EcoRI restriction endonuclease cleavage site map of bacteriophage P22DNA.

The F plasmid is able to co-transfer (mobilize) the small, chimeric R plasmid pBR322 during conjugation only at a very low frequency (Bolivar et al., 1977). Mobilization has been found here to be invariably (> 99%) associated with a structural alteration of pBR322. The alteration was shown, by restriction endonuclease analysis and electron microscopy, to be an insertion of the F attachment sequence λδ (2.8 to 8.5F). λδ is, therefore, an insertion sequence.     

The bacteriophage lambda O and P protein initiators promote the replication of single-stranded DNA.

A soluble enzyme system that specifically initiates lambda dv plasmid DNA replication at a bacteriophage lambda replication origin [Wold et al. (1982) Proc. Natl. Acad. Sci. USA 79, 6176-6180] is also capable of replicating the single-stranded circular chromosomes of phages M13 and phi X174 to a duplex form. This chain initiation on single-stranded templates is novel in that it is absolutely dependent on the lambda O and P protein chromosomal initiators and on several Escherichia coli proteins that are known to function in the replication of the lambda chromosome in vivo, including the host dnaB, dnaG (primase), dnaJ and dnaK replication proteins. Strand initiation occurs at multiple sites following an O and P protein-dependent pre-priming step in which the DNA is converted into an activated nucleoprotein complex containing the bacterial dnaB protein. We propose a scheme for the initiation of DNA synthesis on single-stranded templates in this enzyme system that may be relevant to strand initiation events that occur during replication of phage lambda in vivo.     

A restriction endonuclease cleavage map of bacteriophage P2

Summary A restriction endonuclease cleavage map of phage P2 was constructed. The enzymes used and, within parenthesis, the number of their cleavage sites on the P2 lg cc DNA molecule were: AvaI(3), BalI(1), BamI(3), BglII(3), HaeIII (more than 40; only three were mapped), HindIII(0), HpaI(10), KpnI(3), PstI(3), SalI(2) and SmaI(1). The EcoRI cleavage sites (3), as determined earlier, were used as reference points for this study. The DNAs of a variety of P2 mutants carrying chromosomal aberrations (del1, del2, del3, del6, vir22, vir37(2), vir79 and vir94) were also similarly examined.     

The role of bacteriophage T7 gene 2 protein in DNA replication.

The in vivo function of the gene 2 protein of bacteriophage T7 has been examined. The gene 2 protein appears to modulate the activity of the gene 3 endonuclease in order to prevent the premature degradation of any newly-formed DNA concatemers. This modulation is not however a direct interacton between the two proteins. In single-burst experiments rifamycin can substitute for the gene 2 protein, allowing formation of fast-sedimenting replicative DNA intermediates and progeny phage production. This suggests that the sole function of the gene 2 protein is inhibition of the host RNA polymerase and that the latter enzyme directs or promotes the endonucleolytic action of the gene 3 protein.     

The bacteriophage lambda DNA replication protein P inhibits the oriC DNA- and ATP-binding functions of the DNA replication initiator protein DnaA of Escherichia coli

Under the condition of expression of lambda P protein at lethal level, the oriC DNA-binding activity is significantly affected in wild-type E. coli but not in the rpl mutant. In purified system, the lambda P protein inhibits the binding of both oriC DNA and ATP to the wild-type DnaA protein but not to the rpl DnaA protein. We conclude that the lambda P protein inhibits the binding of oriC DNA and ATP to the wild-type DnaA protein, which causes the inhibition of host DNA synthesis initiation that ultimately leads to bacterial death. A possible beneficial effect of this interaction of lambda P protein with E. coli DNA initiator protein DnaA for phage DNA replication has been proposed.     

In vitro replication of bacteriophage PRD1 DNA. Characterization of the protein-primed initiation site.

Bacteriophage PRD1 replicates its DNA by means of a protein-primed replication mechanism. Using single-stranded oligonucleotide templates carrying the sequence corresponding to the 25 first bases of the 3' end of PRD1 DNA, and Mg2+ as the activating metal ion of the phage DNA polymerase, we show that the fourth base from the 3' end of the template directs, by base complementarity, the dNMP to be linked to the phage terminal protein (TP) in the initiation reaction. This result suggests that phage PRD1 maintains its 3' end DNA sequences via a sliding-back mechanism. The single-stranded DNA templates could not be replicated by the PRD1 DNA polymerase, much in contrast to the natural TP-DNA. Nevertheless, the analysis of the transition products obtained with TP-DNA and origin-containing oligonucleotides suggests that sliding-back occurs stepwise, the fourth base being the directing position during the entire process.     

