Crystallization and preliminary crystallographic characterization of the origin-binding domain of the bacteriophage lambda O replication initiator.

The bacteriophage lambda O protein binds to the lambda replication origin (orilambda) and serves as the primary replication initiator for the viral genome. The binding energy derived from the binding of O to orilambda is thought to help drive DNA opening to facilitate initiation of DNA replication. Detailed understanding of this process is severely limited by the lack of high-resolution structures of O protein or of any lambdoid phage-encoded paralogs either with or without DNA. The production of crystals of the origin-binding domain of lambda O that diffract to 2.5 A is reported. Anomalous dispersion methods will be used to solve this structure.

Functional defects of the DnaK756 mutant chaperone of Escherichia coli indicate distinct roles for amino- and carboxyl-terminal residues in substrate and co-chaperone interaction and interdomain communication.

The first discovery of an Hsp70 chaperone gene was the isolation of an Escherichia coli mutant, dnaK756, which rendered the cells resistant to lytic infection with bacteriophage lambda. The DnaK756 mutant protein has since been used to establish many of the cellular roles and biochemical properties of DnaK. DnaK756 has three glycine-to-aspartate substitutions at residues 32, 455, and 468, which were reported to result in defects in intrinsic and GrpE-stimulated ATPase activities, substrate binding, stability of the substrate-binding domain, interdomain communication, and, consequently, defects in chaperone activity. To dissect the effects of the different amino acid substitutions in DnaK756, we analyzed two DnaK variants carrying only the amino-terminal (residue 32) or the two carboxyl-terminal (residues 455 and 468) substitutions. The amino-terminal substitution interfered with the GrpE-stimulated ATPase activity. The carboxyl-terminal mutations (i) affected stability and function of the substrate-binding domain, (ii) caused a 10-fold elevated ATP hydrolysis rate, but (iii) did not severely affect domain coupling. Surprisingly, DnaK chaperone activity was more severely compromised by the amino-terminal than by the carboxyl-terminal amino acid substitutions both in vivo and in vitro. In the in vitro refolding of denatured firefly luciferase, the defect of the DnaK variant carrying the amino-terminal substitution results from its inability to release, upon GrpE-mediated nucleotide exchange, bound luciferase in a folding competent state. Our results indicate that the DnaK-DnaJ-GrpE chaperone system can tolerate suboptimal substrate binding, whereas the tight kinetic control of substrate dissociation by GrpE is essential.

Recognition, targeting, and hydrolysis of the lambda O replication protein by the ClpP/ClpX protease.

It has previously been established that sequences at the C termini of polypeptide substrates are critical for efficient hydrolysis by the ClpP/ClpX ATP-dependent protease. We report for the bacteriophage lambda O replication protein, however, that N-terminal sequences play the most critical role in facilitating proteolysis by ClpP/ClpX. The N-terminal portion of lambda O is degraded at a rate comparable with that of wild type O protein, whereas the C-terminal domain of O is hydrolyzed at least 10-fold more slowly. Consistent with these results, deletion of the first 18 amino acids of lambda O blocks degradation of the N-terminal domain, whereas proteolysis of the O C-terminal domain is only slightly diminished as a result of deletion of the C-terminal 15 amino acids. We demonstrate that ClpX retains its capacity to bind to the N-terminal domain following removal of the first 18 amino acids of O. However, ClpX cannot efficiently promote the ATP-dependent binding of this truncated O polypeptide to ClpP, the catalytic subunit of the ClpP/ClpX protease. Based on our results with lambda O protein, we suggest that two distinct structural elements may be required in substrate polypeptides to enable efficient hydrolysis by the ClpP/ClpX protease: (i) a ClpX-binding site, which may be located remotely from substrate termini, and (ii) a proper N- or C-terminal sequence, whose exposure on the substrate surface may be induced by the binding of ClpX.

DnaJ dramatically stimulates ATP hydrolysis by DnaK: insight into targeting of Hsp70 proteins to polypeptide substrates.

