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.     

Deletion analysis of the DNA sequence required for the in vitro initiation of replication of bacteriophage lambda

Supercoiled DNA containing the replication origin of bacteriophage lambda can be replicated in vitro. This reaction requires purified lambda O and P replication proteins and a partially purified mixture of Escherichia coli proteins (Tsurimoto, T., and Matsubara, K. (1982) Proc. Natl. Acad. Sci. U.S.A. 79, 7639-7643; Wold, M. S., Mallory, J.B., Roberts, J. D., LeBowitz, J. H., and McMacken, R. (1982) Proc. Natl. Acad. Sci. U.S.A. 79, 6176-6180). The lambda origin region has four repeats of a 19-base pair sequence to which O protein binds. To the right of these sites on the lambda map is a 40-base pair region that is rich in adenine and thymine, followed by a 28-base pair palindromic sequence. To define more precisely the boundaries of the lambda origin, we cloned a 358-base pair piece of lambda DNA containing the origin region into M13mp8 in both orientations. In vitro replication of RF I DNAs prepared from cells infected with these two M13 ori lambda phage was dependent on lambda O and P proteins and a crude protein fraction from uninfected E. coli; with these conditions there was no replication of M13mp8 RF I DNA. We made deletions from the left and the right ends of the lambda origin DNA and determined the deletion end points by DNA sequencing. We have tested RF I DNAs prepared from cells infected with phage carrying ori lambda deletions for their ability to function as templates for O- and P-dependent replication in vitro. Our results show that lambda DNA between nucleotide positions 39072 and 39160 is required for efficient O- and P-dependent replication. This 89-base pair piece of DNA includes only two of the four 19-base pair O protein-binding sites (the two right-most) and the adjoining adenine- and thymine-rich region to the right of the O-binding sites.     

Specialized nucleoprotein structures at the origin of replication of bacteriophage lambda. Protein association and disassociation reactions responsible for localized initiation of replication

Binding of the O protein of phage lambda to the replication origin (ori lambda) results in the formation of an organized nucleoprotein structure termed the O-some. The O-some serves to localize and initiate a six-protein sequential reaction that provides for localized unwinding of the origin region, the critical prepriming step for precise initiation of DNA replication. By the use of electron microscopy of gold-tagged antibody complexes, we have defined four stages of protein association and dissociation reactions that are involved in the prepriming pathway. First, as defined previously, O protein binds to multiple DNA sites and self-associates to form the O-some. Second, lambda P and host DnaB proteins add to the O-some to generate an O.P.DnaB.ori lambda complex. Addition of the DnaK and DnaJ proteins yields a third stage complex containing DnaK, DnaJ, O, P, and DnaB. With the addition of ATP and single-strand binding protein (SSB), the P protein is largely removed, and the DnaB acts as a helicase to generate locally unwound, SSB-coated single strand DNA. Thus, the initiation of lambda DNA replication requires ordered assembly and partial disassembly of specialized nucleoprotein structures. The disassembly activity of DnaK and DnaJ may be their general role in the heat shock response.     

Role of the Escherichia coli grpE heat shock protein in the initiation of bacteriophage lambda DNA replication.

Initiation of replication of lambda DNA requires assembly of the proper nucleoprotein complex consisting of the lambda origin of replication-lambda O-lambda P-dnaB proteins. The dnaJ, dnaK and grpE heat shock proteins destabilize the lambda P-dnaB interaction in this complex permitting dnaB helicase to unwind lambda DNA near ori lambda sequence. First step of this disassembling reaction is the binding of dnaK protein to lambda P protein. In this report we examined the influence of dnaJ and grpE proteins on stability of the lambda P-dnaK complex. Our results show that grpE alone dissociates this complex, but both grpE and dnaJ together do not. These results suggest that, in the presence of grpE protein, dnaK protein has a higher affinity for lambda P protein complexed with dnaJ protein than in the situation where grpE protein is not used.     

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.     

RNA-primed initiation sites of DNA replication in the origin region of bacteriophage lambda genome.

