Binding of DnaA protein to a replication enhancer counteracts the inhibition of plasmid R6K gamma origin replication mediated by elevated levels of R6K pi protein.

Replication of the gamma origin of Escherichia coli plasmid R6K requires pi protein, encoded by the R6K pir gene, and many host factors, including DnaA protein. Pi has dual roles, activating replication at low levels and inhibiting replication at high levels. The inhibitory function of pi is counteracted by integration host factor and a specific sequence of the origin called the enhancer. This 106-bp DNA segment contains a binding site for DnaA protein (DnaA box 1). In this study, we mutated this site to determine if it was required for the enhancer's function. Using gamma origin derivative plasmids with the DnaA box 1 altered or deleted, we show that this site is necessary to protect the origin against levels of wild-type pi protein that would otherwise inhibit replication. To show that the base substitutions in DnaA box 1 weakened the binding of DnaA, we developed a new application of the agarose gel retardation assay. This quick and easy assay has broad applicability, as shown in binding studies with DNA fragments carrying a different segment of the R6K origin, the chromosomal origin (oriC), or the pUC origin. The gel retardation assay suggests a stoichiometry of DnaA binding different from that deduced from other assays.     

Translational options for the pir gene of plasmid R6K: multiple forms of the replication initiator protein pi.

The autogenously controlled pir gene of plasmid R6K was believed to encode a single polypeptide that plays multiple roles in the plasmid's biology. We have isolated an opal (op) mutant at the 18th codon of the pir coding frame which does not totally abolish translation of pir mRNA. In extracts of cells containing this mutation two translational products (35 kDa and 30.2 kDa) have been detected. We propose that the 35-kDa polypeptide produced by the pir18 op mutation contains Trp substituted for Arg18 as the result of an opal readthrough. Translation, which results in the 30.2-kDa polypeptide, originates downstream from the UGA stop signal created by the mutation. Moreover, we realize now that the 30.2-kDa polypeptide is also produced in cells containing a wild-type (wt) pir gene. The shorter variant of the pi protein lacks replication initiation and inhibition functions, as well as autorepressor activity in vivo. We also show that an in-frame fusion of seven N-terminal codons of the trpE gene with a pir gene lacking the first two codons produces two polypeptides which replace the 35-kDa pi protein and are of similar molecular weight. Thus, at least three options exist in the translation of the wt pir mRNA. Start codons are most likely at codon positions 1, 6 or 7, and 36 or 38. Each of these five AUG codons is preceded by a consensus ribosome-binding site (RBS).     

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.     

Binding modes of the initiator and inhibitor forms of the replication protein pi to the gamma ori iteron of plasmid R6K

Discerning the interactions between initiator protein and the origin of replication should provide insights into the mechanism of DNA replication initiation. In the gamma origin of plasmid R6K, the Rep protein, pi, is distinctive in that it can bind the seven 22-bp iterons in two forms; pi monomers activate replication, whereas pi dimers act as inhibitors. In this work, we used wild type and variants of the pi protein with altered monomer/dimer ratios to study iteron/pi interactions. High resolution contact mapping was conducted using multiple techniques (missing base contact probing, methylation protection, base modification, and hydroxyl radical footprinting), and the electrophoretic separation of nucleoprotein complexes allowed us to discriminate between contact patterns produced by pi monomers and dimers. We also isolated iteron mutants that affected the binding of pi monomers (only) or both monomers and dimers. The mutational studies and footprinting analyses revealed that, when binding DNA, pi monomers interact with nucleotides spanning the entire length of the iteron. In contrast, pi dimers interact with only the left half of the iteron; however, the retained interactions are strikingly similar to those seen with monomers. These results support a model in which Rep protein dimerization disturbs one of two DNA binding domains important for monomer/iteron interaction; the dimer/iteron interaction utilizes only one DNA binding domain.     

The replication initiator protein of plasmid R6K tagged with beta-galactosidase shows sequence-specific DNA-binding

We have tagged the replication initiator protein of the plasmid R6K near the C-terminal end by fusion, in the correct reading frame, with the 89 amino acid long N-terminal alpha-donor polypeptide of beta-galactosidase of E. coli. This fusion was carried out with recombinant DNA methods. The protein chimera thus generated retained the activities of both initiation of DNA replication in vivo at the replication origin gamma of R6K and hydrolysis of beta-galactopyranoside when complemented in vivo with the alpha-acceptor polypeptide coded by the lac Z gene containing the M15 deletion. Using the simple and convenient assay for detecting beta-galactosidase, we have partially purified the tagged replication initiator, and have demonstrated that the protein binds to specific DNA sequences of the R6K chromosome. The protein bound to DNA sequences located at two places in the 5' untranslated leader region of the initiator protein cistron.     

