Tn5cos: a transposon for restriction mapping of large plasmids using phage lambda terminase.

A method for the rapid restriction mapping of large plasmids has been developed. A 400-bp fragment of phage lambda DNA containing the cos region has been inserted into Tn5. After in vivo transposition of this Tn5cos element into the plasmid of choice, the plasmid is isolated and linearized at its cos site with phage lambda terminase (Ter). Such Ter linearization was about 70% efficient. After partial digestion of the linear molecules with the appropriate restriction enzyme, the products are selectively labelled at the right or left cohesive phage lambda DNA termini by hybridization with digoxygenin (DIG)-11-dUTP-labelled (using terminal transferase) oligodeoxyribonucleotides complementary to the single-stranded cos ends. After pulsed field gel electrophoresis, the labelled fragments are visualized in the dried gel using a DIG-detection kit. The restriction map can be directly determined from the 'ladder' of partial digestion products.     

Lethal effect of lambda DNA terminase in recombination deficient Escherichia coli

Summary DNA terminase is the enzyme that catalyses the cleavage of DNA concatemers into genome-size molecules and packages them into the capsid. The cleavage (DNA maturation) takes place in a specific site in the phage DNA called cos. Either one of two Escherichia coli proteins, integration host factor (IHF) and terminase host factor (THF), is required, in addition to terminase, for maturation of wild-type DNA in vitro. In vivo, at least some cos cleavage is known to occur in mutants that are unable to synthesize active IHF. No THF-defective mutants have yet been isolated. In order to determine if IHF, THF or any other host protein is involved in DNA maturation in vivo, I devised a selection for host mutants that are unable to support cos cleavage. The selection is based on the assumption that DNA terminase will kill cells by cleaving chromosomally located cos sites. I found that DNA terminase will indeed kill cells provided that they contain a chromosomal cos site and provided also that they are defective in the host recA or recB genes. These two genes are required for certain pathways of genetic recombination and repair of damaged DNA, and I suggest that they prevent terminase-induced killing by repairing broken chromosomes. Interstingly, mutation in a related host gene, recD, did not render cells susceptible to terminase killing. recD and recB both encode subunits of exonuclease V, but recD mutants, unlike recB, remain proficient in genetic recombination and repair. I found mutants that survived the lethal effect of terminase in cos-containing E. coli recA at a frequency of about 5×10^-5. About 90% of these survivors were defective in terminase synthesis, and the rest were defective in IHF function. This result suggests that in the absence of IHF in vivo cos cleavage decreases to a level that permits repair of the damage, and therefore survival, even in recombination deficient cells. The absence of mutations in any other host gene suggests that IHF is the major accessory factor in DNA maturation in vivo. Alternatively, or in addition, mutations in other accessory factors are lethal.Abbreviations gp gene product: e.g. gpA, product of gene A - () prophage state - [] plasmid-carrier state     

The terminal redundant regions of bacteriophage T7 DNA

Summary Some aspects of the involvment of the terminal reduntant regions of T7 DNA on phage production have been studied by transfection experiments with T7 DNA after treatment of the molecules with exonuclease or exonuclease plus exonuclease I. It was found that terminal 5 gaps between 0.08 and 6.4% of the total length did not decrease the infectivity of the molecules although such gaps cannot be filled directly by DNA polymerases. Rather, compared to fully native DNA the infectivity of gapped DNA increased up to 20 fold in rec ⁺ spheroplasts and up to 4 fold in recB spheroplasts. This indicates a protective function of the single-stranded termini against the recBC enzyme in rec ⁺ and possibly another unidentified exonuclease present also in recB. The possibility that spontaneous circularization of the gapped molecules in vivo provides protection against exonucleolytic degradation was tested by transfection with T7 DNA circularization in vitro by thermal annealing. Such molecules were separated from linear molecules by neutral sucrose gradient centrifugation. They displayed a 3 to 6 fold higher infectivity in rec ⁺ and recB compared to linear gapped molecules, which shows that T7 phage production may effectively start from circular DNA.When the 3 single-stranded ends from gapped molecules were degraded by treatment with exonuclease I the infectivity of the molecules was largely abolished in rec ⁺ and recB as soon as 40 to 80 base pairs had been removed per end. It is concluded that the terminal regions of T7 DNA molecules are essential for phage production and that the redundancy comprises probably considerably less than 260 base pairs. The results are discussed with respect to the mode of T7 DNA replication.     

