Integration host factor of Escherichia coli reverses the inhibition of R6K plasmid replication by pi initiator protein.

Integration host factor (IHF) protein is the only host-encoded protein known to bind and to affect replication of the gamma origin of Escherichia coli plasmid R6K. We examined the ability of R6K origins to replicate in cells lacking either of the two subunits of IHF. As shown previously, the gamma origin cannot replicate in IHF-deficient cells. However, this inability to replicate was relieved under the following conditions: underproduction of the wild-type pi replication protein of R6K or production of normal levels of mutant pi proteins which exhibit relaxed replication control. The copy number of plasmids containing the primary R6K origins (alpha and beta) is substantially reduced in IHF-deficient bacteria. Furthermore, replication of these plasmids is completely inhibited if the IHF-deficient strains contain a helper plasmid producing additional wild-type pi protein. IHF protein has previously been shown to bind to two sites within the gamma origin. These sites flank a central repeat segment which binds pi protein. We propose a model in which IHF binding to its sites reduces the replication inhibitor activity of pi protein at all three R6K origins.     

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.     

Conformational changes induced by integration host factor at origin gamma of R6K and copy number control.

We have investigated the role of integration host factor (IHF) in the replication of plasmid R6K by studying the maintainance of the plasmid in a strain of Escherichia coli that lacks both subunits of IHF and in an isogenic wild type strain and found that all three origins, alpha, beta, and gamma, were functional in the absence of IHF; however, loss of IHF reduced the copy number of those replicons initiating solely from ori gamma by 5-fold. Concomitant loss of direct repeats within the origin that bind the R6K replication initiator protein, Pi, resulted in a further reduction in copy number. Using gel mobility shift analysis, we showed that IHF bound specifically only to one site within the A/T rich region of the minimal origin adjacent to the Pi binding sites. The origin region possessed no intrinsic DNA curvature although IHF induced a strong bend upon binding. Combination footprinting with different orders of addition of Pi and IHF suggested that there was no cooperativity between the two proteins with regard to DNA binding. Hydroxyl-radical footprinting revealed hypersensitive asymmetric periodic cleavage sites within the origin region in the presence of IHF that extended over 200 base pairs and a localized perturbation of cleavage chemistry. The presence of periodic cleavages was dependent upon the presence of the wild type R6K origin sequence and was not observed when the IHF binding site was positioned adjacent to a heterologous sequence. We observed that the conformational changes induced by IHF upon binding to the R6K origin were negatively correlated with the observed decrease in copy number, and therefore, origin conformation altered by protein-DNA interaction may play an important role in the regulation of replication initiation.     

Two alternative structures can be formed by IHF protein binding to the plasmid R6K gamma origin.

Escherichia coli integration host factor (IHF) contributes to the regulation of R6K plasmid copy number by counteracting the inhibitory activity of the plasmid-encoded replication protein pi. Two IHF-binding sites (ihf1 and ihf2) flank seven iterons in the origin which bind pi protein. As previously shown by electron microscopy, IHF can compact a large segment of the R6K gamma origin DNA, encompassing site ihf1, an AT-rich domain containing ihf1, and some of the seven iterons located downstream of ihf1. We termed this phenomenon IHF-mediated DNA folding. This folding requires a high IHF concentration, and the region of the origin (replication enhancer) located to the left of the AT-rich domain. However, site ihf2 is not necessary in forming the folded structure. As reported here, IHF binding to ihf2 can be detected in gel mobility shift assays only if the leftmost enhancer region is absent. Sites ihf1 and ihf2 each contain two consensus IHF sequences. Site-directed mutagenesis was performed to determine which sequences are recognized by IHF protein and which sites are involved in forming the various gamma origin-IHF complexes. Finally, we define the boundaries of protection from DNaseI digestion when IHF is bound to ihf2. We propose a model in which IHF protein bound to ihf1, in the absence of the enhancer region, facilitates IHF binding to ihf2.     

