The dnaB-dnaC replication protein complex of Escherichia coli. II. Role of the complex in mobilizing dnaB functions

The dnaC protein of Escherichia coli, by forming a complex with the dnaB protein, facilitates the interactions with single-stranded DNA that enable dnaB to perform its ATPase, helicase, and priming functions. Within the dnaB-dnaC complex, dnaB appears to be inactive but becomes active upon the ATP-dependent release of dnaC from the complex. With adenosine 5'-(gamma-thio)triphosphate substituted for ATP, the dnaB-dnaC complex does not direct dnaB to its targeted actions. Excess dnaC inhibits dna beta actions and augments the ATP gamma S effects. In the dnaA protein-driven initiation of duplex chromosome replication, dnaB is introduced for its essential helicase role via the dnaB-dnaC complex. Similarly, when the dnaA protein interacts nonspecifically with single-stranded DNA, the dnaB-dnaC complex is essential to introduce dnaB for its role in primer formation by primase.     

The ABC-primosome. A novel priming system employing dnaA, dnaB, dnaC, and primase on a hairpin containing a dnaA box sequence

A priming mechanism requiring dnaA, dnaB, and dnaC proteins operates on a single-stranded DNA coated with single-stranded DNA-binding protein. This novel priming, referred to as "ABC-priming," requires a specific hairpin structure whose stem carries a dnaA protein recognition sequence (dnaA box). In conjunction with primase and DNA polymerase III holoenzyme, ABC-priming can efficiently convert single-stranded DNA into the duplex replicative form. dnaA protein specifically recognizes and binds the single-stranded hairpin and permits the loading of dnaB protein to form a prepriming protein complex containing dnaA and dnaB proteins which can be physically isolated. ABC-priming can replace phi X174 type priming on the lagging strand template of pBR322 in vitro, suggesting a possible function of ABC-priming for the lagging strand synthesis and duplex unwinding. Similar to the phi X174 type priming, a mobile nature of ABC-priming was indicated by helicase activity in the presence of ATP of a prepriming protein complex formed at the hairpin. The implications of this novel priming in initiation of replication at the chromosomal origin, oriC, and in its contribution to the replication fork are discussed.     

The dnaA initiator protein binds separate domains in the replication origin of Escherichia coli

After binding to its four 9-mer boxes in the 245-base pair Escherichia coli replication origin (oriC), dnaA protein effects the formation of an "open complex" in an adjacent region made up of three 13-mers (Bramhill, D., and Kornberg, A. (1988) Cell 52, 743-755). This open complex formation requires the ATP form of dnaA protein assisted by HU protein (Sekimizu, K., Bramhill, D., and Kornberg, A. (1987) Cell 50, 259-265). We now provide direct evidence that dnaA protein binds the 13-mers, sequences that bear no resemblance to the 9-mer box. The evidence is (i) displacement of dnaA protein from the open complex by oriC or by a synthetic oligonucleotide containing the 13-mers, but not by a mutant of oriC lacking the 13-mers; (ii) filter binding of the synthetic (13-mer) oligonucleotide by dnaA protein; and (iii) requirement for the ATP form of dnaA protein assisted by HU protein for temperature-dependent binding to the 13-mer region. Controlled proteolysis of dnaA protein results in a prompt loss of oriC binding; an NH2-terminal 30-kDa peptide contains the domain that binds ATP and phospholipids known to destabilize the tightly bound ATP.     

In vitro assembly of a prepriming complex at the origin of the Escherichia coli chromosome

During initiation of DNA replication of plasmids containing the origin of the Escherichia coli chromosome (oriC), the proteins dnaA, dnaB, and dnaC interact and assemble a complex at oriC. The complex is larger and more asymmetric than that formed by dnaA protein and embraces an extra 50 base pairs at the left side of the minimal oriC sequence. Both dnaA and dnaB proteins have been identified in the complex by electron microscopy and antibody binding; dnaC protein was not detected. HU protein, which stimulates the activity of the initiation reaction, was often present. Entry of dnaB protein required dnaA and dnaC proteins and a supercoiled template. Thus, a complex structure, involving multiple proteins and a large region of DNA, must be formed at the origin to prepare the template for priming and replication.     

