Restriction mapping of T4 bacteriophage late gene region which contains the origins of DNA replication

DNAs of lambda T4 recombinants 596-27 (genes 50-5), 596-30 (genes 50-8), 596-29 (genes 50-12), 591-16 (genes 6-8), 591-1 (genes 9-12), 596-13 (genes 13-16), 596-17 (genes 18-20) and 596-11 (genes 25-29) were mapped with the use of EcoRI, HindIII, SmaI, SalI and BamHI restriction enzymes. T4 dcDNA was digested with HindIII restriction endonuclease and resulting fragments were cloned into HindIII lambda vector 761. The recombinants 761-7, 761-17, 761-19, 761-24, 761-44, 761-50, 761-55 contained the region of genes 25-48 and 761-42, 761-26 and 761-16 contained a single HindIII-fragment with genes 6-12 in both orientations. Data obtained with the DNA of the latter recombinants allowed to show the correctness of the map established earlier which did not contain a full set of overlapping sequences. As a result of the experiments reported, the position of EcoRI and HindIII recognition sites in the region of genes 50-20 and 25-48 was determined and in the region of genes 25-48 BglII and XhoI restriction sites were mapped. The location of a single BamHI restriction site in the region of gene 8 was also established.     

Physical mapping and cloning of bacteriophage T4 anti-restriction endonuclease gene.

We have proposed that the ability of T4 to produce non-glucosylated progeny after a single cycle of growth on a galU rglA rglB+ mutant of Escherichia coli is due to the initiation of the rglB+ function by a phage-coded, anti-restriction endonuclease protein. Based on this hypothesis, we screened T4 deletion mutants for failure to give a burst in this host. The absence of an arn gene in phage mutants lacking the 55.5- to 58.4-kilobase region is verified by their inability to protect secondary infecting non-glucosylated phage from rglB-controlled cleavage. A functional arn gene was cloned on plasmid pBR325, and the 0.8-kilobase insert DNA was shown to be homologous to the DNA missing in the arn deletion phage.     

The complex of T4 bacteriophage gene 44 and 62 replication proteins forms an ATPase that is stimulated by DNA and by T4 gene 45 protein

The bacteriophage T4 genome is believed to encode all of the proteins needed for the replication of its own DNA. Included among these proteins are the "polymerase accessory proteins", the products of T4 genes 44, 62 and 45. The first two of these genes specify the synthesis of the 44/62 protein complex, which is here shown to be a DNA-dependent ATPase, hydrolyzing either ATP or dATP to the corresponding nucleoside diphosphate and releasing inorganic phosphate. This nucleotide hydrolysis is greatly stimulated by addition of the gene 45 protein and by single-stranded DNA termini. A rapid micro DNA-cellulose assay is introduced and used to measure accessory protein binding to the complex of T4 gene 32 protein and single-stranded DNA. In the presence of ATP, the 44/62 protein binds to this complex but not to naked DNA, while the 45 protein requires both the 32 protein and the 44/62 protein for detectable binding.     

ADP ribosylation of Escherichia coli RNA polymerase is nonessential for bacteriophage T4 development.

The bacteriophage T4-induced alt and mod gene products covalently add ADP-ribose to the Escherichia coli RNA polymerase alpha polypeptides; phage carrying either an alt or a mod mutation are viable. A genetic cross between T4alt and T4mod phages yielded alt mod recombinant progeny which could not ADP ribosylate RNA polymerase at all, yet grew apparently normally. Thus, ADP ribosylation of RNA polymerase appeared to be nonessential for T4 development (at least in E. coli B/r and E. coli CR63), even though the phage has evolved two distinct enzymes to catalyze this reaction.     

Plasmid models for bacteriophage T4 DNA replication: requirements for fork proteins.