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.     

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.     

DNA of Bacillus subtilis bacteriophage SPP1: physical mapping and localization of the origin of replication.

The genome of Bacillus subtilis bacteriophage SPP1, a linear, 28.5-megadalton DNA duplex, was mapped by analysis with the restriction endonucleases endo R.Sal I, Sma I, Xba I, Bgl I, Bgl II, and EcoRI. The SPP1 genome, like that of the Salmonella typhimurium phage, P22, was found to be a terminally repetitious, circularly permuted molecule. 6-(p-Hydroxyphenylazo)uracil, a selective, reversible inhibitor of SPP1 DNA synthesis, was exploited to synchronize the initiation of genome replication and to selectively label the site of its initiation with radioactive thymidine. Restriction endonuclease analysis of the distribution of the label located the origin of replicative synthesis at an area approximately 0.2 genome length from one molecular terminus.     

Cnr protein, the negative regulator of bacteriophage P4 replication, stimulates specific DNA binding of its initiator protein alpha.

Bacteriophage P4 DNA replication depends upon the phage-encoded alpha protein, which has DNA helicase and DNA primase activity and can specifically bind to the replication origin (ori) and to the cis replicating region (crr). The P4 Cnr protein functions as a negative regulator of P4 replication, and P4 does not replicate in cells that overexpress cnr. We searched for P4 mutants that suppressed this phenotype (Cnr resistant [alpha cr]). Eight independent mutants that grew in the presence of high levels of Cnr were obtained. None of these can establish the plasmid state. Each of these mutations lies in the DNA binding domain of gp alpha that occupies the C terminus of the protein. Five different sequence changes were found: T675M, G732V (three times), G732W (twice), L733V, and L737V. A TrxA-Cnr fusion protein does not bind DNA by itself but stimulates the ori and crr binding abilities of alpha protein in vitro. The alpha cr mutant proteins were still able to bind specifically to ori or crr, but specific DNA binding was less stimulated by the TrxA-Cnr protein. We present evidence that Cnr protein interacts with the gp alpha domain that binds specifically to DNA and that gp(alpha)cr mutations impair this interaction. We hypothesize that gp alpha-Cnr interaction is essential for the control of P4 DNA replication.     

Studies on the cleavage of bacteriophage lambda DNA with EcoRI restriction endonuclease

The five EcoRI² restriction sites in bacteriophage lambda DNA have been mapped at 0.445, 0.543, 0.656, 0.810, and 0.931 fractional lengths from the left end of the DNA molecule. These positions were determined electron-microscopically by single-site cleavage of hydrogen-bonded circular λ DNA molecules and by cleavage of various DNA heteroduplexes between λ DNA and DNA from well defined λ mutants. The DNA lengths of the EcoRI fragments are in agreement with their electrophoretic mobility on agarose gels but are not in agreement with their mobilities on polyacrylamide gels. These positions are different from those previously published by Allet et al. (1973). Partial cleavage of pure λ DNA by addition of small amounts of EcoRI endonuclease does not lead to random cleavage between molecules. Also, the first site cleaved is not randomly distributed among the five sites within a molecule. The site nearest the right end is cleaved first about ten times more frequently than either of the two center sites.     

Characterization of a DNA binding protein of bacteriophage PRD1 involved in DNA replication.

Escherichia coli phage PRD1 protein P12, involved in PRD1 DNA replication in vivo, has been highly purified from E. coli cells harbouring a gene XII-containing plasmid. Protein P12 binds to single-stranded DNA as shown by gel retardation assays and nuclease protection experiments. Binding of protein P12 to single-stranded DNA increases about 14% the contour length of the DNA as revealed by electron microscopy. Binding to single-stranded DNA seems to be cooperative, and it is not sequence specific. Protein P12 also binds to double-stranded DNA although with an affinity 10 times lower than to single-stranded DNA. Using the in vitro phage phi 29 DNA replication system, it is shown that protein P12 stimulates the overall phi 29 DNA replication.     

Identification of the site of interruption in relaxed circles producing during bacteriophage lambda DNA circle replication.

