The DnaK-DnaJ-GrpE chaperone system activates inert wild type pi initiator protein of R6K into a form active in replication initiation

The plasmid R6K is an interesting model system for investigating initiation of DNA replication, not only near the primary binding sites of the initiator protein pi but also at a distance, caused by pi -mediated DNA looping. An important milestone in the mechanistic analysis of this replicon was the development of a reconstituted replication system consisting of 22 different highly purified proteins (Abhyankar, M. A., Zzaman, S., and Bastia, D. (2003) J. Biol. Chem. 278, 45476-45484). Although the in vitro reconstituted system promotes ori gamma-specific initiation of replication by a mutant form of the initiator called pi*, the wild type (WT) pi is functionally inert in this system. Here we show that the chaperone DnaK along with its co-chaperone DnaJ and the nucleotide exchange factor GrpE were needed to activate WT pi and caused it to initiate replication in vitro at the correct origin. We show further that the reaction was relatively chaperone-specific and that other chaperones, such as ClpB and ClpX, were incapable of activating WT pi. The molecular mechanism of activation appeared to be a chaperone-catalyzed facilitation of dimeric inert WT pi into iteron-bound monomers. Protein-protein interaction analysis by enzyme-linked immunosorbent assay revealed that, in the absence of ATP, DnaJ directly interacted with pi but its binary interactions with DnaK and GrpE and with ClpB and ClpX were at background levels, suggesting that pi is recruited by protein-protein interaction with DnaJ and then fed into the DnaK chaperone machine to promote initiator activation.     

Binding of purified wild-type and mutant pi initiation proteins to a replication origin region of plasmid R6K

The three replication origins of the antibiotic resistance plasmid R6K require for their activity in Escherichia coli a DNA segment containing seven 22 base-pair direct repeats and a plasmid-encoded initiation protein (pi). The pi protein functions in the negative control of R6K replication, in addition to its requirement for the initiation of replication. Construction of a plasmid containing the pi structural gene (pir) downstream from the inducible pR promoter of bacteriophage lambda provided high levels of production of pi protein in E. coli. The pi protein was purified and shown to possess general DNA binding properties with a preference for DNA fragments containing the gamma origin of replication, the operator region of the pir gene and the R6K beta-origin region. Velocity sedimentation analysis indicates that the pi protein exists as a dimer in its native form. Agarose gel electrophoresis analysis of pi-gamma-origin complexes suggests that one pi dimer binds to each copy of the 22 base-pair direct repeats in the gamma origin region. Purified mutant pi protein obtained from a temperature-sensitive initiation mutant (pir 105-ts) exhibited temperature-sensitive binding activity to the gamma-origin region, whereas two mutant proteins exhibiting a high copy number phenotype were unaltered (pir104-cop) or slightly reduced (pir1-cop) in binding activity. The patterns of DNase I protection and enhancement were similar for the wild-type and mutant proteins examined.     

Regulation of DNA replication by iterons: an interaction between the ori2 and incC regions mediated by RepE-bound iterons inhibits DNA replication of mini-F plasmid in Escherichia coli.

In bacteria, plasmids and some DNA viruses, DNA replication is initiated and regulated by binding of initiator proteins to repetitive sequences. To understand the control mechanism we used the plasmid mini-F, whose copy number is stringently maintained in Escherichia coli, mainly by its initiator protein RepE and the incC region. The monomers of RepE protein bound to incC iterons, which exert incompatibility in trans and control the copy number of mini-F plasmid in cis. Many incompatibility defective mutants carrying mutations in their incC iterons had lost the affinity to bind to RepE, while one mutant retained high level binding affinity. The mutated incC mini-F plasmids lost the function to control the copy number. The copy number of the wild-type mini-F plasmid did not increase in the presence of excess RepE. These results suggested that the control of replication by incC iterons does not rely on their capacity to titrate RepE protein. Using a ligation assay, we found that RepE proteins mediated a cross-link structure between ori2 and incC, for which the dimerization domain of RepE and the structure of incC seem to be important. The structure probably causes inhibition of extra rounds of DNA replication initiation on mini-F plasmids, thereby keeping mini-F plasmid at a low copy number.     

An initiator protein for plasmid R6K DNA replication. Mutations affecting the copy-number control

Two kinds of mutations affecting the copy-number control of plasmid R6K were isolated and identified in an initiator pi protein by DNA sequencing. Firstly, a temperature-sensitive replication mutation, ts22, with decreased copy number results in a substitution of threonine to isoleucine at position 138 of the 305-amino-acid pi protein. Secondly, a high-copy-number (cop21) mutant was isolated from this ts mutant and was identified by an alteration of alanine to serine at position 162. This cop21 mutation suppressed the Ts character and was recessive to the wild-type allele in the copy control.     