Molecular analysis of the packaging signal in bacteriophage CP-T1 of Vibrio cholerae

Summary We have previously identified a unique site, pac, from which packaging of precursor concatameric viral DNA into proheads starts during the maturation process of bacteriophage CP-T1. The direction of this packaging was determined from restriction enzyme cleavage patterns of CP-T1 DNA. A restriction enzyme generated fragment containing pac was cloned and the surrounding DNA region sequenced. Analysis of the nucleotide sequence revealed numerous repeat regions related to the consensus sequence PuagttGAT.AAT.aa.t. Within the sequenced region an open reading frame encoding a 12260 M_r protein was also identified. This protein appears to share homology with the binding domains of known DNA binding proteins and may represent a putative Pac terminase possessing the specific endonuclease activity required for cleavage at the pac site. Minicell analysis of deletion derivatives of the pac-containing clone revealed a protein of approximately 12900 M_r encoded within this same region, confirming that this Pac protein is phage encoded.     

Structure and role of the terminal repeats of Epstein-Barr virus in processing and packaging of virion DNA.

The linear virion Epstein-Barr virus (EBV) DNA is terminated at both ends by a variable number of direct, tandemly arranged terminal repeats (TRs) which are approximately 500 bp in size The number of TRs at each terminus can vary. After infection of host cells, the EBV DNA circularizes via the TRs by an unknown mechanism, and replication of the viral DNA during the lytic phase of the EBV life cycle leads to large DNA concatemers which need to be cleaved into virion DNA units, eventually. This cleavage event occurs at an unknown locus within the TRs of EBV, which are the cis-acting elements essential for cleavage of the concatemers and encapsidation of the virion DNA. To investigate the mechanism of DNA processing during genome circularization and cleavage of concatemeric DNA, the genomic termini of EBV were cloned, sequenced, and analyzed by direct labeling of the virion DNA. Both termini ended with identical 11-bp elements; the right end has acquired an additional 9-bp stretch that seemed to originate from the leftmost unique sequences. The left terminus is blunt, whereas the right terminus appears to have a 3' single-base extension. In a transient packaging assay, a single terminal repeat was found to be sufficient for encapsidation of plasmid DNA, and mutagenesis of the TR element defined a region of 159 bp, including the 11-bp element, which is essential for packaging. These results indicate that the genomic termini of EBV are not generated by a simple cut of a hypothetical terminase. The mechanism for cleavage of concatemers seems to involve recombination events.     

Protein-primed DNA replication: role of inverted terminal repeats in the Escherichia coli bacteriophage PRD1 life cycle.

Escherichia coli bacteriophage PRD1 and its relatives contain linear double-stranded DNA genomes, the replication of which proceeds via a protein-primed mechanism. Characteristically, these molecules contain 5'-covalently bound terminal proteins and inverted terminal nucleotide sequences (inverted terminal repeats [ITRs]). The ITRs of each PRD1 phage species have evolved in parallel, suggesting communication between the molecule ends during the life cycle of these viruses. This process was studied by constructing chimeric PRD1 phage DNA molecules with dissimilar end sequences. These molecules were created by combining two closely related phage genomes (i) in vivo by homologous recombination and (ii) in vitro by ligation of appropriate DNA restriction fragments. The fate of the ITRs after propagation of single genomes was monitored by DNA sequence analysis. Recombinants created in vivo showed that phages with nonidentical genome termini are viable and relatively stable, and hybrid phages made in vitro verified this observation. However, genomes in which the dissimilar DNA termini had regained identical sequences were also detected. These observations are explained by a DNA replication model involving two not mutually exclusive pathways. The generality of this model in protein-primed DNA replication is discussed.     

