Bacteriophage T4 DNA Replication Research Articles

In vitro replication of bacteriophage T7 DNA damaged by ultraviolet radiation

The effect of ultraviolet radiation on DNA replication has been examined with an in vitro system capable of replicating intact chromosomes of T7 DNA from an exogenous template. Exposure of the template DNA to ultraviolet radiation resulted in a sharp drop in the amount of in vitro DNA synthesis. The residual replication detected when irradiated templates were used was found to proceed semiconservatively and to result in the production of pieces of duplex DNA approximately the same size as the average distance between pyrimidine dimers. It was also found that prior irradiation of the template inhibits formation of fast-sedimenting concatemer-like DNA structures normally synthesized in vitro. Hybridization studies demonstrated that the product synthesized in vitro from ultraviolet-irradiated templates includes DNA from both the left and right halves of the T7 chromosome. This may mean that after ultraviolet irradiation more than one origin of replication exists.

Bacteriophage T7 DNA replication in vitro. Electron micrographic analysis of T7 DNA synthesized with purified proteins.

Extensive replication of duplex T7 DNA is catalyzed in reactions contining T7 DNA polymerase, T7 gene 4 protein, and T7 RNA polymerase. When the product of this reaction is analyzed in the electron microscope, many eye form and Y form replication intermediates are observed. Replication in vitro is not initiated at a single region of the T7 genome. However, we tentatively conclude that initiation does occur preferentially at a few specific sites along the DNA, and that these sites may be near promoters at which the T7 RNA polymerase initiates transcription.

Bacteriophage T7 DNA replication in vitro. Stimulation of DNA synthesis by T7 RNA polymerase.

Four T7 products, DNA polymerase, gene 4 protein, RNA polymerase, and DNA binding protein, have been purified from phage-infected cells. It has been previously shown (Hinkle, D. C., and Richardson, C. C. (1975) J. Biol. Chem. 250, 5523-5529; Kolodner, R., and Richardson, C. C. (1978) J. Biol. Chem. 253, 574-584) that two T7 products, DNA polymerase and gene 4 protein, catalyze extensive synthesis on duplex T7 DNA containing single strand breaks. However, the T7 DNA polymerase purified by our procedure does not efficiently contribute in this reaction, although the preliminary evidence suggests that this enzyme may be the native form of the DNA polymerase. Such inefficient T7 DNA synthesis is greatly augmented by adding the third T7 product, namely T7 RNA polymerase. This DNA synthesis apparently requires transcription, since each of the four rNTPs must be present. The rate of synthesis is increased about 2-fold by the addition of T7 DNA binding protein. In contrast to the results obtained when DNA synthesis is initiated at single strand breaks in a duplex DNA molecule, essentially none of the DNA synthesized in the presence of T7 RNA polymerase is covalently attached to the T7 DNA template. We postulate that in this in vitro system, T7 DNA replication is initiated using an RNA primer synthesized by the T7 RNA polymerase.

Nucleotide sequence of the primary origin of bacteriophage T7 DNA replication: relationship to adjacent genes and regulatory elements.

The 682-base-pair nucleotide sequence between positions 14.45 and 16.15 on the bacteriophage T7 DNA molecule has been determined. We can identify not only the sequence of the primary origin of DNA replication but also the termination of gene 1, all of genes 1.1 and 1.2, the start of gene 1.3, and a number of regulatory sequences. The endpoints of four deletion mutations that extend into this region have been determined. These mutations are inferred to have arisen by recombination between short homologous sequences, three of which ar T7 RNA polymerase promoters. The base changes of four point mutations in gene 1.2 have been identified. The sequence essential for initiation at the primary origin is located between the left endpoints of the two deletions D2 and D303. Sequence analysis of these mutants assigns the primary origin to a 129-base-pair segment between positions 14.73 and 15.05. This intergenic segment is A+T-rich (75%) and contains a single T7 gene 4 protein recognition site; it is preceded by two tandem T7 RNA polymerase promoters. A model for initiation of T7 DNA replication is presented.

Physical mapping of primary and secondary origins of bacteriophage T7 DNA replication.

Deletion mutants of bacteriophage T7 have been used to identify and to map, by electron microscopy, the origins of T7 DNA replication. The primary origin of phage T7 DNA replication lies within a 100-base-pair region located 14.75-15.0% of the distance from the genetic left end of the DNA molecule. T7 phage whose DNA contains a deletion of this region initiate replication at secondary origins, the predominant one of which is located at a distance approximately 4% from the left end of the molecule.

