In vivo replication and recombination of T4 bacteriophage DNA.
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DNA Replication↗
Biomedical subjects
A W Kozinski
Publications and source records attributed to A W Kozinski.
Effects of UV irradiation on the fate of 5-bromodeoxyuridine-substituted bacteriophage T4 DNA.
We have carried out a series of experiments designed to characterize the impact of UV irradiation (260 nm) on 5-bromodeoxyuridine-labeled (heavy) T4 bacteriophage, both before and after infection of Escherichia coli. In many respects, these effects differ greatly from those previously described for non-density-labeled (light) phage. Moreover, our results have led us to propose a model for a novel mechanism of host-mediated repair synthesis, in which excision of UV-damaged areas is followed by initiation of replication, strand displacement, and a considerable amount of DNA replication. UV irradiation of 5-bromodeoxyuridine-labeled phage results in single-stranded breaks in a linear, dose-dependent manner (1.3 to 1.5 breaks per genomic strand per lethal hit). This damage does not interfere with injection of the phage genome, but some of the UV-irradiated heavy phage DNA undergoes additional intracellular breakdown (also dose dependent). However, a minority (25%) of the injected parental DNA is protected, maintaining its preinjection size. This protected moiety is associated with a replicative complex of DNA and proteins, and is more efficiently replicated than is the parental DNA not so associated. Most of the progeny DNA is also found with the replicative complex. The 5-bromodeoxyuridine of heavy phage DNA is debrominated by UV irradiation, resulting in uracil which is removed by host uracil glycosylase. Unlike the simple gap-filling repair synthesis after infection with UV-irradiated light phage, the repair replication of UV-irradiated heavy phage is extensive as determined by density shift of the parental label in CsC1 gradients. The newly synthesized segments are covalently attached to the parental fragments. The repair replication takes place even in the presence of chloramphenicol, a protein synthesis inhibitor, suggesting it is host mediated. Furthermore, the extent of the repair replication is greater at higher doses of UV irradiation applied to the heavy phage. This abundant synthesis results ultimately in dispersion of the parental sequences as short stretches in the midst of long segments of newly synthesized progeny DNA. Together, the extensive replication and the resulting distribution pattern of parental sequences, without significant solubilization of parental label, are most consistent with a model of repair synthesis in which the leading strand displaces, rather than ligates to, the encountered 5' end.
Bromodeoxyuridine↗
Late events in T4 bacteriophage DNA replication. III. Specificity of DNA reinitiation as revealed by hybridization to cloned genetic fragments.
Through the use of the technique of hybridization to cloned genes, the site specificity of the reinitiation of T4 DNA replication was examined at late times after infection, when a large amount of DNA had accumulated in the infected cell. Replication was examined under two conditions; (i) when there was recombination but the repair of the recombinants was inhibited, and (ii) when recombination was followed by covalent joining. When no covalent repair of recombinant was allowed, reinitiation occurred in the areas known to be also involved in the initiation of replication of the parental molecule: thus late reinitiation, if covalent joining is prevented, is site specific. When there was covalent joining, reinitiation displayed no apparent site specificity. The results are discussed in light of the possibility that at late times after infection recombinant intersections act as primers. The similarity of the model proposed to the "break-and-copy" model for lambda phage and the fitness of the proposed model to the genetic phenomena described by others are emphasized.
Base Sequence↗
Genetic specificity of DNA synthesized in the absence of T4 bacteriophage gene 44 protein.
Upon infection of Escherichia coli B with T4 phage with DO amber mutation in gene 44, a minimal amount of phage DNA is synthesized. This progeny DNA is, for the most part, covalently attached to the parental DNA. Analysis of the genetic representation of this DNA was performed by hybridization to cloned genetic segments. It was shown that areas preferentially replicated differ from origins observed in "normal" replication: under normal conditions, there is a strong origin in the genetic area of genes 50-5 and lack of initiation within the group of genes 40-43 and 35-52. In contrast, in the absence of the gene 44 protein, the genetic area of 50-5 is underrepresented, genes 35-36, tRNA, and genes 40-41 are the most prominent among progeny DNA, and the area of gene 39 is least represented. Since the area of gene 35 is known from the genetic data or other to be a high-frequency recombination area, and since the area of gene 39 is known to display a low frequency of recombination, we postulate that the observed uptake of label occurs at the site-specific recombinational intersections.
