DNA bending of pSC101 ori induced by Rep,a replication initiator protein and IHF

Purified Rep (or RepA) protein, a replication initiator of plasmid pSC101, is present almost solely in the dimer form, and its binding activity for the directly repeated sequences (iterons) in the replication origin (ori) is very low. When Rep protein was treated with guanidine hydrochloride followed by renaturation, it was shown to bind to the iterons with very high efficiency. A gel shift experiment suggested that guanidine-treated Rep bound to iterons as a monomer form. The Rep monomer bound noncooperatively to the three iterons and induced bending of the DNA helix axis in the same direction (about 100 degrees ). The configuration of the IHF box that is a binding site of another DNA bending protein IHF, the three iterons and an AT-rich region between these sequences was important for efficient bending of the ori region. Furthermore, a mutant Rep protein (Rep(IHF)) which can support the plasmid replication in IHF-deficient host cells was purified, and it was found that affinity of the Rep(IHF) monomer for iterons was similar to that of wild-type Rep and bent DNA only 14 degrees more strongly than did the wild-type Rep. Rep(IHF)-dependent plasmid replication, however, required both enhancer regions, par and IR-1, in addition to "core ori" as a minimal essential ori, whereas only one of these two enhancers was necessary for wild-type Rep-dependent replication. How Rep(IHF) can support plasmid replication in the absence of IHF is 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.     

Bending of the origin of replication of E. coli by binding of IHF at a specific site

In studies of DNA replication in Escherichia coli, an important question concerns the role of the initiator protein DnaA. This protein is known to bind to a specific 9-bp sequence in the origin of replication, but it is not understood how it can recognize another, relatively distant, 13-bp sequence that has no homology to the binding site but is where the DnaA protein serves its catalytic function in the initiation of DNA replication. This effect of DnaA might be achieved by bending of DNA in this region. I have searched for putative binding sites for integration host factor (IHF), a protein known to bend DNA. Here I report the finding of an IHF binding site in the E. coli origin and present direct evidence that IHF binds and causes DNA bending in this region. On the basis of these results I propose a model wherein formation of a higher-order nucleoprotein structure would facilitate the action of DnaA protein in the initiation events.     

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.     

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.     

Replication enhancer-independent mutation increases the co-operativity with which an initiator protein binds its origin

The plus-strand replication origin of bacteriophage fl has a bipartite structure consisting of a required core origin region and an adjacent A + T-rich enhancer sequence that potentiates replication approximately 100-fold. The core origin binds the initiator protein (gpII) and the enhancer binds the Escherichia coli integration host factor (IHF). gpII binds the core origin in two steps, forming a binding intermediate (complex I) and a functional complex for nicking (complex II). We have used a double-label gel binding assay to determine the stoichiometry of the gpII-origin interaction. The results indicate that complex I contains two gpII molecules per origin, and complex II contains four gpII molecules per origin. Enhancer-independent mutations in gpII allow wild-type levels of replication in the absence of either the enhancer or IHF. We have examined the binding of an enhancer-independent gpII mutant (mp1) protein to the replication origin. The mp1 mutation in gpII (Met40----Ile) increases the co-operativity with which the protein binds to form complex II. In addition, the mutant gpII forms both complexes with a DNA fragment containing only two (beta-gamma) of the three repeats from the core origin sequence, while the wild-type protein forms only complex I with this fragment. We discuss how a mutation that increases the co-operativity of binding of an initiator protein might stimulate DNA replication.     

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.     

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.     

Replication of pSC101: effects of mutations in the E. coli DNA binding protein IHF

Summary We have shown that the plasmid pSC101 is unable to be maintained in strains of E. coli carrying deletions in the genes himA and hip which specify the pleitropic heterodimeric DNA binding protein, IHF. We show that this effect is not due to a modulation of the expression of the pSC101 RepA protein, required for replication of the plasmid. Inspection of the DNA sequence of the essential replication region of pSC101 reveals the presence of a site, located between the DnaA binding-site and that of RepA, which shows extensive homology with the consensus IHF binding site. The proximity of the sites suggests that these three proteins, IHF, DnaA, and RepA may interact in generating a specific DNA structure required for initiation of pSC101 replication.     