Using DNA molecules synthesized in the early stage of lambda phage infection, deoxynucleotides at the transition sites from primer RNA to DNA synthesis have been mapped in the 1.5 kbase area of the lambda phage genome containing the genetically defined replication origin (ori lambda). Sites in the 1-strand (the polarity of the 1-strand is 5' to 3' from the left to the right direction of the lambda phage genetic map) were distributed both inside and outside of the ori lambda, whereas the sites in the r-strand (the strand in the opposite polarity) were mainly distributed more than three hundred nucleotides apart from the ori lambda to the right. A CPuPu sequence was found at -12 to -10 region of transition sites of the r- and the 1-strands in the frequency of 80% and 70%, respectively, and over 60% of the CPuPu sequences were CAG. Properties of the transition sites are discussed in relation to the primer synthesis.     

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.     

The nucleoprotein is required for efficient coronavirus genome replication

The construction of a set of transmissible gastroenteritis coronavirus (TGEV)-derived replicons as bacterial artificial chromosomes is reported. These replicons were generated by sequential deletion of nonessential genes for virus replication, using a modified TGEV full-length cDNA clone containing unique restriction sites between each pair of consecutive genes. Efficient activity of TGEV replicons was associated with the presence of the nucleoprotein provided either in cis or in trans. TGEV replicons were functional in several cell lines, including the human cell line 293T, in which no or very low cytopathic effect was observed, and expressed high amounts of heterologous protein.     

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.     

Escherichia coli dnaA initiation function is required for replication of plasmids derived from coliphage lambda

The dnaA gene function, indispensable for the initiation of Escherichia coli replication from oriC is not essential for the growth of phage lambda. The in-vitro replication of plasmids derived from phage lambda does not seem to require DnaA protein either. However, we present evidence that in vivo the normal replication of lambda plasmids is dnaA-dependent. After inactivating the dnaA gene function, half of the plasmid molecules may enter a single round of replication. Rifampicin sensitivity of this abortive, as well as normal, replication indicates involvement of RNA polymerase. The rifampicin resistance of the normal replication of lambda plasmids in E. coli carrying the dnaAts46 or dnaAts5, but not the dnaAts204 allele at 30 degrees C implies the interaction of DnaA protein and RNA polymerase in this process. We propose that DnaA protein co-operates with RNA polymerase in the initiation of replication at ori lambda. The dispensability of DnaA in the growth of phage lambda and in lambda plasmid replication in vitro is discussed.     

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     

Xenopus CDC7/DRF1 complex is required for the initiation of DNA replication

The Cdc7 kinase is essential for the initiation of DNA replication in eukaryotes. Two regulatory subunits of the Xenopus Cdc7 kinase have been identified: XDbf4 and XDrf1. In this study we determined the expression pattern of XDbf4 and XDrf1 and examined their involvement in DNA replication. We show that XDrf1 expression is restricted to oogenesis and early embryos, whereas XDbf4 is expressed throughout development. Immunodepletion from Xenopus egg extracts indicated that both proteins are only found in complexes with XCdc7 and there is a 5-fold molar excess of the XCdc7/Drf1 over SCdc7/Dbf4 complexes. Both complexes exhibit kinase activity and are differentially phosphorylated during the cell cycle. Depletion of the XCdc7/Drf1 from egg extracts inhibited DNA replication, whereas depletion of XCdc7/Dbf4 had little effect. Chromatin binding studies indicated that XCdc7/Drf1 is required for pre-replication complex activation but not their assembly. XCdc7/Dbf4 complexes bound to the chromatin in two steps: the first step was independent of pre-replication complex assembly and the second step was dependent on pre-replication complex activation. By contrast, binding of XCdc7/Drf1 complexes was entirely dependent on pre-replication complex assembly. Finally, we present evidence that the association of the two complexes on the chromatin is not regulated by ATR checkpoint pathways that result from DNA replication blocks. These data suggest that Cdc7/Drf1 but not Cdc7/Dbf4 complexes support the initiation of DNA replication in Xenopus egg extracts and during early embryonic development.     