Bacteriophage T4 DNA replication initiates from origins at early times of infection and from recombinational intermediates as the infection progresses. Plasmids containing cloned T4 origins replicate during T4 infection, providing a model system for studying origin-dependent replication. In addition, recombination-dependent replication can be analyzed by using cloned nonorigin fragments of T4 DNA, which direct plasmid replication that requires phage-encoded recombination proteins. We have tested in vivo requirements for both plasmid replication model systems by infecting plasmid-containing cells with mutant phage. Replication of origin and nonorigin plasmids strictly required components of the T4 DNA polymerase holoenzyme complex. Recombination-dependent plasmid replication also strictly required the T4 single-stranded DNA-binding protein (gene product 32 [gp32]), and replication of origin-containing plasmids was greatly reduced by 32 amber mutations. gp32 is therefore important in both modes of replication. An amber mutation in gene 41, which encodes the replicative helicase of T4, reduced but did not eliminate both recombination- and origin-dependent plasmid replication. Therefore, gp41 may normally be utilized for replication of both plasmids but is apparently not required for either. An amber mutation in gene 61, which encodes the T4 RNA primase, did not eliminate either recombination- or origin-dependent plasmid replication. However, plasmid replication was severely delayed by the 61 amber mutation, suggesting that the protein may normally play an important, though nonessential, role in replication. We deleted gene 61 from the T4 genome to test whether the observed replication was due to residual gp61 in the amber mutant infection. The replication phenotype of the deletion mutant was identical to that of the amber mutant. Therefore, gp61 is not required for in vivo T4 replication. Furthermore, the deletion mutant is viable, demonstrating that the gp61 primase is not an essential T4 protein.     

Purification and properties of the bacteriophage T4 gene 31 protein required for prehead assembly.

A low molecular weight (approximately 16,000), early protein is characterized as the product of the essential T4 head assembly gene 31. This gene is known to be required to allow formation of any ordered head structure from the major T4 capsid protein, P23 (Laemmli, U.K., Beguin, F., and Gujer-Kellenberger, G. (1970) J. Mol. Biol. 47, 69-85). In wild type infection P31 synthesis ceases at late times; in contrast, P31 is overproduced in certain early or regulatory T4 mutant infections, particularly gene 55 mutant infections. P31 was purified preparatively from Escherichia coli infected with the latter mutant, but could only be obtained for the most part in modified form, possibly due to unusual sensitivity to a proteolytic activity. P31 is not cleaved in vivo during normal head assembly, nor does it become a part of the mature head or any ordered prehead structure as determined by an immunological assay using antiserum prepared against the purified protein. However P31 does appear to become a part of the unordered P23 aggregates (lumps) which accumulate when ordered P23 assembly is prevented. We cound find no evidence for P31 association with T4 DNA or the host membrane. Our experiments favor the hypothesis that P31 directly affects the aggregation state and solubility properties of P23.     

A hotspot for transition mutations in the rIIB gene of bacteriophage T4

Summary We have previously demonstrated that the sequence 5TGGCAA 3 located at codons 32–33 of the rIIB gene of bacteriophage T4 is a hotspot for transition mutations (Nelson et al. 1981). Here I report the properties of the same TGGCAA sequence introduced into the gene at codons 11–12. The sequence is highly mutable in both locations, suggesting that its high mutability is due to features of the TGGCAA itself and is not dependent on the immediate juxtaposition of additional external sequences. Within this sequence, at either location, only the transition at the central G:C pair frequently arises spontaneously or by 2-aminopurine or ethylmethane sulfonate mutagenesis. However, the 3 G:C pair, in addition, is highly mutable after nitrous acid or hydroxylamine treatment. This suggests that, within the TGGCAA sequence, there are two hotspots which are targeted by different mutagens.     

Recombination-dependent DNA replication stimulated by double-strand breaks in bacteriophage T4.