Most, if not all, of the cellular functions of Hsp70 proteins require the assistance of a DnaJ homologue, which accelerates the weak intrinsic ATPase activity of Hsp70 and serves as a specificity factor by binding and targeting specific polypeptide substrates for Hsp70 action. We have used pre-steady-state kinetics to investigate the interaction of the Escherichia coli DnaJ and DnaK proteins, and the effects of DnaJ on the ATPase reaction of DnaK. DnaJ accelerates hydrolysis of ATP by DnaK to such an extent that ATP binding by DnaK becomes rate-limiting for hydrolysis. At high concentrations of DnaK under single-turnover conditions, the rate-limiting step is a first-order process, apparently a change of DnaK conformation, that accompanies ATP binding and proceeds at 12-15 min-1 at 25 degrees C and 1-1.5 min-1 at 5 degrees C. By prebinding ATP to DnaK and subsequently adding DnaJ, the effects of this slow step may be bypassed, and the maximal rate-enhancement of DnaJ on the hydrolysis step is approximately 15 000-fold at 5 degrees C. The interaction of DnaJ with DnaK.ATP is likely a rapid equilibrium relative to ATP hydrolysis, and is relatively weak, with a KD of approximately 20 microM at 5 degrees C, and weaker still at 25 degrees C. In the presence of saturating DnaJ, the maximal rate of ATP hydrolysis by DnaK is similar to previously reported rates for peptide release from DnaK.ATP. This suggests that when DnaK encounters a DnaJ-bound polypeptide or protein complex, a significant fraction of such events result in ATP hydrolysis by DnaK and concomitant capture of the polypeptide substrate in a tight complex with DnaK.ADP. Furthermore, a broadly applicable kinetic mechanism for DnaJ-mediated specificity of Hsp70 action arises from these observations, in which the specificity arises largely from the acceleration of the hydrolysis step itself, rather than by DnaJ-dependent modulation of the affinity of Hsp70 for substrate polypeptides.

Kinetic characterization of the ATPase cycle of the DnaK molecular chaperone.

DnaK, the prototype Hsp70 protein of Escherichia coli, functions as a molecular chaperone in protein folding and protein disassembly reactions through cycles of polypeptide binding and release that are coupled to its intrinsic ATPase activity. To further our understanding of these processes, we sought to obtain a quantitative description of the basic ATPase cycle of DnaK. To this end, we have performed steady-state and pre-steady-state kinetics experiments and have determined rate constants corresponding to individual steps in the DnaK ATPase cycle at 25 degrees C. Hydrolysis of ATP proceeds very slowly with a rate constant (khyd approximately 0.02 min-1) at least 10-fold smaller than the rate constant for any other first-order step in the forward reaction pathway. The ATP hydrolysis step has an activation energy of 26.2 +/- 0.4 kcal/mol and is rate limiting in the steady-state under typical in vitro conditions. ATP binds with unusual strength to DnaK, with a measured KD approximately 1 nM. ADP binds considerably less tightly than ATP and dissociates from DnaK with a koff of approximately 0.4 min-1 (compared with a koff of approximately 0.008 min-1 for ATP). However, in the presence of physiologically relevant concentrations of inorganic phosphate (Pi), the release of ADP from DnaK is greatly slowed, approximately to the rate of ATP hydrolysis. Under these conditions, the ADP-bound form of DnaK, the form that binds substrate polypeptides most tightly, was found to represent a significant fraction of the DnaK population. The slowing of ADP release by exogenous Pi is due to thermodynamic coupling of the binding of the two ligands, which produces a coupling energy of approximately 1.6 kcal/mol. This result implies that product release is not strictly ordered. In the absence of exogenous inorganic phosphate, Pi product, by virtue of its higher koff, is released prior to ADP. However, at physiological concentrations of inorganic phosphate, the alternate product release pathway, whereby ADP dissociates from a ternary DnaK.ADP.Pi complex, becomes more prominent.

Functional properties of replication fork assemblies established by the bacteriophage lambda O and P replication proteins.

