Cloning of bacterial DNA replication genes in bacteriophage lambda

Summary Recombinant lambda phages containing the genes for dnaZ protein (the subunit of DNA polymerse III holoenzyme), primase (dnaG protein) and dnaC protein from Escherichi coli and Salmonella typhimurium were isolated. Each gene cloned from S. typhimurium has extensive DNA sequence homology to the corresponding E. coli gene. Clones selected by complementation of a dnaA temperature-sensitive mutant appear similar to other isolated suppressors of dnaA (Projan and Wechsler 1981). Derivatives of each cloned fragment suitable for overproduction of the protein were constructed. Of those tested, only the phage containing the E. coli dnaZ gene resulted in significant overproduction.Abbreviations DTT dithiothreitol - Ec Escherichia coli - EDTA ethylene diamine tetra acetic acid - kb kilobase 1,000 bases or base-pairs - moi multiplicity of infection - pol I E. coli DNA polymerase I - pol III holoenzyme E. coli DNA polymerase III holoenzyme - pri dnaG, primase-coding gene - SSB single-strand binding protein - St Salmonella typhimurium - sup gene coding for suppressor - ts temperature-sensitive     

Specificity determinants for bacteriophage lambda DNA replication: I. A chain of interactions that controls the initiation of replication

The genetic elements which control autonomous DNA replication differ in functional specificity among coliphage λ, the coliphages φ80 and 82, and the Salmonella phage P22. Hybrid phages derived by genetic recombination between λ and each of these related phages have been used to define and to localize specificity determinants for DNA replication.In λ-P22 hybrid phages (Hilliker & Botstein, 1976) the replication control elements segregate as an intact unit. By contrast, some viable λ-φ80 and λ.82 hybrid phages arise by recombination within the replication control region, in a small interval inside structural gene O. From the properties of such hybrid phages, we infer that the O gene product of λ and the functionally equivalent proteins of φ80 and 82 each interact with a specific nucleotide sequence in the cognate ori site, the DNA target for control of the origin of replication. With respect to this interaction, both the O products and the receptor sequences within ori show stringent type specificity. The donor and receptor specificity determinants for the ori-O interaction lie within an interval of less than 400 base-pairs.The O gene product also interacts with the product of replication gene P (Tomizawa, 1971). The O-P interaction displays limited type specificity; the P-like protein of φ80 can function together with the O protein of λ, but the P protein of λ cannot function with the O-like protein of φ80. The specificity determinants for the O-P interaction can be separated from those for the ori-O interaction.We propose that a chain of interactions between ori, O product, P product, and replication functions of the bacterial host, Escherichia coli, controls specific template selection and the assembly of the essential replication apparatus in the initiation of λ DNA replication.     

Instability of bacteriophage lambda initiator O and P proteins in DNA replication

Lambda dv plasmids having an amber mutation in an initiator gene, O or P, were constructed from mutant lambda phages by recombinant DNA techniques and several properties of such derivatives were investigated. These plasmids are perpetuated in suppressor-plus (amber-permissive) cells, but not in non-suppressor cells. The plasmid copy number in the suppressor-plus cells was low as compared to that of the plasmid without the amber mutation. In cells carrying a thermosensitive suppressor 2, raising the temperature is expected to block new production of amber proteins, but should not affect conservation of the protein made prior to heating. It was observed, however, that replication of the plasmids carrying an amber mutation in the O or P gene was abolished soon after raising the temperature, suggesting that neither of the initiator proteins can continue functioning unless replenished. Pulse-chase experiments demonstrated that O protein decays with a half-life of 8 min. Several lines of evidence suggest that this degradation occurs independently of the protein function. On the other hand, P protein was not degraded under the same experimental conditions. These observations are discussed in connection with functional instability of the initiator molecules. It appears that they do not work catalytically.     

Lack of a unique termination site for the first round of bacteriophage lambda DNA replication

From previous data on the first round of bacteriophage λcIIcIII DNA replication (Schnös & Inman, 1970) it is possible to estimate, by extrapolation, the position on circular λ DNA where bidirectional growing points meet. In the present study we have investigated whether this position occurs at a genetically defined site. To this end, replicative intermediates of λ mutants containing either deletions to the left of the replication origin, or one deletion plus a duplication to the right, were analyzed in the electron microscope. Our results indicate that: (i) leftward growing points can traverse the extrapolated termination point calculated from the λcIIcIII data, (ii) no discontinuity of either right or leftward growing fork position is observed, and (iii) the extrapolated termination points for these mutants are well removed from those calculated for λcIIcIII DNA. From these data we conclude that there is probably no unique termination site for the first round of λ DNA replication and that termination occurs simply by collision of the growing forks.     