We analyzed the mechanism of recombination-dependent DNA replication in bacteriophage T4-infected Escherichia coli using plasmids that have sequence homology to the infecting phage chromosome. Consistent with prior studies, a pBR322 plasmid, initially resident in the infected host cell, does not replicate following infection by T4. However, the resident plasmid can be induced to replicate when an integrated copy of pBR322 vector is present in the phage chromosome. As expected for recombination-dependent DNA replication, the induced replication of pBR322 required the phage-encoded UvsY protein. Therefore, recombination-dependent plasmid replication requires homology between the plasmid and phage genomes but does not depend on the presence of any particular T4 DNA sequence on the test plasmid. We next asked whether T4 recombination-dependent DNA replication can be triggered by a double-strand break (dsb). For these experiments, we generated a novel phage strain that cleaves its own genome within the nonessential frd gene by means of the I-TevI endonuclease (encoded within the intron of the wild-type td gene). The dsb within the phage chromosome substantially increased the replication of plasmids that carry T4 inserts homologous to the region of the dsb (the plasmids are not themselves cleaved by the endonuclease). The dsb stimulated replication when the plasmid was homologous to either or both sides of the break but did not stimulate the replication of plasmids with homology to distant regions of the phage chromosome. As expected for recombination-dependent replication, plasmid replication triggered by dsbs was dependent on T4-encoded recombination proteins. These results confirm two important predictions of the model for T4-encoded recombination-dependent DNA replication proposed by Gisela Mosig (p. 120-130, in C. K. Mathews, E. M. Kutter, G. Mosig, and P. B. Berget (ed.), Bacteriophage T4, 1983). In addition, replication stimulated by dsbs provides a site-specific version of the process, which should be very useful for mechanistic studies.     

Bacteriophage T4 DNA polymerase mutations that confer sensitivity to the PPi analog phosphonoacetic acid.

Mutations that conferred sensitivity to the pyrophosphate analog phosphonoacetic acid in bacteriophage T4 DNA polymerase were identified. The mutations were loosely clustered in four regions of the gene. As found for herpes simplex virus DNA polymerase, T4 mutations that altered sensitivity to phosphonoacetic acid also altered sensitivity to nucleotide analogs. Some of the T4 DNA polymerase mutations also altered the ability of the enzyme to translocate from one template position to the next and affected DNA replication fidelity. Kornberg (A. Kornberg, Science 163:1410-1418, 1969) envisioned a DNA polymerase active center which accommodates primer terminus and template DNAs and the incoming nucleotide. Some mutations identified on the basis of sensitivity to phosphonoacetic acid may be part of such an active center because single amino acid substitutions simultaneously alter several DNA polymerase functions.     

The bacteriophage T4 dexA gene: sequence and analysis of a gene conditionally required for DNA replication

Summary We have cloned and sequenced a bacteriophage T4 EcoRI fragment that complements T4 del (39-56) infections of an optA defective Escherichia coli strain. Bacteria containing this recombinant plasmid synthesize two new proteins with molecular weights of 9 and 26 kilodaltons. We have identified the gene encoding the 26 kilodalton protein as essential for T4 infections of optA defective E. coli. Genetic and biochemical results are consistent with the identification of this protein as the product of the dexA gene, which encodes a 3 to 5 exonuclease.     

Spectrum of spontaneous frameshift mutations. Sequences of bacteriophage T4 rII gene frameshifts

The DNA sequences of 185 independent spontaneous frameshift mutations in the rIIB gene of bacteriophage T4 are described. Approximately half of the frameshifts, including those at hot spot sites, are fully consistent with classical proposals that frameshift mutations are produced by a mechanism involving the misaligned pairing of repeated DNA sequences. However, the remaining frameshifts are inconsistent with this model. Correlations between the positions of two base-pair frameshifts and the bases of DNA hairpins suggest that local DNA topology might influence frameshift mutation. Warm spots for larger deletions share the property of having endpoints adjacent to DNA sequences whose complementarity to sequences a few base-pairs away suggest that non-classical DNA misalignments may participate in deletion mutation. A model for duplication mutation as a consequence of strand displacement synthesis is discussed. In all, 15 frameshifts were complex combinations of frameshifts and base substitutions. Three of these were identical, and have extended homology to a sequence 256 base-pairs away that is likely to participate in the mutational event; the remainder are unique combinations of frameshifts and transversions. The frequency and diversity of complex mutants suggest a challenge to the assumption that the molecular evolution of DNA must depend primarily upon the accumulation of single nucleotide changes.     