Studies on the binding of integration host factor (IHF) and TraM to the origin of transfer of the IncFV plasmid pED208

The origin of transfer (oriT) of the IncFV plasmid pED208 contains a region with three binding sites for both the plasmid-encoded TraM protein and the integration host factor (IHF) of Escherichia coli, a sequence-specific DNA-binding protein. One region, containing overlapping TraM and IHF binding sites, could be interpreted as containing two binding sites for each protein. Using gel retardation assays, an affinity constant for IHF binding to the three main sites was estimated in the presence and absence of 0.1 M potassium glutamate, which increased the avidity of IHF binding to the weaker sites by two orders of magnitude. DNase I protection analyses and electron microscopy were used to determine the affinity of IHF for oriT-containing DNA in the presence and absence of TraM. The binding of IHF and TraM was found to be non-cooperative by the two techniques employed. Electron microscopy also demonstrated that IHF bent the oriT region in a manner consistent with its previously determined mode of action, while TraM had no discernible effect on the appearance of the DNA. This suggested that IHF and TraM interact with a 295 by sequence in the oriT region and organize it into a higher order structure that may have a role in the initiation of DNA transfer and control of traM expression.     

Specific interaction between the initiator protein (Rep) and origin of plasmid ColE2-P9

The replication initiator protein (Rep) of plasmid ColE2-P9 (ColE2) is multifunctional. We are interested in how Rep binds to the origin (Ori) to perform various functions. We used the wild type and variants of Rep to study the Rep-Ori interaction by both in vitro and in vivo approaches, including biochemical analyses of protein-DNA interactions and an in vivo replication assay. We identified three regions (I, II, and III) of Rep, located in the C-terminal half, and three corresponding binding sites (I, II, and III) in Ori which are important for Rep-Ori interaction. We showed that region I, containing a putative helix-turn-helix motif, is necessary and sufficient for specific Ori recognition, interacting with site I of the origin DNA from the major groove. Region II interacts with site II of the origin DNA, from the adjacent minor groove in the left half of Ori, and region III interacts with site III, next to the template sequence for primer synthesis, which is one and one-half turn apart from site I on the opposite surface of the origin DNA. A putative linker region located between the two DNA binding domains (regions II and III) was identified, which might provide Rep an extended conformation suitable for binding to the two separate sites in Ori. Based on the results presented in this paper, we propose a model for Rep-Ori interaction in which Rep binds to Ori as a monomer.     

Distinct functions of the two specificity determinants in replication initiation of plasmids ColE2-P9 and ColE3-CA38

The plasmid ColE2-P9 Rep protein specifically binds to the cognate replication origin to initiate DNA replication. The replicons of the plasmids ColE2-P9 and ColE3-CA38 are closely related, although the actions of the Rep proteins on the origins are specific to the plasmids. The previous chimera analysis identified two regions, regions A and B, in the Rep proteins and two sites, alpha and beta, in the origins as specificity determinants and showed that when each component of the region A-site alpha pair and the region B-site beta pair is derived from the same plasmid, plasmid DNA replication is efficient. It is also indicated that the replication specificity is mainly determined by region A and site alpha. By using an electrophoretic mobility shift assay, we demonstrated that region B and site beta play a critical role for stable Rep protein-origin binding and, furthermore, that 284-Thr in this region of the ColE2 Rep protein and the corresponding 293-Trp of the ColE3 Rep protein mainly determine the Rep-origin binding specificity. On the other hand, region A and site alpha were involved in the efficient unwinding of several nucleotide residues around site alpha, although they were not involved in the stable binding of the Rep protein to the origin. Finally, we discussed how the action of the Rep protein on the origin involving these specificity determinants leads to the plasmid-specific replication initiation.     