RNA priming of DNA replication by bacteriophage T4 proteins.

Bacteriophage T4 DNA replication proteins have been shown previously to require ribonucleoside triphosphates to initiator new DNA chains on unprimed single-stranded DNA templates in vitro. This DNA synthesis requires a protein controlled by T4 gene 61, as well as the T4 gene 41, 43 (DNA polymerase), 44, 45, and 62 proteins, and is stimulated by the gene 32 (helix-destabilizing) protein. In this paper, the nature of the RNA primers involved in DNA synthesis by the T4 proteins has been determined, using phi X174 and f1 DNA as model templates. The T4 41 and "61" proteins synthesize pentanucleotides with the sequence pppA-C(N)3 where N in positions 3 and 4 can be G, U, C, or A. The same group of sequences is found in the RNA at the 5' terminus of the phi X174 DNA product made by the seven T4 proteins. The DNA product chains begin at multiple discrete positions on the phi X174 DNA template. The characteristics of the T4 41 and "61" protein priming reaction are thus appropriate for a reaction required to initiate the synthesis of discontinuous "Okazaki" pieces on the lagging strand during the replication of duplex DNA.

Relation between bacteriophage T4 DNA replication and late transcription in Vitro and in Vivo

An in vitro system for DNA and late RNA synthesis in extracts of phage T4-infected Escherichia coli has been devised. Here we describe the DNA synthesis properties of the system, as derived from T4 wild type- or replication mutant-infected cells. When the system is prepared from wild type phage-infected cells, DNA synthesis in vitro is sustained for a longer time at 25 than at 37°; both dCMP and hmdCMP can be incorporated into DNA. When the system is derived from E. coli infected with replication-defective T4 mutants, DNA synthesis in vitro parallels, at least qualitatively, the in vivo replication properties of these infections. T4 late RNA synthesis in vitro, even that RNA synthesis which should be initiated in vitro, can occur in the absence of concurrent DNA synthesis, both at 25 and at 40°. Nevertheless, bacteria infected with a temperature-sensitive T4 DNA polymerase (gp 43) mutant, tsP36, generate cell-free systems with the property of temperature-sensitive T4 late RNA synthesis in the absence of concurrent DNA synthesis. Bacteria-infected with a temperature-sensitive T4 dCMP hydroxymethylase (gp 42) mutant, tsLB3, do not show a comparable temperature sensitivity. Stimulated by this finding, certain aspects of the connection between T4 DNA replication and late RNA synthesis in vivo have been analyzed (or reanalyzed) The quantitative and qualitative effects, on T4 late RNA synthesis, of blocking DNA synthesis with FUdR and of inactivating temperature-sensitive T4 DNA replication proteins (gp 42 and gp 43) are shown to differ. The outcome of these in vitro and in vivo experiments is consistent with the notion that at least some of the T4 DNA replication proteins interact with the late gene transcription system.

Inhibition of initiation of bacteriophage T4 DNA replication by perturbation of Escherichia coli host membrane composition

3-Decynoyl-N-acetylcysteamine (3-decynoyl-NAC) is an analog which specifically causes the immediate cessation of the biosynthesis of unsaturated fatty acids in Escherichia coli, whereas the synthesis of saturated fatty acids is actually stimulated. As a result, the cell membrane accumulates saturated fatty acids in its phospholipid. Addition of the inhibitor at the time of infection of E. coli by T4 phage had no effect on normal phage replication and development, implying that the synthesis of unsaturated fatty acids per se has little effect on T4 DNA replication. However, if the integrity and composition of the bacterial membrane was grossly perturbed by first treating the cells with the inhibitor for 60 min before infection, the proper initiation and the attainment of a rapid rate of T4 DNA synthesis were not observed. Under these conditions, a full complement of T4 early proteins was synthesized. The membrane associability of the known DNA delay proteins induced by wild-type T4 phage in the treated cells resembled that expected of a culture of untreated cells infected with a DNA delay mutant. When any one of three DNA delay mutants was used to infect 3-decynoyl-NAC-treated cells, T4 DNA replication was aborted. These findings suggest that some kind of specific interactions among the initiation proteins defined by the DNA delay mutants and the bacterial membrane may be necessary to facilitate the normal initiation and rapid rate of T4 DNA replication. A model for the involvement of the three different initiation proteins and the subsequent attainment of rapid DNA synthesis is discussed.