DNA Replication↗
Partial replication of UV-irradiated T4 bacteriophage DNA results in amplification of specific genetic areas.
Upon infection of Escherichia coli with bromodeoxyuridine-labeled t4 phage that had received 10 lethal hits of UV irradiation, a sizable amount of phage DNA was synthesized (approximately 36 phage equivalent units of DNA per infected bacterium), although very little multiplicity reactivation occurs. This progeny DNA was isolated and analyzed. This DNA was biased in its genetic representation, as shown by hybridization to cloned segments of the T4 genome immobilized on nitrocellulose filters. Preferentially amplified areas corresponded to regions containing origins of T4 DNA replication. The size of the progeny DNA increased with time after infection, possibly due to recombination between partial replicas and nonreplicated subunits or due to the gradual overcoming of the UV damage. As the size of the progeny DNA increased, all of the genes were more equally represented, resulting in a decrease in the genetic bias. Amplification of specific genetic areas was also observed upon infection with UV-irradiated, nonbromodeoxyuridine-substituted (light) phage. However, the genetic bias observed in this case was not as great as that observed with bromodeoxyuridine-substituted phage. This is most likely due to the higher efficiency of multiplicity reactivation of the light phage.
Cloning, Molecular↗
Differential amplification of specific areas of phage T4 genome as revealed by hybridization to cloned genetic segments.
Under various conditions, specific genetic areas of the phage T4 DNA molecule are preferentially and repeatedly replicated, resulting in the amplification of these areas. These areas are found to lie in the vicinity of the known origins of DNA replication.
Centrifugation, Density Gradient↗
Partial replicas of UV-irradiated bacteriophage T4 genomes and their role in multiplicity reactivation.
A physicochemical study was made of the replication and transmission of UV-irradiated T4 genomes. The data presented in this paper justify the following conclusions. (i) For both low and high multiplicity of infection there was abundant replication from UV-irradiated parental templates. It exceeded by far the efficiency predicted by the hypothesis that a single lethal hit completely prevents replication of the killed phage DNA: i.e., some dead phage particles must replicate parts of thier DNA. (ii) Replication of the UV-irradiated DNA was repetitive as shown by density reversal experiments. (iii) Newly synthesized progeny DNA originating from UV-irradiated templates appeared as significantly shorter segments of the genomes than progeny DNA produced from non-UV-irradiated templates. A good correlation existed between the number of UV hits and the number of random cuts that would be needed to reduce replication fragments to the length observed. (iv) The contribution of UV-irradiated parental DNA among progeny phage in multiplicity reactivation was disposed in shorter subunits than was the DNA from unirradiated parental phage. It is important to emphasize that it was mainly in the form of replicative hybrid. These conclusions appear to justify excluding interparental recombination as a prerequisite for multiplicity reactivation. They lead directly to some form of partial replica hypothesis for multiplicity reactivation.
Centrifugation, Density Gradient↗
Origins of phage T4 DNA replication as revealed by hybridization to cloned genes.
[3H]Thymidine-labeled progeny DNA was isolated after infection of Escherichia coli with two different bacteriophage T4 mutants. These strands were isolated shortly after the initiation of DNA replication and hybridized to 15 different (EcoRI) T4 restriction fragments cloned in plasmid vectors. Uniformly labeled T4 [32P]DNA extracted from phage particles was cohybridized as a normalizing reference. The results obtained lead to the conclusions that, among the loci tested, initiation occurs predominantly in the area of genes 50-5 and less prominently in the area of genes 25-29. However, our data do not support the idea of initiation in the area of genes 40-43. In contrast, this area displays the least replication among the genes tested.