Mutations of the coat protein gene of bacteriophage λ that overcome the necessity for the FI gene; the EFi domain

The functions of most of the 10 genes involved in phage λ capsid morphogenesis are well understood. The function of the FI gene is one of the exceptions. Mutants in FI fail to mature and package DNA. The gene product (gpFI) seems to act as a catalyst for the formation of an intermediate in capsid assembly called complex II, which contains a procapsid (an empty capsid precursor), terminase (the enzyme that cleaves the DNA precursor and packages it into the procapsid) and DNA. The mechanism for this stimulation remains unknown. It has also been reported that gpFI appeared to stimulate terminase-mediated cos cleavage, in the absence of procapsids, by increasing enzyme turnover. In comparison with other head-gene mutants, FI mutants are leaky, producing approx. 0.1 phage per infected cell. Some second-site revertants of FI ^? phages, called ‘fin’, that bypass the necessity for gpFI, have been isolated and found to harbour a mutation in the genes that code for the two subunits of terminase. In the course of mapping additional fin mutants, it was discovered that some mapped outside the terminase genes. To localize the mutations, restriction fragments of fin mutant DNAs were subcloned into plasmids and their ability to contribute to fin function was determined by marker-rescue analysis. The location of the fin mutation was further delineated by deletion analysis of a plasmid that was positive for fin. This showed that some fin mutations mapped to a region comprising genes E, D and a portion of C. The sequencing of this entire region in several fin isolates showed that the fin mutations are clustered in a small region of gene E corresponding to a portion of 26 amino acid residues of the coat protein (gpE). We have called this region of the protein the EFi domain. All the mutations result in an increase in positive charge relative to the wild-type protein. These results suggest that DNA maturation and packaging are in part controlled by an interaction between gpFI and capsid gpE.     

Processive action of terminase during sequential packaging of bacteriophage lambda chromosomes

Bacteriophage lambda chromosomes are packaged in a polarized, sequential fashion from a multimeric DNA substrate. Mature chromosomes are generated when terminase introduces staggered nicks in the cohesive end sites (cos sites) bounding a chromosome. Packaging is polarized, to the initial and terminal cos sites for packaging a chromosome can be defined. To initiate packaging, terminase binds to cos at cosB, and subsequently cuts at cosN. To terminate packaging of a chromosome, a functional cosB is not required at the terminal cos. To explain this finding, it was proposed earlier that terminase scans for the terminal cosN, rather than any subsequent cosB, during packaging. In the work described here we performed helper packaging experiments to see whether processive action of terminase occurs during sequential packaging of lambda chromosomes. The helper packaging experiments involve trilysogens; strains carrying three prophages in tandem. Infection by a hetero-immune helper phage results in packaging of the repressed prophage chromosomes, since the prophage structure is analogous to the normal DNA substrate. Two chromosomes can be packaged from between the three cos sites of the prophages of a trilysogen. Both chromosomes are packaged even when the central cos is cosB-. Our interpretation of these data is that terminase is brought to the central cos by packaging; following cleavage of the central cos, the terminase remains bound to the distal chromosome; and terminase acts to begin packaging of the distal chromosome. The frequency at which terminase reads across the central cos to initiate packaging of the distal chromosome is in the range from 0.3 to 0.5 in our experiments. Reading across cos was found not to be greatly dependent on the state of cosB, indicating that cosB binding is only needed for packaging the first chromosome in a packaging series. A multilysogen was constructed in which the initial cos was cos+ and the distal cos sites were all cosB-. The initial and downstream chromosomes were found to be packaged. This result indicates that terminase that is brought to the central cos by packaging is not only able to initiate packaging of a downstream chromosome, but can also scan and terminate packaging of the downstream chromosome. A model is presented in which processive action of terminase is the basis for sequential packaging of lambda chromosomes.     

Synthesis of a trans-Acting Inhibitor of DNA Maturation by Prohead Mutants of Phage λ