We have used a set of bacteriophage lambda and Escherichia coli replication proteins to establish rolling circle DNA replication in vitro to permit characterization of the functional properties of lambda replication forks. We demonstrate that the lambda replication fork assembly synthesizes leading strand DNA chains at a physiological rate of 650-750 nucleotides/s at 30 degrees C. This rate is identical to the fork movement rate we obtained using a minimal protein system, composed solely of E. coli DnaB helicase and DNA polymerase III holoenzyme. Our data are consistent with the conclusion that these two key bacterial replication proteins constitute the basic functional unit of a lambda replication fork. A comparison of rolling circle DNA replication in the minimal and lambda replication systems indicated that DNA synthesis proceeded for more extensive periods in the lambda system and produced longer DNA chains, which averaged nearly 200 kilobases in length. The higher potency of the lambda replication system is believed to result from its capacity to mediate efficient reloading of DnaB helicase onto rolling circle replication products, thereby permitting reinitiation of DNA chain elongation following spontaneous termination events. E. coli single-stranded DNA-binding protein and primase individually stimulated rolling circle DNA replication, but they apparently act indirectly by blocking accumulation of inhibitory free single-stranded DNA product. Finally, in the course of this work, we discovered that E. coli DNA polymerase III holoenzyme is itself capable of carrying out significant strand displacement DNA synthesis at about 50 nucleotides/s when it is supplemented with E. coli single-stranded DNA-binding protein.

Cryptic single-stranded-DNA binding activities of the phage lambda P and Escherichia coli DnaC replication initiation proteins facilitate the transfer of E. coli DnaB helicase onto DNA.

The bacteriophage lambda P and Escherichia coli DnaC proteins are known to recruit the bacterial DnaB replicative helicase to initiator complexes assembled at the phage and bacterial origins, respectively. These specialized nucleoprotein assemblies facilitate the transfer of one or more molecules of DnaB helicase onto the chromosome; the transferred DnaB, in turn, promotes establishment of a processive replication fork apparatus. To learn more about the mechanism of the DnaB transfer reaction, we investigated the interaction of replication initiation proteins with single-stranded DNA (ssDNA). These studies indicate that both P and DnaC contain a cryptic ssDNA-binding activity that is mobilized when each forms a complex with the DnaB helicase. Concomitantly, the capacity of DnaB to bind to ssDNA, as judged by UV-crosslinking analysis, is suppressed upon formation of a P x DnaB or a DnaB x DnaC complex. This novel switch in ssDNA-binding activity evoked by complex formation suggests that interactions of P or DnaC with ssDNA may precede the transfer of DnaB onto DNA during initiation of DNA replication. Further, we find that the lambda O replication initiator enhances interaction of the P x DnaB complex with ssDNA. Partial disassembly of a ssDNA:O x P x DnaB complex by the DnaK/DnaJ/GrpE molecular chaperone system results in the transfer in cis of DnaB to the ssDNA template. On the basis of these findings, we present a general model for the transfer of DnaB onto ssDNA or onto chromosomal origins by replication initiation proteins.

A bipartite signaling mechanism involved in DnaJ-mediated activation of the Escherichia coli DnaK protein.

The DnaK and DnaJ heat shock proteins function as the primary Hsp70 and Hsp40 homologues, respectively, of Escherichia coli. Intensive studies of various Hsp70 and DnaJ-like proteins over the past decade have led to the suggestion that interactions between specific pairs of these two types of proteins permit them to serve as molecular chaperones in a diverse array of protein metabolic events, including protein folding, protein trafficking, and assembly and disassembly of multisubunit protein complexes. To further our understanding of the nature of Hsp70-DnaJ interactions, we have sought to define the minimal sequence elements of DnaJ required for stimulation of the intrinsic ATPase activity of DnaK. As judged by proteolysis sensitivity, DnaJ is composed of three separate regions, a 9-kDa NH2-terminal domain, a 30-kDa COOH-terminal domain, and a protease-sensitive glycine- and phenylalanine-rich (G/F-rich) segment of 30 amino acids that serves as a flexible linker between the two domains. The stable 9-kDa proteolytic fragment was identified as the highly conserved J-region found in all DnaJ homologues. Using this structural information as a guide, we constructed, expressed, purified, and characterized several mutant DnaJ proteins that contained either NH2-terminal or COOH-terminal deletions. At variance with current models of DnaJ action, DnaJ1-75, a polypeptide containing an intact J-region, was found to be incapable of stimulating ATP hydrolysis by DnaK protein. We found, instead, that two sequence elements of DnaJ, the J-region and the G/F-rich linker segment, are each required for activation of DnaK-mediated ATP hydrolysis and for minimal DnaJ function in the initiation of bacteriophage lambda DNA replication. Further analysis indicated that maximal activation of ATP hydrolysis by DnaK requires two independent but simultaneous protein-protein interactions: (i) interaction of DnaK with the J-region of DnaJ and (ii) binding of a peptide or polypeptide to the polypeptide-binding site associated with the COOH-terminal domain of DnaK. This dual signaling process required for activation of DnaK function has mechanistic implications for those protein metabolic events, such as polypeptide translocation into the endoplasmic reticulum in eukaryotic cells, that are dependent on interactions between Hsp70-like and DnaJ-like proteins.