Chromosome Mapping↗
Late replication and recombination in the vegetative pool of T4.
The rates and extents of replication are the same for all members of the vegetative pool, whether already residing (progeny) or newly entered (superinfecting). Thus, no member of the pool is sequestered in a replicative complex. Amber N82 infections of nonpermissive host result in extensive breakdown of phage DNA. The extent of fragmentation observed depends on the multiplicity of infection and whether phage ligase is present. Hence, parental DNA suffers single-strand nicks which can be repaired by ligase only if recombination does not interfere. The physiological role of ligase in compensating for such nicks is reemphasized. Superinfecting genomes recombine very rapidly with progeny molecules whose combined lengths are approximately six times that of the superinfecting genomic fragment. The superinfecting phage does not replicate before recombining. Therefore, the lack of replication poses no barrier to efficient recombination.
DNA Ligases↗
Late events in T4 bacteriophage production. I. Late DNA replication is primarily exponential.
The possibility of a switch in the mechanism of T4 DNA replication, from an exponential-bidirectional mode at early stages to a nonexponential (rolling circle) mode at later stages of phage development, has been investigated. The conclusion that DNA replication does not involve such a change in mechanism for the majority of replicating molecules is based on the analysis of the clonal distribution of mutants specifically induced at late times after infection. The clonal distribution of mutants, induced by adding 5-bromodeoxyuridine to infected cells at a time when 100 phage equivalents of DNA had accumulated, fits the pattern predicted by exponential replication.
Bromodeoxyuridine↗
Late events in T4 bacteriophage production. II. Giant bacteriophages contain concatemers generated by recombination.
Analysis of "giant" phage, which package concatenated DNA into their capsids, shows that they are predominantly heterozygous. The results are compatible with the hypothesis that concatemers are generated by recombination.
Coliphages↗
Absence of interparental recombination in multiplicity reconstitution from incomplete bacteriophage T4 genomes.
Interparental recombination between injected T4 DNA molecules is indetectable for incomplete petite phages (carrying a terminally deficient genome and therefore unable to circularize) as well as for genetically complete phages. The nonvialbe petite phages can individually replicate their DNA repeatedly, and they aso undergo multiplicity reconstitution, producing complete phages, provided that a host bacterium is infected by several petite particles that carry genetically complementary segments of DNA. The formation of complete phages in multiplicity reconstitution must be due to recombination among incomplete progeny fragments, i.e., partial replicas of the T4 genomes. It evidently does not result from interparental recombination. To test for interparental recombination, light bacteria (containing no bromouracil) were simultaneously infected in light medium with light radioactive phage in minority (usually less than one per cell) and heavy (bromouracil-labeled) phage in majority (usually about nine per cell). Any interparental recombination should, under these circumstances of infection, head to movement of the radioactive label of the minority light phage DNA to a position of higher density. That possibility was not observed.
Coliphages↗
Repetitive DNA replication of the incomplete genomes of phage T4 petite particles.
The genomes of petite T4 phage particles presumably cannot circularize because they are deficient for a significant terminal segment and hence not terminally redundant like normal T4 genomes. Combined density- and 32P-labeling shows that the majority of such deficient DNA molecules can nevertheless replicate their entire length. Furthermore, the density-shift technique shows that replicated parental strands can exchange their partners for new light strands, indicating that noncircularized T4 DNA molecules replicate repeatedly. When taken together with previously published data, these results indicate that T4 replication is bidirectional from multiple, genetically fixed points of origin. Rolling circle models can, therefore, not be considered as an essential mechanism for the early rounds of T4 replication.
Coliphages↗
Nonreplicated DNA and DNA fragments in T4 r- bacteriophage particles: phenotypic mixing of a phage protein.