Bacteriophage lambda with mutations in genes that control prohead assembly and other head precursors cannot mature their DNA. In this paper we present evidence that the failure of these phage mutants to mature DNA is a reflection of a mechanism that modulates terminase nicking activity during normal phage development. We have constructed plasmids that contain the lambda-cohesive end site (cos) and the genes that code for DNA terminase, the enzyme that matures DNA by cutting at cos. The DNA terminase genes are under control of a thermosensitive cI repressor. These plasmids lack most of the genes involved in prohead morphogenesis and other head precursors. However, when repression is lifted by destruction of the thermosensitive repressor, the terminase synthesized is able to cut almost 100% of the plasmids. Therefore, these plasmids can mature in the absence of proheads and other head gene products. The plasmids are also able to complement mutants of lambda deficient in terminase and DNA maturation. However, in these complementation experiments, if the phage carry mutations in prohead genes E or B, not only is phage DNA maturation blocked, but the plasmid also fails to mature. These experiments show that, in the absence of proheads, phage lambda produces a trans-acting inhibitor of maturation. The genetic determinant of this inhibitor maps in a region extending from the middle of gene B to the end of gene C. A model is proposed in which the nicking activity of DNA-bound terminase is inhibited by the trans-acting inhibitor. Prohead (and other factors) binding to this complex would release the block to allow DNA cleavage and packaging.     

Design of molecules that specifically recognize and cleave apurinic sites in DNA

We have prepared a series of a tailor-made molecules that recognize and cleave DNA at apurinic sites in vitro. These molecules incorporate in their structure different units designed for specific function: an intercalator for DNA binding, an nucleic base for abasic site recognition and a linking chain of variable length and nature (including amino and/or amido functions). The cleavage efficiency of the molecules can be modulated by varying successively the nature of the intercalating agent, the nucleic base and the chain. All molecules bind to native calf thymus DNA with binding constants ranging from 10⁴ to 10⁶ M^?1. Their cleavage activity was determined on plasmid DNA (pBR 322) containing 1.8 AP-sites per DNA-molecule. The minimum requirements for cleavage are the presence of the three units, the intercalator, the nucleic base and at least one amino function in the chain. The most efficient molecules cleaved plasmid DNA at nanomolar concentrations. Enzymatic experiments on the termini generated after cleavage of AP-DNA suggest a strand break induced by a β-elimination reaction. In order to get insight into the mode of action (efficiency, selectivity, interaction), we have used synthetic oligonucleotides containing either a true abasic site at a determined position to analyse the cleavage parameters of the synthetic molecules by HPLC or a chemically stable along (tetrahydrofuran) of the abasic site for high field ¹H NMR spectrometry and footprinting experiments. All results are consistent with a β-elimination mechanism in which each constituent of the molecule exerts a specific function as indicated in the scheme: DNA targeting, abasic site nucleases and can be used advantageously as substitutes for the natural enzyme for in vitro cleavage of AP-sites containing DNA.     

The termini of bacteriophage T7 DNA

The termini of Escherichia coli phage T7 DNA have been labeled with ³²P by the polynucleotide kinase reaction. The DNA was fragmented, denatured, and annealed to denatured T7 DNA embedded in agar; elution was measured as a function of temperature. The terminal fragments were eluted from the gel at temperatures well below that of the bulk of the DNA, suggesting that these regions have a very high adenine-plus-thymine content. However, when DNA doubly labeled throughout at random by growth of the phage in [³H]thymidine and ³²PO₄, was denatured, annealed to the gel, and eluted as a function of temperature, the material eluting from the gel in this low-temperature range was about 50% adenine and thymine. Hence the melting behavior of the terminal fragments is not a result of a high adenine plus thymine content. By electrophoretic analysis of exonucleolytic digests of the T7 DNA it was shown that no unusual bases were present. It is suggested that the low thermal stability of the annealed terminal fragments is a consequence of the high guanine·cytosine regions being unavailable for hybridization, possibly because they are involved in intra-strand hydrogen bonding.     

DNA analysis of temperate bacteriophage Aa/23 isolated from Actinobacillus actinomycetemcomitans

The DNA of the temperate bacteriophage Aaφ23 isolated from the oral bacterium Actinobacillus actinomycetemcomitans was examined structurally both in the phage head and in the prophage. The DNA in phage particles comprises 44 kb linear molecules with a terminal redundancy of 1.6 kb, which represent circular permutations. Thus, DNA is packaged into phage heads by the headful mechanism. The Aaφ23 prophage is integrated into the host chromosome. Received: 15 September 1997 / Accepted: 10 December 1997     

Physical mapping of the BglI, BglII, PstI and EcoRI restriction fragments of staphylococcal phage phi 11 DNA

Summary A cleavage map of the generalized transducing staphylococcal phage 11 DNA has been constructed by reciprocal double digestion. All three BglI, the six BglII, the three PstI, and 11 out of 15 EcoRI sites have been mapped. The map is circular, with a total length of 42 kb, and has been divided into 100 map units. The phage DNA is cyclically permuted and has a terminal redundancy of about 11 kb. The preferential starting point and direction for packaging DNA lies at map unit 79 and proceeds towards higher map units.     