Modulation of the ATPase activity of the molecular chaperone DnaK by peptides and the DnaJ and GrpE heat shock proteins.

Previous studies have demonstrated that the Escherichia coli DnaK, DnaJ, and GrpE heat shock proteins participate in the initiation of bacteriophage lambda DNA replication by mediating the required disassembly of a preinitiation nucleoprotein structure that is formed at the phage replication origin. To gain some understanding in a simpler system of how the DnaJ and GrpE cochaperonins influence the activity of DnaK, we have examined the effect of the cochaperonins on the weak intrinsic ATPase activity of the molecular chaperone DnaK in the presence and absence of peptide effectors. We have found that random sequence peptide chains of 8 or 9 amino acid residues in length yield optimal (10-fold) activation of the DnaK ATPase, whereas peptides with 5 or fewer residues fail to stimulate the ATPase of this bacterial hsp70 homologue. Furthermore, we have discovered that those peptides that interact best with DnaK, as judged by their KA as activators of ATP hydrolysis by DnaK, also act as strong inhibitors of lambda DNA replication in vitro. The inhibitory effect of peptides on lambda DNA replication was overcome by increasing the concentration of DnaK in the replication system. Diminished inhibition was also found when the replication system was supplemented with GrpE cochaperonin, a protein known to increase the effectiveness of DnaK action in lambda DNA replication. These and other results suggest that the peptide-binding site of DnaK is required for its function in lambda DNA replication. Apparently, peptides sequester free DnaK protein and block lambda DNA replication by reducing the amount of DnaK that is free to mediate disassembly of nucleoprotein preinitiation structures. In related studies, we have found that DnaJ, like short peptides, activates the intrinsic ATPase activity of DnaK. DnaJ, however, is substantially more potent in this regard, since it activates DnaK at concentrations 1000-fold below those required for a peptide of random sequence. By itself, the GrpE cochaperonin has no effect on the peptide-independent ATPase activity of DnaK, but GrpE does vigorously stimulate the peptide-dependent ATPase of the DnaK chaperone. Under steady-state conditions, the Vmax of ATP hydrolysis by DnaK was elevated approximately 40-fold by the presence of GrpE and saturating levels of peptides.

The chaperone function of DnaK requires the coupling of ATPase activity with substrate binding through residue E171.

Central to the chaperone function of Hsp70 stress proteins including Escherichia coli DnaK is the ability of Hsp70 to bind unfolded protein substrates in an ATP-dependent manner. Mg2+/ATP dissociates bound substrates and, furthermore, substrate binding stimulates the ATPase of Hsp70. This coupling is proposed to require a glutamate residue, E175 of bovine Hsc70, that is entirely conserved within the Hsp70 family, as it contacts bound Mg2+/ATP and is part of a hinge required for a postulated ATP-dependent opening/closing movement of the nucleotide binding cleft which then triggers substrate release. We analyzed the effects of dnaK mutations which alter the corresponding glutamate-171 of DnaK to alanine, leucine or lysine. In vivo, the mutated dnaK alleles failed to complement the delta dnaK52 mutation and were dominant negative in dnaK+ cells. In vitro, all three mutant DnaK proteins were inactive in known DnaK-dependent reactions, including refolding of denatured luciferase and initiation of lambda DNA replication. The mutant proteins retained ATPase activity, as well as the capacity to bind peptide substrates. The intrinsic ATPase activities of the mutant proteins, however, did exhibit increased Km and Vmax values. More importantly, these mutant proteins showed no stimulation of ATPase activity by substrates and no substrate dissociation by Mg2+/ATP. Thus, glutamate-171 is required for coupling of ATPase activity with substrate binding, and this coupling is essential for the chaperone function of DnaK.