Activation of distant replication origins in vivo by DNA looping as revealed by a novel mutant form of an initiator protein defective in cooperativity at a distance.

We have isolated mutants of the pi initiator protein of the plasmid R6K that are defective in DNA looping in vitro but retain their normal DNA binding affinity for the primary binding sites (iterons) at the gamma origin/enhancer. One such looping defective mutant called R6 was determined to be a proline to leucine change at position 46 near the N terminus of the pi protein. Using a set of genetic assays that discriminate between the activation of the gamma origin/enhancer from those of the distantly located alpha and beta origins, we show that the looping defective initiator protein fails to activate the alpha and beta origins but derepresses initiation from the normally silent gamma origin in vivo. The results conclusively prove that DNA looping is required to activate distant replication origins located at distances of up to 3 kb from the replication enhancer.     

Conformational changes in a replication origin induced by an initiator protein

The replication initiator protein of the plasmid R6K binds to seven contiguous 22 bp direct repeats that form an indispensable part of the three replication origins alpha, beta, and gamma. Binding of the initiator to the direct repeats induced a marked bending of the region of gamma replication origin. Binding of the initiator also promoted unwinding of the origin DNA by at least two turns. Distamycin appeared to antagonize the binding of the initiator to the seven 22 bp direct repeats. At the appropriate DNA and protein concentrations the initiator enhanced topoisomerase-induced catenation of the origin containing supercoiled DNA but not of DNA lacking the origin sequence. Thus, the initiator protein caused significant changes in the secondary and tertiary structures of the replication origin.     

Mechanistic studies of initiator-initiator interaction and replication initiation.

Unlike the chromosome of Escherichia coli that needs only one replication initiator protein (origin recognition protein) called DnaA, many plasmid replicons require dual initiators: host-encoded DnaA and a plasmid-encoded origin recognition protein, which is believed to be the major determinant of replication control. Hitherto, the relative mechanistic roles of dual initiators in DNA replication were unclear. Here, we present the first evidence that DnaA communicates with the plasmid-encoded pi initiator of R6K and contacts the latter at a specific N-terminal region. Without this specific contact, productive unwinding of plasmid ori gamma and replication is abrogated. The results also show that DnaA performs different roles in host and plasmid replication as revealed by the finding that the ATP-activated form of DnaA, while indispensable for oriC replication, was not required for R6K replication. We have analyzed the accessory role of the DNA bending protein, integration host factor (IHF), in promoting initiator-origin interaction and have found that IHF significantly enhances the binding of DnaA to its cognate site. Collectively, the results further advance our understanding of replication initiation.     

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.     

Cooperative binding of initiator protein to replication origin conferred by single amino acid substitution.

The replication initiator protein pi of plasmid R6K binds seven 22 bp direct repeats (DR) in the gamma origin. The pi protein also binds to an inverted repeat (IR) in the operator of its own gene, pir, which lies outside the gamma origin sequences. A genetic system was devised to select for pi protein mutants which discriminate between IR and DR (York et al., Gene (Amst.) 116, 7-12, 1992; York and Filutowicz, J. Biol. Chem. 268, 21854-21861, 1993). From this selection the mutant pi S87N protein was isolated which is deficient in repressing the pir gene's expression because it cannot bind to IR at the pir gene operator. Remarkably, we discovered that pi S87N binds to DR cooperatively under conditions where wt pi binds independently. Moreover, the pi S87N is more active as a replication initiator in vivo when supplied at the same level as wt pi. Quantitative binding assays showed that both wt pi and pi S87N bind a DNA fragment containing a single DR unit with a similar affinity (Kd = 0.3 x 10(-12) M). Thus, cooperativity of pi S87N is most likely achieved through altered interactions between promoters bound at adjacent DR units.     

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.     