Multipartite regulation of rctB, the replication initiator gene of Vibrio cholerae chromosome II

Replication initiator proteins in bacteria not only allow DNA replication but also often regulate the rate of replication initiation as well. The regulation is mediated by limiting the synthesis or availability of initiator proteins. The applicability of this principle is demonstrated here for RctB, the replication initiator for the smaller of the two chromosomes of Vibrio cholerae. A strong promoter for the rctB gene named rctBp was identified and found to be autoregulated in Escherichia coli. Promoter activity was lower in V. cholerae than in E. coli, and a part of this reduction is likely to be due to autorepression. Sequences upstream of rctBp, implicated earlier in replication control, enhanced the repression. The action of the upstream sequences required that they be present in cis, implying long-range interactions in the control of the promoter activity. A second gene specific for chromosome II replication, rctA, reduced rctB translation, most likely by antisense RNA control. Finally, optimal rctBp activity was found to be dependent on Dam. Increasing RctB in trans increased the copy number of a miniplasmid carrying oriCII(VC), implying that RctB can be rate limiting for chromosome II replication. The multiple modes of control on RctB are expected to reduce fluctuations in the initiator concentration and thereby help maintain chromosome copy number homeostasis.     

Negative control of plasmid pSC101 replication by increased concentrations of both initiator protein and iterons

Increased intracellular concentrations of the initiator protein Rep (or RepA) interfere with pSC101 DNA replication, and mutated Rep proteins that result in an increase in plasmid copy numbers do not inhibit the replication. A rep mutant (rep(inh)) defective in the inhibitory activity was isolated and found to be a new high copy number mutant. The inhibitory function of Rep was enhanced by the coexistence of directly repeated sequences (DR; iterons) in the replication origin region (ori), but not by the inverted repeat sequences (IR) in ori and the rep promoter. This synergistic effect of Rep and DR sequences for the replication inhibition was dependent on their intracellular concentrations. Considering that DR sequences are the specific binding sites of the Rep monomer form, the Rep monomer-DR complex might be responsible for the inhibition of the plasmid replication. Furthermore, the Rep monomer in the crude cell extracts facilitated dimerization of DR DNA fragments by DNA ligase. Neither synergistic inhibitory function with DR nor Rep mediated dimerization of DR DNA was observed in high copy number mutant Rep proteins. The role of the Rep-iteron complex in the copy number control of pSC101 is discussed.     

Participation of the Escherichia coli heat shock proteins DnaJ, DnaK, and GrpE in autorepression of the P1 plasmid repA promoter

The replicon of the low copy number plasmid P1 uses the three Escherichia coli heat shock proteins DnaJ, DnaK, and GrpE for the efficient initiation of its DNA replication. The only P1-encoded protein required for plasmid replication is the initiator, RepA. Binding of RepA to the origin also represses the promoter for the repA gene, which is located within the origin. We found that repression is incomplete in E. coli strains with mutations in the dnaJ, dnaK, or grpE genes. Since there is no decrease in RepA concentration in the mutant strains, the mutations are likely to affect the protein-DNA or protein-protein reactions required for repression, thereby decreasing RepA binding at its promoter. We also showed that the deficit in repression can be overcome by providing excess RepA, implying that the mechanism of repression is not altered in the mutant strains. Since repression requires RepA binding to the origin, a binding deficit might account for the replication defect in the heat shock mutants.     

Association of cyclin A and cdk2 with SV40 DNA in replication initiation complexes is cell cycle dependent

The cell cycle is driven by the sequential activation of a family of cyclin-dependent kinases (CDK) in association with cyclins. In mammalian cells the timing of activation of cyclin A-associated kinase activity coincides with the onset of DNA synthesis in S-phase. Using in vitro replication of SV40 origin-containing DNA as a model system, we have analyzed the proteins associated with DNA during initiation of DNA replication in S-phase cell extracts. This analysis reveals that, in addition to replication initiation proteins, cyclin A and cdk2 are also specifically associated with DNA. The association of cyclin A and cdk2 with DNA during initiation is cell cycle regulated and occurs specifically in the presence of SV40 origin-containing plasmid and SV40 T antigen (the viral replication initiator protein). The interactions among proteins involved in initiation play an important role in DNA replication. We therefore investigated the ability of cyclin A and cdk2 to associate with replication initiation proteins. Under replication initiation conditions, cyclin A and cdk2 from S-phase extracts specifically associate with SV40 T antigen. Further, the interaction of cyclin A-cdk2 with SV40 T antigen is mediated via cyclin A, and purified recombinant cyclin A associates directly with SV40 T antigen. Taken together, our results suggest that cyclin A and cdk2 are components of the SV40 replication initiation complex, and that protein-protein interactions between cyclin A-cdk2 and T antigen may facilitate the association of cyclin A-cdk2 with the complex. Received: 30 July 1996; in revised form: 25 September 1996 / Accepted: 8 October 1996     

Role of papillomavirus E1 initiator dimerization in DNA replication

Viral initiator proteins are polypeptides that form oligomeric complexes on the origin of DNA replication (ori). These complexes carry out a multitude of functions related to initiation of DNA replication, and although many of these functions have been characterized biochemically, little is understood about how the complexes are assembled. Here we demonstrate that loss of one particular interaction, the dimerization between E1 DNA binding domains, has a severe effect on DNA replication in vivo but has surprisingly modest effects on most individual biochemical activities in vitro. We conclude that the dimer interaction is primarily required for initial recognition of ori.     