Bacteriophage T7 DNA replication. An electron microscopic study of the growing point and the role of the T7 gene 4 protein in the formation of DNA fragments.

Bacteriophage T7 DNA replication. An electron microscopic study of the growing point and the role of the T7 gene 4 protein in the formation of DNA fragments.

Purification of the gene 43, 44, 45, and 62 proteins of the bacteriophage T4 DNA replication apparatus.

A procedure has been developed which allows the T4 bacteriophage proteins corresponding to the products of genes 43, 44, 45, and 62 to be purified to near homogeneity from a single T4-infected cell lysate (greater than 90% single species as judged by sodium dodecyl sulfate polyacrylamide elctrophoresis). In these preparations, the major problem of removing all contaminating nucleases has been overcome. Each of the above proteins is known from genetic analysis to be essential for phage DNA replication. The protein product of gene 43 is T4 DNA polymerase, and its recovery can be monitored using a standard DNA polymerase assay. The other three gene products have been designated as "polymerase accessory proteins," since they directly enhance polymerase function on both single- and double-stranded DNA templates. Their activities were monitored by an "in vitro complementation assay," which measures the stimulation of DNA synthesis observed in a concentrated lysate of T4 mutant-infected Escherichia coli cells when the missing T4 wild type protein is added. Starting from 300 g of infected cell paste, we obtained 9.3 mg of gene 43 protein, 21 mg of gene 45 protein, and 70 mg of a tight complex made up of 44 and 62 proteins; final yields were estimated at 30%, 14%, and 28%, respectively, of the initial activity present in the lysate. When the above purified proteins are incubated with preparations of two other T4 DNA replication proteins (gene 41 and gene 32 proteins) plus deoxyribonucleoside and ribonucleoside triphosphates, extensive DNA synthesis occurs on both single- and double-stranded DNA templates. As reported elsewhere, this synthesis mimicks that catalyzed by the T4 DNA replication apparatus in vivo.

Cloning and localization of the in vitro functional origin of replication of bacteriophage T7 DNA.

Cloning and localization of the in vitro functional origin of replication of bacteriophage T7 DNA.

Involvement of DNA gyrase in replication and transcription of bacteriophage T7 DNA

Growth of bacteriophage T7 is inhibited by the antibiotic coumermycin A1, an inhibitor of the Escherichia coli DNA gyrase. Since growth of the phage is insensitive to the antibiotic in strains containing a coumermycin-resistant DNA gyrase, this enzyme appears to be required for phage growth. We have investigated the effect of coumermycin on the kinetics of DNA, RNA, and protein synthesis during T7 infection. DNA synthesis is completely inhibited by the antibiotic. In addition, coumermycin significantly inhibits transcription of late but not early genes. Thus, E. coli DNA gyrase may play an important role in transcription as well as in replication of T7 DNA.

Replication of Bacteriophage T7 DNA by Purified Proteins

Bacteriophage T7 provides a system for understanding the enzymatic and molecular mechanisms involved in the replication of a linear, duplex DNA molecule. Each T7 particle contains an entirely double-stranded DNA molecule, each strand consisting of an uninterrupted sequence of 40,000 nucleotides (Fig. 1a). The DNA molecules isolated from T7, like those from T-even phages, contain a terminal repetition but, unlike the circularly permuted collection isolated from T-even phages, they form a unique collection of sequences (for reviews, see Thomas 1966; Studier 1972).

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.

Marker rescue and partial replication of bacteriophage T7 DNA.

Experiments reported here show that some UV-irradiated wild-type T7 phage markers can be rescued efficiently by coinfection with T7 amber mutant phage in a permissive host. Other results show that the segments of a UV-irradiated genome that replicate efficiently are those that also are rescued efficiently during a marker rescue experiment. At higher doses, fewer markers are rescued efficiently and fewer segments of the genome replicate efficiently. The results clearly indicate that the probability of marker rescue is correlated with the ability of the DNA containing the marker to replicate. Sucrose density gradient analysis shows that UV irradiation does not produce double-strand scissions in T7 DNA at doses used here. Therefore, the partial replication and rescue of markers from the left end of the genome is not due simply to injection of only the left end of the T7 DNA.

The structure and maturation of intermediates in bacteriophage T7 DNA replication.

