-repair endonuclease. Therefore, these enzymes are not vital for repair of nitrous acid induced damage in bacteriophage T7.     

Specific fragmentation of T7 phage DNA at low-melting sites.

A method has been developed for selective fragmentation of T7 DNA at AT-rich regions. The molecules have been subjected to complete digestion with single-strand-specific SI endonuclease after fixation of DNA AT-rich regions in the denatured state by glyoxal. The treatment resulted in three fragments having molecular weights of 13.6 +/- 0.4, 8.2 +/- 0.4 and 3.5 +/- 0.16 megadaltons as determined by electron microscopy. The position of these fragments along the T7 DNA molecule has been determined by means of analysis of the intermediates during SI-cleavage.     

Evidence that a phage T4 DNA packaging enzyme is a processed form of the major capsid gene product

A phage T4 DNA packaging enzyme appears to arise as a processed form of the major T4 capsid structural protein gp23. The enzyme activity and antigen are missing from all head gene mutants that block the morphogenetic proteolytic processing reactions of the head proteins in vivo. The enzyme antigen can be formed in vitro by T4 (gp21) specific processing of gp23 containing extracts. Enzyme antigen is found in active processed proheads but not in full heads. The enzyme and the major capsid protein show immunological cross-reactivity, produce common peptides upon proteolysis, and share an assembly-conformation-dependent ATP binding site. The packaging enzyme and the mature capsid protein (gp23*) both appear to arise from processing of gp23, the former as a minor product of a specific gp23 structure in the prohead, acting in DNA packaging as a DNA-dependent ATPase, and a headful-dependent terminase.     

RNA polymerase synthesis in vitro directed by T7 phage DNA

Summary T7 RNA polymerase is synthesized in vitro, dependent on T7 DNA. The in vitro synthesized T7 polymerase has the characteristic properties: resistance to rifampicin and streptolydigin and the typical template specificity.     

Length determination of the terminal redundant regions in the DNA of phage T7

Summary The length of the terminal redundant regions in T7 DNA has been determined by two methods. One involved the specific labeling and isolation of the redundant DNA fragment and determination of the molecular weight by polyacrylamide gel electrophoresis. A value of 150±10 nucleotide pairs was obtained. The other determination based on a correlation of the melting temperature of the redundant region to that of whole T7 DNA confirmed the result obtained by the first method.     

Mechanistic aspects of the DNA junction-resolving enzyme T7 endonuclease I

The chemical mechanism of phosphodiester bond hydrolysis catalyzed by a junction-resolving enzyme has been investigated. Endonuclease I of phage T7 is a member of the nuclease superfamily of proteins that include many restriction enzymes, and the structure of the active site is very similar to that of BglI in particular. It contains three acidic amino acids that coordinate two divalent metal ions. Using mass spectrometry we have shown that endonuclease I catalyzes the breakage of the P-O3' bond, in common with restriction enzymes. We have found that the pH dependence of the hydrolysis reaction is log-linear, with a gradient of 0.9. Substitution of the scissile phosphate by an electrically neutral methylphosphonate significantly impairs the rate of bond cleavage. However, the introduction of chirally pure methylphosphonate groups shows that the effect of substitution of the proS oxygen atom is much greater than that for the proR. This is consistent with our current model of the structure of the DNA bound in the active site of endonuclease I, where the proS oxygen atom is coordinated directly to both metal ions as it is in BglI. The activity is also very sensitive to repositioning of the carboxylate groups of Asp 55 and Glu 65 in the active site, although some restoration of activity in endonuclease I E65D was observed in the presence of Mn2+ ions. A mechanism of hydrolysis consistent with all of these data is proposed.     

Flow birefringence of T7 phage DNA: Dependence on salt concentration

We have determined extinction angles and flow birefringence of T7 bacteriophage DNA over a wide range of shear, polymer concentration, and solvent ionic strength. From these data, information on the simple salt dependence of coil permeability to solvent and on short-range intrachain interactions (persistence length) was obtained. At all ionic strengths, our results are consistent with a partially draining coil in the Gaussian subchain dynamical theory of Rouse-Zimm-Tschoegl-Bloomfield. Salt dependence of persistence length is comparable to, although somewhat less than, that obtained previously using similar methods with a fivefold higher-molecular-weight DNA (T2 bacteriophage DNA). Possible reasons for observed discrepancies are analyzed, and the results of this work are compared in detail to other current studies of solvent ionic strength dependence in persistence length and hydrodynamic properties of DNA.