The maturation of coliphage lambda DNA in the absence of its packaging

In vivo, λ DNA cannot be cleaved at cos (matured) if proheads are not present; in vitro, however, cos cleavage readily takes place in the absence of proheads. In order to investigate this paradox, we have constructed plasmids that synthesize λ terminase in vivo upon induction. The plasmids also contain cos at the normal position, about 190 bp upstream of λ gene Nul. One of the plasmids, pFM3, produces levels of terminase comparable to those found after phage induction. If cells carrying pFM3 are thermoinduced, almost 100% of the intracellular plasmid DNA has a double-strand interruption at or near cos.

Since the only λ genes that pFM3 carries are Nul, A, W and B, this in vivo cleavage is occurring in the absence of proheads. Previous failure to observe 2 maturation with phages carrying prohead mutations may be due to exonucleolytic degradation of the unprotected DNA ends, a different DNA topology or compartmentalization, or terminase inhibition in the absence of prohead by the product of another λ gene that maps to the right of gene B.     

Mutations in the terminase genes of bacteriophage λ that bypass the necessity for FI

DNA maturation in bacteriophage λ is the process by which the concatemeric precursor DNA is cleaved at sites called cos to generate mature λ DNA molecules. These DNA molecules are then packaged into procapsids, the empty capsid precursors. The enzyme that catalyses these events is λ DNA terminase. It is composed of two subunits, made of 181 and 641 amino acids, the products of genes Nu1 and A, respectively. The product of the FI gene (gpFI ) stimulates the formation of an intermediate in capsid assembly called complex II, which contains a procapsid, terminase and DNA. The mechanism of stimulation remains unknown. It has been suggested that gpFI may also stimulate terminase-mediated cos cleavage, in the absence of procapsids, by increasing enzyme turnover. Mutants in FI fail to mature and package DNA but, in comparison with other capsid gene mutants, FI mutants are leaky. Second site mutants of FI phages, called ‘fin’ (for FI independence), bypass the necessity for gpFI. These mutants were originally localized to the region of Nu1 and A and are of two classes: finA includes those that induce the synthesis of fourfold more gene A product (gpA ) than wild-type phages, and finB includes those that produce normal amounts of gpA. Whereas all finA mutants analysed map to Nu1, finB mutants have been found both in E and in Nu1. The existence of E mutants able to bypass the necessity for gpFI in vivo shows that gpE and gpFI interact, directly or indirectly. Here we have analysed and sequenced two finA mutants and one finB mutant. All of these map in Nu1. Of the two finA mutants, one corresponds to an Ala163Ser change and the other is a silent mutation. It is likely that the finA mutations alter mRNA conformation in a manner that results in an increase in the efficiency of A mRNA translation. The fourfold increase in gpA synthesis translates into a 10-fold increase in terminase activity. These results show that terminase overproduction is sufficient to bypass the necessity for gpFI and that such an overproduction can be achieved by changes in the efficiency of translation of A due to subtle changes in the sequence upstream of the gene. The finBcs₁₀₃ mutation is a His-87→Tyr change in Nu1. Therefore, an alternative way in which to bypass the requirement for gpFI involves an alteration in the structure of gpNu1. It is likely that the altered gpNu1 would increase cleavage and packaging efficiency directly or indirectly. We have determined that DNA cleavage in vivo does not occur in the absence of gpFI. Therefore it seems that gpFI somehow facilitates an otherwise latent capacity of terminase to autoactivate its nucleolytic activity.     