A compact nucleoprotein structure is produced by binding of Escherichia coli integration host factor (IHF) to the replication origin of plasmid R6K.

Understanding the role of Escherichia coli histone-like protein integration host factor (IHF) in replication of R6K plasmid (Dellis, S., and Filutowicz, M. (1991) J. Bacteriol. 173, 1279-1286) requires detailed analyses of the interaction of IHF protein with the plasmid's replication origin (gamma ori). We describe an electron microscopic analysis which shows that a compact structure can be formed in the presence of IHF, in which, on average, a 102-base pair (bp) ori segment is involved. IHF.gamma ori complexes also undergo a two-step conformational change in an IHF concentration-dependent manner when analysed by band shift assay. We believe that the DNA is bent at low IHF concentrations, but folded at high IHF concentrations. This idea is supported by the fact that electrophoretic mobility of the IHF.gamma ori complexes is faster at higher concentrations of IHF. Furthermore, it is shown that the formation of a compact nucleoprotein structure depends on the two regions flanking the AT-rich segment; the iterons to the right and the 106-bp ori domain to the left. Finally we show that IHF protects the entire AT-rich segment of the ori against nuclease cleavage. In addition to the protection, an altered cleavage pattern by DNase I, in the presence of high levels of IHF, was observed within the iterons but not within the 106-bp domain of the ori. Implications of the IHF-mediated gamma ori folding as a possible mechanism protecting the ori from replication inhibition by R6K initiator protein tau are discussed.     

The integration host factor of Escherichia coli binds to multiple sites at plasmid R6K gamma origin and is essential for replication.

Examination of the effect of the himA and himD mutants of E. coli on the maintenance of plasmid R6K has revealed that the gamma origin-containing replicons cannot be established in any of the mutants deficient in the production of E. coli Integration Host Factor (IHF). Contrary, the R6K derivatives containing other origins of the plasmid (alpha and/or beta) replicate in a host lacking functional IHF protein. We show that IHF protein binds specifically to a segment of the replication region which is essential for the activity of all three R6K origins. Mapping the IHF binding sequence with neocarzinostatin showed that the protein protects three segments of the origin: two strong binding sites reside within an AT-rich block, while the third, considerably weaker site is separated from the other two by a cluster of the seven 22 bp direct repeats. These seven repeats have been shown previously to bind the R6K-encoded initiator protein pi. We also demonstrate that the establishment of pi-origin complexes prior to IHF addition prevents the binding of the IHF protein to the gamma origin. The binding sequences of IHF and pi proteins do not overlap, therefore, we propose that the binding of pi protein alters the structure of the DNA and thereby prevents the subsequent binding of IHF protein.     

Activity of the replication terminus of plasmid R6K in hybrid replicons in Escherichia coli.

Hybrids between the antibiotic resistance plasmid R6K and RSF2124, a derivative of plasmid ColEl were constructed in vetro. These hybrids exhibit the replication properties of both parents in Escherichia coli, including the use of either the R6K or the ColEl origin of replication during logarithmic phase growth. Incompatibility properties of both parental plasmids also are expressed by the hybrid plasmids. Analysis of replicative intermediates showed that the asymmetric terminus of R6K was functional in the hybrids. In the absence of protein synthesis where replication of the hybrid plasmid is initiated only from the ColE1 origin, the R6K terminus either prevents or severely impedes the progress of the replication fork. The activity of the R6K terminus region is expressed independent of the direction of DNA replication and in the absence of the R6K replication origin.     

Roles of a 106-bp origin enhancer and Escherichia coli DnaA protein in replication of plasmid R6K.

A dnaA 'null' strain could not support replication of intact plasmid R6K or derivatives containing combinations of its three replication origins (alpha, gamma, beta). DnaA binds in vitro to sites in two functionally distinct segments of the central gamma origin. The 277-bp core segment is common to all three origins and contains DnaA box 2, which cannot be deleted without preventing replication. Immediately to the left of the core lies the 106-bp origin enhancer, which contains DnaA box 1. When the origin enhancer is deleted, the core alone can still initiate replication if levels of wt pi protein are decreased or if copy-up pi mutant proteins are provided in trans. DnaA does not effect expression of R6K replication initiator protein pi, although several DnaA boxes were identified in the coding segment of the pir gene, which encodes pi. Together these data suggest that a single DnaA box, 2, is sufficient for initiation from the gamma origin and might be sufficient for initiation from the gamma origin and might be sufficient and required for the activity of the alpha and beta origins as well. Implications of the DnaA protein binding to two domains of the gamma origin and the role of the 106-bp origin enhancer in replication are discussed.     