Monomer/dimer ratios of replication protein modulate the DNA strand-opening in a replication origin

DNA opening is an essential step in the initiation of replication via the Cairns mode of replication. The opening reaction was investigated in a gamma ori system by using hyperactive variants of plasmid R6K-encoded initiator protein, pi. Reactivity to KMnO4 (indicative of opening) within gamma ori DNA occurred in both strands of a superhelical template upon the combined addition of wt pi, DnaA and integration host factor (IHF), each protein known to specifically bind gamma ori. IHF, examined singly, enhanced reactivity to KMnO4. The IHF-dependent reactive residues, however, are distinct from those dependent on pi (wt and hyperactive variants). Remarkably, the DNA helix opening does not require IHF and/or DnaA when hyperactive variants of pi were used instead of wt protein. We present three lines of evidence consistent with the hypothesis that DNA strand separation is facilitated by pi monomers despite the fact that both monomers and dimers of the protein can bind to iterons (pi binding sites). Taken together, our data suggest that pi elicits its ability to modulate plasmid copy number at the DNA helix-opening step.     

Plasmid-encoded initiation protein is required for activity at all three origins of plasmid R6K DNA replication in vitro

DNA replication of plasmid R6K initiates at three unique sites, ori alpha, ori beta, and ori gamma. Replicating DNA molecules of a deletion derivative of R6K were synthesized in an in vitro system containing pi protein fraction from cells carrying a mini-R6K derivative that produced only this initiation protein as an R6K-encoded protein and analyzed by electron miscroscopy. Requirement of pi protein for the activity of all these three replication origins in vitro was verified. Frequencies of initiation at the three origins were almost equal.     

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.     

DNA segments of the IncX plasmid R485 determining replication and incompatibility with plasmid R6K

The expression of incompatibility properties between the IncX plasmids R6K and R485 of Escherichia coli was examined. For small autonomously replicating derivatives of both plasmid elements, the requirements for incompatibility expression include a functional R485 replicon and an active R6K beta-origin region. Functional R6K alpha and gamma origins are not directly involved in incompatibility expression between R6K and R485. A trans-acting replication system was constructed for plasmid R485. It consists of a 3.2-(kb) DNA fragment of R485 that specifies a product(s) in trans which supports replication from an R485 origin plasmid. A minimal R485 origin region of 591 bp was derived utilizing this trans-acting replication system and the nucleotide sequence of this origin region determined. The most striking feature of the sequence is the presence of six tandem 22-bp nucleotide sequence direct repeats.     

Structural properties of the beta origin of replication of plasmid R6K

The beta origin of replication of plasmid R6K, one of three active R6K origins of replication, requires most or all of a 1962-base pair (bp) sequence for activity. The nucleotide sequence of a portion of this functional beta origin was determined in an earlier study (Stalker, D., Kolter, R., and Helinski, D. (1982) J. Mol. Biol. 161, 33-43). In this work, the sequence of the remaining portion of this 1964-bp segment was obtained. In addition to its activity as an origin of replication, this sequence also contains sufficient information for autonomous replication in Escherichia coli. A 277-bp region containing seven 22-bp direct repeats is present at one end of the beta origin segment (Stalker, D., Kolter, R., and Helinski, D. (1979) Proc. Natl. Acad. Sci. U. S. A. 76, 1150-1154) while the other end contains a 140-bp sequence that includes a relaxation complex site. The 277-bp direct repeat region is required for activity of the beta origin. The start of the beta origin of replication as mapped by electron microscopy (Crosa, J. (1980) J. Biol. Chem. 255, 11075-11077) lies approximately 1000 bp away from the 277-bp region. The pi structural gene, which makes up most of the sequence between the direct repeats and the beta origin, is required in cis for beta origin activity. The pi protein also is required for beta origin activity but can be provided in trans. The nucleotide sequence just beyond the pi structural gene and within or near the start of beta origin of replication contains an open reading frame for a 151-amino acid protein. Deletions ranging from 94 bp to 1590 bp were obtained within the 1964-bp beta origin region. In every case, the deletion results in loss of origin activity even when the deleted sequence plus adjacent regions are provided in trans. These observations suggest a requirement for a specific secondary structure over an extensive region for beta origin activity.     

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.     

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.