Physical studies of RNA involvement in bacteriophage lambda DNA replication and prophage excision

Non-diffusible genetic elements in bacteriophage λ DNA replication and λ prophage excision have been analyzed by the DNA-cutting assay of Freifelder and Kirschner (1971) and Freifelder et al. (1972). The mutant ti12, which affects a unique site for replication in or near the origin of replication (Dove et al., 1971), makes λ DNA partially refractory to replicative DNA-cutting. RNA synthesis in the vicinity of the origin, of replication seems to control the susceptibility of λ DNA to replicative DNA-cutting (Dove et al., 1969). Analogously, RNA synthesis in the vicinity of the left-hand prophage terminus seems to control excisional DNA-cutting of derepressed λ DNA, as predicted by the studies of Davies et al. (1972). These physical studies confirm previous genetic analyses and imply that the elements involved act at a very early stage in replication and in excision.     

Ordered assembly of nucleoprotein structures at the bacteriophage lambda replication origin during the initiation of DNA replication

Replication of the chromosome of bacteriophage lambda depends on the cooperative action of two phage-coded proteins and seven replication and heat shock proteins from its Escherichia coli host. As previously described, the first stage in this process is the binding of multiple copies of the lambda O initiator to the lambda replication origin (ori lambda) to form the nucleosomelike O-some. The O-some serves to localize subsequent protein-protein and protein-DNA interactions involved in the initiation of lambda DNA replication to ori lambda. To study these interactions, we have developed a sensitive immunoblotting protocol that permits the protein constituents of complex nucleoprotein structures to be identified. Using this approach, we have defined a series of sequential protein assembly and protein disassembly events that occur at ori lambda during the initiation of lambda DNA replication. A second-stage ori lambda.O (lambda O protein).P (lambda P protein).DnaB nucleoprotein structure is formed when O, P, and E. coli DnaB helicase are incubated with ori lambda DNA. In a third-stage reaction the E. coli DnaJ heat shock protein specifically binds to the second-stage structure to form an ori lambda.O.P.DnaB.DnaJ complex. Each of the nucleoprotein structures formed in the first three stages was isolated and shown to be a physiological intermediate in the initiation of lambda DNA replication. The E. coli DnaK heat shock protein can bind to any of these early stage nucleoprotein structures, and in a fourth-stage reaction a complete ori lambda.O.P.DnaB.DnaJ.DnaK initiation complex is assembled. Addition of ATP to the reaction enables the DnaK and DnaJ heat shock proteins to mediate a partial disassembly of the fourth-stage complex. These protein disassembly reactions activate the intrinsic helicase activity of DnaB and result in localized unwinding of the ori lambda template. The protein disassembly reactions are described in the accompanying articles.     

Role of genes O and P in the replication of bacteriophage lambda DNA.

DNA replication in coliphage λ occurs in two stages. The first round of replication generates mainly circular progeny DNA by a double-branched θ-type replicative form (Ogawa et al., 1968; Schnös &; Inman, 1970). In the late stage of λ DNA replication, however, σ-type rolling-circle replicative form DNA molecules, which produce multigenomic linear concatemers, are primarily found (Takahashi, 1974).At both early and later times, a temperature shift of λ Ots or Pts infected cells from 32 °C (permissive) to 43 °C (non-permissive temperature) caused a rapid reduction of the rate of radioactive precursor incorporation into λ DNA, showing that the gene O and P products are essential for the continuation of λ DNA synthesis. Observations on the molecular fine structure of the replicating fork after a temperature shift revealed characteristic long “single-strand connections” and single-strand “whiskers” at the branch point. These observations suggest that λ gene O and P products are directly involved in the propagation of daughter strands.     

Nucleotide sequence of bacteriophage lambda DNA

The nucleotide sequence of the DNA of bacteriophage λ has been determined using the dideoxy chain termination method in conjunction with random cloning in M13 vectors. Various methods were studied for sequencing specific regions to complete the sequence, but all were much slower than the random approach. The DNA in its circular form contains 48,502 base-pairs. Open reading frames were identified and, where possible, ascribed to genes by comparing with the previously determined genetic map. The reading frames for 46 genes were clearly identified, though in about 20 the position of the protein initiation site could not be rigorously established. Probable positions for the kil, cIII and lom genes are suggested but remain uncertain. There are about 20 other unidentified reading frames that may code for proteins.The genome is fairly compact with comparatively little non-coding DNA. In many cases the translation terminators and initiators overlap, particularly in the sequence A-T-G-A where the TGA terminates one gene and the ATG initiates the next. Such structures seem to be characterized by a purine-rich sequence, rather than by a specific “Shine and Dalgarno” sequence, before the initiator. In the whole of the left arm the codon CTA, which is normally read by a minor leucine tRNA, is absent. The distribution of other rare codons in the genes of the left arm suggests that they may have a controlling function on the relative amounts of the proteins produced.     

The role of template superhelicity in the initiation of bacteriophage lambda DNA replication.