The DNA that accumulates in the lambda infection restricted to the early (circular) stage of replication consists of approximately two-thirds covalently closed circles and one-third relaxed circles bearing a single interruption in either strand of the duplex. The latter molecules are presumed to be a unique class in that the interruption is not repairable by DNA polymerase and ligase. Preferential radioisotopic labeling of the region immediately adjacent to the interruption, followed by hybridization to sheared fragments of the lambda chromosome with varying guanine plus cytosine content, suggested that the nick resides at the position of the mature molecular ends of the lambda chromosome. Digestion of the labeled molecules with restriction enzymes and reconstruction experiments in which Hershey circles were generated by annealing of interrupted strands isolated from the relaxed circles support this interpretation. The results indicate that the relaxed circles consist of a population containing one interruption in either of the two strands of the duplex jointly representing the two "nicks" contained in Hershey circles (in which the cohesive ends are annealed). These molecules could result from the inability of the maturation function to make the required staggered endonucleolytic cuts when the DNA substrate is a monomeric circle rather than a multimeric linear molecule. Alternatively, this interruption could be the result of an endonucleolytic cutting event critical to DNA replication.     

Drosophila topoisomerase II double-strand DNA cleavage: analysis of DNA sequence homology at the cleavage site.

In order to study the sequence specificity of double-strand DNA cleavage by Drosophila topoisomerase II, we have mapped and sequenced 16 strong and 47 weak cleavage sites in the recombinant plasmid p pi 25.1. Analysis of the nucleotide and dinucleotide frequencies in the region near the site of phosphodiester bond breakage revealed a nonrandom distribution. The nucleotide frequencies observed would occur by chance with a probability less than 0.05. The consensus sequence we derived is 5'GT.A/TAY decrease ATT.AT..G 3', where a dot means no preferred nucleotide, Y is for pyrimidine, and the arrow shows the point of bond cleavage. On average, strong sites match the consensus better than weak sites.     

The DNA site utilized by bacteriophage P22 for initiation of DNA packaging

Virion proteins recognize their cognate nucleic acid for encapsidation into virions through recognition of a specific nucleotide sequence contained within that nucleic acid. Viruses like bacteriophage P22, which have partially circularly permuted, double-stranded virion DNAs, encapsidate DNA through processive series of packaging events in which DNA is recognized for packaging only once at the beginning of the series. Thus a single DNA recognition event programmes the encapsidation of multiple virion chromosomes. The protein product of P22 gene 3, a terminase component, is thought to be responsible for this recognition. The site on the P22 genome that is recognized by the gene 3 protein to initiate packaging series is called the pac site. We report here a strategy for assaying pac site activity in vivo, and the utilization of this system to identify and characterize the site genetically. It is an asymmetric site that spans 22 basepairs and is located near the centre of P22 gene 3.     

The replication of bacteriophage P4 DNA in vitro. Partial purification of the P4 alpha gene product

A soluble enzyme system has been prepared from a phage P4-infected Escherichia coli strain that supports the replication of exogenous, supercoiled P4 DNA. This DNA synthesis in vitro depends upon the four deoxyribonucleotides and ATP, but is enhanced about four- to fivefold by the presence of other ribonucleotides. E. coli DNA polymerase III holoenzyme, the E. coli single-strand DNA binding protein, and the partially purified P4 alpha gene product are required for replication in vitro. Rifamycin does not inhibit P4 replication in vitro. Since the P4 alpha gene codes for a rifamycin-resistant RNA polymerase (Barrett et al., 1983), and since P4 DNA replication is independent of the host primase (Bowden et al., 1975), we believe the alpha gene product is functioning as a P4-specific DNA primase.     

Host cell DNA chain initiation protein requirements for replication of bacteriophage G4 replicative-form DNA.

Bacteriophages G4ev1 and G4bs1 are simple temperature-resistant derivatives of wild-type G4 as demonstrated by restriction endonuclease analyses. The rate of replication of the duplex replicative-form DNA of these phages was normal in dnaB and dnaC mutants of the host, whereas the rate was markedly reduced in a dnaG host mutant at the restrictive temperature. We conclude that G4 duplex DNA replication requires the host cell dnaG protein, but not the dnaB and dnaC proteins. The reasons for the differences between our conclusions and those based on previously published data are documented and discussed.