Membrane-bound fractions of R6K plasmid DNA in Escherichia coli.

The intracellular location of plasmid DNA has been of interest in an effort to understand the maintenance of these molecules. We have employed a simple procedure which enables us to isolate from exponentially grown cells on sucrose gradients membrane-complexed forms of R6K plasmid DNA. Electron micrographs identified the complexing of membrane fractions to circular forms of R6K DNA. Biochemical studies of the complexed R6K molecules showed the presence of membrane-specific proteins and suggested that complexing of R6K DNA was primarily with inner membrane fractions of Escherichia coli.     

Altered (copy-up) forms of initiator protein pi suppress the point mutations inactivating the gamma origin of plasmid R6K.

The R6K gamma origin core contains the P2 promoter, whose -10 and -35 hexamers overlap two of the seven binding sites for the R6K-encoded pi protein. Two mutations, P2-201 and P2-203, which lie within the -35 region of P2, are shown to confer a promoter-down phenotype. We demonstrate here that these mutations prevent replication of a gamma origin core plasmid. To determine whether or not the reduced promoter activity caused by these mutations is responsible for their effect on replication, we generated two new mutations (P2-245-6-7 and P2-246) in the -10 hexamer of the P2 promoter. Although these new mutations inhibit P2 activity as much as the P2-201 and P2-203 mutations, they do not prevent replication of the gamma origin core. Therefore, activity of the P2 promoter does not appear to be required for replication. We also show that the inability of the gamma origin to function in the presence of the P2-201 and P2-203 mutations is reversed by the hyperactive variants of pi protein called copy-up pi. This suppression occurs despite the fact that in vivo dimethyl sulfate methylation protection patterns of the gamma origin iterons are identical in cells producing wild-type pi and those producing copy-up pi variants. We discuss how the P2-201 and P2-203 mutations could inhibit replication of the gamma origin core and what mechanisms might allow the copy-up pi mutants to suppress this deficiency.     

Interaction of the plasmid R6K-encoded replication initiator protein with its binding sites on DNA

Initiation of DNA replication in plasmid R6K is potentiated by the plasmid-encoded 35 kd replication initiator protein. We had previously reported that the initiator bound to two regions of R6K DNA called Site I and Site II. Using DNAase I footprinting technique we have demonstrated that the initiator bound to seven tandem repeats of a 22 bp long sequence in Site I. In Site II, the initiator bound to a single repeat having the same consensus sequence and to two partial repeats that most likely overlap the promoter of the initiation protein cistron. Using dimethyl sulfate as a chemical probe, we have determined the purine residues of Site I and Site II that make contact with the initiator protein. The results show that eight out of nine contact points per repeat in Site I were located on one of the two strands of the DNA. The binding of the initiator to the Site II sequence could explain the observed autoregulation of the synthesis of the initiator protein by promoter occlusion.     

Structure-based functional analysis of the replication protein of plasmid R6K: key amino acids at the pi/DNA interface

In previous work, we characterized the bases in an iteron of plasmid R6K that are important for the binding of pi protein monomers and dimers. Here we investigate the following six amino acids of pi, encoded by pir, hypothesized to be important for DNA contact: Ser71, Try74, Gly131, Gly211, Arg225, and Arg254.     

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.     

Replication of plasmid R6K in DNA polymerase deficient mutants of Escherichia coli.

The plasmid R6K has been introduced into a number of Escherichia coli polymerase deficient (pol) mutants. In polCts mutants transferred to the nonpermissive temperature to inactivate polymerase III, R6K replicates but the replication products have a density in dye-CsCl gradients intermediate between supercoiled and linear forms. This aberrant replication requires normal cellular levels of polymerase I since it does not occur in polA polCts mutants. Normal R6K replication and maintenance occur in a polA polB polC+ host, however, we cannot tell from our experiments wheather polymerase I or III replicates R6K in polA+ polC+ host. Polymerase II, the polB gene product, has no detectable role in R6K replication.