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.     

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.     

Mechanism of origin activation by monomers of R6K-encoded pi protein

One recurring theme in plasmid duplication is the recognition of the origin of replication (ori) by specific Rep proteins that bind to DNA sequences called iterons. For plasmid R6K, this process involves a complex interplay between monomers and dimers of the Rep protein, pi, with seven tandem iterons of gamma ori. Remarkably, both pi monomers and pi dimers can bind to iterons, a new paradigm in replication control. Dimers, the predominant form in the cell, inhibit replication, while monomers facilitate open complex formation and activate the ori. Here, we investigate a mechanism by which pi monomers out-compete pi dimers for iteron binding, and in so doing activate the ori. With an in vivo plasmid incompatibility assay, we find that pi monomers bind cooperatively to two adjacent iterons. Cooperative binding is eliminated by insertion of a half-helical turn between two iterons but is diminished only slightly by insertion of a full helical turn between two iterons. These studies show also that pi bound to a consensus site promotes occupancy of an adjacent mutated site, another hallmark of cooperative interactions. pi monomer/iteron interactions were quantified using a monomer-biased pi variant in vitro with the same collection of two-iteron constructs. The cooperativity coefficients mirror the plasmid incompatibility results for each construct tested. pi dimer/iteron interactions were quantified with a dimer-biased mutant in vitro and it was found that pi dimers bind with negligible cooperativity to two tandem iterons.     

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.     

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.     

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.     

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.     

AT-rich region and repeated sequences - the essential elements of replication origins of bacterial replicons

Repeated sequences are commonly present in the sites for DNA replication initiation in bacterial, archaeal, and eukaryotic replicons. Those motifs are usually the binding places for replication initiation proteins or replication regulatory factors. In prokaryotic replication origins, the most abundant repeated sequences are DnaA boxes which are the binding sites for chromosomal replication initiation protein DnaA, iterons which bind plasmid or phage DNA replication initiators, defined motifs for site-specific DNA methylation, and 13-nucleotide-long motifs of a not too well-characterized function, which are present within a specific region of replication origin containing higher than average content of adenine and thymine residues. In this review, we specify methods allowing identification of a replication origin, basing on the localization of an AT-rich region and the arrangement of the origin's structural elements. We describe the regularity of the position and structure of the AT-rich regions in bacterial chromosomes and plasmids. The importance of 13-nucleotide-long repeats present at the AT-rich region, as well as other motifs overlapping them, was pointed out to be essential for DNA replication initiation including origin opening, helicase loading and replication complex assembly. We also summarize the role of AT-rich region repeated sequences for DNA replication regulation.     

Positioning and the specific sequence of each 13-mer motif are critical for activity of the plasmid RK2 replication origin

The minimal replication origin of the broad-host-range plasmid RK2, oriV, contains five iterons which are binding sites for the plasmid-encoded replication initiation protein TrfA, four DnaA boxes, which bind the host DnaA protein, and an AT-rich region containing four 13-mer sequences. In this study, 26 mutants with altered sequence and/or spacing of 13-mer motifs have been constructed and analysed for replication activity in vivo and in vitro. The data show that the replacement of oriV 13-mers by similar but not identical 13-mer sequences from Escherichia coli oriC inactivates the origin. In addition, interchanging the positions of the oriV 13-mers results in greatly reduced activity. Mutants with T/A substitutions are also inactive. Furthermore, introduction of single-nucleotide substitutions demonstrates very restricted sequence requirements depending on the 13-mer position. Only two of the mutants are host specific, functional in Pseudomonas aeruginosa but not in E. coli. Our experiments demonstrate considerable complexity in the plasmid AT-rich region architecture required for functionality. It is evident that low internal stability of this region is not the only feature contributing to origin activity. Our studies suggest a requirement for sequence-specific protein interactions within the 13-mers during assembly of replication complexes at the plasmid origin.     