Helicase action of dnaB protein during replication from the Escherichia coli chromosomal origin in vitro

Initiation of bidirectional replication from the origin of the Escherichia coli chromosome (oriC) proceeds through stages in which the components of the two replication forks are assembled. From a complex containing proteins dnaA, dnaB, and dnaC bound at oriC, the dnaB helicase moves in both directions to unwind the duplex. In the absence of replication, this unwinding generates a bubble at oriC coated by single strand binding protein. Addition of gyrase allows unwinding to proceed extensively in both directions from oriC at 60 base pairs/s/fork at 37 degrees C. This rate is sharply dependent on temperature and also stimulated by both primase and DNA polymerase III holoenzyme, even in the absence of DNA synthesis. Primer and DNA synthesis are efficient when coupled to template unwinding. DNA synthesis proceeds bidirectionally from oriC at a rate limited by unwinding. With extensive unwinding preceding DNA synthesis, initiations are not limited to oriC.     

A novel protein binds a key origin sequence to block replication of an E. coli minichromosome

A sequence of three tandem repeats of a 13-mer in the replication origin (oriC) of E. coli is the highly conserved site of opening of the duplex for initiation of DNA synthesis. A protein that binds this sequence has been discovered in E. coli and purified to homogeneity. This novel 33 kd polypeptide behaves as a dimer. Binding to the 13-mers is specific and limited to this region. At a ratio of 10-20 monomers per oriC plasmid, the binding blocks initiation by preventing the opening of the 13-mer region by dnaA protein. Once the 13-mers are opened by dnaA protein action, the 33 kd protein is without effect on the subsequent stages of replication. The specificity of binding and profound inhibitory effect suggest a regulatory role for this protein at an early stage of chromosome initiation.     

Fate of the DnaA initiator protein in replication at the origin of the Escherichia coli chromosome in vitro

The dnaA initiator protein binds specific sequences in the 245-base pair Escherichia coli origin (oriC) to form a series of complexes which eventually are opened enough to admit dnaB helicase into a prepriming complex (Bramhill, D., and Kornberg, A. (1988) Cell 52, 743-755). ATP bound to a high-affinity site on dnaA protein is the preferred form for one or more of the early stages, but an elevated level of ATP is needed for a later stage; further evidence for a low-affinity site has now been obtained. We find that at limiting levels of dnaA protein only the ATP form produces an active initial complex; neither the ADP nor the non-nucleotide forms are effective. Augmentation of the activity of a limiting level of the ATP form of dnaA protein by the otherwise inert ADP form implies that at some stage of initiation both forms are active. The dnaA protein is essential up to the stage of forming the prepriming complex; upon salt dissociation from an oriC complex, the protein can be recycled to function at a fresh origin. Distinctive conformational states of the ATP form are implied by interactions with oriC DNA, by the influence of phospholipids on accelerating nucleotide exchange, and by the susceptibility to proteolytic cleavage.     

Polar arrest of the simian virus 40 tumor antigen-mediated replication fork movement in vitro by the tus protein-terB complex of Escherichia coli.

The effect of the tus protein-terB sequence complex of Escherichia coli on the movement of the SV40 large tumor antigen (T antigen)-mediated replication fork during SV40 DNA replication in vitro has been examined. In the monopolymerase and dipolymerase systems, the tus protein-terB complex efficiently blocked the replication fork movement in a polar fashion, as observed in prokaryotic replication systems. With crude cytosolic extracts of HeLa cells, the same polarity of fork arrest was observed, but the block of replication fork movement was inefficient. These results indicate that the structure of the prokaryotic tus protein-terB complex allows it to block replication fork movement in an orientation-dependent manner. We also show that the tus protein-terB complex blocks the 3'----5' helicase action of T antigen in a polar fashion, using substrates comprised of single-stranded M13 DNA with either a 52-base pair (bp) or 29-bp duplex containing the terB sequence. The tus protein-terB complex formed on the 52-bp duplex was less effective than the complex formed on the 29-bp duplex in blocking the helicase action of T antigen. With the 52-bp duplex substrate, T antigen movement was only partially (30%) blocked by the tus protein-terB sequence complex in the active orientation, whereas the E. coli dnaB helicase moving 5'----3' was blocked more than 90% by the complex in the active orientation. However, with the shorter 29-bp duplex substrate, the complex blocked the T antigen helicase activity about 75%, whereas the dnaB helicase activity was completely blocked. Altogether, these results suggest that the T antigen helicase activity, when coupled to DNA replication, is more susceptible to arrest by the tus protein-terB complex than the T antigen functioning as a helicase alone.     