"Conservative phage" containing a genome derived from an infecting phage particle which has not undergone replication in the cell but nevertheless has become encapsulated and released in a normal phage particle, are found after infection of Escherichia coli with rII(-) or rI(-) mutants under conditions which result in rapid lysis. If such conservative phage are derived from a mixed infection with v(+) and v(1) phage, they display phenotypic mixing of the v gene product (an endonuclease carried in the phage particle). Populations of rI and rII mutant phage grown under conditions of rapid lysis include particles containing short DNA fragments. It is suggested that a "maturation defect", common to rI and rII mutants, but absent in rIII mutants, may account for the encapsulation of nonreplicated DNA as well as that of the DNA fragments.
Bacteriolysis↗
Injection of ultraviolet-damage-specific enzyme by T4 bacteriophage.
When UV-irradiated T4 bacteriophage (v(+)) infects in the presence of chloramphenicol, the phage DNA rapidly acquires single-stranded breaks proportional to the dose of UV. In contrast, when UV-irradiated T4 v(1) (radiation sensitive mutant) infects under identical conditions, the phage DNA remains integral. A series of coinfections with v(+) and v(1) phage (UV-v(1) + majority non-UV-v(+) and UV-v(+) and majority non-UV-v(1)) show that the enzyme responsible for breakage is injected by the phage. It is also demonstrated that the v(1) phage injects an inactive enzyme that delays breakage by the v(+) enzyme and interferes with subsequent repair. The cross of v(+) and v(1) phage produces mixed progeny that contain both active and inactive enzyme in a single capsid. The possible function of this breaking enzyme, necessitating injection of multiple copies, is considered.
Bacterial Proteins↗
Multiple and specific initiation of T4 DNA replication.
Partially replicated T4 DNA molecules (PRM) whose parental or progeny DNA was labeled with bromodeoxyuridine BUdR was analyzed by gradual shearing followed by CsCl banding of the sheared product. Analysis of PRM containing 18-mum replicated DNA showed that each replicated region was 3- to 6-mum long, indicating three to 6 replicative sites per molecule. Analysis of PRM containing 9-mum replicated DNA similarly indicated two to three replicated regions per molecule. DNA from the replicated regions of PRM containing 10-mum replicated DNA ("donor") was hybridized to DNA from mature phage ("recipient"), and the resulting hybrid was subjected to digestion with exonuclease I. The extent of protection of the recipient and more efficient self-annealing of progeny fragments from PRM indicated that the replicated regions represented 8 to 10 nonrandom locations of the genome. Possible significance of multiple sites for initiation of DNA replication is discussed.
Bromodeoxyuridine↗
Host-mediated repair of discontinuities in DNA from T4 bacteriophage.
Discontinuities of T4 DNA which are caused by excision of UV-damaged areas, by decay of (32)P atoms, or which are present in DNA from rII(-)lig(am) (-) phage produced in a host nonpermissive for amber mutants are all repaired by bacterial enzymes after infection in the presence of chloramphenicol. Escherichia coli DNA polymerase I participates in the host-mediated repair, but an approximately 20-fold variation in the levels of host polynucleotide ligase does not affect either the kinetics or the extent of repair observed. Upon removal of chloramphenicol, host-repaired DNA from UV-irradiated phage undergoes a secondary cycle of breakage, which ultimately results in solubilization of most of the phage DNA. If the cells are co-infected with nonirradiated helper phage, the secondary breaks are repaired and the continuity of the polynucleotide chain is restored. The close coincidence in the extent of primary and secondary breakage suggests that phage-coded enzymes recognize and excise areas improperly repaired by the host. In contrast to host-mediated repair, repair mediated by rescuing phage probably restored functionality to the damaged DNA.
Centrifugation, Density Gradient↗
In vivo production of an RNA-DNA copolymer after infection of Escherichia coli by bacteriophage T4.
An RNA-DNA copolymer was isolated from Escherichia coli infected with bacteriophage T4. The RNA and DNA are covalently linked, and in the same polynucleotide strand. The DNA of the copolymer hybridizes specifically to the left strand of phage T4 DNA. The copolymer is produced in cells infected with amber mutants of phage T4 deficient in DNA replication and is not inhibited by the addition of chloramphenicol.
Bromodeoxyuridine↗