Rifampicin-resistant initiation of chromosome replication from oriC in ihf mutants

IHF (integration host factor) mutants exhibit asynchronous initiation of chromosome replication from oriC as determined from flow cytometric analysis of cultures where RNA synthesis was inhibited with rifampicin. However, the run-out kinetics of chromosome replication in ihf mutants shows that they continue to produce oriCs for some time in the absence of RNA synthesis resulting in a twofold increase in the oriC per mass ratio. An ihf dnaA double mutant did not exhibit this continued increase of the oriC per mass ratio. This indicates that ihf mutants can initiate replication from oriC in a rifampicin-resistant initiation mode but requires fully functional DnaA protein. The origin per mass ratio, determined by a quantitative Southern blotting technique, showed that the ihf mutants had an origin per mass ratio that was 60% of the wild type although it had a normal DnaA protein concentration. This shows that the initiation mass was substantially higher in the ihf mutants. The oriC per terminus ratio, which was also determined by Southern blotting, was very low in the ihf mutant, although it grew with the same doubling times as the wild-type strain. This indicates that cells lacking IHF replicate their chromosome(s) very fast.     

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.     

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.     

The integration host factor of Escherichia coli binds to bent DNA at the origin of replication of the plasmid pSC101

The integration host factor (IHF) of Escherichia coli is necessary for maintenance of pSC101. The protein binds specifically to the replication origin of the plasmid, in the AT-rich region located immediately adjacent to the left, weak binding site for the plasmid-encoded initiator protein. DNAase I and OH- radical footprinting experiments showed that IHF protects 49 bp of the DNA at the origin region. Methylation protection analyses revealed that IHF contacts purine residues in both the major and minor grooves of the DNA. Electrophoretic analyses showed that IHF binds to bent DNA, and the protein binding further enhances the degree of DNA bending. Site-directed mutagenesis of three of the contact points not only abolished binding of the protein to the DNA but also inactivated the replication origin. Therefore, binding of IHF to the ori sequence most probably is necessary for initiation of plasmid replication.     

Structure-function analysis of the SaPIbov1 replication origin in Staphylococcus aureus

The SaPIs and their relatives are phage satellites and are unique among the known bacterial pathogenicity islands in their ability to replicate autonomously. They possess a phage-like replicon, which is organized as two sets of iterons arrayed symmetrically to flank an AT-rich region that is driven to melt by the binding of a SaPI-specific initiator (Rep) to the flanking iterons. Extensive deletion analysis has revealed that Rep can bind to a single iteron, generating a simple shift in a gel mobility assay; when bound on both sides, a second retarded band is seen, suggesting independent binding. Binding to both sites of the ori is necessary but not sufficient to melt the AT-rich region and initiate replication. For these processes, virtually the entire origin must be present. Since SaPI replication can be initiated on linear DNA, it is suggested that bilateral binding may be necessary to constrain the intervening DNA to enable Rep-driven melting.     

A DNA segment conferring stable maintenance on R6K gamma-origin core replicons.

The plasmid R6K gamma origin consists of two adjacent modules, the enhancer and the core, and requires R6K initiator protein pi for replication. While the core alone can replicate at a low level of wild-type pi protein, we show here that host cells do not stably maintain core plasmids. The presence of the enhancer segment confers stable inheritance on core plasmids without a significant change in average plasmid copy number. Deletions and site-directed mutagenesis indicated that the stability of core plasmids is not mediated by binding sites or consensus sequences in the enhancer for DnaA, pi protein, gyrase, Fis, or Dcm methylase. Proper segregation of core plasmids requires only the R6K stb or stability-related region, which includes the 20-bp segment of the 100-bp enhancer adjacent to the core. The use of the pi 116 mutant protein, which increases plasmid copy number fourfold, does not stabilize core plasmids lacking the enhancer. We also show that at an elevated level of wild-type pi, the gamma-origin plasmid is unstable, even in the presence of the enhancer. We discuss the differences and similarities between the R6K stability system and those found in other plasmids.