DNA helicase requirements for DNA replication during bacteriophage T4 infection.

The lytic bacteriophage T4 uses multiple mechanisms to initiate the replication of its DNA. Initiation occurs predominantly at replication origins at early times of infection, but there is a switch to genetic recombination-dependent initiation at late times of infection. The T4 insertion-substitution system was used to create a deletion in the T4 dda gene, which encodes a 5'-3' DNA helicase that stimulates both DNA replication and recombination reactions in vitro. The deletion caused a delay in T4 DNA synthesis at early times of infection, suggesting that the Dda protein is involved in the initiation of origin-dependent DNA synthesis. However, DNA synthesis eventually reached nearly wild-type levels, and the final number of phages produced per bacterium was similar to that of the wild type. When the dda mutant phage also contained a mutation in T4 gene 59 (a gene normally required only for recombination-dependent DNA replication), essentially no DNA was synthesized. Recent in vitro studies have shown that the gene 59 protein loads a component of the primosome, the T4 gene 41 DNA helicase, onto DNA. A molecular model for replication initiation is presented that is based on our genetic data.     

Suppression of gene 49 mutations of bacteriophage T4 by a second mutation in gene X: structure of pseudorevertant DNA.

Mutations in gene 49 of bacteriophage T4 were suppressed by a second mutation in gene X. Mapping studies located gene X between genes 41 and 42. Complementation results indicated that mutations in FdsA gene (a suppressor of gene 49 mutants) were in gene X. The intracellular pseudorevertant DNA was examined for unusual properties which could explain its successful encapsidation. After the in vivo inactivation of a temperature-sensitive gene 32 (DNA unwinding) protein, the intracellular pseudorevertant DNA was converted into DNA pieces of approximately genome size. A similar conversion was observed after in vitro digestion of pseudorevertant DNA with single-strand-specific S1 endonuclease. Appreciable quantities of oligomeric intermediates were not produced during this conversion process. These data indicate that pseudorevertant DNA contains sizable single-stranded gaps and has a conformation similar to that of wild-type DNA. The results further suggest that the suppression of gene 49 mutant abnormal DNA phenotype and the encapsidation defect by a second mutation in gene X is associated with the formation of sizable single-stranded gaps. These studies raise the possibility that single-stranded gaps may be involved directly in the DNA encapsidation process, or may act indirectly as a consequence of their effect on the organization of intracellular DNA.     

Physical mapping and complete nucleotide sequence of the denV gene of bacteriophage T4.

Phage T4 deletion mutants that are folate analog resistant (far) and contain deletions in the region of the T4 genome near denV have been isolated previously. We showed that one of these mutants (T4farP12) expressed normal denV gene activity, whereas another mutant (T4farP13) was defective in the denV gene. The rII-distal (right) physical endpoints of these deletions defined the limits of the interval in which the rII-proximal (left) endpoint of the denV gene should be located. The deletion endpoints were identified by restriction and Southern hybridization analyses of phage derivatives containing deoxycytidine instead of hydroxymethyldeoxycytidine in their DNAs. The results of these analyses localized the rII-proximal (left) end of the denV gene to a region between 62.4 and 64.3 kilobases on the T4 physical map. denV+ phage resulted from marker rescue with two of five denV- alleles tested, using plasmids containing a 1.8-kilobase fragment from this region or a 179-base-pair terminal fragment derived from it. Sequencing of the 179-base-pair fragment from wild-type DNA showed a 130-base-pair open reading frame with its termination codon at the rII-proximal end. Confirmation that this open reading frame is part of the denV coding sequence was obtained by identifying a TAG amber codon in the homologous DNA derived from a denV amber mutant strain. This mutant strain rescued the denV+ allele from plasmids containing the wild-type sequence. An adjacent overlapping restriction fragment was also cloned, permitting determination of the remaining denV gene sequence. Based on these results, the 3' end of the coding region of the denV locus was mapped to kilobase position 64.07 on the T4 physical map, and the 5' end was mapped to position 64.48.