The prepriming steps in the initiation of bacteriophage lambda DNA replication depend on the action of the lambda O and P proteins and on the DnaB helicase, single-stranded DNA binding protein (SSB), and DnaJ and DnaK heat shock proteins of the E. coli host. The binding of multiple copies of the lambda O protein to the phage replication origin (ori lambda) initiates the ordered assembly of a series of nucleoprotein structures that form at ori lambda prior to DNA unwinding, priming and DNA synthesis steps. Since the initiation of lambda DNA replication is known to occur only on supercoiled templates in vivo and in vitro, we examined how the early steps in lambda DNA replication are influenced by superhelical tension. All initiation complexes formed prior to helicase-mediated DNA-unwinding form with high efficiency on relaxed ori lambda DNA. Nonetheless, the DNA templates in these structures must be negatively supertwisted before they can be replicated. Once DNA helicase unwinding is initiated at ori lambda, however, later steps in lambda DNA replication proceed efficiently in the absence of superhelical tension. We conclude that supercoiling is required during the initiation of lambda DNA replication to facilitate entry of a DNA helicase, presumably the DnaB protein, between the DNA strands.     

Host virus interactions in the initiation of bacteriophage lambda DNA replication. Recruitment of Escherichia coli DnaB helicase by lambda P replication protein

The bacteriophage lambda P protein promoters replication of the phage chromosome by recruiting a key component of the cellular replication machinery to the viral origin. Specifically, P protein delivers one or more molecules of Escherichia coli DnaB helicase to a nucleoprotein structure formed by the lambda O initiator at the lambda replication origin. Using purified proteins, we have examined the features of the pivotal host virus interaction between P and DnaB. These two proteins interact in vitro to form a P.DnaB protein complex that can be resolved by sedimentation or by chromatography on DEAE-cellulose from the individual free proteins. The sedimentation coefficient of the P.DnaB complex, 13 S, suggests a size larger than that of free DnaB hexamer (Mr = 313,600). The P.DnaB complex isolated by glycerol gradient sedimentation contains approximately three protomers of P/DnaB hexamer, consistent with a molecular weight of 393,000. The isolated P.DnaB complex functions in vitro in the initiation of lambda DNA replication. Interaction of P with DnaB strongly suppressed both the intrinsic DNA-dependent ATPase activity of DnaB, as well as the capacity of DnaB to assist E. coli primase in the general priming reaction. Formation of a P.DnaB protein complex also blocked DnaB from functioning in the initiation of E. coli DNA replication in vitro. The physical and functional properties of lambda P protein suggest that it is a viral analogue of the E. coli DnaC replication protein. Like P, DnaC also binds to DnaB (Wickner, S., and Hurwitz, J. (1975) Proc. Natl. Acad. Sci. U. S. A. 72, 921-925), but unlike P, DnaC stimulates DnaB-mediated general priming. When viral P and bacterial DnaC replication proteins were placed in direct competition with one another for binding to DnaB, the viral protein was clearly predominant. For example, a 5-fold molar excess of DnaC protein only partially reversed the inhibitory effect of P on general priming. Furthermore, when a preformed DnaC.DnaB protein complex was incubated briefly with P protein, it was readily converted into a P.DnaB protein complex and the bulk of the bound DnaC was released as free protein. It is likely that the capacity of the lambda P protein to outcompete the analogous host protein for binding to the bacterial DnaB helicase is the critical molecular event enabling infecting phage to recruit cellular replication proteins required for initiation of DNA synthesis at the viral origin.     

Isolation and analysis of Escherichia coli mutants that allow increased replication of bacteriophage lambda.

Escherichia coli mutants were isolated that supported the growth of a lambda Ots and, in at least one case, a lambda Bts phage at the normally nonpermissive temperature of 39 degrees C. In one such strain, Ots and Bts suppression ability appeared to be a function of the guaB gene. Ots suppression by the mutant guaB strain was prevented if high levels of guanine or xanthine were present in the medium. No other base had any effect on Ots suppression in this strain. Other strains carrying spontaneous mutations resulting in guanine or xanthine auxotrophy (guaA or guaB lesions, respectively) all allowed lambda Ots replication at 39 degrees C; Ots suppression in these strains was also abolished by addition of guanine to the medium. Thus, reduced intracellular guanine levels resulting from guaA or guaB mutations appeared to suppress the inability of lambda Ots and, at least in some cases, Bts bacteriophage to form plaques at 39 degrees C. In burst size experiments, a guaB mutant produced a larger phage yield per infected cell of both lambda Ots and lambda O+ phage at 39 degrees C than did a similar guaB+ strain. It appeared that a lower-than-normal level of guanine (or a guanine derivative) in these cells may permit unusually efficient lambda replication. The fact that O+ and lambda Ots bursts in the guaB mutant were reduced significantly by addition of exogenous guanine to the medium is consistent with this suggestion. Another strain that suppresses the Ots allele has no known auxotrophic requirements, and suppression in this strain was unaffected by addition of guanine to the medium; however, addition of cytidine to the medium specifically eliminated Ots suppression in this strain. The mutation responsible for allowing Ots replication in this strain is unknown.     

The rolling-circle replicative structure of a bacteriophage lambda DNA

Intermediates of λ DNA replication in the second half of the latent period have been isolated and investigated in the electron microscope. The isolated replicative structures were predominantly single-branched “$\text{rolling-circle}$” replicative forms. The long linear tails (concatemers) may be the precursor of mature λ DNA.