Parental strand recognition of the DNA replication origin by the outer membrane in Escherichia coli.

The outer membrane of Escherichia coli binds the origin of DNA replication (oriC) only when it is hemimethylated. We report here the results of a footprinting analysis with the outer membrane which demonstrate that its interaction with oriC occurs mainly at the left moiety of the minimal oriC, where 10 out of 11 Dam methylation sites are concentrated. Two regions, flanking the Integration Host Factor (IHF) sites, are preferentially recognized at the minimum membrane concentration at which oriC plasmid replication is inhibited in vitro. We have identified the putative proteins involved in hemimethylated oriC binding and cloned one of the corresponding genes (hobH). The purified LacZ-HobH fusion protein specifically binds oriC DNA at the same preferential sites as the membrane. A mutant of the hobH gene reveals partial asynchronous initiation of DNA replication.     

Identification of cellular factors that bind specifically to the Epstein-Barr virus origin of DNA replication.

The specific binding of HeLa cell factors to DNA sequences at the Epstein-Barr virus (EBV) latent origin of DNA replication was detected by gel shift experiments and DNase I footprinting analysis. These cellular proteins protected at least five discrete regions of the DNA replication origin. The viral protein required for EBV plasmid replication, EBV nuclear antigen 1 (EBNA-1), binds to specific sequences within the origin region. The HeLa cell proteins competed with EBNA-1 for binding to EBV origin DNA in vitro, leading to the possibility that these cellular proteins regulate EBV DNA replication by displacing EBNA-1 at the origin sites.     

Conserved response regulator CtrA and IHF binding sites in the alpha-proteobacteria Caulobacter crescentus and Rickettsia prowazekii chromosomal replication origins

CzcR is the Rickettsia prowazekii homolog of the Caulobacter crescentus global response regulator CtrA. CzcR expression partially compensates for developmental defects in ctrA mutant C. crescentus cells, and CzcR binds to all five CtrA binding sites in the C. crescentus replication origin. Conversely, CtrA binds to five similar sites in the putative R. prowazekii replication origin (oriRp). Also, Escherichia coli IHF protein binds over a central CtrA binding site in oriRp. Therefore, CtrA and IHF regulatory proteins have similar binding patterns in both replication origins, and we propose that CzcR is a global cell cycle regulator in R. prowazekii.     

Boundaries of the pSC101 minimal replicon are conditional.

The DNA segment essential for plasmid replication commonly is referred to as the core or minimal replicon. We report here that host and plasmid genes and sites external to the core replicon of plasmid pSC101 determine the boundaries and competence of the replicon and also the efficiency of partitioning. Missense mutations in the plasmid-encoded RepA protein or mutation of the Escherichia coli topoisomerase I gene enable autonomous replication of a 310-bp pSC101 DNA fragment that contains only the actual replication origin plus binding sites for RepA and the host-encoded DnaA protein. However, in the absence of a repA or topA mutation, the DNA-bending protein integration host factor (IHF) and either of two cis-acting elements are required. One of these, the partitioning (par) locus, is known to promote negative DNA supercoiling; our data suggest that the effects of the other element, the inverted repeat (IR) sequences that overlap the repA promoter, are mediated through the IR's ability to bind RepA. The concentrations of RepA and DnaA, which interact with each other and with plasmid DNA in the origin region (T. T. Stenzel, T. MacAllister, and D. Bastia, Genes Dev. 5:1453-1463, 1991), also affect both replication and partitioning. Our results, which indicate that the sequence requirements for replication of pSC101 are conditional rather than absolute, compel reassessment of the definition of a core replicon. Additionally, they provide further evidence that the origin region RepA-DnaA-DNA complex initiating replication of pSC101 also mediates the partitioning of pSC101 plasmids at cell division.