DNA replication initiation is required for mid-cell positioning of FtsZ rings in Caulobacter crescentus

Polymerization of the GTPase FtsZ to form a structure called the Z-ring is the earliest known step in bacterial cell division. Mid-cell Z-ring assembly coincides with the beginning of the replication cycle in the differentiating bacterium Caulobacter crescentus. Z-ring disassembly occurs at the end of the division cycle, resulting in the complete degradation of FtsZ from both stalked and swarmer progeny cells. New Z-rings can only form in the replicative stalked cell. Conditional mutants in DNA replication were used to determine what role DNA replication events play in the process of Z-ring assembly at different stages in the cell cycle. Z-ring assembly occurred even when early stages of DNA replication were blocked; however, the Z-rings were localized at a subpolar region of the cell. Z-rings only assembled at the proper mid-cell location if DNA replication had initiated. Z-ring assembly coincided with areas containing little or no DNA, and Z-rings could not form over an unreplicated chromosome. Overexpressed FtsZ in the absence of DNA replication did not stimulate productive mid-cell Z-ring assembly but, instead, caused the ends of cells to constrict over an extended area away from the nucleoid. These results indicate that the state of chromosome replication is a major determinant of Z-ring localization in Caulobacter.     

Defining cosQ, the site required for termination of bacteriophage lambda DNA packaging

Bacteriophage lambda is a double-stranded DNA virus that processes concatemeric DNA into virion chromosomes by cutting at specific recognition sites termed cos. A cos is composed of three subsites: cosN, the nicking site; cosB, required for packaging initiation; and cosQ, required for termination of chromosome packaging. During packaging termination, nicking of the bottom strand of cosN depends on cosQ, suggesting that cosQ is needed to deliver terminase to the bottom strand of cosN to carry out nicking. In the present work, saturation mutagenesis showed that a 7-bp segment comprises cosQ. A proposal that cosQ function requires an optimal sequence match between cosQ and cosNR, the right cosN half-site, was tested by constructing double cosQ mutants; the behavior of the double mutants was inconsistent with the proposal. Substitutions in the 17-bp region between cosQ and cosN resulted in no major defects in chromosome packaging. Insertional mutagenesis indicated that proper spacing between cosQ and cosN is required. The lethality of integral helical insertions eliminated a model in which DNA looping enables cosQ to deliver a gpA protomer for nicking at cosN. The 7 bp of cosQ coincide exactly with the recognition sequence for the Escherichia coli restriction endonuclease, EcoO109I.     

Formation of oligomeric structures from plasmid DNA carrying cos lambda that is packaged into bacteriophage lambda heads.

Plasmids that carry cos lambda, the region necessary for lambda phage packaging and that are as small as four kilobases in size can be packaged into lambda phage heads in head-to-tail tandem oligomeric structures. Multimeric oligomers as large as undecamers have been detected. Oligomer formation depends upon the products of red and gam of lambda, and the general recombination occurs between different plasmids that share homologous DNA regions. The packaging efficiency of plasmids depends on its copy number in cells and its genome size. Upon injection into a cell, the DNA establishes itself as a plasmid in a tandem structure. When such a plasmid in a high oligomeric structure is used as the source of packaging DNA, the packaging efficiency of the plasmids is elevated. The oligomers are stable in recA cells, whereas they drift toward lower oligomers in recA+ cells.     

The form of the DNA substrate required for excisive recombination of bacteriophage lambda.

We have studied the excision reaction of bacteriophage lambda, both in vivo and in vitro, using as a substrate a λatt²(L × R) phage carrying both the right and left-hand prophage attachment sites. Int and Xis are provided by induction of the heat-inducible defective prophage, λc1857 ΔH1. After a brief induction (5 min) of these cells, excisive recombination is blocked in the presence of the DNA gyrase inhibitor, coumermycin. However, after a longer induction (greater than 30 min) excisive recombination occurs efficiently under conditions where λ integrative recombination is inhibited by coumermycin. In such extensively induced coumermycin-treated cells, infecting λatt²(L × R) DNA is not supercoiled, and recombinants are found among the relaxed covalently closed circular DNA.In vitro, starting with a hydrogen-bonded λatt² DNA substrate, excision is insensitive to high concentrations of coumermycin and novobiocin. To study the DNA substrate requirements for excisive recombination in more detail, we have developed a restriction fragment assay for excisive recombination. With this assay, we demonstrate that supercoiled, hydrogen-bonded, and linear λatt² DNA molecules are all efficient substrates in the in vitro excision reaction. Spermidine is required but ATP and Mg²⁺ are not. We conclude that supercoiling is not an absolute requirement for site-specific recombination of λ.