Thermodynamic and structural analysis of the folding/unfolding transitions of the Escherichia coli molecular chaperone DnaK.

The thermal unfolding of the Escherichia coli 70 kDa heat shock protein, DnaK, exhibits three well defined transitions. At pH 7.6, these transitions are centered at 45.2, 58.0 and 73.3 degrees C. High sensitivity calorimetric scans as a function of pH indicate that the folding/unfolding behavior is well described by a four-state model which includes a delta H, tm and delta Cp for each state. Calorimetric scans of a 44 kDa N-terminal proteolytic fragment show a major transition centered at 47.5 degrees C (N1) and a minor transition at 79.4 degrees C (N2). A calorimetric scan of a 23 kDa C-terminal proteolytic fragment exhibits a low temperature peak at 58.5 degrees C (C1) and a high temperature peak at 70.6 degrees C (C2). Deconvolution analysis of the low temperature peak reveals that it is actually composed of two transitions of roughly equal delta H centered at 50.4 degrees C (C1a) and 58.2 degrees C(C1b). These experiments have allowed us to assign the transitions of the intact protein as follows. The low temperature transition of DnaK can be assigned to the N-terminal region on the basis of the similarity between the delta H and tm values for the low temperature transition and those obtained for the N1 transition of the isolated N-terminal fragment. This assignment is also supported by measurements of the intrinsic fluorescence emission as a function of temperature. DnaK contains a single tryptophan localized at residue 102 in the N-terminal domain of the protein. Additionally, calorimetric scans show that the tm of the low temperature transition increases by 9.2 degrees C in the presence of excess ADP, which is known to bind to the N-terminal domain. The middle transition can be assigned to the C1a and C1b transitions of the C-terminal fragment on the basis of the similarity of delta H and tm. In the intact protein C1a and C1b form a single cooperative unit; however, the cooperative interactions between these folding/unfolding domains are disrupted in the isolated fragment. The high temperature transition of the intact protein is composed of contributions from both the N-terminal and C-terminal regions of the protein. These studies have allowed us to develop a quantitative model of the folding/unfolding behavior of DnaK.

Different conformations for the same polypeptide bound to chaperones DnaK and GroEL.

The proteins DnaK (hsp70) and GroEL (cpn60) from Escherichia coli are prototypes of two classes of molecular chaperones conserved throughout evolution. The analysis of transferred nuclear Overhauser effects in two-dimensional NMR spectra is ideally suited to determine chaperone-bound conformations of peptides. The peptide vsv-C (amino-acid sequence KLIGVLSSLFRPK) stimulates the ATPase of BiP and Hsc70 (ref. 3) and the intrinsic ATPase of DnaK. The affinity of the vsv-C peptide for DnaK is greatly reduced in the presence of ATP. Here we analyse transferred nuclear Overhauser effects and show that the peptide is in an extended conformation while bound to DnaK but is helical when bound to GroEL. NMR also indicates that the mobility of the peptide backbone is reduced more by binding to DnaK than by binding to GroEL, whereas the side chains are less mobile when bound to GroEL.

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.

Transcriptional activation of bacteriophage lambda DNA replication in vitro: regulatory role of histone-like protein HU of Escherichia coli.