The Escherichia coli dnaB replication protein is a DNA helicase

Genetic and biochemical analyses indicate that the Escherichia coli dnaB replication protein functions in the propagation of replication forks in the bacterial chromosome. We have found that the dnaB protein is a DNA helicase that is capable of unwinding extensive stretches of double-stranded DNA. We constructed a partially duplex DNA substrate, containing two preformed forks of single-stranded DNA, which was used to characterize this helicase activity. The dnaB helicase depends on the presence of a hydrolyzable ribonucleoside triphosphate, is maximally stimulated by a combination of E. coli single-stranded DNA-binding protein and E. coli primase, is inhibited by antibody directed against dnaB protein, and is inhibited by prior coating of the single-stranded regions of the helicase substrate with the E. coli single-stranded DNA-binding protein. It was determined that the dnaB protein moves 5' to 3' along single-stranded DNA, apparently in a processive fashion. To invade the duplex portion of the helicase substrate, the dnaB protein requires a 3'-terminal extension of single-stranded DNA in the strand to which it is not bound. Under optimal conditions at 30 degrees C, greater than 1 kilobase pair of duplex DNA can be unwound within 30 s. Based on these findings and other available data, we propose that the dnaB protein is the primary replicative helicase of E. coli and that it actively and processively migrates along the lagging strand template, serving both to unwind the DNA duplex in advance of the leading strand and to potentiate synthesis by the bacterial primase of RNA primers for the nascent (Okazaki) fragments of the lagging strand.     

Localized melting of duplex DNA by Cdc6/Orc1 at the DNA replication origin in the hyperthermophilic archaeon Pyrococcus furiosus

The initiation step is a key process to regulate the frequency of DNA replication. Although recent studies in Archaea defined the origin of DNA replication (oriC) and the Cdc6/Orc1 homolog as an origin recognition protein, the location and mechanism of duplex opening have remained unclear. We have found that Cdc6/Orc1 binds to oriC and unwinds duplex DNA in the hyperthermophilic archaeon Pyrococcus furiosus, by means of a P1 endonuclease assay. A primer extension analysis further revealed that this localized unwinding occurs in the oriC region at a specific site, which is 12-bp long and rich in adenine and thymine. This site is different from the predicted duplex unwinding element (DUE) that we reported previously. We also discovered that Cdc6/Orc1 induces topological changes in supercoiled oriC DNA, and that this process is dependent on the AAA+ domain. These results indicate that topological alterations of oriC DNA by Cdc6/Orc1 introduce a single-stranded region at the 12-mer site, that could possibly serve as an entry point for Mcm helicase.     

Interactions of DNA replication factors in vivo as detected by introduction of suppressor alleles of dnaA into other temperature-sensitive dna mutants.

Suppressor mutations located within dnaA can suppress the temperature sensitivity of a dnaZ polymerization mutant, indicating in vivo interaction of the products of these genes. The suppressor allele of dnaA [designated dnaA(SUZ, Cs)] could not be introduced, even at the permissive temperature, by transduction into temperature-sensitive (Ts) dnaC or dnaG recipients; it was transduced into dnaB(Ts) and dnaE(Ts) strains but at very low frequency. Recipient cells which were dnaA+ dnaE(Ts) were killed by the incoming dnaA(SUZ, Cs) allele, and it is presumed that combinations of dnaA(SUZ, Cs) with dnaB(Ts), dnaC(Ts), or dnaG(Ts) are lethal also. In one specific case, the lethality required the presence of three alleles: the incoming dnaA suppressor mutation, the resident dnaA+ gene, and the dnaB(Ts) gene. This was shown by the fact that dnaB(Ts) could readily be introduced into a dnaA(SUZ, Cs) dnaB+ recipient. That is, in the absence of dnaA+, the dnaA suppressor and dnaB(Ts) double mutant was stable. One model to explain these results proposes that the dnaA protein functions not only in initiation but also in the replication complex which contains multiple copies of dnaA and other replication factors.     