Concatemeric DNA from T7-infected cells consists of phage genomes in a linear head-to-tail arrangement. Adjacent genomes within a concatemer overlap for the length of the terminal repetition. Fast-sedimenting T7 DNA contains single-stranded regions at roughly unit-lentth intervals but these interruptions are heterogeneously distributed and do not occur at the genetic termini. Mutations in either bacteriophage genes 9, 18, or 19 (required for DNA maturation and packaging) lead to the synthesis and persistence of DNA with fewer interruptions than normal.

Role of deoxycytidylate deaminase in deoxyribonucleotide synthesis in bacteriophage T4 DNA replication

Role of deoxycytidylate deaminase in deoxyribonucleotide synthesis in bacteriophage T4 DNA replication

Involvement of DNA gyrase in bacteriophage T7 DNA replication.

NOVOBIOCIN and the related antibiotic coumermycin A1 (referred to as coumermycin) inhibit the supercoiling of circular double-stranded DNA catalysed by Escherichia coli DNA gyrase1. They also inhibit the replication of chromosomal DNA in E. coli cells2, and of circular double-stranded DNA in cell-free systems from E. coli3–6. The activity of DNA gyrase obtained from a coumermycin-resistant (cour) mutant and the replication of colicin El plasmid (Col El) DNA in a cell extract of the mutant are resistant to both drugs7. These results show that DNA gyrase is an essential component of these systems for the replication of the circular double-stranded DNA. It is interesting to examine whether DNA gyrase also has a role in replication of linear double-stranded DNA. The DNA of E. coli bacteriophage T7 is linear and double stranded8. Linear replicating molecules have been isolated from infected cells and it has been claimed that the phage DNA replicates as a linear form, at least in the first round of replication9. We show here that the replication of phage T7 DNA, including the first round, is inhibited by coumermycin.

Inhibitory effect of agents altering the structure of DNA on the synthesis of pyrimidine deoxyribonucleotides in bacteriophage T4 DNA replication.

Inhibitory effect of agents altering the structure of DNA on the synthesis of pyrimidine deoxyribonucleotides in bacteriophage T4 DNA replication.

Regulation of deoxyribonucleotide biosynthesis during in vivo bacteriophage T4 DNA replication. Intrinsic control of synthesis of thymine and 5-hydroxymethylcytosine deoxyribonucleotides at precise ratio found in DNA.

The kinetics of the de novo formation of pyrimidine deoxyribonucleotides is the same after infection by wild type bacteriophage T4, which generate very low steady state levels of deoxytibonucleotides, and by T4 DNA synthesis-negative mutatants (Dna-), which accumulate high levels, suggesting that the control is not by a feedback mechanism. In this study, the ratio of the de novo synthesis of dTMP to HmdCMP derivatives was measured by determining the total thymine and 5-hydroxylxytosine (HmCyt) deoxyribonucleotides synthesized by the reductive pathways from [6-3H]uracil including those in DNA and any degradation products excreted into the medium. The ratio of the de novo synthesis of Thy/HmCyt derivatives remained constant at 2.1 +/- 0.1 for at least 45 min after infection by wild type phage, i.e. precisely at the Thy/HmCyt ratio in T4 DNA. On infection by phage mutated in the Dna-genes 32, 41, 44, or 45, the ratio still remained close to 2 to 1 for at least 25 min. Only after the pyrimidine deoxyribonucleotide concentrations reached levels about 100-fold greater than the initial values did the ratio begin to increase. However, a mutant of the structural gene for T4 DNA polymerase showed some increase in ratio by 15 min. Mutants of gene 1 (HmdCMP kinase) were distinct in that the Thy/HmCyt ratio dropped to about 1.0 by 25 min, and then remained quite constant. Uniquely, in these mutants a significant quantity of 5-hydroxymethyluracil or a derivative was found, about 40% being in the medium. The product was shown to be derived by deamination of a 5-HmCyt derivative. All Dna- mutants tested excreted 35 to 50% of their thymine derivatives, mostly as thymine, into the medium. Neither thymine nor 5-hydroxymethyluracil derivates were excreted after wild type phage infection. We propose that pyrimidine deoxyribonucleotide synthesis is regulated at a Thy:HmCyt ratio of 2:1 as an intrinsic property of a complex of enzymes synthesizing and channeling deoxyribonucleotides for T4 DNA replication and not exclusively by effector-sensitive mechanisms.