rctB mutations that increase copy number of Vibrio cholerae oriCII in Escherichia coli

RctB serves as the initiator protein for replication from oriCII, the origin of replication of Vibrio cholerae chromosome II. RctB is conserved between members of Vibrionaceae but shows no homology to known replication initiator proteins and has no recognizable sequence motifs. We used an oriCII based minichromosome to isolate copy-up mutants in Escherichia coli. Three point mutations rctB(R269H), rctB(L439H) and rctB(Y381N) and one IS10 insertion in the 3'-end of the rctB gene were obtained. We determined the maximal C-terminal deletion that still gave rise to a functional RctB protein to be 165 amino acids. All rctB mutations led to decreased RctB-RctB interaction indicating that the monomer is the active form of the initiator protein. All mutations also showed various defects in rctB autoregulation. Loss of the C-terminal part of RctB led to overinitiation by reducing binding of RctB to both rctA and inc regions that normally serve to limit initiation from oriCII. Overproduction of RctB(R269H) and RctB(L439H) led to a rapid increase in oriCII copy number. This suggests that the initiator function of the two mutant proteins is increased relative to the wild-type.     

Control of plasmid DNA replication by iterons: no longer paradoxical

Replication origins of a family of bacterial plasmids have multiple sites, called iterons, for binding a plasmid-specific replication initiator protein. The iteron-initiator interactions are essential for plasmid replication as well as for inhibition of plasmid over-replication. The inhibition increases with plasmid copy number and eventually shuts plasmid replication off completely. The mechanism of inhibition appears to be handcuffing, the coupling of origins via iteron-bound initiators that block origin function. The probability of a trans-reaction such as handcuffing is expected to increase with plasmid copy number and diminish with increases in cell volume, explaining how the copy number can be maintained in a growing cell. Control is also exerted at the level of initiator synthesis and activation by chaperones. We propose that increases in active initiators promote initiation by overcoming handcuffing, but handcuffing dominates when the copy number reaches a threshold. Handcuffing should be ultrasensitive to copy number, as the negative control by iterons can be stringent (switch-like).     

A single amino acid alteration in the initiation protein is responsible for the DNA overproduction phenotype of copy number mutants of plasmid R6K.

A novel type of high copy-number (cop) mutants of a mini-R6K plasmid were isolated. The mutations were mapped in the pir gene which encodes the pi initiation protein for plasmid R6K DNA replication. They resulted in an alteration by substitution of a single amino acid: threonine to isoleucine at the 108th position for the cop41, and proline to serine at the 113th position for the cop50, of the 305 amino acid pi protein. The cop41 mutation in the pi protein was found to be trans-dominant over the wild-type allele in the copy control of plasmid R6K. Moreover, it was shown that the altered pi protein was not overproduced in maxicells carrying this mutant plasmid and had a higher affinity to the repeated sequence which is present in the pir promoter region. Most likely the mutated pi protein also interacts more efficiently with the same repeated sequences, a target of pi, in the replication origin region and increases the frequency of the initiation event per cell division.     

Measurement of gene expression by translational coupling: effect of copy mutations on pT181 initiator synthesis.

We have prepared and analyzed two types of gene fusion between the replication initiator gene, repC, and the reporter gene, blaZ, in order to investigate the relationship between pT181 plasmid copy number and RepC initiator protein production. A series of pT181 copy mutant plasmids, with copy numbers ranging from 70 to 800 copies per cell, were analyzed. In one type of gene fusion used in this study, blaZ was translationally coupled to the C-terminal end of the repC coding sequence such that native forms of both proteins were produced. This gene fusion arrangement, which permitted monitoring of RepC production (as BlaZ activity) by plasmids using the protein for their own replication, demonstrated a linear relationship, with one exception, between RepC production and plasmid copy number over a 20-fold range. In the second type of fusion, blaZ was translationally fused to the C-terminal end of repC. As the translational fusion did not produce active RepC protein, the fusion-containing pT181 derivatives were maintained in a strain which provided RepC in trans, and were thus analyzed at constant copy number. In contrast to previous analyses of this type, our translational fusion constructs expressed repC at levels proportional to the copy numbers of the plasmids from which the fusions were prepared. Using these data, we have calculated a minimum figure for the number of RepC molecules synthesized per replication event.     

Regulation of the dnaA product in Escherichia coli.

Summary When an E. coli mutant (CRT46, dnaA46), thermosensitive in the initiation of DNA replication, grows at intermediate temperatures its DNA/mass ratio is somewhat lower than normal, but the cells possess an excess of initiation capacity, which can be expressed in the absence of proteins synthesis and lead to the accumulation of anomalously high amounts of DNA. A shift-up in temperature causes inhibition of initiation, and at the same time the production of initiation capacity is accelerated. After a shift-down in temperature initiation is released but the production of capacity is inhibited. The initiation capacity is thermolabile.The simplest explanation of these observations is that the dnaA product has a dual role: a positive function as an initiator of replication and a negative control function in its own synthesis.