ATP activates dnaA protein in initiating replication of plasmids bearing the origin of the E. coli chromosome

ATP is bound to dnaA protein with high affinity (KD = 0.03 microM) and hydrolyzed slowly to ADP in the presence of DNA. ADP is also bound tightly to dnaA protein and exchanges with ATP very slowly. The ATP form is active in replication; the ADP form is not. A unique conformation of oriC, formed in an early initiation stage, depends on dnaA protein being in the ATP form. The subsequent entry of dnaB protein to form a prepriming complex also requires ATP binding and is blocked by bound ADP. Inasmuch as hydrolysis of ATP is far slower than these initiation reactions and since the poorly hydrolyzable analogue ATP gamma S can replace ATP, the ATP function appears to be allosteric. The extraordinary affinity of ATP for dnaA protein, its slow hydrolysis to ADP, the profound inhibition of dnaA functions by ADP, and the very slow exchange of ADP all point to a possible regulatory role for these nucleotides in the cell cycle.     

Opening of the replication origin of Escherichia coli by DnaA protein with protein HU or IHF.

Opening of the three tandem repeats of a 13-mer in the replication origin (oriC) of Escherichia coli is a prime event in the replication in vitro of minichromosomes (Bramhill, D., and Kornberg, A. (1988) Cell 54, 915-918). DnaA, the initiator protein, requires protein HU or IHF, along with a millimolar level of ATP and negative superhelical density in the plasmid to open this region. The extent of opening, as judged by cleavage by a single-strand-specific endonuclease (i.e. P1 nuclease), correlated closely with replication of the oriC plasmid. In an initial complex, preceding opening of the 13-mers, the footprint of DnaA protein bound by ATP covered its four 9-mer recognition sequences. The footprint of the nucleotide-free form of the protein, by contrast, was more extensive and thus, less specific.     

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.     

Extensive unwinding of the plasmid template during staged enzymatic initiation of DNA replication from the origin of the Escherichia coli chromosome

Early in the staged initiation of enzymatic replication of plasmids containing the unique origin of the E. coli chromosome (oriC), the plasmid is converted to a new topological form which is highly underwound, two to 15 times more than native supercoiled DNA. The underwinding reaction precedes priming of DNA synthesis and follows an initial complex formation, requiring ATP and proteins dnaA, dnaB, and dnaC; underwinding depends on the further addition of gyrase and SSB. DnaB protein as a helicase and gyrase as a topoisomerase drive the underwinding with the energy of ATP hydrolysis. The underwound template, extensively single-stranded and complexed with proteins, is an active form for priming by primase and elongation by DNA polymerase III holoenzyme.     

Suppression of tif-mediated induction of SOS functions in Escherichia coli by an altered dnaB protein.

The tif-1 mutation in the Escherichia coli recA gene is known to cause induction of the various "SOS" functions at high temperature, including massive synthesis of the recA protein, lethal filamentation, elevated mutagenesis, and, in lambda lysogens, induction of prophage. It is shown here that the deoxyribonucleic acid initiation mutation dnaB252 suppresses all these manifestations of tif expression. Induction of lambda by ultraviolet irradiation, however, is not affected by the dnaB252 mutation. No similar suppression of tif is observed with other dnaB mutations affecting deoxyribonucleic acid elongation or with other deoxyribonucleic acid initiation mutations at the dnaA and dnaC loci. The fact that an alteration of the dnaB protein specifically suppresses tif-mediated SOS induction implies a role of the replication apparatus in this process, as has been suggested for ultraviolet induction. The induction of lambda is known to proceed via repressor cleavage, presumably promoted by an activated (protease) form of the recA protein. Since lambda induction is normal after ultraviolet irradiation of the tif-1 dnaB252(lambda) strain, tif-mediated induction in this strain may be blocked in a tif-specific step leading to activation of the recA (tif) protein. It is possible that the recA (tif) mutant protein may be directly involved in the replication complex in processes leading to this activation.     

Replication initiation at the Escherichia coli chromosomal origin

To initiate DNA replication, DnaA recognizes and binds to specific sequences within the Escherichia coli chromosomal origin (oriC), and then unwinds a region within oriC. Next, DnaA interacts with DnaB helicase in loading the DnaB-DnaC complex on each separated strand. Primer formation by primase (DnaG) induces the dissociation of DnaC from DnaB, which involves the hydrolysis of ATP bound to DnaC. Recent evidence indicates that DnaC acts as a checkpoint in the transition from initiation to the elongation stage of DNA replication. Freed from DnaC, DnaB helicase unwinds the parental duplex DNA while interacting the cellular replicase, DNA polymerase III holoenzyme, and primase as it intermittently forms primers that are extended by the replicase in duplicating the chromosome.