Cloning of restriction and modification genes in E. coli: The HhaII system from Haemophilus haemolyticus

The genes for a Class II restriction-modification system (HhaII) from Haemophilus haemolyticus have been cloned in Escherichia coli. The vector used for cloning was plasmid pBR322 which confers resistance to tetracycline and ampicillin and contains a single endonuclease R·PstI site, (5′)C-T-G-C-A^↓-G (3′), in the ampicillin gene. The procedure developed by Bolivar et al. (1977) was used to form DNA recombinants. H. haemolyticus DNA was cleaved with PstI endonuclease and poly(dC) extensions were added to the 3′-OH termini using terminal deoxynucleotidyl transferase. Circular pBR322 DNA was cleaved to linear molecules with PstI endonuclease and poly(dG) extensions were added to the 3′-OH termini, thus regenating the PstI cleavage site sequence. Recombinant molecules, formed by annealing the two DNAs, were used to transfect a restriction and modification-deficient strain of E. coli (HB101 r^?m^?recA). Tetracycline-resistant clones were tested for acquisition of restriction phenotype (as measured by growth on plates seeded with phage λcI·O). A single phage-resistant clone was found. The recombinant plasmid, pDI10, isolated from this clone, had acquired 3 kilobases of additional DNA which could be excised with PstI endonuclease. In addition to the restriction function, cells carrying the plasmid expressed the HhaII modification function. Both activities have been partially purified by single-stranded DNA-agarose chromatography. The cloned HhaII restriction activity yields cleavage patterns identical to HinfI. A restriction map of the cloned DNA segment is presented.     

Specificity of cleavage in replicative-form DNA of bovine herpesvirus 1.

The linear double-stranded DNA genome of herpesvirus as it is present in infectious virions needs to be circularized after infection of host cells and before DNA replication. Replicative-form genomes have to be cleaved into linear unit-length molecules during virion maturation and are most probably the substrate for inversion of the short segment relative to the long segment of the bovine herpesvirus 1 (BHV-1) genome. Those regions of the BHV-1 genome which are functionally involved in these processes have been analyzed at the molecular level by cloning and sequencing the genomic termini, the fusion of both termini from replicative-form molecules, and the junction between the short and the long genome segment. On the basis of the simple genome arrangement of BHV-1, it was inferable that the cleavage of replicative-form genomes by a hypothetical BHV-1 terminase activity may be specified by a sequence at the left end of UL (An element), which is located proximal to a reiterated beta element that makes up the cleavage site itself. The relationship of those elements in BHV-1 and the comparison to similar regions of other herpesviruses indicate consensus sequence elements which are functionally important for cleavage and isomerization of viral DNA during maturation of virions.     

The molecular structure of the transducing particles of Salmonella phage P22. II. Density gradient analysis of DNA

Summary CsCl density gradient analysis showed that the DNA of plaque forming particles ofSalmonella phageP22 is lighter than the host DNA. The DNA of transducing phages exhibits an intermediate density, but close to host DNA. BU labelling of DNA synthesized in the cells after phage infection resulted in a density increase of transducing DNA of about 0.004 gxcm^-3, whereas infectious DNA increased by about 0.045 gxcm^-3. Shearing of isolated DNA molecules from unlabelledP22 lysates demonstrated that transducing DNA consists of two pieces of DNA of different density: 90% stem from the bacterial host whereas 10% are phage DNA and therefore responsible for the BU lable in transducing phages.     

The genome of B. subtilis phage SPP1:

Summary DNA molecules of B. subtilis phage SPP1 exhibit terminal redundancy and are partially circularly permuted. This was established by the hybridization of selected EcoRI restriction fragments to single strands of SPP1 DNA and by an analysis of the distribution of denaturation loops in partially denatured SPP1 DNA molecules. Deletions in SPP1 DNA are not compensated by an increase in terminally repetitious DNA. This finding, which is unique to SPP1, is discussed in terms of a modification of the Streisinger/Botstein model of phage maturation.     

Physical characterization of the genome of the Myxococcus xanthus bacteriophage MX-8

Summary We have constructed a restriction map for the genome of bacteriophage MX-8 from Myxococcus xanthus using the enzymes PvuII, MboI, and EcoRI. The phage genome size, as determined by restriction analysis, is 51.7±0.6 Kb. Double digestions, redigestions of isolated fragments, and crossed-contact hybridization of partial digestion products show that the restriction map is circular. Restriction analysis and Southern hybridization show that the phage DNA molecules are packaged sequentially from a concatemer starting from a specific site which we have mapped. The DNA molecules have an average terminal redundancy of approximately 8% and are circularly permuted over at least 40% of the genome.