Initiation of bacteriophage lambda DNA replication in vivo and in crude in vitro systems is strongly dependent on transcription at or near the lambda replication origin (ori lambda). Through its capacity to prevent RNA polymerase-mediated 'transcriptional activation' of lambda DNA replication, the lambda cI repressor is capable of negatively regulating initiation of lambda DNA replication, even when all required replication proteins are present. Surprisingly, the strict requirement for transcriptional activation of lambda DNA replication was lost when lambda replication was initiated in an in vitro system composed of nine purified replication proteins [Mensa-Wilmot et al. (1989) J. Biol. Chem., 264, 2853-2861]. We have found that crude extracts of Escherichia coli contain proteins that are capable of restoring the physiological linkage between transcription and ori lambda-dependent replication when they are added to the nine-protein replication system. The protein primarily responsible for this effect has been purified and identified as protein HU, a histone-like protein that is a major constituent of the bacterial nucleoid. HU, when present at a 1:1 weight ratio with supercoiled ori lambda plasmid, is a potent inhibitor of lambda DNA replication in the nine-protein replication system. However, when the ori lambda template is transcribed by E. coli RNA polymerase, the HU-mediated inhibition of lambda DNA replication is abolished. HU does not inhibit propagation of lambda replication forks. Instead, HU apparently interferes with the assembly or function of nucleoprotein structures containing the E. coli DnaB helicase that are formed at ori lambda prior to priming and DNA synthesis. We suggest that the chromatin structure of the template DNA in the region surrounding ori lambda plays a central role in the negative regulation of the initiation of lambda DNA replication in vivo.

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.

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.

Heat shock protein-mediated disassembly of nucleoprotein structures is required for the initiation of bacteriophage lambda DNA replication.

Three Escherichia coli heat shock proteins, DnaJ, DnaK, and GrpE, are required for replication of the bacteriophage lambda chromosome in vivo. We show that the GrpE heat shock protein is not required for initiation of lambda DNA replication in vitro when the concentration of DnaK is sufficiently high. GrpE does, however, greatly potentiate the action of DnaK in the initiation process when the DnaK concentration is reduced to a subsaturating level. We demonstrate in the accompanying articles (Alfano, C. and McMacken, R. (1989) J. Biol. Chem. 264, 10699-10708; Dodson, M., McMacken, R., and Echols, H. (1989) J. Biol. Chem. 264, 10719-10725) that DnaJ and DnaK bind to prepriming nucleoprotein structures that are assembled at the lambda replication origin (ori lambda). Binding of DnaJ and DnaK completes the ordered assembly of an ori lambda initiation complex that also contains the lambda O and P initiators and the E. coli DnaB helicase. With the addition of ATP, the DnaJ and DnaK heat shock proteins mediate the partial disassembly of the initiation complex, and the P and DnaJ proteins are largely removed from the template. Concomitantly, on supercoiled ori lambda plasmid templates, the intrinsic helicase activity of DnaB is activated and DnaB initiates localized unwinding of the DNA duplex, thereby preparing the template for priming and DNA chain elongation. We infer from our results that DnaK and DnaJ function in normal E. coli metabolism to promote ATP-dependent protein unfolding and disassembly reactions. We also provide evidence that neither the lambda O and P initiators nor the E. coli DnaJ and DnaK heat shock proteins play a direct role in the propagation of lambda replication forks in vitro.

Reconstitution of a nine-protein system that initiates bacteriophage lambda DNA replication.

We have established an in vitro system, composed of highly purified bacteriophage lambda and Escherichia coli proteins, that specifically replicates supercoiled templates bearing the lambda replication origin (ori lambda). The complete system is composed of three groups of proteins: the virus-encoded initiator proteins (the lambda O and P proteins), the E. coli replication fork propagation machinery (single-stranded DNA-binding protein, dnaB helicase, dnaG primase, DNA polymerase III holoenzyme, and DNA gyrase), and two bacterial heat shock proteins (dnaJ and dnaK proteins). DNA replication in this system is initiated at or near ori lambda and proceeds unidirectionally rightwards through theta-structure intermediates, ultimately yielding a pair of intertwined daughter circles as the final product. In striking contrast to the situation in vivo and in crude in vitro systems, initiation of lambda DNA replication in the purified protein system does not require "transcriptional activation" of the origin region by E. coli RNA polymerase. We conclude that E. coli primase generates the primers for all leading and lagging strand DNA chains synthesized in this reconstituted lambda replication system.

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.